Patent Application
20160270647
The present disclosure relates generally to an assessment of central nervous system inflammation, and more specifically, to exemplary embodiments of systems, methods and computer-accessible media for assessing nervous system inflammation using, e.g., leukocyte-endothelial interaction in the retina.
Inflammation is the immune reaction of a tissue to injury or infection that often involves an increase in blood flow with an influx of white blood cells (leukocytes) and chemokines that facilitate healing. Leukocyte infiltration can be a hallmark of inflammation, and a leukocyte-endothelial interaction (“LEI”) can be a first step in the recruitment of leukocytes from the circulation to the inflamed tissue. Therefore, a method to image LEI noninvasively can be useful for rapid assessment of the time course of brain injury and response to intervention. A method to detect LEI can be especially useful where sample collection and analysis (e.g., blood or spinal fluid) can be difficult, and access to heavy imaging machinery, such as MRI, may not be available, such as in a military setting or in rural, underserved areas.
Traumatic brain injury (“TBI”) can usually be accompanied, and can be exacerbated, by inflammation of or in a central nervous system (“CNS”). Survivors of TBI suffer from long-term disabilities, and even mild TBI can cause cognitive impairment, fatigue and pain. In experimental models of TBI, it has been shown that integrin on leukocytes and soluble intercellular cellular adhesion molecules (“ICAM”) can be elevated in TBI. Furthermore, aggregates of leukocytes and platelets can be found within hours after TBI, indicating that activation of leukocytes and platelets can be among the earliest signs of neuro-inflammation. Activated leukocytes can interact with endothelial cells that may themselves not express inflammatory signals, as demonstrated in leukocytes extravasating in the contralateral (e.g., uninjured) brain hemisphere after experimental TBI.
Similarly, multiple sclerosis is an autoimmune disease that is characterized by inflammation in the brain and spinal chord. For example, infiltration of the brain by autoreactive immune cells that originate in the peripheral circulation damages the axonal myelin sheath, thus resulting in a demyelination of the neurons. Demyelination interferes with a neuronal signal transmission, which in turn results in a number of physical or cognitive disabilities. Since myelin once damaged likely cannot be repaired, it is important to combat multiple sclerosis as early as possible.
Thus, it may be beneficial to provide an exemplary system, method and computer-accessible medium that can overcome at least some of the deficiencies described herein above, and provide, for example, an assessment of a nervous system inflammation.
In reviewing a radiation-induced injury in the mouse retina, persistent inflammation (e.g., microglia activation, breakdown of blood-retinal barrier, and LEI) following a single dose of gamma irradiation was observed. Microglia activation was also visualized after controlled focal damage in retinal blood vessels using a CX3CR1 reporter mouse whose microglial cells (e.g., the resident immune sentinels of the CNS) express the green fluorescent protein (“GFP”). (See, e.g.,
For example, using exemplary embodiments of systems, methods and computer-accessible mediums according to the present disclosure, activated leukocytes, either autoreactive or after activation through interaction with the inflamed blood-brain barrier, can be detected interacting in the retinal vasculature by in vivo imaging. The integrity of the blood-retina barrier can be compromised by LEI, and become detectable by leakage of fluorescein. Thus, according to such exemplary embodiments, it is possible to characterize LEI and integrity of the blood-retina barrier in retinal vasculature in, e.g., autoimmune encephalomyelitis (EAE), a rodent model of MS, as potential markers that can be assessed by noninvasive imaging of the retina. It is also possible to determine if a decrease of clinical score during treatment, for example, with integrin alpha4 blocker (natalizumab) corresponds to a reduction of LEI and fluorescein leakage.
The inflammation associated with injury in the CNS, e.g., the eye, brain, spinal chord, etc., as well as in other locations and/or areas of anatomy can be assessed by imaging LEI, e.g., in the retina near the optic disc. For example, after an induction of inflammation in the brain, for example in TBI, Multiple Sclerosis (MS), a number of activated leukocytes remain in circulation for some time because not all leukocyte can infiltrate the site of inflammation at once. With an increasing circulation time, the probability that leukocytes pass the retinal vasculature, where they can be detected, can increase. Likewise, LEI associated with ocular diseases, such as diabetic retinopathy or Glaucoma, can be detected by an exemplary retinal imaging procedure according to an exemplary embodiment of the present disclosure. A retinal flow cytometer was previously developed for detection and quantification of fluorescently labeled leukocytes in the circulation of live animals. The exemplary systems, methods and computer-accessible mediums, according to an exemplary embodiment of the present disclosure, can be used to visualize activated leukocytes interacting with the retinal vasculature following CNS injury.
Exemplary embodiments of the systems, methods and computer accessible medium according to the present disclosure can be provided to quantify LEI in the retina and in the brain using an established model of radiation-induced CNS inflammation. Chimeric mice can be generated whose leukocytes can express the red fluorescent protein (“DsRed”), and whose microglia expresses the green fluorescent protein (“GFP”). LEI in the retinal vasculature can be assessed using an SLO developed specifically for mouse eye imaging, while LEI in the brain vasculature can be imaged through the thinned skull using a custom-built video rate laser scanning confocal/multiphoton microscope.
Microglia activation can serve as an independent marker for inflammation at these two locations. The exemplary systems, methods and computer-accessible mediums, according to an exemplary embodiment of the present disclosure, can be used to assess LEI following TBI. For example, the correlation (e.g., kinetics and dose response) between LEI in the retina and in the brain can be examined using the Marmarou model of TBI, or by controlled cortical impact.
An exemplary procedure using the exemplary SLO can be performed for human eye imaging. The SLO can be optimized for high resolution label free imaging of the optic disc by implementing adaptive optics and speckle reduction techniques. The optimum wavelength for imaging leukocyte based on intrinsic backscattering contrast can also be determined. Pilot studies to image LEI in human eyes can be initiated that can use optical coherence tomography (“OCT”) to image the retinal vasculature as a biomarker for TBI.
These and other objects of the present disclosure can be achieved by provisions of exemplary system, method and computer-accessible medium according to exemplary embodiments of the present disclosure that can, for example, receive regarding at least one portion of ophthalmic sample(s) based on a radiation(s) provided from the sample, determine whether an inflammation marker(s) is present in the portion(s) of the sample based on the received information, and identify that an abnormality(s) exists in a further anatomical structure based on the determination. The further anatomical structure can be different from the sample(s).
According to further exemplary embodiments of the present disclosure, the further structure can include a portion(s) of a central nervous system. For example, the radiation(s) can be provided from the retina of the sample(s). Imaging the retina of the sample(s) can be performed, and the determination can be made regarding the retina based on the image. The marker(s) can be measurable, and can include an interaction of white blood cells with a blood vessel wall. The marker(s) can also be or include an identification of blood vessel leakage. The information can be obtained from a confocal reflectance system, a fluorescence system, an optical coherence tomography system, or an optical frequency domain imaging system. In certain embodiments of the present disclosure the abnormality(s) can include (i) a brain injury, (ii) a spinal cord injury, (iii) multiple sclerosis, (iv) a stroke, or (v) a brain tumor. The abnormality(s) can also include (i) a brain abnormality, (ii) a spinal cord abnormality, (iii) or an ophthalmic abnormality.
These and other objects, features and advantages of the exemplary embodiments of the present disclosure will become apparent upon reading the following detailed description of the exemplary embodiments of the present disclosure, when taken in conjunction with the appended claims.
Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying Figures showing illustrative embodiments of the present disclosure, in which:
Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the present disclosure will now be described in detail with reference to the figures, it is done so in connection with the illustrative embodiments and is not limited by the particular embodiments illustrated in the figures or in the appended claims.
The exemplary system, method and computer-accessible medium, according to an exemplary embodiment of the present disclosure, can facilitate a longitudinal in-vivo tracking of both the resident microglial (e.g., disappearing) and the BMDC (e.g., engrafting) populations that can assist to obtain a more complete picture of cellular radiation response. For example, the inner retina, as an optically-directly accessible gray matter compartment of the CNS, lends itself to in vivo imaging and cell tracking, facilitating disease progression in individual animals to be followed serially over time. The exemplary system, method and computer-accessible medium according to an exemplary embodiment of the present disclosure can utilize a multi-color SLO specifically for confocal imaging of the mouse retina with the compatibility of simultaneously acquiring up to three fluorescence channels at video rate. The dynamics of the native microglia population and the engrafting BMDCs after BMT have been investigated. For this purpose, heterozygous CX3CR1 mice that express GFP in retinal microglia were exposed to a lethal dose of gamma radiation and rescued with a bone marrow transplant from universal DsRed donor mice. The numbers of native GFP+ cells and donor DsRed+ BMDCs in the retina were quantified by in vivo imaging before and over a time course of four months after the irradiation and BMT. Progressive loss of GFP+ microglia was observed with delayed engraftment of DsRed+ BMDCs. The total cell number was below the baseline value of resident microglial cells for most of the observation period, even after four months. Leukocyte endothelial interaction, which can be essential for circulating cells to recognize their homing site, was observed throughout this period, suggesting prolonged inflammation in the retina. Fluorescein angiography demonstrated that the integrity of the blood-retina barrier can be compromised after irradiation.
Exemplary Results
An exemplary SLO has been developed for a retinal imaging of a mammal (e.g., mouse, human, etc.) based on the video-rate confocal microscope. (See, e.g., References 15 and 16). Multiple laser sources and a multi-edge dichroic beam splitter facilitated up to three channels to be acquired simultaneously (See, e.g.,
Reflectance imaging can be accomplished using, e.g., a polarization rotation with a quarter-wave plate 150 placed between the Volk lens 145 and the eye 140. Further, a polarizing beam splitter cube 151 can separate backscattering from the retina from the incident light and direct it onto a focusing lens 152 that focuses the reflected light through a confocal pinhole 153 into the reflectance detector 154. The fluorescence emitted in the retina of the eye 140 can be separated from the excitation light, and directed into the fluorescence detection arm by the main, triple-edge dichroic beam splitter. Inside the fluorescence detection arm, e.g., 560 nm and 650 longpass dichroic beam splitters at 560 nm (e.g., FF560-Di01, Semrock) 121 and 650 nm (e.g., FF650-Di01, Semrock) 122 can separate light into three (or more) distinct fluorescence detectors. Each fluorescence detector can be or include an assembly including, e.g., a bandpass filter 123 to further narrow the fluorescence detection (e.g., red=650-825 nm, green=550-650 nm and blue=500-550 nm) and an approximately 75 mm achromatic lens 124 that can focus the light through an approximately 50 μm confocal pinhole 125 (e.g., corresponding to about 3.2 to 4.1 times the Airy disc size) into a photomultiplier tube (“PMTs”) 126 (e.g., R3896). Three or more channels, for example, can be acquired simultaneously when the PMTs 126 and 154 are connected to one or more computers, thereby, e.g., facilitating the observation of up to three distinct cell populations in real-time at video-rate. It should be understood that other type of electro-magnetic radiations (e.g., other than light) can be used with the exemplary embodiments described herein. In addition, the above-described configuration is merely exemplary, and it should be understood that other configuration can be implemented in accordance with the exemplary embodiments of the present disclosure.
Pinholes of about 50 μm in diameter, corresponding to 3 to 4 Airy discs, resulted in a depth of focus of approximately 40 μm, thus facilitating a thick optical section of retinal tissue to be imaged at once, without the need for axial movement. In its current configuration, the SLO does not use adaptive optics. (See, e.g., References 17, 18). GFP and DsRed were excited with the 491 and 532 nm excitation laser(s) (e.g., see exemplary configuration 110 of
An alignment of the exemplary (e.g., laser or other electro-magnetic radiation producing) sources 105, 110 and exemplary respective telescopes 106, 111 can be undertaken to reduce or minimize chromatic focusing error(s) among the multiple (e.g., three) wavelengths. To verify the results of the exemplary system alignment, the inner retina was imaged in reflectance mode with each of the three lasers in the same mouse. The exemplary results indicate that the three lasers image the same optical section in the mouse retina (See e.g., exemplary reflectance images shown in
GFP+ resident microglia and DsRed+ BMDCs were tracked over a time course of four months after BMT from universal DsRed donors into lethally irradiated CX3CR1 recipient mice (n=3).
This number decreased by 0% at the first measured time point 15 days after irradiation and transplantation (See e.g., exemplary graph of
A breakdown of the blood-retina barrier can be and was detected by an exemplary fluorescein angiography procedure, where fluorescein leaks into the retinal parenchyma were present, e.g., only where the BRB is damaged (as shown in
One of the important aspects of in vivo imaging can be the ability to detect dynamic interactions between sells and their environment. Thus, in addition to longitudinal cell tracking, in vivo imaging can facilitate “zooming in” on the short-term dynamics at each time point to visualize cell behavior and cellular interactions that may not be available using histological methods. Circulating BMDCs that can temporarily interact with the vascular endothelium can be observed by acquiring time-lapse images.
Exemplary Discussion
Consistent with the notion of inflammation, e.g., leukocyte endothelial interaction can be observed after the irradiation and bone marrow transplant. Leukocyte endothelial interaction can be the result of adhesion molecule mediated signaling that can enable circulating leukocytes to roll, arrest and eventually extravasate near the site of an inflammation. (See, e.g., Reference 31). Adhesion molecule upregulation and leukocyte endothelial interaction can be considered to last approximately one week after injury. (See, e.g., References 32 and 33). While a peak of interacting leukocytes was observed seven days after irradiation, the exemplary results can indicate that leukocyte endothelial interaction continues throughout the observation period. The interaction of leukocytes and endothelial cells can be frequently accompanied by a disruption of the blood-retinal barrier. Fluorescein angiography can demonstrate that the blood retinal barrier (BRB) was compromised during the first few days after irradiation. (See, e.g.,
In the protected environment of the CNS and the retina, e.g., leukocyte endothelial interaction and leakage through the blood-retina barrier may not be observed under physiological condition, but can be considered to be signs of inflammation. (See, e.g., References 34 and 35). For example, a subset of resident monocytes can also patrol the intact vasculature of the mesentery and brain under physiological conditions without extravasating. (See, e.g., References 36 and 37). Thus, a fraction of LEI at later time points can be a fact physiological LEI of such resident monocytes.
In vivo imaging can provide long-term and short-term dynamic information regarding, e.g., the behavior and interactions of cells that cannot be gathered with ex vivo methods. The exemplary system, method and computer-accessible medium according to an exemplary embodiment of the present disclosure can track and/or quantify the endogenous microglia and engrafting BMDC populations simultaneously over months in the living mouse retina by in vivo retinal imaging. For example, an engraftment of DsRed+ BMDC after lethal irradiation and bone marrow transplant in CX3CR1GFP/+ mice can be accompanied by loss of the resident GFP+ microglia. Leukocyte endothelial interaction, thought to be absent under homeostatic conditions and commonly associated with CNS inflammation, can be observed even months after the irradiation. It is possible to directly correlate the effects of ionizing radiation on retinal vascular integrity, microglia and BMDCs in dependence of the irradiation dose directly delivered to the head.
Exemplary Materials and Methods
The exemplary system according to the present disclosure has been described with reference to
Attention to the axial alignment of the imaging lasers can be provided to compensate for the chromatic aberrations of, e.g., the mouse eye that have been reported to be approximately 7D across the visible wavelength range. (See, e.g., Reference 25). The instrument can initially be aligned with the red laser as a reference beam. The exemplary lengths of the various telescopes can be optimized to minimize divergence and times-diffraction-limit factor of the reference beam as measured with a beam propagation analyzer (e.g., ModeMaster, Coherent). The confocal pinhole of the reflectance channel can be conjugated by placing a mirror in the last intermediate image plane of the system and optimizing the confocal throughput. To match the focal plane of the other laser wavelengths (e.g., about 488 and about 532 nm) to that of the red laser, the respective source telescope can be slightly adjusted to introduce a small beam divergence. The exemplary alignment of the three laser beams can be optimized in three dimensions in an artificial eye, built from a 2 mm focal length lens (e.g., NA=0.5, 2 mm clear aperture, Geltech 350150) held in a brass housing with a target placed in the focal plane of the lens. An exemplary source telescope of the 491 and 532 laser can be adjusted until best possible images of the target can be acquired with all three wavelengths. As an additional alignment procedure, the confocal pinholes of the three fluorescence channels can be conjugated for each excitation wavelength.
Mice expressing GFP in microglia under the control of the fractalkine receptor promoter CX3CR1 (e.g., B6.129P-Cx3cr1tm1Litt/J) were purchased from Jackson Laboratory. The fractalkine receptor can be specifically expressed on microglia, a population of blood monocytes, NK and dendritic cells. (See, e.g., Reference 38, 18). The mice were maintained as heterozygotes by crossing homozygous CX3CR1-GFP mice with the parental C57BL/6 strain to ensure proper function of the fractalkine receptor on microglia. (See, e.g., Reference 39). Mice were exposed to a single dose of 9 Gy gamma radiation with a Cesium source (e.g., Gammacell 40 Exactor, MDS Nordion). Lethally irradiated mice were rescued five hours after the exposure by bone marrow transplantation of 4×106 cells harvested from homozygous actin-DsRed donor mice [B6. Cg-Tg(CAG-DsRed*MST)1Nagy/J].
For the exemplary imaging procedure, the mice were held in a heated holding tube that integrated a nose cone for delivery of 1-2% isoflurane mixed in oxygen for inhalation anesthesia. The tube was mounted on a six-axis stage that aided the positioning of the mouse eye in the SLO imaging beam. The pupil was dilated with a drop of Tropicamide. A contact lens was placed on the mydriatic eye and a drop of GenTeal eye drops prevented the cornea from drying. In vivo images were recorded at baseline prior to the irradiation and at days 15, 28, 42, 70, 90 and 120 after the irradiation. At each time point, the numbers of resident GFP+ cells and DsRed+ bone marrow derived cells were evaluated.
Exemplary Image Analysis
To identify interactions between bone marrow derived leukocytes with the blood vessel endothelial wall, exemplary time-lapse imaging procedure(s) can be performed. One exemplary image can be taken, e.g., once every 30 sec for several minutes. The exemplary images of the resulting temporal stack can be aligned to compensate for motion artifacts such as rotation or drifting of the eye. Cells moving in the major blood vessels can be tracked, e.g., manually in ImageJ using the MTrackJ plugin, or automatically.
Exemplary Fluorescein Angiography
C57BL/6 mice were injected with 25 μl of 5% fluorescein solution, diluted in Phosphate Buffered Saline from 10% Fluorescein (e.g., USP, IMS Ltd.) via the tail vein while under isofluorane anesthesia and on the SLO stage. Immediately, images were taken at 30 sec intervals over a 3 min period to follow leakage of the fluorescein into the retinal parenchyma. As a measure of fluorescein leakage, the contrast between capillaries and non-vascular tissue was evaluated within segments delineated by the major blood vessels. Michelson contrast, defined as the ratio of the difference between maximum and minimum pixel values over their sum, was used for the measurement. Contrast value was normalized to baseline values taken before irradiation for each mouse.
As shown in
Further, the exemplary processing arrangement 902 can be provided with or include an input/output arrangement 914, which can include, for example, a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. For example, anatomical data 920 can be provided to the input/output arrangement 914. As shown in
For example, the further structure can include a portion(s) of a central nervous system. The radiation(s) can be provided from the retina of the sample(s). Imaging the retina of the sample(s) can be performed, and the determination can be made regarding the retina based on the image. The marker(s) can be measurable, and can include an interaction of white blood cells with a blood vessel wall. The marker(s) can also include or be an identification of blood vessel leakage. The information regarding the portions of procedure 1010 can be obtained from a confocal reflectance system, a fluorescence system, an optical coherence tomography system, and/or an optical frequency domain imaging system. In certain embodiments of the present disclosure, the abnormality(s) can include (i) a brain injury, (ii) a spinal cord injury, (iii) multiple sclerosis, (iv) a stroke, or (v) a brain tumor. The abnormality(s) can also include (i) a brain abnormality, (ii) a spinal cord abnormality, (iii) or an ophthalmic abnormality.
The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not limited to, for example, data and information. It should be understood that, while these words, and/or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.
The following references are hereby incorporated by reference in their entirety.
This application relates to and claims priority from U.S. Patent Application No. 61/895,749, filed on Oct. 25, 2013, the entire disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/62381 | 10/27/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61895749 | Oct 2013 | US |
