Patent Application
20220296108
This application claims priority to Korean Patent Application No. 10-2019-0061415 field on May 24, 2019, the entire contents of which are incorporated herein by reference. In addition, this application claims priority to Korean Patent Application No. 10-2019-0061416 field on May 24, 2019, the entire contents of which are incorporated herein by reference. In addition, this application claims priority to Korean Patent Application No. 10-2019-0061417 field on May 24, 2019, the entire contents of which are incorporated herein by reference.
This research was conducted by the Korea Advanced Institute of Science and Technology with the support of the Ministry of Health & Welfare's disease recovery technology development project, under the research title “Development of in-vivo microscopy for imaging of pathophysiology of pulmonary hypertension at cellular scale” (project number: HI15C0399030017). In addition, this research was conducted by the Korea Advanced Institute of Science and Technology with the support of the Ministry of Science and ICT's individual basic research project, under the research title “Ultrafast laser scanning intravital microscopy needle probe-based deep-tissue microvisualization technology for human disease pathophysiology analysis and diagnosis” (project number: NRF-2017R1E1A1A01074190).
The present specification discloses a method for quantitation of microcirculation in a subject, in which a functional capillary ratio is calculated from a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject, and an apparatus for measuring microcirculation in a subject. In addition, the present specification discloses a method for providing information on microcirculatory disorder in a subject, in which a dynamic element in target factors is analyzed from a plurality of motion images of the target factors over time in a second blood stream passing through the capillaries of the subject, and an apparatus for diagnosis of microcirculatory disorder in a subject. In addition, the present specification discloses a composition for prevention or treatment of lung injury, which contains an inhibitor against the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries and alleviates microcirculatory disorder in the lung, a screening method, a method for providing information for diagnosis of lung injury and disorder, and a composition and a for diagnosis of pulmonary microcirculatory disorder.
Microcirculation is the circulation of blood in small blood vessels such as arterioles, venules, capillaries, lymph capillaries, etc. It is the core of metabolism, where supply and discharge of materials occur. The quantitation of microcirculation has been achieved by measurement of functional capillary density (FCD), by counting the number of functional capillaries by allocating 1 if a red blood cell that pass through the blood vessel within 30 seconds and allocating 0 otherwise. The existing method of measuring functional capillary density is limited in that the difference in functionality is not distinguished for the case where one red blood cell passes through one capillary in 30 seconds and the case where hundreds of red blood cell pass therethrough. In addition, since the pulmonary capillary has a network structure, there is limitation in calculating the density because it is difficult to define the beginning and end of each capillary. Furthermore, the method of measuring functional capillary density merely informs the functional capillary density of the microcirculatory system as a numerical value and fails to visualize the change in functional capillaries as images.
Sepsis is one of the foremost contributor to hospital deaths (Torio C M, Moore B J. National Inpatient Hospital Costs: The Most Expensive Conditions by Payer, 2013: Statistical Brief #204. Healthcare Cost and Utilization Project (HCUP) Statistical Briefs, Rockville (Md.), 2016; Hall M J, Levant S, DeFrances C J. Trends in inpatient hospital deaths: National Hospital Discharge Survey, 2000-2010. NCHS Data Brief 2013 (118): 1-8). It is a syndrome characterized by a dysregulated response of the host to invading pathogens, which involves hemodynamic alterations that lead to multiple life-threatening organ dysfunctions (Singer M, Deutschman C S, Seymour C W, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard G R, Chiche J D, Coopersmith C M, Hotchkiss R S, Levy M M, Marshall J C, Martin G S, Opal S M, Rubenfeld G D, van der Poll T, Vincent J L, Angus D C. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016: 315(8): 801-810; Angus D C, van der Poll T. Severe sepsis and septic shock. N Engl J Med 2013: 369(9): 840-851). Among the organs damaged by sepsis, the lung is the first and most frequent organ to fail, and acute respiratory failure syndrome (ARDS) or acute lung injury (ALI) is one of the most critical prognostic factors for mortality in patients with sepsis (Lagu T, Rothberg M B, Shieh M S, Pekow P S, Steingrub J S, Lindenauer P K. Hospitalizations, costs, and outcomes of severe sepsis in the United States 2003 to 2007. Crit Care Med 2012: 40(3): 754-761). Despite intense research efforts aimed at treating sepsis-induced acute lung injury, no effective therapy aimed at microcirculation is available (Thompson B T, Chambers R C, Liu K D. Acute Respiratory Distress Syndrome. N Engl J Med 2017: 377(6): 562-572). Although it has been identified that dead space assessment could provide significant clinical data in acute lung injury (Nuckton T J, Alonso J A, Kallet R H, Daniel B M, Pittet J F, Eisner M D, Matthay M A. Pulmonary dead-space fraction as a risk factor for death in the acute respiratory failure syndrome. N Engl J Med 2002: 346(17): 1281-1286), until now, it has remained a hypothesis in terms of the impairment of the lung alveoli that are ventilated but not perfused. Admittedly, acute respiratory failure syndrome is a poorly understood syndrome with regard to the association between lung injury and microcirculation (Ryan D, Frohlich S, McLoughlin P. Pulmonary vascular dysfunction in ARDS. Ann Intensive Care 2014: 4: 28). Recently, a study reported the evidence of thrombi in the pulmonary vasculature which was limited as an ex-vivo study, and to date, the in-vivo process of neutrophil influx and the consequent disorder of pulmonary microcirculation remain to be investigated (Matthay M A, Ware L B, Zimmerman G A. The acute respiratory failure syndrome. J Clin Invest 2012: 122(8): 2731-2740; Yuan Y, Alwis I, Wu MCL, Kaplan Z, Ashworth K, Bark D, Jr., Pham A, McFadyen J, Schoenwaelder S M, Josefsson E C, Kile B T, Jackson S P. Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia. Sci Transl Med 2017: 9(409)).
Unregulated recruitment and activation of neutrophils could induce organ injury through release of inflammatory mediators, including cytokines and reactive oxygen species (ROS) (Grommes J, Soehnlein O. Contribution of neutrophils to acute lung injury. Mol Med 2011: 17(3-4): 293-307; Matute-Bello G, Downey G, Moore B B, Groshong S D, Matthay M A, Slutsky A S, Kuebler W M, Acute Lung Injury in Animals Study G. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 2011: 44(5): 725-738). Yet, the existing knowledge on the detailed dynamic behavior of neutrophils in the pulmonary microcirculation is mostly limited to speculation gleaned from observations in the systemic circulation (Phillipson M, Kubes P. The neutrophil in vascular inflammation. Nat Med 2011: 17(11): 1381-1390). Because the diameter of neutrophils is greater than that of the pulmonary capillaries, neutrophils should be deformed to pass through the capillaries, which is a relatively time-consuming process (Doerschuk C M. Mechanisms of leukocyte sequestration in inflamed lungs. Microcirculation 2001: 8(2): 71-88). This process, referred to as neutrophil sequestration, was originally described for cells other than the freely circulating group of neutrophils within the lung and has been observed, to some extent, using macroscopic radiolabeling imaging devices (MacNee W, Selby C. New perspectives on basic mechanisms in lung disease; Neutrophil traffic in the lungs: role of hemodynamics, cell adhesion, and deformability. Thorax 1993: 48(1): 79-88). Indeed, previous studies have demonstrated neutrophil sequestration in lung capillaries; however, the mechanism of how the neutrophil sequestration event leads to acute lung injury or acute respiratory failure syndrome remains unknown (Kuebler W M, Borges J, Sckell A, Kuhnle G E, Bergh K, Messmer K, Goetz A E. Role of L-selectin in leukocyte sequestration in lung capillaries in a rabbit model of endotoxemia. Am J Respir Crit Care Med 2000: 161(1): 36-43; Lien D C, Henson P M, Capen R L, Henson J E, Hanson W L, Wagner W W, Jr., Worthen G S. Neutrophil kinetics in the pulmonary microcirculation during acute inflammation. Lab Invest 1991: 65(2): 145-159). Therefore, when considering the importance and obscurity of the pulmonary microcirculation in acute lung injury or acute respiratory failure syndrome, understanding the changes in the pulmonary microcirculation including the dynamic behavior of neutrophils is imperative for elucidation of the pathophysiology, which may lead to novel treatment strategies for sepsis-induced acute lung injury or acute respiratory failure syndrome (Looney M R, Bhattacharya J. Live imaging of the lung. Annu Rev Physiol 2014: 76: 431-445).
The inventors of the present disclosure have studied on a method for quantitation of microcirculation in a subject based on area rather than density, and have completed the present disclosure. To investigate pulmonary microcirculation in sepsis-induced lung injury, the inventors of the present disclosure used a custom-designed video-rate laser scanning confocal microscope in combination with a micro-suction-based lung imaging window (Kim P, Puoris′haag M, Cote D, Lin C P, Yun S H. In vivo confocal and multiphoton microendoscopy. J Biomed Opt 2008: 13(1): 010501; Han S, Lee S J, Kim K E, Lee H S, Oh N, Park I, Ko E, Oh S J, Lee Y S, Kim D, Lee S, Lee D H, Lee K H, Chae S Y, Lee J H, Kim S J, Kim H C, Kim S, Kim S H, Kim C, Nakaoka Y, He Y, Augustin H G, Hu J, Song P H, Kim Y I, Kim P, Kim I, Koh G Y. Amelioration of sepsis by TIE2 activation-induced vascular protection. Sci Transl Med 2016: 8(335): 335ra355). Using the intravital lung imaging system, they directly identified the alteration of microcirculatory perfusion in a sepsis-induced acute lung injury (ALI) model, and completed the present disclosure. Furthermore, for development of a composition for preventing or treating lung injury, the inventors of the present disclosure observed the neutrophils of a model having pulmonary microcirculatory disorder using the custom-designed video-rate laser scanning confocal microscope and completed the present disclosure by identifying a target for improvement of pulmonary microcirculatory disorder in the neutrophils.
In an aspect, the present disclosure is directed to providing a method and an apparatus for quantitation of microcirculation in a subject based on a functional capillary ratio (FCR), which is the ratio of functional capillary area measured from a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject to the total capillary area, and a computer program executing the method.
In another aspect, the present disclosure is directed to providing a method and an apparatus for providing information for diagnosis of microcirculatory disorder in a subject, which allows fast and accurate diagnosis of microcirculatory disorder in a subject using a functional capillary ratio (FCR) of the subject calculated by the method for quantitation of microcirculation described above, and a computer program and a system executing the method.
In another aspect, the present disclosure is directed to providing a method for providing information for diagnosis of microcirculatory disorder in a subject by analyzing dynamic elements of target factors such as sequestration time, track displacement length, track length, track velocity or track meandering index from a plurality of motion images of the target factors over time in a second blood stream passing through the capillaries of the subject, and an apparatus for diagnosis of microcirculatory disorder.
In another aspect, the present disclosure is directed to providing a composition for prevention or treatment of lung injury, which is capable of alleviating microcirculatory disorder in the lung by inhibiting the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries, thereby allowing erythrocyteserythrocytes to smoothly pass through the pulmonary capillaries and increasing gas exchange in a subject suffering from pulmonary microcirculatory disorder, and a method for screening a substance for preventing or treating lung injury.
In another aspect, the present disclosure is directed to providing a method for providing information useful for diagnosis of pulmonary microcirculatory disorder, and a composition and a kit for diagnosis of pulmonary microcirculatory disorder.
In an aspect, the present disclosure provides a method for quantitation of microcirculation in a subject, which includes: a step of obtaining a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject; a step of measuring functional capillary area in which the target factors move in the first blood stream from the plurality of motion images; and a step of calculating functional capillary ratio (FCR) according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area. [Formula 1]
In another aspect, the present disclosure provides an apparatus for measuring microcirculation in a subject, which acquires quantitative data on the microcirculation in the subject based on a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject according to Formula 1. Specifically, the apparatus may include: an imaging unit imaging target factors in a first blood stream passing through the capillaries of the subject; and a measuring unit acquiring quantitative data on the microcirculation in the subject according to Formula 1 based on the images imaged by the imaging unit.
In another aspect, the present disclosure provides a method for providing information for diagnosis of microcirculatory disorder in a subject, which includes a step of acquiring information for diagnosing microcirculatory disorder in the subject from the functional capillary ratio (FCR) calculated by the method for quantitation of microcirculation in a subject described above.
In another aspect, the present disclosure provides a computer program stored in a computer-readable medium, which is associated with a hardware and executes the method for quantitation of microcirculation or the method for providing information for diagnosis of microcirculatory disorder in a subject.
In another aspect, the present disclosure provides a method for providing information for diagnosis of microcirculatory disorder, which includes: a step of obtaining a plurality of motion images of target factors over time in a second blood stream flowing through the capillaries of the subject; a step of analyzing one or more dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of the target factors in the second blood stream from the plurality of motion images; and a step of acquiring information for diagnosis of microcirculatory disorder in the subject from the dynamic element analysis result.
In another aspect, the present disclosure provides an apparatus for diagnosis of microcirculatory disorder, which includes: an imaging unit imaging target factors in a second blood stream passing through the capillaries of a subject; and an analysis unit analyzing one or more dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of the target factors in the second blood stream based on the plurality of motion images imaged by the imaging unit.
In another aspect, the present disclosure provides a composition for preventing, alleviating or treating lung injury, which contains an inhibitor against the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries as an active ingredient, and prevents, alleviates or treats lung injury through alleviation of microcirculatory disorder in the lung.
In another aspect, the present disclosure provides a method for screening a substance for preventing, alleviating or treating lung injury, which includes: (a) a step of preparing a lung injury model; (b) a step of the lung injury model with a test substance; (c) a step of measuring the change in the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries of the lung injury model caused by the test substance; and (d) a step of identifying whether the test substance increases a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocyteserythrocytes pass to the total capillary area of the lung injury model.
In another aspect, the present disclosure provides a method for providing information for diagnosis of pulmonary microcirculatory disorder, which includes: a step of measuring the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils isolated from the pulmonary capillaries of a test subject; and a step of identifying a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocyteserythrocytes pass to the total capillary area of the lung of the test subject.
In another aspect, the present disclosure provides a composition for diagnosis of pulmonary microcirculatory disorder, which contains a reagent for detecting mRNAs or proteins of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries.
In another aspect, the present disclosure provides a kit for diagnosis of pulmonary microcirculatory disorder, which contains a reagent for detecting mRNAs or proteins of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries.
The present disclosure relates to quantification of microcirculation of a subject based on a functional capillary ratio (FCR), or the ratio of functional capillary area, which is the ratio of functional capillary area measured from a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject to the total capillary area. Because microcirculation can be quantified based on area rather than density, the area where through which one red blood cell passes and the area where through which a plurality of erythrocyteserythrocytes pass can be distinguished.
Through this, microcirculation can be quantified more easily, conveniently and accurately because the space (area) through which erythrocyteserythrocytes pass actually can be reflected, and it is possible to quantify the microcirculation network which is difficult to quantify based on density.
In addition, because the ratio of the functional capillary area to the total capillary area can be visually identified with one image, the location of functional capillaries, i.e., the capillaries through which more erythrocyteserythrocytes pass, can be identified conveniently, and microcirculatory disorder can be identified accurately and quickly based on the quantification result.
In addition, the present disclosure relates to a method for providing information for diagnosis of microcirculatory disorder in a subject by analyzing a dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of target factors in a second blood stream flowing through the capillaries of the subject from a plurality of motion images of the target factors over time, and an apparatus for diagnosis of microcirculatory disorder. The method and apparatus provide the advantage that microcirculatory disorder in a subject can be diagnosed more accurately and quickly by acquiring information on the motion of neutrophils within capillaries easily and conveniently.
In addition, the composition according to an embodiment of the present disclosure exhibits superior effect as a composition for prevention or treatment of lung injury because it can alleviate microcirculatory disorder in the lung by inhibiting the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries, thereby allowing erythrocyteserythrocytes to smoothly pass through the pulmonary capillaries and increasing gas exchange in a subject suffering from pulmonary microcirculatory disorder. In addition, it allows faster, more convenient and more accurate diagnosis of pulmonary microcirculatory disorder through measurement of the expression or activity of macrophage-1 antigen in neutrophils isolated from the pulmonary capillaries.
In an aspect of the present specification, the terms “unit”, “module”, “device”, “system”, etc. may refer to not only a hardware but also a combination of softwares executed by the hardware. For example, the hardware may be a CPU or other data-processing devices including processors. And, the software executed by the hardware may be a process, an object, an executable file, a thread of execution, a program such as a calculation program, etc.
In an aspect of the present specification, “microcirculation” is the circulation of blood in small blood vessels such as arterioles, venules, capillaries, lymph capillaries, etc. It is the core of metabolism, where supply and discharge of materials occur. In an aspect of the present disclosure, the microcirculation may be quantified based on a functional capillary area measured form of a plurality of motion images of target factors in a first blood stream, information for diagnosis of microcirculatory disorder in the subject may be provided, or the microcirculatory disorder of a subject may be diagnosed by analyzing a dynamic element in target factors in a second blood stream from a plurality of motion images of the target factors in the second blood stream, although the present specification is not limited thereto. And, in an aspect of the present disclosure, the microcirculation may be, for example, microcirculation in the lung, microcirculation in the eye, microcirculation in the kidney or microcirculation in skin, and the skin may be hand, foot, etc., although not being limited thereto.
In an aspect of the present specification, the subject for quantitation of microcirculation or diagnosis of microcirculatory disorder may be any subject without limitation. Specifically, the subject may be a non-human animal such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, etc. or human, although not being limited thereto. In addition, the subject may be a subject having microcirculatory disorder, capillary circulatory disorder or peripheral circulatory disorder, although not being limited thereto.
In an aspect of the present specification, the capillary of the subject is not particularly limited as long as microcirculation may be quantified based on a functional capillary area measured form of a plurality of motion images of target factors in a first blood stream, information for diagnosis of microcirculatory disorder in the subject may be provided, or the microcirculatory disorder of the subject may be diagnosed by analyzing a dynamic element in target factors in a second blood stream from a plurality of motion images of the target factors in the second blood stream, and may be one or more capillary selected from a group consisting of the lung, kidney, skin and eye of the subject, although not being limited thereto.
In an aspect of the present specification, the plurality of motion images over time may be a plurality of images imaged with time intervals of 1/900 to 1 second. For example, when the plurality of images are an image (M) at one time point (T) and images (M−1, M+1) at time points (T−1, T+1) before and after the time point (T) of the same time interval (t), because the three images (M−1, M, M+1) are respectively images at first time point (T−1), second time point (T) and third time point (T+1) with the same time interval (t), a flow path of target factors in a first blood stream or a second blood stream passing through the capillaries of the subject may occur over time in the three images (M−1, M, M+1). Therefore, quantitative data about microcirculation can be acquired by measuring the functional capillary area in which the target factor pass in the first blood stream. Specifically, the functional capillary area may be measured by identifying the same target factors in the three images (M−1, M, M+1) imaged with the time interval (t). Also, information for diagnosis of microcirculatory disorder may be provided by analyzing dynamic elements of the target factor in the second blood stream, e.g., the sequestration time, track displacement length, track length, track velocity or track meandering index of the target factors. Specifically, the dynamic elements of the target factors may be analyzed by identifying the same target factors in the three images (M−1, M, M+1) imaged with the time interval (t).
In an aspect of the present specification, the time interval (t) of the three images (M−1, M, M+1) may be from 1/900 second to 1 second, specifically from 1/300 second to ⅓ second, more specifically 1/900 second or longer, 1/800 second or longer, 1/700 second or longer, 1/600 second or longer, 1/500 second or longer, 1/400 second or longer, 1/300 second or longer, 1/200 second or longer, 1/100 second or longer, 1/90 second or longer, 1/80 second or longer, 1/70 second or longer, 1/60 second or longer, 1/50 second or longer, 1/45 second or longer, 1/40 second or longer, 1/35 second or longer, 1/30 second or longer, 1/25 second or longer, 1/20 second or longer, 1/15 second or longer, 1/10 second or longer or ⅕ second or longer, and 1 second or shorter, ⅕ second or shorter, 1/10 second or shorter, 1/15 second or shorter, 1/20 second or shorter, 1/25 second or shorter, 1/30 second or shorter, 1/35 second or shorter, 1/40 second or shorter, 1/45 second or shorter, 1/50 second or shorter, 1/60 second or shorter, 1/70 second or shorter, 1/80 second or shorter, 1/90 second or shorter, 1/100 second or shorter, 1/200 second or shorter, 1/300 second or shorter, 1/400 second or shorter, 1/500 second or shorter, 1/600 second or shorter, 1/700 second or shorter, 1/800 second or shorter or 1/900 second or shorter. However, the time interval is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the first blood stream, or the dynamic elements of the target factor can be analyzed from the plurality of motion images of the target factors in the second blood stream.
In another aspect of the present specification, the plurality of motion images may be a plurality of images imaged at a frame rate or speed of 1-900 frames/second. The frame rate or speed may be specifically 3-300 frames/second, more specifically 1 frame/second or higher, 5 frames/second or higher, 10 frames/second or higher, 15 frames/second or higher, 20 frames/second or higher, 25 frames/second or higher, 30 frames/second or higher, 35 frames/second or higher, 40 frames/second or higher, 45 frames/second or higher, 50 frames/second or higher, 60 frames/second or higher, 70 frames/second or higher, 80 frames/second or higher, 90 frames/second or higher, 100 frames/second or higher, 200 frames/second or higher, 300 frames/second or higher, 400 frames/second or higher, 500 frames/second or higher, 600 frames/second or higher, 700 frames/second or higher or 800 frames/second or higher, and 900 frames/second or lower, 800 frames/second or lower, 700 frames/second or lower, 600 frames/second or lower, 500 frames/second or lower, 400 frames/second or lower, 300 frames/second or lower, 200 frames/second or lower, 100 frames/second or lower, 90 frames/second or lower, 80 frames/second or lower, 70 frames/second or lower, 60 frames/second or lower, 50 frames/second or lower, 45 frames/second or lower, 40 frames/second or lower, 35 frames/second or lower, 30 frames/second or lower, 25 frames/second or lower, 20 frames/second or lower, 15 frames/second or lower, 10 frames/second or lower or 5 frames/second or lower. However, the frame rate or speed is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the first blood stream, or the dynamic elements of the target factor can be analyzed from the plurality of motion images of the target factors in the second blood stream.
In an aspect of the present specification, the plurality of images may be images imaged by confocal scanning laser microscopy, fluorescence microscopy, two-photon microscopy or three-photon microscopy, although not being limited thereto.
In an aspect of the present specification, the functional capillary refers to a capillary where the function of the capillary, e.g., exchange of oxygen, carbon dioxide, nutrients and other substances between blood and tissue through diffusion, occurs actively. The functional capillary may be a capillary through which the target factors in a first blood stream such as leukocytes, erythrocytes, blood platelets, lymphocytes, etc. pass or a capillary through which the target factors in a second blood stream such as neutrophils, etc. pass. A higher ratio of the functional capillary to the total capillary means that microcirculation in the subject is smooth or there is no microcirculatory disorder.
Hereinafter, the present disclosure is described in detail.
In an aspect, the present disclosure provides a method for quantitation of microcirculation in a subject, which includes: a step of obtaining a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject; a step of measuring functional capillary area in which the target factors move in the first blood stream from the plurality of motion images; and a step of calculating functional capillary ratio (FCR) according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area. [Formula 1]
The quantitation of microcirculation in a subject means the quantification of the degree of microcirculation in the subject.
The method for quantitation of microcirculation in a subject may include a step of obtaining a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject.
The target factors in the first blood stream are factors that pass through the microcirculatory capillaries of the subject. The quantitative data about the microcirculation may be acquired by measuring the area traveled by the target factors in the first blood stream, e.g., functional capillary area, over time from the plurality of motion images. The target factors in the first blood stream may be the factors that pass through the capillaries of the subject through microcirculation. The factors may be specifically constituent factors of blood that can practically reflect the flow rate or flow amount through the capillaries of the subject, more specifically one or more selected from a group consisting of leukocytes, erythrocytes, blood platelets and lymphocytes, although not being limited thereto. In addition, the target factors in the first blood stream may be labeled to obtain motion images. Specifically, the labeling may be achieved with one or more selected from a group consisting of a fluorescent dye, a transgenic probe and an antibody label. More specifically, the transgenic probe may be one or more selected from a group consisting of CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), GFP (green fluorescent protein) and RFP (red fluorescent protein), although not being limited thereto. More specifically, the antibody label may be one conjugated with a fluorescent probe, e.g., an antibody conjugated with one or more fluorescent probe selected from a group consisting of Alexa 405, Alexa 488, Alexa 555 and Alexa 647, although not being limited thereto. In an example of the present disclosure, for the cease where the target factors in the first blood stream are erythrocytes, microcirculation was quantified by fluorescence-staining the erythrocytes with Vybrant DiD (V22887, ThermoFisher Scientific) and measuring the area traveled by the erythrocytes (functional capillary area) from a plurality of motion images thereof.
The method for quantitation of microcirculation in a subject may include a step of measuring the functional capillary area in which the target factors move in the first blood stream from the plurality of motion images.
The functional capillary refers to a capillary where the function of the capillary, e.g., exchange of oxygen, carbon dioxide, nutrients and other substances between blood and tissue through diffusion, occurs actively. The functional capillary may be a capillary through which the target factors in a first blood stream such as leukocytes, erythrocytes, blood platelets, lymphocytes, etc. pass or a capillary through which the target factors in a second blood stream such as neutrophils, etc. pass. A higher ratio of the functional capillary to the total capillary means that microcirculation in the subject is smooth or there is no microcirculatory disorder.
The functional capillary area may be measured by identifying the same target factors from the plurality of motion images, and may be calculated by measuring the area traveled by the target factors in the first blood stream from the change in their location over time. Specifically, the flow path of the target factors in the first blood stream passing through the capillaries of the subject over time may be measured from the plurality of motion images, and the functional capillary area may be measured from the flow path of the target factors in the first blood stream. More specifically, after measuring the flow path of the target factors in the first blood stream by comparing the plurality of images imaged with a time interval (t), the functional capillary area may be measured from the flow path of the target factors in the first blood stream.
In an example of the present disclosure, the flow path of erythrocytes as target factors in a first blood stream passing through capillaries could be measured by fluorescence-staining the erythrocytes, acquiring the motion images of the erythrocytes with different time intervals (0.000 second, 0.033 second and 0.066 second) and merging the plurality of motion images of the erythrocytes (Experimental Example 1-1 and
The method for quantitation of microcirculation in a subject may include a step of calculating a functional capillary ratio (FCR) according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area. [Formula 1]
The quantitation of microcirculation may be accomplished by the calculation of the functional capillary ratio according to Formula 1. Because microcirculation can be quantified based on area rather than density, the area where through which one red blood cell passes and the area where through which a plurality of erythrocytes pass can be distinguished. Through this, microcirculation can be quantified more easily, conveniently and accurately because the space (area) through which erythrocytes pass actually can be reflected, and it is possible to quantify the microcirculation network which is difficult to quantify based on density.
In an example of the present disclosure, the functional capillary ratio was calculated according to Formula 1 from the area traveled by DiD-stained erythrocytes as the functional capillary area and the vessel area detected by Tie2 or dextran signaling as the total capillary area. Through this, the microcirculation of the subject could be quantified (Experimental Example 2 and
In another aspect, the present disclosure provides an apparatus for measuring microcirculation in a subject, which acquires quantitative data on the microcirculation in the subject based on a plurality of motion images of target factors over time in a first blood stream passing through the capillaries of the subject according to Formula 1. Specifically, the apparatus may include: an imaging unit imaging target factors in a first blood stream passing through the capillaries of the subject; and a measuring unit acquiring quantitative data on the microcirculation in the subject according to Formula 1 based on the images imaged by the imaging unit. The subject, the microcirculation, the target factors in the first blood stream, the images and the quantitation of microcirculation are the same as described above.
Functional capillary ratio=functional capillary area/total capillary area. [Formula 1]
The measurement of microcirculation in the subject may be accomplished by measurement of quantitative data about the microcirculation in the subject, and the quantitative data about the microcirculation may be acquired by calculating the functional capillary ratio according to Formula 1.
The imaging unit may image a plurality of motion images of the target factors in the blood stream passing through the capillaries over time. For example, when the plurality of images are an image (M) at one time point (T) and images (M−1, M+1) at time points (T−1, T+1) before and after the time point (T) of the same time interval (t), because the three images (M−1, M, M+1) are respectively images at first time point (T−1), second time point (T) and third time point (T+1) with the same time interval (t), a flow path of target factors in a first blood stream or a second blood stream passing through the capillaries of the subject may occur over time in the three images (M−1, M, M+1). Therefore, quantitative data about microcirculation can be acquired by measuring the functional capillary area in which the target factor pass in the first blood stream. Specifically, the functional capillary area may be measured by identifying the same target factors in the three images (M−1, M, M+1) imaged with the time interval (t).
The time interval (t) of the three images (M−1, M, M+1) may be from 1/900 second to 1 second, specifically from 1/300 second to ⅓ second, more specifically 1/900 second or longer, 1/800 second or longer, 1/700 second or longer, 1/600 second or longer, 1/500 second or longer, 1/400 second or longer, 1/300 second or longer, 1/200 second or longer, 1/100 second or longer, 1/90 second or longer, 1/80 second or longer, 1/70 second or longer, 1/60 second or longer, 1/50 second or longer, 1/45 second or longer, 1/40 second or longer, 1/35 second or longer, 1/30 second or longer, 1/25 second or longer, 1/20 second or longer, 1/15 second or longer, 1/10 second or longer or ⅕ second or longer, and 1 second or shorter, ⅕ second or shorter, 1/10 second or shorter, 1/15 second or shorter, 1/20 second or shorter, 1/25 second or shorter, 1/30 second or shorter, 1/35 second or shorter, 1/40 second or shorter, 1/45 second or shorter, 1/50 second or shorter, 1/60 second or shorter, 1/70 second or shorter, 1/80 second or shorter, 1/90 second or shorter, 1/100 second or shorter, 1/200 second or shorter, 1/300 second or shorter, 1/400 second or shorter, 1/500 second or shorter, 1/600 second or shorter, 1/700 second or shorter, 1/800 second or shorter or 1/900 second or shorter. However, the time interval is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the first blood stream.
The plurality of motion images may be a plurality of images imaged at a frame rate or speed of 1-900 frames/second. The frame rate or speed may be specifically 3-300 frames/second, more specifically 1 frame/second or higher, 5 frames/second or higher, 10 frames/second or higher, 15 frames/second or higher, 20 frames/second or higher, 25 frames/second or higher, 30 frames/second or higher, 35 frames/second or higher, 40 frames/second or higher, 45 frames/second or higher, 50 frames/second or higher, 60 frames/second or higher, 70 frames/second or higher, 80 frames/second or higher, 90 frames/second or higher, 100 frames/second or higher, 200 frames/second or higher, 300 frames/second or higher, 400 frames/second or higher, 500 frames/second or higher, 600 frames/second or higher, 700 frames/second or higher or 800 frames/second or higher, and 900 frames/second or lower, 800 frames/second or lower, 700 frames/second or lower, 600 frames/second or lower, 500 frames/second or lower, 400 frames/second or lower, 300 frames/second or lower, 200 frames/second or lower, 100 frames/second or lower, 90 frames/second or lower, 80 frames/second or lower, 70 frames/second or lower, 60 frames/second or lower, 50 frames/second or lower, 45 frames/second or lower, 40 frames/second or lower, 35 frames/second or lower, 30 frames/second or lower, 25 frames/second or lower, 20 frames/second or lower, 15 frames/second or lower, 10 frames/second or lower or 5 frames/second or lower. However, the frame rate or speed is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the first blood stream, or the dynamic elements of the target factor can be analyzed from the plurality of motion images of the target factors in the second blood stream.
The imaging unit may be a confocal scanning laser microscope, a fluorescence microscope, a two-photon microscope or a three-photon microscope, although not being limited thereto.
The measuring unit may measure functional capillary area from the change in the location of the target factors in the first blood stream over time interval by identifying the same target factors from the plurality of motion images of the target factors in the first blood stream imaged by the imaging unit and may calculate the functional capillary ratio according to Formula 1. Specifically, after measuring the flow path of the target factors in the first blood stream passing through the capillaries of the subject over time from the plurality of motion images of the target factors in the first blood stream, the functional capillary area may be determined form the path of the target factors in the first blood stream. More specifically, by comparing the plurality of images imaged with the time interval (t), the flow path of the target factors in the first blood stream may be easily identified and traced, and the functional capillary ratio may be calculated according to Formula 1 by measuring the functional capillary area form the path of the target factors in the first blood stream. For measurement of the area depending on the flow path, the area may be determined through pixel analysis of the flow path. In an example of the present disclosure, an analysis program such as ImageJ was used.
In another aspect, the present disclosure provides a method for providing information for diagnosis of microcirculatory disorder in a subject, which includes a step of acquiring information for diagnosing microcirculatory disorder in the subject from the functional capillary ratio (FCR) calculated by the method for quantitation of microcirculation in a subject described above. The method for quantitation of microcirculation in a subject, the functional capillary ratio and the microcirculation are the same as described above.
In another aspect, the present disclosure provides an apparatus for providing information for diagnosis of microcirculatory disorder in a subject, which acquires information for diagnosing microcirculatory disorder in the subject from the calculated functional capillary ratio (FCR). The apparatus may include: a microcirculation quantitation unit acquiring quantitative data about the microcirculation of the subject according to Formula 1 based on the plurality of motion images of target factors in the first blood stream passing through the capillaries of the subject over time; and a microcirculatory disorder identification unit identifying microcirculatory disorder based on the acquired functional capillary ratio (FCR).
The microcirculatory disorder refers to abnormal microcirculation in which the target factors in the first blood stream such as leukocytes, erythrocytes, blood platelets, lymphocytes, etc. cannot smoothly pass through the capillaries. Specifically, the microcirculatory disorder may refer to a state where the functional capillary ratio is 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower or 5% or lower as compared to the functional capillary ratio of a normal group with no microcirculatory disorder, or the functional capillary ratio is 0.4 or lower, 0.38 or lower, 0.36 or lower, 0.34 or lower, 0.32 or lower, 0.3 or lower, 0.28 or lower, 0.26 or lower, 0.24 or lower, 0.22 or lower, 0.2 or lower, 0.18 or lower, 0.16 or lower, 0.14 or lower, 0.12 or lower, 0.1 or lower, 0.08 or lower, 0.06 or lower, 0.04 or lower or 0.02 or lower. However, the range of the functional capillary ratio for identification of microcirculatory disorder may vary depending on the organ of the subject in which the capillaries are distributed, and is not limited to the above ranges.
In an example of the present disclosure, a control group model (treated with PBS) and a sepsis-induced acute lung injury mouse model treated with LPS showed no difference in total capillary area. However, the acute lung injury mouse model had a functional capillary ratio (FCR) decreased by 50% or more as compared to the control group model as the functional capillary area in which erythrocytes pass was decreased rapidly (Experimental Example 3,
In another aspect, the present disclosure provides a method for providing information for diagnosis of microcirculatory disorder, which includes: a step of obtaining a plurality of motion images of target factors over time in a second blood stream flowing through the capillaries of the subject; a step of analyzing one or more dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of the target factors in the second blood stream from the plurality of motion images; and a step of acquiring information for diagnosis of microcirculatory disorder in the subject from the dynamic element analysis result. Previously, endothelial dysfunction and vasoconstriction were suggested as potential central mechanisms in impaired systemic microcirculation, and it was known that sequestration in pulmonary capillaries functions as an immune surveillance system for detecting pathogens in the circulation. However, they could not explain how neutrophil sequestration progresses to microcirculatory disorder, particularly pulmonary microcirculatory disorder such as acute respiratory failure syndrome. In contrast, according to an exemplary embodiment of the present disclosure, it can be seen that cluster formation of neutrophils recruited during the early stage of sepsis-induced acute lung injury plays a key role in pulmonary microcirculatory disorder. Specifically, the neutrophils form clusters and act as an obstacle in capillaries and arterioles, leading to the redistribution and obstruction of the pulmonary microcirculation.
The method for providing information for diagnosis of microcirculatory disorder may include a step of obtaining a plurality of motion images of the target factors in the second blood stream flowing through the capillaries of the subject over time.
The target factors in the second blood stream are factors that pass through the microcirculatory capillaries of the subject. Information for diagnosis of microcirculatory disorder may be provided by analyzing the dynamic elements of the target factors, e.g., the sequestration time, track displacement length, track length, track velocity or track meandering index of the target factors, from the plurality of motion images of the target factors in the second blood stream over time. The target factors in the second blood stream may be the factors that pass through the capillaries of the subject along the microcirculation. They may be specifically the constituent factors of blood that can practically reflect the flow rate or flow amount through the capillaries of the subject, more specifically neutrophils, although not being limited thereto. In addition, the target factors in the second blood stream may be labeled to obtain motion images. Specifically, when the target factors in the second blood stream are neutrophils, the neutrophils may be labeled with a fluorophore-conjugated nucleic acid (DNA or RNA) encoding a peptide expressed in the neutrophils. In addition, the neutrophils may be labeled with an antibody specific for the neutrophils, and the antibody may be conjugated with a fluorophore. The nucleic acid encoding a peptide expressed in the neutrophils may be specifically one or more selected from a group consisting of a nucleic acid encoding a lysine motif (LysM) domain, a nucleic acid encoding leukocyte 6G (Ly6G), a nucleic acid encoding cluster of differentiation molecule 11B (CD11b) and a nucleic acid encoding cluster of differentiation molecule 18 (CD18). However, any nucleic acid allowing the analysis of dynamic elements from images by labeling the neutrophils may be used without limitation. And, the antibody specific for the neutrophils may be an antibody specific for a peptide expressed in the neutrophils, specifically an antibody specific for one or more selected from a group consisting of a lysine motif (LysM) domain, leukocyte 6G (Ly6G), cluster of differentiation molecule 11B (CD11b) and cluster of differentiation molecule 18 (CD18). However, any antibody allowing the analysis of dynamic elements from images by labeling the neutrophils may be used without limitation. In addition, the fluorophore may be specifically a transgenic probe or a fluorescent probe. More specifically, the transgenic probe may be one or more selected from a group consisting of CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), GFP (green fluorescent protein) and RFP (red fluorescent protein), and the fluorescent probe may be one or more selected from a group consisting of Alexa 405, Alexa 488, Alexa 555 and Alexa 647, although not being limited thereto.
According to an exemplary embodiment of the present disclosure, when the target factors in the second blood stream are neutrophils, information for diagnosis of microcirculatory disorder may be provided by injecting an anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) conjugated with a fluorophore Alexa Fluor 555 or 647 (A-20005/A-20006, ThermoFisher Scientific) into the subject and monitoring the motion of neutrophils labeled with the antibody by measuring fluorescence signals. In addition, as previously known, the neutrophil-induced obstruction of blood flow increases the mismatching area of ventilation and perfusion, thereby intensifying hypoxia due to sepsis-induced acute respiratory failure syndrome (ARDS). Compared to the previous intravital imaging studies on the adhesion of neutrophils in the pulmonary capillaries (Yang N, Liu Y Y, Pan C S, Sun K, Wei X H, Mao X W, Lin F, Li X J, Fan J Y, Han J Y. Pretreatment with andrographolide pills® attenuates lipopolysaccharide-induced pulmonary microcirculatory disorder and acute lung injury in rats. Microcirculation 2014: 21(8): 703-716; Gill S E, Rohan M, Mehta S. Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo. Respir Res 2015: 16: 109), the method for providing information for diagnosis of microcirculatory disorder according to an exemplary embodiment of the present disclosure shows how dead space with ventilation/perfusion mismatch is created in the microcirculation by neutrophils. Moreover, the information can be provided more conveniently and accurately by directly imaging dead space fraction, which has been estimated indirectly by the difference in the partial pressure of arterial versus exhaled carbon dioxide using volumetric capnography.
The method for providing information for diagnosis of microcirculatory disorder may include a step of analyzing one or more dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of the target factors in the second blood stream from the plurality of motion images.
The sequestration time refers to the duration of time during which the target factors in the second blood stream are retained (sequestered) in a specific area of capillaries while passing through the capillaries. If the target factor in the second blood stream needs to be deformed to pass thorough the capillary because it has a large diameter, it requires longer time to pass thorough the capillary as compared to the blood flow rate. In this case, the target factor may be sequestered in a specific area of the capillary rather than passing through the capillary, leading to microcirculatory disorder. In an example of the present disclosure, whereas neutrophils passed through capillaries in a control mouse model (PBS), the flow of neutrophils was interrupted in numerous spots in a lung injury mouse model (ALI mouse model) (Experimental Example 4 and
The track displacement length refers to the amount of change of the location (unit: μm) of the target factors in the second blood stream over time. A larger value of the track displacement length means a higher motility of the target factors.
The track length refers to the distance (unit: μm) actually traveled by the target factors in the second blood stream over time. A larger value of the track length means a higher motility of the target factors.
The track velocity refers to the distance traveled the target factors in the second blood stream by per unit time (unit: μm/m). A larger value of the track velocity means a higher motility of the target factors.
The track meandering index refers to the tendency of the target factors in the second blood stream to move toward a target site or along a specific direction (unit: a.u., or arbitrary unit). A larger value of the track meandering index means a higher tendency of the target factors in the second blood stream to move toward a target site or along a specific direction and arrive at the target site within the fastest time. The track meandering index (a.u.) may be calculated using the Spots & Tracking function of the IMARIS program.
In an example of the present disclosure, lung injury mouse models (ALI mouse model, LPS 3h mouse model and LPS 6h mouse model) showed difference in the sequestration time, track displacement length, track length, track velocity and track meandering index when compared with a control group (PBS), suggesting that information for diagnosis of microcirculatory disorder in the subject can be provided through analysis of dynamic elements (Experimental Example 6 and
The analysis of dynamic elements may be accomplished by identifying the same target factors from the plurality of motion images. Specifically, the flow path of the target factors in the second blood stream passing through the capillaries of the subject over time may be measured from the plurality of motion images, and the functional capillary area may be measured from the flow path of the target factors in the second blood stream. More specifically, after measuring the flow path of the target factors in the second blood stream by comparing the plurality of images imaged with a time interval (t), dynamic elements may be measured from the flow path of the target factors in the second blood stream.
The method for providing information for diagnosis of microcirculatory disorder may include a step of acquiring information for diagnosis of microcirculatory disorder in the subject from the dynamic element analysis result.
If the sequestration time of the target factors in the second blood stream as the information for diagnosis of microcirculatory disorder in the subject is 5 minutes or longer, it may be diagnosed as microcirculatory disorder. As described above, when the subject has microcirculatory disorder, the sequestration time is increased as compared to a control group because the tendency of the target factors in the second blood stream remaining (entrapment) in specific area rather than passing through the capillaries is increased. Accordingly, if the sequestration time of the target factors in the second blood stream is 5 minutes or longer, it may be diagnosed as microcirculatory disorder. Specifically, if the sequestration time is 5 minutes or longer, 5 minutes and 10 seconds or longer, 5 minutes and 20 seconds or longer, 5 minutes and 30 seconds or longer, 5 minutes and 40 seconds or longer, 5 minutes and 50 seconds or longer, 6 minutes or longer, 6 minutes and 10 seconds or longer, 6 minutes and 20 seconds or longer, 6 minutes and 30 seconds or longer, 6 minutes and 40 seconds or longer, 6 minutes and 50 seconds or longer, 7 minutes or longer, 7 minutes and 10 seconds or longer, 7 minutes and 20 seconds or longer, 7 minutes and 30 seconds or longer, 7 minutes and 40 seconds or longer, 7 minutes and 50 seconds or longer, 8 minutes or longer, 8 minutes and 10 seconds or longer, 8 minutes and 20 seconds or longer, 8 minutes and 30 seconds or longer, 8 minutes and 40 seconds or longer, 8 minutes and 50 seconds or longer, 9 minutes or longer, 9 minutes and 10 seconds or longer, 9 minutes and 20 seconds or longer, 9 minutes and 30 seconds or longer, 9 minutes and 40 seconds or longer, 9 minutes and 50 seconds or longer, 10 minutes or longer, 11 minutes or longer, 12 minutes or longer, 13 minutes or longer, 14 minutes or longer, 15 minutes or longer, 16 minutes or longer, 17 minutes or longer, 18 minutes or longer or 19 minutes or longer, it may be diagnosed as microcirculatory disorder. However, because the range of the sequestration time for diagnosis of microcirculatory disorder may vary depending on the particular subject, the type of the capillary, the age, sex and body weight of the subject, the particular disease or pathological condition of the subject, or the severity of the disease or pathological condition and diagnosis of microcirculatory disorder based on these factors is within the level of those skilled in the art, the present disclosure is not limited to the above ranges. In an example of the present disclosure, neutrophil sequestration time was longer for lung injury mouse models (LPS administration groups) with about 8 minutes for a LPS 3h mouse model and about 18 minutes for a LPS 6h mouse model as compared to a control group (PBS, about 3 minutes). The sequestration time was about 2 times longer at 6 hours after the administration of LPS (LPS 6h mouse model) than at 3 hours after the administration of LPS (LPS 3h mouse model) (Experimental Example 6 and
If the track meandering index of the target factors in the second blood stream as the information for diagnosis of microcirculatory disorder in the subject is 0.4 a.u. or lower, it may be diagnosed as microcirculatory disorder. If the subject has microcirculatory disorder, the target factors in the second blood stream tend to be sequestered in specific area or move very slowly without directionality rather than passing through capillaries along the blood flow, when compared with a control group. Accordingly, it may be diagnosed as microcirculatory disorder if the track meandering index of the target factors in the second blood stream is 0.4 a.u. or lower. Specifically, it may be diagnosed as microcirculatory disorder if the track meandering index is 0.4 a.u. or lower, 0.39 a.u. or lower, 0.38 a.u. or lower, 0.37 a.u. or lower, 0.36 a.u. or lower, 0.35 a.u. or lower, 0.34 a.u. or lower, 0.33 a.u. or lower, 0.32 a.u. or lower, 0.31 a.u. or lower, 0.3 a.u. or lower, 0.29 a.u. or lower, 0.28 a.u. or lower, 0.27 a.u. or lower, 0.26 a.u. or lower, 0.25 a.u. or lower, 0.24 a.u. or lower, 0.23 a.u. or lower, 0.22 a.u. or lower, 0.21 a.u. or lower or 0.2 a.u. or lower. However, because the range of the track meandering index for diagnosis of microcirculatory disorder may vary depending on the particular subject, the type of the capillary, the age, sex and body weight of the subject, the particular disease or pathological condition of the subject, or the severity of the disease or pathological condition and diagnosis of microcirculatory disorder based on these factors is within the level of those skilled in the art, the present disclosure is not limited to the above ranges. In an example of the present disclosure, the track meandering index of neutrophils was lower lung injury mouse models (LPS administration groups) with about 0.4 a.u. for a LPS 3h mouse model and about 0.2 a.u. for a LPS 6h mouse model as compared to a control group (PBS, about 0.5 a.u.). The track meandering index was decreased to about ½ at 6 hours after the administration of LPS (LPS 6h mouse model) than at 3 hours after the administration of LPS (LPS 3h mouse model) (Experimental Example 6 and
When the dynamic element is one or more selected from a group consisting of track displacement length, track length and track velocity, a plurality of motion images of the target factors in the second blood stream over time may be a plurality of motion image sets imaged with a time interval (U) of 2 hours or longer as described below: (1) a plurality of first image sets (SET_M1) at one time point (T1), wherein the first image sets include an image (M1) at the time point (T1) and images (M1−1, M1+1) at time points (T1−1, T1+1) before and after the same time interval (t) with respect to the one time point (T1);
(2) a plurality of second image sets (SET_M2) at one time point (T2) which is after the one time point (T1) by a time (U) which is 2 hours or longer, wherein the second image sets include an image (M2) at the time point (T2) and images (M2−1, M2+1) at time points (T2−1, T2+1) before and after the same time interval (t) with respect to the time point (T2); and
(3) a plurality of third image sets (SET_M3) at a time point (T3) which is after the one time point (T2) by a time (U) which is 2 hours or longer, wherein the third image sets include an image (M3) at the time point (T3) and image (M3−1, M3+1) at time points (T3-1, T3+1) before and after the same time interval (t) with respect to the one time point (T3).
The plurality of motion image sets may be obtained as described above in (1)-(3), and the plurality of motion image sets may be 2 or more, 3 or more, 4 or more, 5 or more or 6 or more image sets.
The time interval (U) between the plurality of image sets may be 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer or 6 hours or longer. However, the time interval is not limited to the above ranges as long as information for diagnosis of microcirculatory disorder can be provided by analyzing one or more dynamic element selected from a group consisting of the track displacement length, track length and track velocity of the target factors in the second blood stream from the plurality of image sets.
When the dynamic element is one or more selected from a group consisting of track displacement length, track length and track velocity, the dynamic elements may be analyzed sequentially in time from the plurality of motion image sets, and, if the dynamic element is decreased with time as a result of the analysis of the dynamic elements, it may be diagnosed as microcirculatory disorder. For a subject with microcirculatory disorder, e.g., if lung injury occurs due to endotoxin, the motility of neutrophils is increased during the early stage of lung injury but, as inflammation is aggravated due to microcirculatory disorder, the motility of neutrophils is decreased, thereby resulting in decreased track displacement length, track length and track velocity, as compared to a control group. Accordingly, if the track displacement length, track length or track velocity of the target factors in the second blood stream is decreased over time, it may be diagnosed as microcirculatory disorder. In an example of the present disclosure, the track displacement length, track length and track velocity were increased in a lung injury mouse model 3 hours after LPS administration (LPS 3h mouse model), when compared with a control group (PBS), and then were decreased to a level similar to that of the control group in a lung injury mouse model 6 hours after LPS administration (LPS 6h mouse model) (Experimental Example 6 and
The method for providing information for diagnosis of microcirculatory disorder may further include a step of detecting the generation of reactive oxygen species in the target factors in the second blood stream passing through the capillaries. Specifically, the detection of the generation of reactive oxygen species may be achieved by dihydroethidium (DHE) staining. However, any method capable of detecting the generation of reactive oxygen species in vivo or in situ may be used without limitation. If reactive oxygen species are generated in the target factors in the second blood stream, it may be diagnosed as microcirculatory disorder.
In an example of the present disclosure, whereas reactive oxygen species were not generated in the temporarily sequestered neutrophils of a control group (PBS), reactive oxygen species were generated in the neutrophils in capillaries of a lung injury mouse model (ALI mouse model), and the proportion of reactive oxygen species-generating neutrophils with respect to the total neutrophils was remarkably increased (Experimental Example 8 and
In another aspect, the present disclosure provides an apparatus for diagnosis of microcirculatory disorder, which includes: an imaging unit imaging target factors in a second blood stream passing through the capillaries of a subject; and an analysis unit analyzing one or more dynamic element selected from a group consisting of sequestration time, track displacement length, track length, track velocity and track meandering index of the target factors in the second blood stream based on the plurality of motion images imaged by the imaging unit. The subject, microcirculation, the microcirculatory disorder, the target factors in the second blood stream, the plurality of motion images, the dynamic elements, the analysis of the dynamic elements and the information for diagnosis of microcirculatory disorder are the same as described above.
The information for diagnosis of microcirculatory disorder may be acquired from the dynamic element analysis result of the target factors in the second blood stream of the subject.
The imaging unit may image a plurality of motion images of the target factors in the second blood stream passing through the capillaries over time. For example, when the plurality of images are an image (M) at one time point (T) and images (M−1, M+1) at time points (T−1, T+1) before and after the time point (T) of the same time interval (t), because the three images (M−1, M, M+1) are respectively images at second time point (T−1), second time point (T) and third time point (T+1) with the same time interval (t), a flow path of target factors in a first blood stream or a second blood stream passing through the capillaries of the subject may occur over time in the three images (M−1, M, M+1).
The time interval (t) of the three images (M−1, M, M+1) may be from 1/900 second to 1 second, specifically from 1/300 second to ⅓ second, more specifically 1/900 second or longer, 1/800 second or longer, 1/700 second or longer, 1/600 second or longer, 1/500 second or longer, 1/400 second or longer, 1/300 second or longer, 1/200 second or longer, 1/100 second or longer, 1/90 second or longer, 1/80 second or longer, 1/70 second or longer, 1/60 second or longer, 1/50 second or longer, 1/45 second or longer, 1/40 second or longer, 1/35 second or longer, 1/30 second or longer, 1/25 second or longer, 1/20 second or longer, 1/15 second or longer, 1/10 second or longer or ⅕ second or longer, and 1 second or shorter, ⅕ second or shorter, 1/10 second or shorter, 1/15 second or shorter, 1/20 second or shorter, 1/25 second or shorter, 1/30 second or shorter, 1/35 second or shorter, 1/40 second or shorter, 1/45 second or shorter, 1/50 second or shorter, 1/60 second or shorter, 1/70 second or shorter, 1/80 second or shorter, 1/90 second or shorter, 1/100 second or shorter, 1/200 second or shorter, 1/300 second or shorter, 1/400 second or shorter, 1/500 second or shorter, 1/600 second or shorter, 1/700 second or shorter, 1/800 second or shorter or 1/900 second or shorter. However, the time interval is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the second blood stream.
The plurality of motion images may be a plurality of images imaged at a frame rate or speed of 1-900 frames/second. The frame rate or speed may be specifically 3-300 frames/second, more specifically 1 frame/second or higher, 5 frames/second or higher, 10 frames/second or higher, 15 frames/second or higher, 20 frames/second or higher, 25 frames/second or higher, 30 frames/second or higher, 35 frames/second or higher, 40 frames/second or higher, 45 frames/second or higher, 50 frames/second or higher, 60 frames/second or higher, 70 frames/second or higher, 80 frames/second or higher, 90 frames/second or higher, 100 frames/second or higher, 200 frames/second or higher, 300 frames/second or higher, 400 frames/second or higher, 500 frames/second or higher, 600 frames/second or higher, 700 frames/second or higher or 800 frames/second or higher, and 900 frames/second or lower, 800 frames/second or lower, 700 frames/second or lower, 600 frames/second or lower, 500 frames/second or lower, 400 frames/second or lower, 300 frames/second or lower, 200 frames/second or lower, 100 frames/second or lower, 90 frames/second or lower, 80 frames/second or lower, 70 frames/second or lower, 60 frames/second or lower, 50 frames/second or lower, 45 frames/second or lower, 40 frames/second or lower, 35 frames/second or lower, 30 frames/second or lower, 25 frames/second or lower, 20 frames/second or lower, 15 frames/second or lower, 10 frames/second or lower or 5 frames/second or lower. However, the frame rate or speed is not specially limited as long as microcirculation can be quantified from a plurality of motion images of the target factors in the first blood stream, or the dynamic elements of the target factor can be analyzed from the plurality of motion images of the target factors in the second blood stream.
The imaging unit may be a confocal scanning laser microscope, a fluorescence microscope, a two-photon microscope or a three-photon microscope, although not being limited thereto.
The plurality of motion images imaged by the imaging unit may be a plurality of motion image sets imaged with a time interval (U) of 2 hours or longer as described below:
(1) a plurality of first image sets (SET_M1) at one time point (T1), wherein the first image sets include an image (M1) at the time point (T1) and images (M1−1, M1+1) at time points (T1−1, T1+1) before and after the same time interval (t) with respect to the one time point (T1);
(2) a plurality of second image sets (SET_M2) at one time point (T2) which is after the one time point (T1) by a time (U) which is 2 hours or longer, wherein the second image sets include an image (M2) at the time point (T2) and images (M2−1, M2+1) at time points (T2−1, T2+1) before and after the same time interval (t) with respect to the time point (T2); and
(3) a plurality of third image sets (SET_M3) at a time point (T3) which is after the one time point (T2) by a time (U) which is 2 hours or longer, wherein the third image sets include an image (M3) at the time point (T3) and image (M3−1, M3+1) at time points (T3−1, T3+1) before and after the same time interval (t) with respect to the one time point (T3).
The plurality of motion image sets may be obtained as described above in (1)-(3), and the plurality of motion image sets may be 2 or more, 3 or more, 4 or more, 5 or more or 6 or more image sets.
The time interval (U) between the plurality of image sets may be 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer or 6 hours or longer. However, the time interval is not limited to the above ranges as long as information for diagnosis of microcirculatory disorder can be provided by analyzing one or more dynamic element selected from a group consisting of the track displacement length, track length and track velocity of the target factors in the second blood stream from the plurality of image sets.
The apparatus for diagnosis of microcirculatory disorder may further include a reactive oxygen species detection unit detecting the generation of reactive oxygen species in the target factors in the second blood stream. Specifically, the detection unit may detect the generation of reactive oxygen species by dihydroethidium (DHE) staining. However, any method capable of detecting the generation of reactive oxygen species in vivo or in situ may be used without limitation.
In another aspect, the present disclosure provides a composition for preventing, alleviating or treating lung injury, which contains an inhibitor against the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries as an active ingredient, and prevents, alleviates or treats lung injury through alleviation of microcirculatory disorder in the lung. In addition, the present disclosure provides a composition for alleviating microcirculatory disorder in the lung, which contains an inhibitor against the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries as an active ingredient.
In an aspect of the present disclosure, a subject which is a target for the prevention or treatment may be any subject requiring prevention or treatment of lung injury without limitation. Specifically, the subject may be a non-human animal such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, etc. or human, although not being limited thereto. In addition, the subject may be a subject having microcirculatory disorder, capillary circulatory disorder or peripheral circulatory disorder, although not being limited thereto.
The neutrophils are a type of granular leukocytes (granulocytes) produced mainly in the bone marrow. They account for 50-70% of leukocytes and about 90% of granulocytes in human blood, and are also called neutrocytes or heterophils. The neutrophils are leukocytes that arrive at the damaged or infected site first when tissue damage or microbial infection occurs. It is known that various chemotactic factors such as interleukin 8 (IL8), etc. are produced at the damaged or infected site and, as a result, neutrophils are recruited and cause acute inflammatory responses. The most distinguished functions of the neutrophils are phagocytosis and killing of bacteria. In an aspect of the present disclosure, the neutrophils may be neutrophils within pulmonary capillaries.
The macrophage-1 antigen (Mac-1) is an adhesion molecule belonging to the integrin family. It is a glycoprotein expressed in neutrophils, monocytes, macrophages, activated lymphocytes, etc., and is also called CD11b/CD18, which is a heterodimer wherein CD11b (αM chain, molecular weight ˜170,000 Da) and CD18 (β2 chain, molecular weight ˜95,000 Da) are noncovalently bound. The macrophage-1 antigen is stored in secretory granules in cells and can be rapidly expressed on cell surface following cell activation. The αM chain has three binding sites for divalent metal ions. The adhesion of this molecule is dependent on the divalent metal ions, and its ligands are ICAM−1, iC3b, fibrinogen, blood coagulation factor X, LPS (lipopolysaccharide), etc. The adhesion mediated by the molecule is involved in the adhesion of leukocytes to vascular endothelial cells, infiltration into tissues, phagocytosis, etc., and the molecule is known to associate with cytoskeletal structures, protein kinases, etc. in the cytoplasm and to be involved in signaling in the respiratory burst (oxidative burst) of leukocytes, etc.
Although an example of the present disclosure implies that neutrophil depletion alleviates pulmonary microcirculatory disorder, the effect of treatment-induced neutrophil depletion in sepsis is unclear because the effects on bacterial clearance and the response to systemic inflammation are unclear. Accordingly, the inventors of the present disclosure evaluated the subpopulation of neutrophils that may ameliorate lung injury. Flow cytometry showed that Mac-1 (CD11b/CD18), which interacts with ICAM−1 in endothelial cells and various coagulation factor, was significantly upregulated in the sequestered neutrophils in the lung of a lung injury model.
In the present specification, the term “gene expression” is used in the broadest concept, including transcription, translation, post-translational modification, etc.
The composition according to an aspect of the present disclosure may contain an inhibitor against the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries as an active ingredient.
The inhibitor against the expression or activity of macrophage-1 antigen may be a substance that inhibits the translation of an mRNA which encodes macrophage-1 antigen. Specifically, it may be an oligonucleotide binding to at least a portion of an mRNA which encodes macrophage-1 antigen, and may be one or more of a siRNA, a shRNA and a miRNA. The inhibitor against the expression or activity of macrophage-1 antigen may be one or more of a siRNA, a shRNA and a miRNA, which induce RNA interference (RNAi), and may provide an effect of preventing or treating lung injury through the RNAi phenomenon of interfering with an mRNA encoding the macrophage-1 antigen in order to inhibit the mRNA expression of a gene encoding the macrophage-1 antigen. The miRNA is an endogenous small RNA existing in cells. It is derived from a DNA that does not synthesize proteins and is generated from a hairpin-shaped transcript. The miRNA binds to the complementary sequence of the 3′-UTR of a target mRNA to induce the inhibition of the translation or destabilization of the mRNA, ultimately serving as a repressor to inhibit protein synthesis of the target mRNA. It is known that one miRNA can target several mRNAs and an mRNA can also be regulated by several miRNAs. Other RNAs that induce RNAi include the short interfering RNA (siRNA), which is a short RNA of about 19-27 mer, and the shRNA, which has a short hairpin structure.
The inhibitor against the expression or activity of macrophage-1 antigen may include a peptide binding specifically to macrophage-1 antigen in neutrophils, specifically an antibody binding specifically to macrophage-1 antigen in neutrophils. Specifically, the antibody may be an antibody binding specifically to CD11b or CD18, more specifically an antibody binding specifically to CD11 b, represented by an amino acid sequence of SEQ ID NO 1 or 2, more specifically an antibody binding specifically to a peptide having 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher or 99% or higher identity to the amino acid sequence of SEQ ID NO 1 or 2, further more specifically BD Biosciences' BD Pharmingen™ (Catalog Number: BD 553307), although not being limited thereto.
Alternatively, the antibody may be specifically abciximab, and the abciximab may be ISU Abxis's abciximab (Clotinab), although not being limited thereto.
The inhibitor against the expression or activity of macrophage-1 antigen may be contained at a concentration of 0.2-20 mg/mL, specifically 0.2 mg/mL or higher, 0.3 mg/mL or higher, 0.4 mg/mL or higher, 0.5 mg/mL or higher, 0.6 mg/mL or higher, 0.7 mg/mL or higher, 0.8 mg/mL or higher, 0.9 mg/mL or higher, 1 mg/mL or higher, 1.1 mg/mL or higher, 1.2 mg/mL or higher, 1.3 mg/mL or higher, 1.4 mg/mL or higher, 1.5 mg/mL or higher, 1.6 mg/mL or higher, 1.7 mg/mL or higher, 1.8 mg/mL or higher, 1.9 mg/mL or higher, 2 mg/mL or higher, 3 mg/mL or higher, 4 mg/mL or higher, 5 mg/mL or higher, 6 mg/mL or higher, 7 mg/mL or higher, 8 mg/mL or higher, 9 mg/mL or higher, 10 mg/mL or higher, 15 mg/mL or higher or 20 mg/mL or higher and 20 mg/mL or lower, 15 mg/mL or lower, 10 mg/mL or lower, 9 mg/mL or lower, 8 mg/mL or lower, 7 mg/mL or lower, 6 mg/mL or lower, 5 mg/mL or lower, 4 mg/mL or lower, 3.9 mg/mL or lower, 3.8 mg/mL or lower, 3.7 mg/mL or lower, 3.6 mg/mL or lower, 3.5 mg/mL or lower, 3.4 mg/mL or lower, 3.3 mg/mL or lower, 3.2 mg/mL or lower, 3.1 mg/mL or lower, 3 mg/mL or lower, 2.9 mg/mL or lower, 2.8 mg/mL or lower, 2.7 mg/mL or lower, 2.6 mg/mL or lower, 2.5 mg/mL or lower, 2.4 mg/mL or lower, 2.3 mg/mL or lower, 2.2 mg/mL or lower, 2.1 mg/mL or lower, 2 mg/mL or lower, 1.5 mg/mL or lower, 1 mg/mL or lower, 0.5 mg/mL or lower or 0.2 mg/mL or lower, based on the total volume of the composition, although not being limited thereto.
The microcirculatory disorder refers to abnormal microcirculation in which leukocytes, erythrocytes, blood platelets, lymphocytes, etc. cannot smoothly pass through the capillaries. Specifically, the microcirculatory disorder may refer to a state where the functional capillary ratio (FCR) according to Formula 1 is 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower or 5% or lower as compared to the functional capillary ratio of a normal group with no microcirculatory disorder, or the functional capillary ratio is 0.4 or lower, 0.38 or lower, 0.36 or lower, 0.34 or lower, 0.32 or lower, 0.3 or lower, 0.28 or lower, 0.26 or lower, 0.24 or lower, 0.22 or lower, 0.2 or lower, 0.18 or lower, 0.16 or lower, 0.14 or lower, 0.12 or lower, 0.1 or lower, 0.08 or lower, 0.06 or lower, 0.04 or lower or 0.02 or lower. However, the range of the functional capillary ratio for identification of microcirculatory disorder may vary depending on the organ of the subject in which the capillaries are distributed, and is not limited to the above ranges.
Functional capillary ratio=functional capillary area/total capillary area [Formula 1]
The alleviation of microcirculatory disorder may be increasing a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung. Specifically, it may be increasing the functional capillary ratio (FCR) according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area [Formula 1]
The functional capillary area may be measured by identifying the same target factors from a plurality of motion images of the target factors such as leukocytes, erythrocytes, blood platelets, lymphocytes, neutrophils, etc. in a first blood stream or a second blood stream, and may be calculated by measuring the area traveled by the target factors in the first blood stream from the change in their location over time. Specifically, the flow path of the target factors in the first blood stream or second blood stream passing through the capillaries of the subject over time may be measured from the plurality of motion images, and the functional capillary area may be measured from the flow path of the target factors in the first blood stream or second blood stream. More specifically, after measuring the flow path of the target factors in the first blood stream or second blood stream by comparing the plurality of images imaged with a time interval (t), the functional capillary area may be measured from the flow path of the target factors in the first blood stream or second blood stream. More specifically, after measuring the flow path of the target factors in the first blood stream or second blood stream by comparing the plurality of images imaged with a time interval (t), the functional capillary area may be measured from the flow path of the target factors in the first blood stream or second blood stream.
The lung injury may be a disease caused by microcirculatory disorder in the lung, specifically one or more disease selected from a group consisting of pulmonary vasoconstriction, asthma, respiratory failure, respiratory distress syndrome (RDS), acute respiratory failure syndrome (ARDS), cystic fibrosis (CF), allergic rhinitis (AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), pulmonary graft rejection, lung infection, bronchitis and cancer. However, the disease is not specially limited as long as it is a disease caused by microcirculatory disorder in the lung.
The inhibitor against the expression or activity of macrophage-1 antigen according to an aspect of the present disclosure may increase the functional capillary ratio (FCR) in the pulmonary capillaries of the subject as compared to a normal control group or a control group prior to the administration of the inhibitor. Specifically, it may increase the functional capillary ratio (FCR) by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more or 3 times or more. However, the range of the increase of the functional capillary ratio is not limited to the above ranges as long as the pulmonary microcirculatory disorder of the subject can be alleviated. In an example of the present disclosure, it was confirmed that the FCR was decreased by 50% or higher in a CLP mouse model (CLP mouse model or pre-Abc mouse model) having pulmonary microcirculatory disorder as compared to a normal group (Sham), and pulmonary microcirculatory disorder was alleviated as the FCR was increased by about 2 times or more when the expression or activity of Mac-1 was inhibited (anti-Mac-1 mouse model, abciximab mouse model or post-Abc mouse model), suggesting that the composition according to an exemplary embodiment of the present disclosure can prevent or treat lung injury (Experimental Example 12-2,
The inhibitor against the expression or activity of macrophage-1 antigen according to an aspect of the present disclosure may decrease the number of sequestered neutrophils per unit area (512×512 μm) of pulmonary capillaries in a subject as compared to a normal control group or a control group prior to the administration of the inhibitor. Specifically, it may decrease the number of sequestered neutrophils per unit area (512×512 μm) of pulmonary capillaries by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more or 55% or more as compared to a normal control group or a control group prior to the administration of the inhibitor. However, the range of the decrease of the number of sequestered neutrophils per unit area (512×512 μm) of pulmonary capillaries is not limited to the above ranges as long as the pulmonary microcirculatory disorder of the subject can be alleviated.
In this aspect, the composition may be a pharmaceutical composition or a food composition.
The pharmaceutical composition may be prepared into a solid, semisolid or liquid form for oral administration by adding a commonly used inorganic or organic carrier to the active ingredient.
Preparations for oral administration may include a tablet, a pill, a granule, a soft/hard capsule, a dust, a fine granule, a powder, an emulsion, a syrup, a pellet, etc. The active ingredient of the present disclosure may be prepared into a preparation form by a method commonly used in the art, and a surfactant, an excipient, a colorant, a fragrance, a preservative, a stabilizer, a buffer, a suspending agent or other adjuvants commonly used in the art may be used adequately.
The pharmaceutical composition according to the present disclosure may be usefully used for preventing or treating lung injury, specifically lung injury caused by pulmonary microcirculatory disorder, more specifically lung injury in which the number of sequestered neutrophils in pulmonary capillaries is increased, dead space is increased or the number of erythrocytes passing through capillaries is decreased due to pulmonary microcirculatory disorder. The lung injury may be pulmonary vasoconstriction, asthma, respiratory failure, respiratory distress syndrome (RDS), acute respiratory failure syndrome (ARDS), cystic fibrosis (CF), allergic rhinitis (allergic rhinitis, AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), pulmonary graft rejection, lung infection, bronchitis, cancer, etc., although not being limited thereto.
The pharmaceutical composition may be administered orally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, etc.
In addition, the administration dosage of the composition or the active ingredient in the composition will vary depending on the age, sex and body weight of a subject to be treated, the particular disease or pathological condition to be treated, the severity of the disease or pathological condition, administration route and the discretion of a diagnoser. Determination of the administration dosage based on these factors is within the level of those skilled in the art. The administration dosage may be 0.001-2000 mg/kg/day, specifically 0.5-1500 mg/kg/day, more specifically 0.001 mg/kg/day or more, 0.01 mg/kg/day or more, 0.1 mg/kg/day or more, 0.5 mg/kg/day or more, 1 mg/kg/day or more, 10 mg/kg/day or more, 50 mg/kg/day or more, 100 mg/kg/day or more, 150 mg/kg/day or more, 200 mg/kg/day or more, 250 mg/kg/day or more, 300 mg/kg/day or more, 350 mg/kg/day or more, 400 mg/kg/day or more, 450 mg/kg/day or more, 500 mg/kg/day or more, 550 mg/kg/day or more, 600 mg/kg/day or more, 650 mg/kg/day or more, 700 mg/kg/day or more, 750 mg/kg/day or more, 800 mg/kg/day or more, 850 mg/kg/day or more, 900 mg/kg/day or more, 950 mg/kg/day or more, 1000 mg/kg/day or more, 1050 mg/kg/day or more, 1100 mg/kg/day or more, 1150 mg/kg/day or more, 1200 mg/kg/day or more, 1250 mg/kg/day or more, 1300 mg/kg/day or more, 1350 mg/kg/day or more, 1400 mg/kg/day or more, 1450 mg/kg/day or more, 1500 mg/kg/day or more, 1550 mg/kg/day or more, 1600 mg/kg/day or more, 1650 mg/kg/day or more, 1700 mg/kg/day or more, 1750 mg/kg/day or more, 1800 mg/kg/day or more, 1850 mg/kg/day or more, 1900 mg/kg/day or more or 1950 mg/kg/day or more and 2000 mg/kg/day or less, 1950 mg/kg/day or less, 1900 mg/kg/day or less, 1850 mg/kg/day or less, 1800 mg/kg/day or less, 1750 mg/kg/day or less, 1700 mg/kg/day or less, 1650 mg/kg/day or less, 1600 mg/kg/day or less, 1550 mg/kg/day or less, 1500 mg/kg/day or less, 1450 mg/kg/day or less, 1400 mg/kg/day or less, 1350 mg/kg/day or less, 1300 mg/kg/day or less, 1250 mg/kg/day or less, 1200 mg/kg/day or less, 1150 mg/kg/day or less, 1100 mg/kg/day or less, 1050 mg/kg/day or less, 1000 mg/kg/day or less, 950 mg/kg/day or less, 900 mg/kg/day or less, 850 mg/kg/day or less, 800 mg/kg/day or less, 750 mg/kg/day or less, 700 mg/kg/day or less, 650 mg/kg/day or less, 600 mg/kg/day or less, 550 mg/kg/day or less, 500 mg/kg/day or less, 450 mg/kg/day or less, 400 mg/kg/day or less, 350 mg/kg/day or less, 300 mg/kg/day or less, 250 mg/kg/day or less, 200 mg/kg/day or less, 150 mg/kg/day or less, 100 mg/kg/day or less, 50 mg/kg/day or less, 10 mg/kg/day or less, 1 mg/kg/day or less, 0.5 mg/kg/day or less, 0.1 mg/kg/day or less or 0.01 mg/kg/day or less.
The food composition may be a health food composition, and may be prepared by adequately adding the inhibitor against the expression or activity of macrophage-1 antigen of the present disclosure to a food either alone or in combination with another food ingredient according to a common method.
The health food is not particularly limited. Examples of the food to which the active ingredient can be added are meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all health foods in the usual meaning.
A health beverage composition of the present disclosure may contain various flavorants, natural carbohydrates, etc. as additional ingredients like conventional beverages. The natural carbohydrate may be a monosaccharide such as glucose or fructose, a disaccharide such as maltose or sucrose, a polysaccharide such as dextrin or cyclodextrin, or a sugar alcohol such as xylitol, sorbitol, erythritol, etc. As a sweetener, a natural sweetener such as thaumatin and stevia extract or a synthetic sweetener such as saccharin and aspartame may be used. The natural carbohydrate may be contained in an amount of 0.01-0.04 wt %, specifically 0.02-0.03 wt %, based on the total weight of the composition of the present disclosure.
In addition, the health food of the present disclosure may contain various nutrients, vitamins, electrolytes, flavorants, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, antiseptics, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. In addition, it may contain a pulp for preparation of natural fruit juice, fruit juice beverages and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives may be 0.01-0.1 wt % based on the total weight of the composition of the present disclosure.
In another aspect, the present disclosure provides a method for screening a substance for preventing, alleviating or treating lung injury, which includes: (a) a step of preparing a lung injury model; (b) a step of treating the lung injury model with a test substance; (c) a step of measuring the change in the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries of the lung injury model caused by the test substance; and (d) a step of identifying whether the test substance increases a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung injury model. The microcirculation, the microcirculatory disorder, the neutrophils, the macrophage-1 antigen and the lung injury are the same as described above.
The lung injury model may be a non-human animal such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, etc., specifically a sepsis-induced lung-injured non-human animal, more specifically a non-human animal in which sepsis has been induced by LPS administration or a CLP (cecal ligation and puncture) non-human animal mode in which the appendix has been punctured and then ligated, although not being limited thereto.
In an exemplary embodiment of the present disclosure, “relative expression level” may refer to the degree of the inhibition of the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries after treatment with a test substance as compared to the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries before treatment with the test substance. Alternatively, the “relative expression level” may refer to the degree of the inhibition of the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries treated with the test substance as compared to the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries not treated with the test substance. For example, the relative expression level may include the relative expression level of an mRNA or the relative expression level of a protein.
In the step (c), the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries may be compared before and after treating the lung injury model with the test substance. Alternatively, in the step (c), the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries may be compared for a lung injury model treated with the test substance and a lung injury model not treated with the test substance.
In addition, the screening method may further include a step of, if the expression or activity of macrophage-1 antigen is decreased as compared to before the treatment with the test substance as a result of measuring the expression or activity in the step (c) or if a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung injury model, is increased, identifying the test substance as a substance for preventing, alleviating or treating lung injury.
In the step of identifying as a substance for preventing, alleviating or treating lung injury, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the expression or activity of macrophage-1 antigen has decreased by about 10% or more as a result of measuring the expression or activity in the step (c). That is to say, if the expression or activity has decreased by about 10% or more when the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model treated with the test substance was compared with the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model before treatment with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury. Alternatively, if the expression or activity has decreased by about 10% or more when the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model treated with the test substance was compared with the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model not treated with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury. For example, when the expression or activity of macrophage-1 antigen has decreased by 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more or 90% or more as compared to before the treatment with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury, although not being limited thereto. The degree of the expression or activity is measured within statistical significance. The statistical significance refers to significant difference achieved through biological statistical analysis, quantitatively with a p-value smaller than 0.05.
In an exemplary embodiment, the degree of the expression or activity of macrophage-1 antigen may be identified by known techniques, e.g., reverse transcription polymerase chain reaction (RT-PCR), ELISA, western blot or immune blot, although not being limited thereto.
In the step of identifying as a substance for preventing, alleviating or treating lung injury, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio, i.e. the ratio of the area passed by erythrocytes to the total capillary area, has increased by about 1.1 times or more in the step (c). Specifically, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio (FCR) according to Formula 1 of a lung injury model has increased by 1.1 times or more after treatment with the test substance as compared to before the treatment with the test substance. Alternatively, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio has increased by about 1.1 times or more in a lung injury model treated with the test substance as compared to a lung injury model not treated with the test substance.
Functional capillary ratio=functional capillary area/total capillary area [Formula 1]
More specifically, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio has increased by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more or 3 times or more in a lung injury model treated with the test substance as compared to a lung injury model not treated with the test substance.
In another aspect, the present disclosure provides a method for screening a substance alleviating microcirculatory disorder in the lung, which includes: (a) a step of preparing a lung injury model; (b) a step of treating the lung injury model with a test substance; (c) a step of measuring the change in the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries of the lung injury model caused by the test substance; and (d) a step of identifying whether the test substance increases a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung injury model. The lung injury model, the microcirculation, the microcirculatory disorder, the neutrophils, the macrophage-1 antigen, the relative expression level and the functional capillary ratio are the same as described above.
The step (c) may include a step of comparing the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries before and after treating the lung injury model with the test substance. Alternatively, the step (c) may include a step of comparing the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of a lung injury model treated with the test substance with that of a lung injury model not treated with the test substance.
In addition, the screening method may further include a step of, if the expression or activity of macrophage-1 antigen is decreased as compared to before the treatment with the test substance as a result of measuring the expression or activity in the step (c) or if a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung injury model, is increased, identifying the test substance as a substance for preventing, alleviating or treating lung injury.
In the step of identifying as a substance for preventing, alleviating or treating lung injury, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the expression or activity of macrophage-1 antigen has decreased by about 10% or more as a result of measuring the expression or activity in the step (c). That is to say, if the expression or activity has decreased by about 10% or more when the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model treated with the test substance was compared with the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model before treatment with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury. Alternatively, if the expression or activity has decreased by about 10% or more when the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model treated with the test substance was compared with the expression or activity of macrophage-1 antigen in neutrophils within pulmonary capillaries of the lung injury model not treated with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury. For example, when the expression or activity of macrophage-1 antigen has decreased by 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more or 90% or more as compared to before the treatment with the test substance, it may be identified as a substance for preventing, alleviating or treating lung injury, although not being limited thereto. The degree of the expression or activity is measured within statistical significance. The statistical significance refers to significant difference achieved through biological statistical analysis, quantitatively with a p-value smaller than 0.05.
In an exemplary embodiment, the degree of the expression or activity of macrophage-1 antigen may be identified by known techniques, e.g., reverse transcription polymerase chain reaction (RT-PCR), ELISA, western blot or immune blot, although not being limited thereto.
In the step of identifying as a substance for preventing, alleviating or treating lung injury, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio, i.e. the ratio of the area passed by erythrocytes to the total capillary area, has increased by about 1.1 times or more in the step (c). Specifically, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio (FCR) according to Formula 1 of a lung injury model has increased by 1.1 times or more after treatment with the test substance as compared to before the treatment with the test substance. Alternatively, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio has increased by about 1.1 times or more in a lung injury model treated with the test substance as compared to a lung injury model not treated with the test substance.
Functional capillary ratio=functional capillary area/total capillary area [Formula 1]
More specifically, the test substance may be identified as a substance for preventing, alleviating or treating lung injury if the functional capillary ratio has increased by 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more or 3 times or more in a lung injury model treated with the test substance as compared to a lung injury model not treated with the test substance.
In an exemplary embodiment, the present disclosure may provide the macrophage-1 antigen in neutrophils within pulmonary capillaries as a biomarker for diagnosis of pulmonary microcirculatory disorder. In another exemplary embodiment, the present disclosure may provide a composition or a kit for diagnosis of pulmonary microcirculatory disorder. In another exemplary embodiment, the present disclosure may provide a method for providing information for diagnosis of pulmonary microcirculatory disorder.
Specifically, the present disclosure may provide a method for providing information for diagnosis of pulmonary microcirculatory disorder, which includes: a step of measuring the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils isolated from the pulmonary capillaries of a test subject; and a step of identifying a functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung of the test subject.
In an exemplary embodiment, the method may further include a step of comparing the degree of expression or activity of macrophage-1 antigen in the neutrophils isolated from the pulmonary capillaries of the test subject with the degree of expression or activity of macrophage-1 antigen in the neutrophils isolated from the pulmonary capillaries of a normal control group.
Also, in an exemplary embodiment, the method may further include a step of providing information that the subject has pulmonary microcirculatory disorder when the degree of expression or activity of macrophage-1 antigen in the neutrophils isolated from the pulmonary capillaries of the test subject is higher than the degree of expression or activity of macrophage-1 antigen in the neutrophils isolated from a normal control group. For example, it may be identified as pulmonary microcirculatory disorder when the expression or activity of macrophage-1 antigen in neutrophils isolated from the pulmonary capillaries of a test subject is higher than the expression or activity of macrophage-1 antigen in neutrophils isolated from the pulmonary capillaries of a normal control group by 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more or 90% or more. The degree of expression or activity is measured within statistical significance. The statistical significance refers to significant difference achieved through biological statistical analysis, quantitatively with a p-value smaller than 0.05.
In an exemplary embodiment, the method may further include a step of comparing the functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung of the test subject, with the functional capillary ratio of a normal control group.
In another exemplary embodiment, the method may include a step of providing information that the test subject has pulmonary microcirculatory disorder when the functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung of the test subject, is lower than that of a normal control group. The functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total pulmonary capillary area, may be a functional capillary ratio (FCR) according to Formula 1. For example, when the functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total capillary area of the lung of the test subject, is 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower or 5% or lower, it may be determined as pulmonary microcirculatory disorder. However, the ratio for determining microcirculatory disorder may vary depending on the organ of the subject in which the capillaries are distributed, and is not limited to the above ranges. The functional capillary ratio, which is the ratio of the area of functional capillaries through which erythrocytes pass to the total pulmonary capillary area, is measured within statistical significance. The statistical significance refers to significant difference achieved through biological statistical analysis, quantitatively with a p-value smaller than 0.05.
In another aspect, the present disclosure may provide a composition for diagnosis of pulmonary microcirculatory disorder, which contains a reagent for detecting mRNAs or proteins of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries.
In another aspect, the present disclosure may provide a kit for diagnosis of pulmonary microcirculatory disorder, which includes a reagent for detecting mRNAs or proteins of macrophage-1 antigen (Mac-1) in neutrophils within pulmonary capillaries. The kit may further include an instruction describing a method for providing the information for diagnosis of microcirculatory disorder.
In an exemplary embodiment, the reagent for detecting mRNAs or proteins of macrophage-1 antigen included in the composition or kit for diagnosis of pulmonary microcirculatory disorder may include one or more of a primer and a probe binding specifically to macrophage-1 antigen. In an exemplary embodiment, the reagent for detecting proteins of macrophage-1 antigen may include one or more of an antibody and a probe binding specifically to macrophage-1 antigen.
In the present specification, the “probe” includes a polynucleotide having a base sequence capable of binding complementarily to the target site of a gene, a variant thereof, or the polynucleotide with a label attached thereto.
In the present specification, the “primer” refers to a polynucleotide having a base sequence capable of binding complementarily to the terminal of a specific region of a gene to amplify the target site of the gene through PCR or a variant thereof. The primer does not have to be completely complementary to the specific area region, and is sufficient as long as it can form a double-stranded structure by hybridizing to the terminal.
In the present specification, the “hybridization” refers to the formation of a duplex structure through pairing of two single-stranded nucleic acids having complementary base sequences.
The hybridization may occur not only when the sequences of the single-stranded nucleic acids are completely complementary to each other (perfect match) but also when there are some mismatches.
In the present specification, the “polynucleotide” refers to a polymer of a plurality of nucleotides. The term is used in a broad concept, including oligonucleotides which are polymers of tens of nucleotides.
Hereinafter, the present disclosure is described more specifically through examples and experimental examples. However, the following example and experimental examples are provided only for the purpose of illustrating the present disclosure and the scope of the present disclosure is not limited by them.
In the following example and experimental example, all animal experiments were performed in accordance with the standard guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of KAIST (Protocol Nos. KA2014-30 and KA2016-55).
Also, in the following example and experimental example, all data are presented as mean±SD or median±interquartile range, as appropriate, to represent the values of each group. The statistical difference between means or medians was determined by unpaired 2-tailed Student's t-test, Mann-Whitney test, one-way ANOVA with post-hoc Holm-Sidak's multiple comparisons, or Kruskal-Wallis test with post-hoc Dunn's multiple comparison tests, as appropriate. Statistical significance was set at P<0.05 and analysis was performed with Prism 6.0 (Graph Pad).
For quantification of microcirculation, a sepsis-induced acute lung injury mouse model was prepared as follows.
All mice used in the examples were individually housed in ventilated and temperature (22.5° C.- and humidity (52.5%)-controlled cages under 12:12h light:dark cycle and were provided with standard diet and water ad libitum. 8- to 20-weeks-old male mouse (20-30 g) were used for the experiment. C57BL/6N mice were purchased from OrientBio (Suwon, Korea) and Tie2-GFP mice (Stock No. 003658, Jackson Laboratory) where GFP is expressed under an endothelium-specific Tie2 promoter were purchased from Jackson Laboratory.
To generate a sepsis-induced acute lung injury (ALI) mouse model, high-dose LPS was administered to the Tie2-GFP mice (hereinafter, Tie2-GFP-ALI mouse model).
For the high-dose LPS model, LPS (10 mg/kg, E. coli serotype 055:B5, L2880, Sigma-Aldrich) was intraperitoneally administered to the peritoneum of the Tie2-GFP mice 3-6 hours before capillary imaging. As a control group, the same amount of PBS was injected into the peritoneum of the mice.
For diagnosis of microcirculatory disorder, a sepsis-induced acute lung injury mouse model was prepared in the same manner as in Example 1-1.
But, all the mice used in this example were LysMGFP/+ mice, rather than Tie2-GFP mice, provided by Professor Minsu Kim at University of Rochester (hereinafter, referred to as LysMGFP/+ mouse model).
To generate a sepsis-induced acute lung injury (ALI) mouse model, high-dose LPS was administered to the LysMGFP/+ mice.
For the high-dose LPS model, LPS (10 mg/kg, E. coli serotype 055:B5, L2880, Sigma-Aldrich) was intraperitoneally administered to the peritoneum of the LysMGFP/+ mice 3-6 hours before capillary imaging (hereinafter, referred to as ALI mouse models; LPS 3h mouse model: 3 hours after administration of LPS, LPS 6h mouse model: 6 hours after administration of LPS). As a control group, the same amount of PBS was injected into the peritoneum of the mice (hereinafter, referred to as control group or PBS mouse model).
For studying a composition for preventing or treating lung injury disease, a sepsis-induced acute lung injury mouse model was prepared in the same manner as in Example 1-1.
But, all the mice used in this example were LysMGFP/+ mice, rather than Tie2-GFP mice, provided by Professor Minsu Kim at University of Rochester (hereinafter, referred to as LysMGFP/+ mouse model).
As a sepsis-induced acute lung injury (ALI) mouse model, a high-dose LPS mouse model administered to the LysMGFP/+ mouse or a CLP (cecal ligation and puncture) mouse model was utilized.
For the high-dose LPS model, LPS (10 mg/kg, E. coli serotype 055:B5, L2880, Sigma-Aldrich) was intraperitoneally administered to the peritoneum of the LysMGFP/+ mice 3-6 hours before capillary imaging (hereinafter, referred to as ALI mouse models; LPS 3h mouse model: 3 hours after administration of LPS, LPS 6h mouse model: 6 hours after administration of LPS). As a control group, the same amount of PBS was injected into the peritoneum of the mice (hereinafter, referred to as control group or PBS mouse model).
CLP model was performed by a single experienced operator according to a previously described method. Specifically, 75% of the cecum of the LysMGFP/+ mouse was ligated with 6-0 black silk and single puncture with a dual hole in the distal cecum was made with a 21-gauge needle. After the puncture, the cecum was gently squeezed to confirm the patency of the puncture hole for extrusion of feces. The cecum was replaced to the abdominal cavity and the abdominal incision was closed with 4-0 black silk. A normal group (sham group) underwent the same surgical procedure except the cecal ligation and puncture.
Neutrophil-depleted mouse models (hereinafter, neutrophil-depleted models) were prepared to investigate the effect of neutrophils on the lung injury caused by pulmonary microcirculatory disorder. Specifically, a neutrophil-depleted mouse model with no lung injury was prepared by intraperitoneally injecting 200 μg of anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) to the LysMGFP/+ mice of Example 1-3 24 hours before intravital lung imaging (hereinafter, N-Dep model). In addition, a neutrophil-depleted lung injury mouse model was prepared by intraperitoneally injecting 200 μg of anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) to the acute lung injury mouse model of Example 1-3 24 hours before the preparation of the acute lung injury mouse model in Example 1-3 (hereinafter, N-Dep+LPS model).
For imaging of microcirculation in vivo, the erythrocytes and vasculature of the mouse model of Example 1-1 were fluorescence-stained. Specifically, erythrocytes were acquired by cardiac puncture and then labeled according to the method described in the product information sheet. At this time, erythrocytes were fluorescence-labeled Vybrant DiD (V22887, ThermoFisher Scientific). Then, adoptive transfer was performed by injecting 50 million counts of DiD-labeled erythrocytes via a vascular catheter into the tail vein of the Tie2-GFP-ALI mouse model of Example 1-1 right before imaging.
In addition, to visualize the blood vessel with a fluorescent dye, FITC (molecular weight 2 MDa, Sigma-Aldrich)- or tetramethylrhodamine (TMR)-conjugated dextran dye was injected to the Tie2-GFP-ALI mouse model of Example 1-1 via the same vascular catheter.
The procedure of injecting the DiD-labeled erythrocytes or the FITC- or TMR-conjugated dextran dye to the mouse model is described below intravital lung imaging.
(2) Labeling of Neutrophils of Mouse Model of Example 1-2
For imaging of the motion of neutrophils in vivo, anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) conjugated with the fluorophore Alexa Fluor 555 or 647 (A-20005/A-20006, ThermoFisher Scientific) was injected via the tail vein of the mouse model of Example 1-2 2 hours before imaging for labeling of neutrophils.
In addition, the erythrocytes and vasculature of the mouse model of Example 1-2 were fluorescence-stained. Specifically, erythrocytes were acquired by cardiac puncture and then fluorescence-labeled Vybrant DiD (V22887, ThermoFisher Scientific) according to the method described in the product information sheet. Then, 50 million counts of DiD-labeled erythrocytes were injected via a vascular catheter into the tail vein of the mouse model of Example 1-2 right before imaging. In addition, to visualize the vasculature, FITC (molecular weight 2 MDa, Sigma-Aldrich)- or tetramethylrhodamine (TMR)-conjugated dextran dye was injected to the Tie2-GFP-ALI mouse model of Example 1-2 via the same vascular catheter.
The procedure of injecting the fluorophore-labeled anti-Ly6G+ monoclonal antibody, DiD-labeled erythrocytes or the FITC- or TMR-conjugated dextran dye to the mouse model is described below intravital lung imaging.
(3) Labeling of Neutrophils of Mouse Model of Example 1-3
To visualize the molecular expression of pulmonary sequestered neutrophils in vivo, 25 μg of CD11b (Clone M1/70, 553307, BD Biosciences) and 25 μg of CD18 (Clone GAME-46, 555280, BD Biosciences) conjugated with the fluorophore Alexa Fluor 555 (A-20005, ThermoFisher Scientific) was injected via the tail vein of the mouse model of Example 1-3 2 hours before imaging for labeling of neutrophils.
In addition, the erythrocytes and vasculature of the mouse model of Example 1-3 were fluorescence-stained. Specifically, erythrocytes were acquired by cardiac puncture and then fluorescence-labeled Vybrant DiD (V22887, ThermoFisher Scientific) according to the method described in the product information sheet. Then, 50 million counts of DiD-labeled erythrocytes were injected via a vascular catheter into the tail vein of the mouse model of Example 1-3 right before imaging. In addition, to visualize the vasculature, FITC (molecular weight 2 MDa, Sigma-Aldrich)- or tetramethylrhodamine (TMR)-conjugated dextran dye was injected to the Tie2-GFP-ALI mouse model of Example 1-3 via the same vascular catheter.
The procedure of injecting the fluorophore-labeled CD11b and CD18, DiD-labeled erythrocytes or the FITC- or TMR-conjugated dextran dye to the mouse model is described below intravital lung imaging.
(4) Intravital Lung Imaging
Intravital lung imaging was performed as follows.
Specifically, after anesthetizing the Tie2-GFP-ALI mouse model, control mouse model and normal (sham) mouse model of Example 1-1, the LysMGFP/+ mouse model, ALI mouse model, LPS 3h mouse model, LPS 6h mouse model and PBS mouse model of Example 1-2, the LysMGFP/+ mouse model, ALI mouse model, PBS mouse model and normal (sham) mouse model of Example 1-3, the N-Dep mouse model and N-Dep+LPS mouse model of Example 1-4, and a Mac-1-inhibited mouse model of Experimental Example 12-1 with ketamine (80 mg/kg) and xylazine (12 mg/kg), intubation was performed using a 20-gauge vascular catheter as a lightning guidewire and was connected to a mechanical ventilator (MouseVent, Kent Scientific). Ventilation was conducted in the setting of an inspiratory pressure 24-30 mmHg, a respiratory rate of 120-130 breaths per minute, and a positive-end expiratory pressure (PEEP) of 2 cmH2O. 2% isoflurane was delivered to maintain anesthesia status, and pulse oximetry was applied to monitor oxygenation and survival status. A thermal probe of a homeothermic system (RightTemp, Kent Scientific) was introduced into the rectum, and a feedback-regulated heating pad was used to maintain body temperature at 37.0° C. The tail vein was cannulated with a 30-gauge needle attached to a PE-10 tube for intravenous injection of the dye, erythrocytes and neutrophils of (1). Then, the mice were positioned in right lateral decubitus, which was followed by left thoracotomy. The skin and muscle were dissected until rib exposure, and incision was made between the 3rd and 4th ribs to expose the pleura. After the thoracotomy, an imaging window described below in the experimental examples was applied to the surface of the pleura, and negative suction pressure was applied by a pump (DOA-P704-AA, GAST) and a regulator (NVC 2300a, EYELA) via a tube connected to the lung imaging window.
To visualize pulmonary microcirculation in vivo through the pulmonary imaging window, a custom-built video-rate laser-scanning confocal microscopy system was implemented.
Imaging System
Specifically, three continuous laser modules (488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt) and 640 nm (MLD640, Cobolt)) were utilized as excitation light sources for multi-color fluorescence imaging. Laser beams were collinearly integrated by diachronic beam splitters (DBS1; FF593-Di03, DSB2; FF520-Di02, Semrock) and transferred to a laser beam scanner by multi-edge diachronic beam splitters (DBS3; Di01-R405/488/561/635, Semrock). The laser scanning section consisted of 2 axes: X-axis scanning with a rotating polygonal mirror with 36 facets (MC-5, aluminum-coated, Lincoln Laser) and Y-axis scanning with galvanometer scanning mirror (6230H, Cambridge Technology). The two-dimensional raster scanning laser beam was transferred to the lung of the Tie2-GFP-ALI mouse model of Example 1-1 through commercial objective lenses (LUCPLFLN, 20×, NA 0.45, Olympus, LUCPLFLN, 40×, NA 0.6, Olympus, LCPLFLN100×LCD, 100×, NA 0.85, Olympus). The fluorescence signals emitted from the lung of the mouse model on a XYZ translational 3D stage (3DMS, Sutter Instrument) were epi-detected by the objective lenses. De-scanned three-color fluorescence signals were spectrally divided by diachronic beam splitters (DBS4; FF560-Di01, DBS5; FF649-Di01, Semrock) and then detected by a photomultiplier (PMT; R9110, Hamamatsu) through bandpass filters (BPF1; FF02-525/50, BPF2; FF01-600/37, BPF3; FF01-685/40, Semrock). The voltage output of each PMT was digitalized by a 3-channel frame grabber (Solios, Matrox) with 8-bit resolution at a sampling rate of 10 MHz. Video-rate movies were displayed and recorded in real time using a customized imaging software based on Matrox Imaging Library (MIL9, Matrox) and Visual C# at a frame rate of 30 Hz and a frame size of 512×512 pixels.
Image Processing
The images imaged using the imaging system were displayed and stored at an acquisition rate of 30 frames per second with 512×512 pixels per frame. The real-time image frames were averaged over 30 frames using a MATLAB (Mathworks) code to improve contrast and signal-to-noise ratio. To minimize motion artifact, each frame was processed with an image registration algorithm prior to the averaging. Image rendering with three-dimensional reconstruction, track analysis of erythrocytes and neutrophils and plotting of track displacement were conducted using IMARIS 8.2 (Bitplane).
A result of imaging the pulmonary microcirculation of the control mouse model of Example 1-1 using the imaging system described above and processing the obtained mages as described above is shown in
As shown in
Pulmonary microcirculation was imaged in the same manner as in Experimental Example 1-1, except that the LysMGFP/+ mouse model of Example 1-2 was used instead of the Tie2-GFP-ALI mouse model of Example 1-1.
A result of imaging the pulmonary microcirculation of the LysMGFP/+ mouse model of Example 1-2 to which LPS was not administered using the imaging system described above and processing the obtained mages as described above is shown in
As shown in
In contrast, in the sepsis-induced acute lung injury mouse model, the functional capillary ratio (FCR; calculated as a ratio of the functional capillary area to the total capillary area) was decreased during the early stage of acute lung injury. The capillary obstruction found in the lung injury mouse model was induced by the objects inside the capillaries that could represent the primary pathophysiological mechanism underlying the decreased FCR. From the result shown in
Therefore, it can be seen that neutrophil in capillaries, specifically the entrapment of neutrophils in capillaries, are associated with microcirculatory disorder, particularly sepsis. Accordingly, the method for providing information and the apparatus for diagnosis of microcirculatory disorder according to an aspect of the present disclosure can provide a plurality of motion images of neutrophils passing through capillaries by clearly visualizing the motion of neutrophils inside the capillaries and allow easy and convenient diagnosis of microcirculatory disorder in a subject by acquiring information about the motion of each neutrophil.
For quantification of microcirculation in a subject based on a functional capillary ratio (FCR), functional capillary imaging analysis was performed using a real-time movie of DiD-labeled erythrocytes flowing in capillaries, which was acquired using the imaging system and image processing of Experimental Example 1-1. After splitting the colors of the movie, sequential images of channels detecting DiD was processed by a median filter with a radius of 2 pixels to enhance the signal-to-noise ratio. The maximal intensity projection of 600-900 frames (20-30 seconds) was generated to show the functional capillaries perfused by erythrocytes. The functional capillary ratio (FCR) was calculated according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area [Formula 1]
In Formula 1, the total capillary area means the vessel area detected by Tie2 or dextran signaling, and the functional capillary area means the area traveled by DiD-labeled erythrocytes. All image processing to calculate the functional capillary ratio was performed with ImageJ (https://imagej.nih.gov/ij/), and the result is shown in
As shown in
The functional capillary ratio was compared for the Tie2-GFP-ALI mouse model in which sepsis was induced by LPS administration in Example 1-1 and a control group to which PBS was administered instead of LPS. Pulmonary microcirculation was imaged also for the control mouse model in the same manner as in Experimental Example 1-1 and Experimental Example 2 and the obtained images were analyzed. As a result, although no significant difference was found in the mean velocity of erythrocytes between the control mouse model and the lung injury mouse model, the erythrocyte perfusion pattern changed dramatically in the lung injury mouse model. In addition, the erythrocytes in sequential images from 600 frames (20 seconds) were presented in a maximal intensity projection to quantify the perfusion area of the erythrocytes.
The functional capillary ratio (FCR) of the control mouse model and the lung injury mouse model (Tie2-GFP-ALI mouse model) was calculated according to Formula 1 as in Experimental Example 2, and the result is shown in
As shown in
Also, as shown in
Furthermore, arterial blood gas analysis was performed to assess the oxygen partial pressure and carbon dioxide partial pressure in the arterial blood of the control group model and the lung injury mouse model (Tie2-GFP-ALI mouse model). Specifically, a 1-mL syringe with a 22-gauge needle was coated with heparin and introduced into the left ventricle of the heart of the control group model (PBS, n=6) and the lung injury mouse model (LPS, n=16). Then, about 200 μL of blood was sampled and analyzed with an i-STAT handheld blood analyzer (G3 cartridge, Abbott Point of Care Inc.). The mice were euthanized in a CO2 chamber right after the blood sampling. The arterial blood gas analysis result is shown in
Accordingly, by using the method for quantitation of microcirculation and the apparatus for measuring microcirculation according to an aspect of the present disclosure, the microcirculation in a subject can be quantified easily and conveniently in vivo based on the functional capillary ratio, and microcirculatory disorder can be identified accurately and quickly based on the quantification result.
The relationship between the motion of neutrophils in capillaries and microcirculatory disorder was confirmed in Experimental Example 1-2. In order to visualize the motion of neutrophils in the ALI mouse model (LPS) and the control group model (PBS) prepared in Example 1-2, a customized video-rate laser scanning confocal microscopy system was implemented in the same manner as in Experimental Examples 1-1 and 1-2. The result of intravital imaging is shown in
As shown in
In addition, based on the wide field image processing result of
Therefore, it can be seen that the sequestration (entrapment) of neutrophils in capillaries is associated with microcirculatory disorder. Accordingly, the method for providing information and the apparatus for diagnosis of microcirculatory disorder according to an aspect of the present disclosure can provide a plurality of motion images of neutrophils passing through capillaries by clearly visualizing the motion of neutrophils inside the capillaries and allow easy and convenient diagnosis of microcirculatory disorder in a subject by acquiring information about the motion of neutrophils.
It was confirmed in Experimental Example 4 that the entrapment of neutrophils in capillaries can lead to microcirculatory disorder such as lung injury. In this example, the motion (track) of neutrophils was comparatively analyzed for the acute lung injury mouse models (LPS 3h mouse model and LPS 6h mouse model) in which sepsis was induced by LPS administration in Example 1-2 and the control mouse model to which PBS was administered instead of LPS.
Tracking of Neutrophils Through Time-Lapse Imaging
Specifically, a customized video-rate laser scanning confocal microscopy system was implemented in the same manner as in Experimental Examples 1-1 and 1-2 and the pulmonary microcirculation of mouse models was intravitally imaged at a slow rate for 30 minutes. The result is shown in
As shown in the track images of the neutrophils time-lapse imaged for 30 minutes (
This is also confirmed from the track displacement of the neutrophils shown in
That is to say, from
Comparison of Degree of Neutrophil Sequestration
The degree of neutrophil sequestration was compared for the control group and the lung injury mouse model from the time-lapse imaging result (
As shown in
It was confirmed in Experimental Example 5 that the motion of neutrophils in capillaries was decreased in the lung injury mouse model as compared to the control group model due to sequestration. Based on the data acquired in Experimental Examples 1, 4 and 5, the dynamic behavior of neutrophils (sequestration time, track displacement length, track length, track velocity and track meandering index) was comparatively analyzed for the acute lung injury mouse models in which sepsis was induced by LPS administration in Example 1-2 (LPS 3h mouse model and LPS 6h mouse model) and the control mouse model to which PBS was administered instead of LPS, and the result is shown in
First, the sequestration time (
The track displacement length (
The track length (
The track velocity (
The track meandering index (
From
Also, as seen from
Taken together, the dynamic behavior of neutrophils shows that, during the early period of endotoxin-induced acute lung injury, neutrophils are activated and become motile inside the capillaries; however, in the late period, they are gradually arrested inside the capillaries. Accordingly, the method for providing information and the apparatus for diagnosis of microcirculatory disorder according to an aspect of the present disclosure can provide a plurality of motion images of neutrophils passing through capillaries by clearly visualizing the motion of neutrophils inside the capillaries and allow easy and convenient diagnosis of microcirculatory disorder in a subject by acquiring information about the motion of neutrophils.
It was confirmed from Experimental Example 6 that neutrophils are sequestered inside capillaries due to acute lung injury. The entire process of dead space formation in the pulmonary microcirculation was investigated through intravital imaging of the ALI mouse model prepared in Example 1-2.
Real-Time Imaging and Time-Lapse Imaging
Specifically, the real-time images of
In addition, cluster formation by neutrophils (Ly6G+ cells) in the branching region of arterioles connected to capillaries was intravital imaged at a slow rate for 10 minutes using a customized video-rate laser scanning confocal microscopy system according to the same method as in Experimental Examples 1-1 and 1-2. As a result, the time-lapse images of
As shown in
In addition, as shown in
Confirmation of Correlation Between Neutrophil Sequestration and Dead Space Formation Through Visualization of Functional Capillaries
To confirm the correlation between neutrophil sequestration and dead space formation, erythrocytes were stained to visualize the functional capillaries where the erythrocytes circulate smoothly. Specifically, the pulmonary microcirculation of the ALI mouse model of Example 2 having DiD-labeled erythrocytes was imaged at a slow rate for 10 minutes according to the method of Experimental Example 5 and the obtained images were processed by the method of Experimental Examples 1-1 and 1-2. The track path of the DiD-labeled erythrocytes was obtained in the same manner as in the neutrophil tracking of Experimental Example 5. The result is shown in
As shown in
Accordingly, since the neutrophils sequestered inside capillaries due to microcirculatory disorder such as lung injury can form dead space, the method for providing information and the apparatus for diagnosis of microcirculatory disorder according to an aspect of the present disclosure can provide a plurality of motion images of neutrophils passing through capillaries by clearly visualizing the motion of neutrophils inside the capillaries and allow easy and convenient diagnosis of microcirculatory disorder in a subject by acquiring information about the motion of neutrophils.
It was confirmed in Experimental Example 6 that neutrophils in capillaries are sequestered due to acute lung injury. The effect of neutrophil sequestration on the release of reactive oxygen species (ROS) in situ was investigated for the ALI mouse model and the control group to which PBS was administered instead of LPS prepared in Example 1-2.
Specifically, DHE (dihydroethidium) staining was performed using high-dose DHE (10 mg/kg) to investigate the generation of reactive oxygen species according to the method previously used in intravital imaging researches (Finsterbusch M, Hall P, Li A, Devi S, Westhorpe C L, Kitching A R, Hickey M J. Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus. Proc Natl Acad Sci USA 2016: 113(35): E5172-5181). A stock solution was prepared by dissolving 15.7 mg of DHE in 1.5 mL of DMSO and was stored at −20° C. Then, after heating the DHE solution up to 60° C. to 10 mg/kg, followed by diluting in 50 μL of saline, it was immediately injected intravenously to the ALI mouse model and the control mouse model of Example 1-2 for intravital microscopy.
The generation of reactive oxygen species (ROS) was investigated 20 minutes after the injection of DHE. The number of neutrophils (ROS+Ly6G+) was determined by counting the cells with naked eyes or using the ImageJ program, or using the Spots function of the IMARIS program. In addition, the number of reactive oxygen species-generating neutrophils (ROS+Ly6G+) was determined from the neutrophils double positive for neutrophils (Ly6G+, red color) and DHE (blue color) using the Colocalization function of the IMARIS program.
The result is shown in
In addition, as shown in
Through this, it was confirmed that, in contrast to the previous understanding that reactive oxygen species are produced by neutrophils at the site of inflammation, the production of reactive oxygen species is initiated at a much early stage because of the development of neutrophil entrapment in capillaries. The findings also imply that the entrapped neutrophils could release reactive oxygen species in situ, which could harm the endothelial cells and adjacent intravascular structure before extravasation.
Accordingly, since the neutrophils sequestered inside capillaries can produce reactive oxygen species during microcirculatory disorder such as lung injury can form dead space, the method for providing information and the apparatus for diagnosis of microcirculatory disorder according to an aspect of the present disclosure can provide a plurality of motion images of neutrophils passing through capillaries by clearly visualizing the motion of neutrophils inside the capillaries and allow easy and convenient diagnosis of microcirculatory disorder in a subject by acquiring information about the motion of neutrophils.
To summarize, the method for quantitation of microcirculation and the apparatus for measuring microcirculation according to an aspect of the present disclosure allow easier and more convenient quantification of microcirculation in a subject in vivo based on the functional capillary ratio, and allow accurate and fast diagnosis of microcirculatory disorder based on the quantification result.
Pulmonary microcirculation was imaged in the same manner as in Experimental Example 1-1 except that the control mouse model (PBS) and the ALI mouse model (LPS) of Example 1-3 and the neutrophil-depleted models of Example 1-4 (N-Dep mouse model and N-Dep+LPS mouse model) were used as mouse models instead of the Tie2-GFP-ALI mouse model of Example 1-1.
After imaging the pulmonary microcirculation of the control mouse model (PBS) and the ALI mouse model (LPS) of Example 1-3 and the neutrophil-depleted models of Example 1-4 (N-Dep mouse model and N-Dep+LPS mouse model) using the imaging system described above, the obtained images were processed according to the image processing process described above. The result is shown in
Comparison of Functional Capillary Ratio (FCR)
It was confirmed in Experimental Example 9 that pulmonary microcirculatory disorder is improved in the neutrophil-depleted models. In order to confirm it through quantified data, functional capillary imaging analysis was performed using the real-time movie of DiD-labeled red blood cell flowing through capillaries obtained using the imaging system and image processing described in Experimental Example 9. After splitting colors of the movie, sequential images of channels detecting DiD were processed by a median filter with a radius of 2 pixels to enhance the signal-to-noise ratio. Maximal intensity projection of 600-900 frames (20-30 seconds) was generated to show the functional capillary perfused by erythrocytes. The functional capillary ratio (FCR) was calculated according to Formula 1.
Functional capillary ratio=functional capillary area/total capillary area. [Formula 1]
In Formula 1, the total capillary area means the vessel area detected by Tie2 or dextran signaling, and the functional capillary area means the area traveled by DiD-labeled erythrocytes. All image processing to calculate the functional capillary ratio was performed with ImageJ (https://imagej.nih.gov/ij/), and the result is shown in
As shown in
Histological Analysis
Histological analysis was performed to compare the number of neutrophils per unit area (512×512 μm) of the control mouse model (PBS) and the ALI mouse model (LPS) of Example 1-3 and the neutrophil-depleted models of Example 1-4 (N-Dep mouse model and N-Dep+LPS mouse model).
Specifically, lung tissues were harvested after intravital imaging of the mouse models. The tissues were perfused and fixed with 4% paraformaldehyde and then further fixed overnight in 4% paraformaldehyde. For H&E (hematoxylin and eosin) staining, the fixed tissues were processed using standard procedures, embedded in paraffin and then sliced into 4-μm sections, followed by conventional H&E staining. The result is shown in
In addition, although the number of neutrophils (LysM+ cells) was decreased to a certain level during the preparation of the neutrophil-depleted models of Example 1-4, as shown in
To summarize the results of
Although it was confirmed in Experimental Example 10 that neutrophil depletion in capillaries can prevent or treat lung injury by alleviating microcirculatory disorder, the neutrophil depletion strategy is not feasible clinically. Therefore, to identify the target for sequestered neutrophils for prevention or treatment of lung injury, experiment was conducted as follows. It was hypothesized that the integrin expression pattern of the neutrophils in the left ventricle that had passed through pulmonary capillaries would be different from the pattern of the neutrophils in the lung.
Flow Cytometry
For investigation of the integrin expression of left ventricle-derived neutrophils and lung-derived neutrophils, neutrophils were isolated from the left ventricle (LV) and lung of the control (PBS) mouse model and the ALI mouse model (LPS) of Example 1-3, respectively, and flow cytometry analysis was conducted for the isolated neutrophils.
First, in order to isolate pulmonary sequestered neutrophils, the lung was harvested and digested without perfusion. The lung was placed in PBS solution, minced, filtered through a 40-μm filter and stained at 4° C. for 30 minutes. Meanwhile, for isolation of neutrophils from left ventricle, 100 μL of blood was taken from the left ventricle of the mouse model using a syringe and 1.0×106 neutrophils were isolated using a flow cytometer (FACS, BD, LSRFortessa™) after hemolyzing erythrocytes. Ly6G-FITC (1A8, 551460, BD Biosciences), CD11a-BV510 (M17/4, 563669, BD Biosciences) CD11b-PE-Cy7 (M1/70, 552850, BD Biosciences), CD18-APC (C71/16, 562828, BD Biosciences), CD62L (MEL-14, 560514, BD Biosciences) and Viability Dye eFluor 506 (65-0866-14, ThermoFisher Scientific) were used as clonal antibodies and the stained cells were analyzed with an LSR Fortessa flow cytometer (BD Biosciences). Then, flow cytometry was performed for the two groups of neutrophils gated on Ly6G+ using Flowjo (FlowJo, LLC). The result is shown in
From
Intravital Imaging
The integrin in the sequestered neutrophils of the control (PBS) mouse model of Example 1-3 and the ALI mouse model (LPS) was imaged and then processed according to the method of Experimental Example 9. The result of visualizing neutrophils and the expression of CD11b and CD18 on the surface of the neutrophils in vivo is shown in
As shown in
Comparison of Number of Neutrophils Expressing CD11b or CD18
The number of neutrophils expressing CD11 b or CD18 was compared for the ALI mouse model (LPS) and control mouse model (PBS) of Example 1-3, and the result is shown in
As shown in
From the above results, it was confirmed that the expression level of Mac-1 (CD11b/CD18) integrin is increased in the neutrophils sequestered due to lung injury as compared to the neutrophils circulating in the microcirculation.
It was confirmed in Experimental Example 11 that the expression level of Mac-1 (CD11b/CD18) integrin is increased in the neutrophils sequestered in the lung due to lung injury. In this example, it was investigated whether lung injury, particularly microcirculatory disorder, is improved by a Mac-1 inhibitor against the expression or activity in a Mac-1-inhibited mouse model prepared in Experimental Example 12-1. The CLP model of Example 1-3 was used as a sepsis model to extend the above findings to a polymicrobial sepsis model.
In order to investigate the effect of the inhibition of Mac-1 activity in lung injury, a mouse model with the activity of Mac-1 inhibited (hereinafter, Mac-1-inhibited model) was prepared. Specifically, the anti-Mac-1 model with the activity of Mac-1 inhibited was prepared by intraperitoneally injecting anti-CD11b antibody (5 mg/kg, Clone M1/70, 553307, BD Biosciences) 1 hour after the preparation of the CLP mouse model of Example 1-3 and 5 hours before intravital. In addition, an abciximab model was prepared as another Mac-1-inhibited model by injecting abciximab (10 mg/kg, Clotinab, ISU Abxis) to the CLP mouse model of Example 1-3 in the same manner as the preparation of the anti-Mac-1 model.
The pulmonary microcirculation of the CLP mouse model of Example 1-3 (Fc in
As shown in
Also, as shown in
The result of histological analysis also showed that the number of sequestered neutrophils (Ly6G+ cells) was increased for the CLP mouse model (Fc) as compared to the normal group (Sham) and then was recovered to a level comparable to that of the normal group by inhibition of Mac-1 (anti-Mac-1 mouse model and abciximab mouse model), as shown in
It was confirmed in Experimental Example 12-2 that microcirculatory disorder can be alleviated by inhibiting the expression or activity of Mac-1. This was confirmed again by investigating the effect before and after administration of abciximab.
Specifically, pulmonary microcirculation was imaged 6 hours after preparation of the CLP mouse model of Example 1-3 (hereinafter, pre-Abc mouse model) in the same manner as in Experimental Example 9, and functional capillary ratio (FCR) was measured in the same manner as in Experimental Example 10. Then, after administering abciximab to the CLP mouse model in the same manner as in Experimental Example 12-1 (hereinafter, post-Abc mouse model), pulmonary microcirculation was imaged and functional capillary ratio (FCR) was measured 30 minutes later. The result is shown in
As shown in
In addition, arterial blood gas analysis was performed to assess the oxygen partial pressure and carbon dioxide partial pressure in the arterial blood of the pre-Abc mouse model and the post-Abc mouse model. Specifically, a 1-mL syringe with a 22-gauge needle was coated with heparin and introduced into the left ventricle of the heart of the normal mouse model (Sham) (n=8), pre-Abc mouse model (Fc) (n=10) and post-Abc mouse model (Abc) (n=6). Then, about 200 μL of blood was sampled and analyzed with an i-STAT handheld blood analyzer (G3 cartridge, Abbott Point of Care Inc.). The mice were euthanized in a CO2 chamber right after the blood sampling. The arterial blood gas analysis result is shown in
As shown in
Accordingly, the composition containing a Mac-1 inhibitor against the expression or activity in neutrophils according to an aspect of the present disclosure has a superior effect of preventing or treating lung injury by alleviating microcirculatory disorder in the lung.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0061415 | May 2019 | KR | national |
| 10-2019-0061416 | May 2019 | KR | national |
| 10-2019-0061417 | May 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/000549 | 1/10/2020 | WO |
