Patent Application
20140074176
The present invention relates generally to neural stimulations, and more particularly to method and apparatus for selectively controlling neural activities of a target of interest with light, and method for identifying spatial and temporal factors that are controllable for enhancing reproducibility of a hybrid electro-optical stimulation, and applications of the same.
Excitation and inhibition are critical for the normal function of neural circuitry. Thus, to analyze the dynamics of neural circuitry, or to create effective brain-computer interfaces, it is essential to be able to excite or inhibit neurons reversibly and with high specificity. Intracellular microelectrodes make it possible to monitor sub-threshold activity and precisely regulate currents or voltages across the membrane of individual neurons. However, this technology is not practical for large-scale recordings from hundreds of neurons simultaneously, especially in intact, behaving subjects, whose movements will dislodge them, damaging both the electrodes and the neurons. Extracellular electrode arrays provide an effective way to stimulate large numbers of neurons simultaneously, and high frequency electrical stimulation has been developed as a means of inhibiting neurons [1]; but because of current spread, it is often difficult to use these techniques for fine control of individual neurons. At the same time, the burgeoning interest in deep brain stimulation, pain management, functional electrical stimulation, and brain-computer interfaces, have all created a demand for higher levels of specificity and control. In the last decade, optogenetics has become a promising new technology for exciting and inhibiting small groups of neurons with high spatial and temporal precision, but the need for genetic manipulation may create barriers to its clinical use in humans [2, 3].
Several years ago, Wells et al. described the use of infrared laser light to transiently excite neural tissue [4]. Subsequent studies have shown that infrared stimulation works through a spatially precise and thermally-mediated process without the need for genetic modifications [5]. Recent studies have suggested that part of the action of infrared stimulation may be through changes in membrane capacitance [6]. In the last few years, infrared simulation has been used to activate a wide range of excitable tissues including peripheral nerves [4, 7, 8], somatosensory cortex [9], the auditory systems [10], and cardiac tissue [11, 12]. Combining both electrical and infrared stimulation modalities (hybrid electro-optical stimulation) has been shown to be an effective means of both enhancing the specificity of electrical stimulation and reducing the amount of thermal energy that must be deposited in tissue [13, 14].
A recent study by us demonstrated that it was possible to use infrared light to reversibly inhibit excitation of peripheral motor axons, but the mechanism of action was unclear [14]. Other studies had noted that pulsed infrared light could cause inhibitory effects in mammalian cortex, but the process was difficult to control reliably and attributed to activation of inhibitory neurons [9]. Global temperature changes leading to inhibition of action potential generation and propagation, a phenomenon known as “heat block”, have been investigated in both unmyelinated and myelinated preparations [15, 16]. Recent modeling studies indicate the potential for block of action potential generation and propagation with local increases in nerve temperature [17]. The underlying mechanism of global and/or local thermal neural inhibition involves the temperature-dependence of the Hodgkin-Huxley voltage-gated channels. At increased temperatures, the rate of inactivation of sodium channels and activation of potassium channels overwhelms the rate of activation of sodium channels [16-18]. Thus, the recovery phase of the action potential overtakes the rising phase, leading to either a faster and weaker response, or complete but reversible block of the action potential generation or propagation [15, 18].
Hybrid neural stimulation was developed as a new stimulation modality combining traditional electrical techniques with novel infrared nerve stimulation methods [49]. The combination of the two techniques utilizes their respective advantages while avoiding their primary limitations. Specifically, hybrid stimulation combines the safety, established characteristics and demonstrated clinical utility of electrical stimulation with the spatial selectivity of infrared neural stimulation (INS). While hybrid stimulation does not provide the contact- and artifact-free aspects of INS, the high spatial selectivity of INS remains and enhances clinical neural interfaces. Additionally, sub-threshold electrical currents should also reduce the problem of electrode corrosion over time. The essence of hybrid stimulation is to combine a sub-threshold electrical stimulus over a broad area, and then bring a spatially selective location to threshold by adding a sub-threshold pulse of infrared light. In doing so, both the electrical current and optical radiant exposures are reduced, effectively achieving spatial selectivity with reduced risk of tissue damage. Previously, hybrid stimulation was shown to reduce optical radiant exposures (J cm−2) by approximately a factor of 3 when compared to INS alone [49]. By offering reduced threshold radiant exposures, hybrid nerve stimulation is attractive for biomedical applications requiring spatial selectivity where laser power constraints and tissue damage are primary concerns. However, further development of this technology requires that the reliability and repeatability of hybrid stimulation be improved.
The experiments demonstrating feasibility of hybrid stimulation in the rat sciatic nerve showed large variations in the reduction of optical radiant exposures [49]. In these experiments, the electrical threshold was set at a chosen sub-threshold current and the additional optical radiant exposure required to achieve stimulation threshold was determined as a percent of the optical threshold radiant exposure when it was applied alone. The reduction in optical radiant exposures and their variability were both shown to increase as the applied electrical stimulus approached threshold. For an electrical stimulus at 95% of the threshold current, the additional optical energy required for stimulation ranged from 6% to 60% of the optical stimulation threshold.
Hybrid electro-optical neural stimulation is a novel paradigm combining the advantages of optical and electrical stimulation techniques while reducing their respective limitations. However, in order to fulfill its promise, this technique requires reduced variability and improved reproducibility.
Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies.
In one aspect, this invention involves the use of optical techniques for inhibiting activity in excitable tissues or target endpoints controlled by the excitable tissue. In embodiments of the invention, infrared wavelengths are used to inhibit neural activity. However, the invention is not constrained to infrared wavelengths or neural applications. This invention works in endogenous tissues, which is fundamentally different from optogenetic techniques that require genetic modifications to allow optical control. The underlying mechanism of this invention is proposed to be a thermally mediated process, whereby a sufficient temperature increase in the excitable tissue changes the rate at which ion channels are opened and closed. While global temperature changes in neurons leading to block of action potential generation and propagation has been known for decades, the invention demonstrates the use of light to create a local temperature change for selective and reversible inhibition. According to the invention, this technology can be used to improve the selectivity of electrical stimulation and to block propagating action potentials away from their site of generation.
In one aspect, the present invention relates to a method of transient and selective suppression of neural activities of a target of interest. The target of interest contains one or more nerves of a living subject, such a human or animal. In one embodiment, the method includes selectively applying at least one light to the target of interest at selected locations with predetermined radiant exposures to create a localized and selective inhibitory response therein. In one embodiment, the localized and selective inhibitory response comprises a local temperature change.
In one embodiment, the neural activities comprise generation and propagation of action potentials. The action potentials are evoked electrically by an electrical stimulus applied to the target of interest.
In one embodiment, the at least one light comprises pulses of a single light generated from a laser source.
In one embodiment, the pulses of the single light are synchronized with the electrical stimulus, such that the pulses of the single light and the electrical stimulus end at the same time.
In another embodiment, the pulses of the single light are applied prior to the start time of the electrical stimulus at a first predetermined time.
In yet another embodiment, the pulses of the single light are applied after the start time of the electrical stimulus at a second predetermined time.
In one embodiment, the at least one light comprises two or more lights, and each of the two or more lights comprises pulses of light generated from a respective laser source.
In one embodiment, the pulses of the two or more lights are synchronized with the electrical stimulus, such that the pulses of the two or more lights and the electrical stimulus end at the same time.
In another embodiment, the pulses of the two or more lights are applied prior to the start time of the electrical stimulus at a first predetermined time.
In yet another embodiment, the pulses of the two or more lights are applied after the start time of the electrical stimulus at a second predetermined time.
In one embodiment, the step of selectively applying the at least one light to the target of interest comprises simultaneously applying the two or more lights to the target of interest at the selected locations,
In another embodiment, the step of selectively applying the at least one light to the target of interest comprises alternately or sequentially applying the two or more lights to the target of interest at the selected locations.
In one embodiment, each of the at least one light comprises an infrared light.
In another aspect, the invention relates to an apparatus for selectively controlling of neural activities of a target of interest. In one embodiment, the apparatus has a source for generating at least one light; and a probe coupled to the at least one light source for selectively delivering the at least one light to the target of interest at selected locations to create a localized and selective inhibitory response therein.
In one embodiment, the neural activities comprise generation and propagation of action potentials. In one embodiment, the action potentials are evoked electrically by an electrical stimulus applied to the target of interest.
In one embodiment, the light source comprises a laser source, and the at least one light comprises pulses of a single light generated from the laser source.
In one embodiment, the pulses of the single light are synchronized with the electrical stimulus, such that the pulses of the single light and the electrical stimulus end at the same time.
In another embodiment, the pulses of the single light are applied prior to the start time of the electrical stimulus at a first predetermined time.
In a further embodiment, the pulses of the single light are applied after the start time of the electrical stimulus at a second predetermined time.
In one embodiment, the light source comprises two or more light laser sources, and the at least one light comprises two or more lights, each light comprising pulses of light generated from a respective laser source of the two or more light laser sources.
In one embodiment, the pulses of the two or more lights are synchronized with the electrical stimulus, such that the pulses of the two or more lights and the electrical stimulus end at the same time.
In another embodiment, the pulses of the two or more lights are applied prior to the start time of the electrical stimulus at a first predetermined time.
In yet another embodiment, the pulses of the two or more lights are applied after the start time of the electrical stimulus at a second predetermined time.
In one embodiment, the probe is configured to simultaneously deliver the two or more lights to the target of interest at the selected locations,
In another embodiment, the probe is configured to alternately or sequentially deliver the two or more lights to the target of interest at the selected locations.
In one embodiment, each of the at least one light comprises an infrared light.
In one embodiment, the probe comprises at least one optical fiber having one end coupled to the at least light source and a working end positioned proximate to the target of interest for selectively delivering the at least one light to the target of interest at the selected locations.
In yet another aspect, the invention relates to a method for identifying spatial factors that are controllable for enhancing reproducibility of a hybrid electro-optical stimulation to a target of interest. In one embodiment, the method includes simultaneously applying electrical pulses at a sub-threshold and optical pulses of a set magnitudes to the target of interest, wherein the optical pulses of a set magnitudes are delivered by an optical fiber; translating the optical fiber back and forth across the target of interest, and measuring a position of the optical fiber when translating; reconstructing the exact position of the optical fiber at the time of the hybrid stimulation; and correlating the working end of the optical fiber with the presence or absence of the hybrid stimulation as indicated by an evoked potential on a nerve recording, so as to obtain the spatial factors.
In one embodiment, The method of claim 33, wherein the sub-threshold is about 90% less than the threshold of the electrical stimulation.
In one embodiment, the method further includes determining existence of a finite region of excitability (ROE) with size altered by the strength of the optical stimulus and recruitment dictated by the polarity of the electrical stimulus.
In one embodiment, the electrical pulses and the optical pulses are synchronized such that they end concurrently.
In a further aspect, the invention relates to a method for identifying temporal factors that are controllable for enhancing reproducibility of a hybrid electro-optical stimulation to a target of interest. In one embodiment, the method includes simultaneously applying electrical pulses and optical pulses to the target of interest; regularly measuring threshold currents of the electrical stimulus to monitor underlying changes in the electrical stimulation with time, and measuring radiant exposures eliciting the hybrid stimulation along with the threshold currents of the electrical stimulus; reducing the stimulus current to a sub-threshold; applying different radiant exposures along with the sub-threshold current pulses to the target of interest, and recording each hybrid stimulus pulse as either a 1 or 0 as determined by the presence (1) or absence (0) of action potentials; repeating the process for the predetermined duration; and processing the recorded data to obtain the temporal factors.
In one embodiment, the electrical pulses and the optical pulses are synchronized such that they end concurrently.
These and other aspects of the present invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some terms used in this specification are more specifically defined below.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
It will be understood that when an element is referred to as being “on” another element, it can be directly on the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being “directly on” another element, there are no intervening elements present. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
It will be understood that, although the terms first, second, third etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the present invention.
Furthermore, relative terms, such as “lower” or “bottom” and “upper” or “top,” may be used herein to describe one element's relationship to another element as illustrated in the Figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation depicted in the Figures. For example, if the device in one of the figures is turned over, elements described as being on the “lower” side of other elements would then be oriented on “upper” sides of the other elements. The exemplary term “lower”, can therefore, encompasses both an orientation of “lower” and “upper,” depending of the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as “below” or “beneath” other elements would then be oriented “above” the other elements. The exemplary terms “below” or “beneath” can, therefore, encompass both an orientation of above and below.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
As used herein, “plurality” means two or more. As used herein, the terms “comprising”, “including”, “carrying”, “having”, “containing”, “involving”, and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
As used herein, the term “inhibition” refers to a transient elimination of action potential initiation or generation, while the term “block”” refers to a transient impediment to action potential propagation.
In one aspect, this invention involves the use of optical techniques for inhibiting activity in excitable tissues or target endpoints controlled by the excitable tissue. In embodiments of the invention, infrared wavelengths are used to inhibit neural activity. However, the invention is not constrained to infrared wavelengths or neural applications. One embodiment of this invention works in endogenous tissues, which is fundamentally different from optogenetic techniques that require genetic modifications to allow optical control. The underlying mechanism of this invention is due to a thermally mediated process, whereby a sufficient temperature increase in the excitable tissue changes the rate at which ion channels are opened and closed. While global temperature changes in neurons leading to block of action potential generation and propagation has been known for decades, the invention demonstrates the use of light to create a local temperature change for selective and reversible inhibition. According to the invention, this technology can be used to improve the selectivity of electrical stimulation and to block propagating action potentials away from their site of generation.
The primary novel element of this invention is the use of light to create a localized and selective inhibitory response. The application of this local inhibition to enhance current interfaces or to control unwanted activity is also novel.
The invention addresses two primary problems. (1) Current interfaces with excitable tissues are limited in their ability to selectively recruit sub-populations spatially and, in the case of neurons, following the physiological recruitment order of smallest neurons before largest neurons. Using light, one is able to selectively inhibit the activation of sub-populations of excitable tissues, thereby enhancing the selectivity of the method used for stimulation. (2) There are many clinical and research applications where it is desirable to block unwanted activity. The invention allows selective block of propagating biopotentials to prevent them from reaching their endpoint. For example, this would allow for titrated control of sensory perception or block of spastic neuromuscular activity.
Potential products and applications of this technology include peripheral nerve interfaces (e.g. nerve cuff), brain-computer interfaces, combination with high-frequency electrical nerve conduction block, control of cardiac function, pain management, functional neuromuscular stimulation, cochlear implants, analysis of neural circuitry and dynamics.
In another aspect, the invention relates to method for identifying spatial and temporal factors that play a role in and are controlled to enhance the reproducibility of hybrid electro-optical stimulation.
The hybrid electro-optical neural stimulation that combines the advantages of optical and electrical stimulation techniques while reducing their respective limitations. However, in order to fulfill its promise, this technique requires reduced variability and improved reproducibility. According to the invention, a comparative physiological approach is used to aid the further development of this technique by identifying the spatial and temporal factors characteristic of hybrid stimulation that may contribute to experimental variability and/or a lack of reproducibility. Using transient pulses of infrared light delivered simultaneously with a bipolar electrical stimulus in either the marine mollusk Aplysia californica buccal nerve or the rat sciatic nerve, we determined the existence of a finite region of excitability with size altered by the strength of the optical stimulus and recruitment dictated by the polarity of the electrical stimulus. Hybrid stimulation radiant exposures yielding 50% probability of firing (REM) were shown to be negatively correlated with the underlying changes in electrical stimulation threshold over time. In Aplysia, but not in the rat sciatic nerve, increasing optical radiant exposures (J cm−2) beyond the REM ultimately resulted in inhibition of evoked potentials. Accounting for the sources of variability identified in this study increased the reproducibility of stimulation from 35% to 93% in Aplysia and 23% to 76% in the rat with reduced variability.
These and other aspects of the present invention are more specifically described below.
Without intent to limit the scope of the invention, exemplary methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.
This example demonstrates that, among other things, infrared light can precisely turn off electrically stimulated neurons. Specifically, pulses of infrared light can be utilized to reversibly inhibit action potential generation and propagation with high temporal and spatial specificity, and to reversibly control functional output, i.e., muscle force. These results could provide the basis for novel techniques for studying neural circuitry, and for selectively controlling peripheral neuronal activity, which could have significant implications for the development of more precise brain-computer interfaces and prosthetic devices.
A detailed investigation was carried out using the unmyelinated buccal nerve 2 (BN2) of the marine mollusk Aplysia californica buccal ganglion. This nerve provides a robust and experimentally tractable ex vivo preparation with substantial length and a distal trifurcation that allows for simultaneous recording of multiple branches, and a muscular target that is known and tractable to study. These results were also validated in the myelinated rat sciatic nerve.
Aplysia californica (n=4) weighing 250-350 g (Marinus Scientific, Long Beach, Calif.) were maintained in an aerated aquarium containing circulating artificial seawater (ASW) (Instant Ocean; Aquarium Systems, Mentor, Ohio) kept at 16-17° C. The animals were fed dried seaweed every 1-3 days.
Aplysia were anesthetized with an injection of 333 mM MgCl2 (˜50% of body weight) prior to dissection. Once anesthetized, animals were dissected and the buccal ganglia were removed and pinned in a recording dish and immersed in Aplysia saline (460 mM NaCl, 10 mM KCl, 22 mM MgCl2, 33 mM MgSO4, 10 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.6). Aplysia buccal ganglia are symmetric, so each hemiganglion has an associated buccal nerve 2 (BN2). Each BN2 was transected just distal to its attachment to its respective hemiganglion and anchored in place by pinning the protective sheath around the nerve to the Sylgard base (Dow Corning, Midland, Mich.) of the recording dish. Once securely pinned, the three distal branches of BN2 were suctioned into nerve-recording electrodes to monitor the response to stimulation. Nerve-recording electrodes were made by hand-pulling polyethylene tubing (1.27 mm outer diameter, 0.86 mm inner diameter; PE90; Becton Dickinson) over a flame to the desired inner diameter. Recording electrodes were suction-filled with Aplysia saline prior to suctioning of the nerve. Nerve signals were amplified (×1000) and band-pass filtered (300-500 Hz) using an AC-coupled differential amplifier (model 1700; A-M Systems), digitized (Axon Digidata 1320A; Molecular Devices, Sunnyvale, Calif.) and recorded (Axograph X; Axograph Scientific).
Extracellular stimulating electrodes were made from thin-wall borosilicate capillary glass (catalogue No. 6150; A-M Systems, Everett, Wash.) pulled to a diameter of about 40 μm and resistances of about 0.1 MΩ (model P-80/PC; Sutter Instruments, Novato, Calif.). For each experiment, an electrode was capillary-filled with Aplysia saline and positioned on the top surface of the nerve, in contact with the nerve sheath, using a micromanipulator. The return electrode was positioned at a distance in the bath to create monopolar stimulation. Monophasic currents supplied by a stimulus isolator (A360; WPI) were used for all experiments.
Two tunable diode laser systems were used throughout the study in this example. Laser 1 includes a prototype tunable diode laser (Capella; Lockheed-Martin-Aculight, Bothwell, Wash.) with wavelength λ=1450 nm coupled to a 200 μm diameter fiber optic (Ocean Optics, Dunedin, Fla.). Laser 2 includes a similar and commercially available diode laser (λ=1860 nm) coupled to a 100 μm diameter fiber optic. Fiber optics was secured in place using micromanipulators.
Amplified and filtered nerve responses were acquired at 5 kHz. AxoGraph X software (AxoGraph X; AxoGraph Scientific, Sydney, Australia) was used to coordinate stimulation and inhibition protocols, and to record acquired data. Post-acquisition data analysis was performed using a combination of AxoGraph X, Matlab (Matlab r2010b; Mathworks, Natick, Mass.) and Microsoft Excel (part of Microsoft Office Professional Plus 2010). Data are expressed as mean plus/minus the standard error of the mean.
Radiant exposures normalize applied optical energy per unit area. Radiant energy was measured using an energy meter and pyroelectric energy detector (Nova II, Ophir; PE50BB-VR-ROHS, Ophir). The radiant exposure was determined by dividing the radiant energy by the area of the circular fiber tip (i.e., 0.0314 mm2). In order to report radiant exposure at the level of the axons, many assumptions and calculations would be necessary. For simplicity and accuracy, the measured value at the tip of the fiber optic prior to any additional assumptions was used.
For the rat, the laser spot size incident on the nerve surface was measured using the knife-edge technique. Thus, we report a measured spot-size (0.0026 cm2) to provide greater accuracy. These methods of radiant exposure determination are consistent with published literature.
Fiber optics from laser systems 1 and 2 were positioned such that they flanked the stimulating electrode transverse to the nerve's longitudinal axis as shown
Each trial (n=5) included a series of repeating 500 msec episodes. For each episode, a monophasic electrical stimulus (τp=0.25 msec) providing current sufficient to generate consistent action potentials on all three recording electrodes (461.4±36.2 μA) was applied at 100 msec. Pulses of infrared inhibition from each laser source were synchronized with the supra-threshold electrical stimulus such that the pulses ended at the same time. This allowed total charge and total heat deposition to occur simultaneously. Each trial typically followed an ABACABACA pattern in which nerves were stimulated electrically (A), then either Laser 1 or Laser 2 was added (B), then the laser was removed leaving only electrical stimulation (A), followed by the other laser being added (C), and then the process was repeated. Nerve responses for each condition were analyzed using the integrated compound nerve action potential (iCNAP): the ensemble average for each condition within a given trial was rectified and summed over 20 msec following the electrical stimulation artifact.
To characterize how the relative timing of the infrared and electrical pulses affects threshold radiant exposures for inhibition of action potential generation, a single infrared pulse (λ=1450 nm, τp=0.5 msec) was delivered at time points before and after an electrical stimulus (τp=0.25 msec). The timing scheme was such that t=0 corresponded to the infrared and electrical pulses ending simultaneously. The infrared pulse was delivered over the range of t=−20 msec to t=0.5 msec (n=4 for each time point). For each trial, the electrical stimulus was 110% of the threshold current, where electrical threshold was defined as the minimum current required to generate 5 consecutive evoked responses. Infrared pulses (n=10) at 5 different radiant exposures were applied for each time point. The presence (1) or absence (0) of an evoked response was recorded and aggregated to achieve the probability of a stimulated response for each radiant exposure. At each time point, the probability versus radiant exposure data is fit to the negative of the cumulative distribution function (CDF). Threshold for infrared thermal inhibition at each time point was defined as the radiant exposure generating <50% of an evoked response [14].
The nerve preparation was as described previously, except a single 200 μm fiber optic coupled to the 1450 nm laser source was positioned approximately 1 cm distal to the site of electrical stimulation, but proximal to the nerve trifurcation (
BN2 of an Aplysia (314 g) was dissected and secured to a recording dish. The saline level of the Sylgard-covered dish was lowered so that it was just covering the surface of the nerve (
To find the temperature change required for nerve conduction block in Aplysia, we averaged all trials (N=3 nerves, n=11 trials) and found the minimum duration of laser exposure for which the BN2c iCNAP was significantly reduced. Significance was determined using p<0.004 in Aplysia and p<0.002 in the rat after correcting for multiple comparisons using the Bonferroni method. This duration was then compared to the measured temperature (
An Aplysia (422 g) was anesthetized with an injection of approximately 50% body weight isotonic MgCl2. The animal's buccal mass was removed and placed in a Petri dish within a solution of 50% Aplysia saline and 50% isotonic MgCl2. Both buccal nerves 2 were severed at their attachment points to the buccal ganglia. Incisions were made through the dorsal and ventral surfaces of the buccal mass, and further incisions were made to remove the radula-odontophore and pharyngeal tissue, leaving the I1/I3 muscle split into two separate halves with each half innervated by its buccal nerve 2. The rest of the buccal mass and the ganglia were discarded. The muscle halves were moved to a recording dish with a Sylgard surface in the back half of the dish. Each I1/I3 half was glued (Duro Quick-Gel superglue, Henkel Corp., Avon, Ohio) by its anterior edge to the glass bottom of the dish just in front of the Sylgard. After gluing, the dish was filled with Aplysia saline. Each buccal nerve 2 was gently stretched and pinned on the Sylgard surface, and polyethylene suction electrodes were attached to the ends of the nerves. A 200 μm diameter fiber optic coupled to the 1450 nm laser source was positioned distal to the suction electrode and proximal to the nerve trifurcation. Force transducers (Grass Technologies, West Warwick, R.I.) were attached to the medial portions of the I1/I3 halves using silk sutures.
Electrical stimulation was applied using the nerve suction electrodes. Control trials included 5 repetitions of electrical stimulation (τp=1 msec, 500 μA) delivered at 10 Hz for 2 sec. Each repetition was followed by an interval of 12 seconds with no stimulation. Experimental trials included the same protocol. In addition, however, infrared pulses (τp=0.2 msec) were applied at 200 Hz for 3 seconds beginning 1 second before the third electrical stimulus. Five sets of control and experimental trials were repeated for a given parameter set with 3 min between each trial to allow the nerve to rest.
All experiments were performed following protocols approved by the Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley rats (n=2) weighing 250-300 g (Charles River) were anesthetized with continuously inhaled isoflurane (induction: 3% isoflurane, 3.0 LPM oxygen; maintenance: 2-2.5% isoflurane, 1.5 LPM oxygen). A rectal probe and heating pad (catalog No. 40-90-8, FHC, Bowdoin, Me.) were used to maintain the rat at a target body temperature of 35-37° C. throughout the experiment. The animals were placed on a polycarbonate platform and their hindlimbs were shaved. The dorsal surface of the foot was then taped to the edge of the platform. An incision was made from the heel to the vertebral column and the skin was separated from the underlying tissue. The biceps femoris was then cut and divided proximal from the Achilles tendon to expose the sciatic nerve. The sural and peroneal branches of the sciatic nerve were transected so only innervation of the planterflexor muscles remained.
Paired EMG electrodes made from perfluoroalkoxy (PFA)-coated silver wire (0.003″ bare, 0.005″ coated; A-M Systems, Sequim, Wash.) were inserted along the length of the medial gastrocnemius and lateral gastrocnemius muscles. EMG signals were amplified (×100) and band-pass filtered (100-1000 Hz) using an AC-coupled differential amplifier (model 1700; A-M Systems), digitized (20 kHz; Axon Digidata 1440A; Molecular Devices, Sunnyvale, Calif.) and recorded (Axograph X; Axograph Scientific).
A monopolar nerve cuff electrode was placed around the trunk of the sciatic nerve. Each trial (n=12) included one 10 sec episode. Monophasic electrical stimuli (τp=0.1 msec; 750 μA) were delivered at 8 Hz for the duration of the trial. At 4 sec, pulses of infrared light (τp=0.2 msec; 75.7 mJ/cm2) were delivered at 200 Hz for 3 seconds. Laser spot size for radiant exposure calculations was measured using the knife-edge technique [32]. Nerve responses were analyzed using the iEMG, which was calculated in the same manner as the iCNAP described above.
To investigate the selective inhibition of electrically evoked action potentials, an extracellular micropipette was used to provide nonspecific supra-threshold stimulation to the main trunk of BN2. Electrically evoked responses were recorded on the three distal branches of BN2: BN2a, BN2b and BN2c [19, 20], allowing the primary compound nerve action potential to be deconvolved and resolved into some of its spatial components. Two optical fibers were positioned on opposite sides of the micropipette and coupled to independent laser sources (
The results obtained in the example indicate some amount of selective inhibition in both location and size of axons. At this time, it is not clear why infrared inhibition predominantly blocked action potential propagation along BN2c without significantly affecting BN2a or BN2b. In some trials, action potentials on BN2b experienced an increase in size during infrared inhibition of BN2c, while in other a slight decrease was observed. These affects were not statistically significant (p>0.05). These results may imply neurophysiological differences in the axonal units projecting to the different branches of BN2. Motor neurons are known to project to BN2c [33]. Unpublished data from our lab imply that BN2b and BN2c together contain the axons of the motor neurons, while BN2a contains sensory neuronal projections. Evoked responses at the soma of motor neurons were found to project to BN2b and/or BN2c, but not BN2a. This conclusion is further suggested by the observed lack of I1/I3 muscle contraction when both BN2b and BN2c are severed and BN2a is left intact. Published studies also show that stimulation of BN2a directly leads to activity in interneurons B4/B5 [19] as well as the elicitation of motor programs [20]. In addition to neurophysiological differences in projected neurons, it is also possible that projections to BN2c are more peripherally located relative to the nerve cross-section. This would allow the infrared-induced temperature gradient to reach axons projecting to BN2c before affecting those projecting to BN2a or BN2b. In the case of increased magnitude of action potentials on BN2b, this may be due to lower temperatures at the periphery of the laser spot size inducing hybrid electro-optical stimulation as opposed to inhibition [5, 34].
To characterize how the relative timing of the infrared and electrical pulses affects threshold radiant exposures for inhibition of action potential generation, a single infrared pulse (λ=1450 nm, τp=0.5 msec) was delivered at time points before and after an electrical stimulus (τp=0.25 msec). With the infrared pulse (τp=0.25 msec) delivered prior to the electrical pulse (τp=0.25 msec), threshold radiant exposures for inhibition slowly increased as the timing between the pulses increased (
Infrared and electrical pulses were synchronized for the purpose of demonstrating temporally precise inhibition of action potential initiation (
The nerve preparation was as described previously, except a single 200 μm fiber optic coupled to the 1450 nm laser source was positioned approximately 1 cm distal to the site of electrical stimulation, but proximal to the nerve trifurcation (
In addition to inhibiting the initiation of electrically evoked action potentials, localized block of propagating responses was also demonstrated. Electrically evoked responses were stimulated at 4 Hz and propagated to BN2a, BN2b and BN2c. A single fiber optic was positioned along the nerve trunk distal to the site of supra-threshold electrical stimulation (at about 1 cm) (
The temperature measured in this study for the Aplysia is an overestimate of the actual temperature reaching BN2. In order to visualize temperature with the IR camera the fiber optic was kept above the surface of the saline/nerve rather than immersed in the saline as during experimentation. Thus, an insulating saline-air interface was present during temperature measurements. When modeling laser-tissue interactions, the tissue-air interface is often considered adiabatic with heat reflecting back into the simulated volume [35]. In the actual experimental preparation, the added saline above the site of infrared absorption would help to conduct heat away and yield a lesser temperature rise than was measured with the IR camera.
The tissue temperature in response to infrared inhibition of propagation action potentials was also measured in the rat. A supra-threshold stimulus (0.12 mJ/cm2) resulted in an approximately 10° C. increase in tissue temperature.
To demonstrate the functional relevance of the inhibition, the effects on muscle force were measured. The distal BN2 muscle innervation was left intact and the contraction force of the I1/I3 muscles was measured with a force transducer (
Infrared inhibition of propagating action potentials was also demonstrated in a myelinated mammalian nerve. Applying infrared pulses to the tibial branch of the rat sciatic nerve, distal to the site of electrical stimulation, reduced evoked EMG amplitude of the lateral gastrocnemius (LG) (
Both inhibition and enhancement of propagated responses were observed in the rat sciatic nerve. Whether inhibition or enhancement occurred depended on the location of the fiber optic relative to the nerve and the portion of the motor pool recruited electrically. By moving the fiber optic to different locations on the nerve we were able to see both inhibition and enhancement, though inhibition occurred the majority of the time. By changing the relative locations of the stimulating and return electrodes we were able to evoke different EMG responses, which correlated to either inhibition or enhancement.
As disclosed above, the results presented in this example demonstrate that infrared light can be used as a non-contact, artifact free and highly reversible form of precise neural inhibition. This technology is conducive to miniaturization for the control of single neurons as well as implementation into a multi-site array for governing larger neuronal populations. Pulsed infrared light is known to achieve spatially and temporally precise neural stimulation [21, 22]. Combining infrared stimulation with infrared inhibition offers the potential for full and precise control of a neural system with a single modality.
The ability to selectively inhibit the initiation and/or propagation of neural activity may have significant implications for neural prostheses and therapies. Primary challenges facing electrical neural prostheses are fractionation of spatial recruitment and mirroring of the physiological recruitment order (i.e., smaller diameter fibers before larger diameter fibers). By inhibiting the generation of selected electrically evoked responses as shown in
The use of infrared light addresses potential limitations of the current alternatives to the block of neural propagation. The use of high frequency alternating current (HFAC) is an electrical method for blocking the propagation of neural potentials that is nearing clinical implementation. However, a challenge to this approach is the electrically evoked activity that occurs at the onset of the blockade [24]. Here we demonstrate selective inhibition of propagating action potentials without inducing increased activity at any point during the block. Rapid nerve cooling is fast acting, reversible and lacks any onset activation, but this technique will be difficult to miniaturize and is unlikely to match the spatial specificity of a laser-based approach [25]. Furthermore, the telecommunications and computing industries are driving the development of advanced laser technologies, and spatially-precise miniaturized implantable laser sources are being developed for infrared stimulation applications [26, 27]. Optogenetic methods have become increasingly popular due to the ability to selectively excite and silence neurons with spatial and temporal precision [2], but these approaches require genetic manipulations that are currently confined to limited species and non-clinical uses [3]. In contrast, pulsed infrared light is also capable of spatial and temporally precise excitation and inhibition, but without the need for viral vectors or transgenic species.
As infrared inhibition is a thermally mediated phenomenon, the ultimate application of this technique will be contingent on the absence of thermally induced changes in tissue morphology or function. While infrared radiant exposures required to inhibit action potential generation are much lower than stimulation thresholds reported previously [14], the local temperature rise required for propagation block in Aplysia is approximately 8° C. (
Mou et al. proposed that thermal block of action potential propagation would require greater temperature increases than for inhibition of action potential initiation [17]. This is likely due to the action potential safety factor, which allows propagation to continue even when local excitability is reduced [30]. The excess current available for action potential propagation may explain why we experienced more robust block of action potential initiation than propagation. As Mou et al. showed, either a greater increase in temperature for one node or a lesser temperature rise distributed over multiple nodes may be required to block propagation.
Thermal neural inhibition using infrared light provides a simple tool for neural control that will aid both neural circuit analysis and the development of therapies for treating neurological disorders. Because of its simplicity, it is likely that there will be widespread and diverse application of this technique across a wide array of species and preparations.
Hybrid electro-optical neural stimulation is a novel paradigm combining the advantages of optical and electrical stimulation techniques while reducing their respective limitations. However, in order to fulfill its promise, this technique requires reduced variability and improved reproducibility.
Among other things, one of the objectives of this example was to identify common factors that play a role in and may be controlled to enhance the reproducibility of hybrid electro-optical stimulation. Using this methodology, relevant sources of variability were identified in an experimentally tractable and relatively simple neurobiological system. These variability sources were tested in a more clinically relevant model, where the complexity of the neural system may obscure their detections. Accordingly, the experimental procedures differ slightly between the two model neural systems. However, the purpose of this example is to analyze and assess the overarching trends rather than the minor differences in stimulation protocols. To accomplish these goals, the choices of neural systems are the buccal nerve of the invertebrate marine mollusk Aplysia californica and the sciatic nerve of the vertebrate mammal Rattus norvegicus (rat). The Aplysia buccal ganglion provides a tractable, robust nervous system with large identified neurons and relatively few axons per nerve [50, 51]. These advantages facilitate the systematic empirical exploration of potential factors underlying the reproducibility of hybrid stimulation. The myelinated rat sciatic nerve is a more clinically relevant model for hybrid stimulation, but it is less robust than Aplysia nerves, and the fundamental interaction between the optical and electrical stimuli is confounded by the presence of myelin and a less stable nerve preparation. Therefore, the example identifies and characterizes factors contributing to the reproducibility of hybrid stimulation in the Aplysia buccal nerve and then evaluates those factors in the rat sciatic nerve to determine whether similar trends are observed. In this exemplary study, both spatial and temporal factors that may be controlled to reduce variability and enhance reproducibility were investigated.
There are two aspects of the spatial component that are addressed: (1) the relative locations of the optical and electrical stimuli and (2) the size of the excitable region as a function of the optical stimulus strength. The mechanism of INS was shown to involve a thermal gradient [52]. Thus, it is assumed that the thermal gradient and the electrical current path must overlap spatially. However, what is not known is where this overlap may occur, or how the two fields may affect each other. The activating function, which describes the transmembrane potentials leading to the electrical activation of a neuron, results in neurons closest to the cathode being activated first (with larger axons recruited before smaller axons) [53, 54]. Experimentally, stimulation threshold current is shown to increase with increasing distance from the cathode [55]. Given that the electrical stimulus preferentially targets neurons nearest the cathode, it is hypothesized that hybrid stimulation requires the lowest optical pulse energies when the optical stimulus is located along the electrical current path and adjacent to the cathode. Like electrical stimulation, increasing INS radiant exposures results in an increase in magnitude of the evoked response, suggesting recruitment of additional axons [56]. Therefore, it is expected that for a given sub-threshold electrical stimulus, an increase in the sub-threshold optical stimulus yields an increase in the size of the excitable region for hybrid stimulation.
It has long been known that electrical stimulation thresholds vary over time [57]. In examining temporal factors, it is evaluated how brief fluctuations (minutes) and long-term trends (minutes to hours) in electrical stimulation thresholds affect optical pulse energies for hybrid stimulation. Correct measures of optical energies for hybrid stimulation require an accurate determination of the electrical ‘priming’ stimulus at the time of the measurement. If one incorrectly assumes that the electrical stimulation threshold is stationary over a fixed period of time, then hybrid stimulation performance will suffer. To address this issue, threshold optical energies for hybrid stimulation is measured while monitoring electrical thresholds over an extended period of time. It is hypothesized that if the electrical threshold is known at any point in time, then the additional optical energy required for stimulation can be predicted for a given sub-threshold stimulus. Additionally, changes in threshold radiant exposures for the optical component of hybrid stimulation are positively correlated with the changes in the underlying electrical stimulation threshold.
In this example, a comparative physiological approach was employed to aid the further development of this technique by identifying the spatial and temporal factors characteristic of hybrid stimulation that contributes to experimental variability and/or a lack of reproducibility. Using transient pulses of infrared light delivered simultaneously with a bipolar electrical stimulus in either the marine mollusk Aplysia californica buccal nerve or the rat sciatic nerve, the existence of a finite region of excitability with size altered by the strength of the optical stimulus and recruitment dictated by the polarity of the electrical stimulus was determined. Hybrid stimulation radiant exposures yielding 50% probability of firing (RE50) were shown to be negatively correlated with the underlying changes in electrical stimulation threshold over time. In Aplysia, but not in the rat sciatic nerve, increasing optical radiant exposures (J/cm2) beyond the RE50 ultimately resulted in inhibition of evoked potentials. Accounting for the sources of variability identified in this study increased the reproducibility of stimulation from 35% to 93% in Aplysia and 23% to 76% in the rat with reduced variability.
Aplysia californica (n=26) weighing 190-250 g (Marinus Scientific, Newport Beach, Calif.) were maintained in an aerated aquarium containing circulating artificial seawater (ASW) (Instant Ocean; Aquarium Systems, Mentor, Ohio) kept at 16-17° C. The animals were fed dried seaweed every 1-3 days.
Aplysia were anesthetized with an injection of 333 mM MgCl2 (50% of body weight) prior to dissection. Once anesthetized, animals were dissected and the buccal ganglia were removed and pinned in a recording dish and immersed in Aplysia saline (460 mM NaCl, 10 mM KCl, 22 mM MgCl2, 33 mM MgSO4, 10 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.6). Once dissected and pinned, Aplysia nerves were left untreated so as not to reduce spontaneous activity. We chose not to discard data from trials where spontaneous activity occurred, as excitability varies with the level of activity. This is an inherent biological factor to be assessed in the exemplary study. For each experiment, the nerve of interest (either buccal nerve 2 (BN2) or buccal nerve 3 (BN3)) was anchored in place by pinning the protective sheath around the nerve to the Sylgard base (Dow Corning, Midland, Mich.) of the recording dish. Once securely pinned, the nerve to be investigated was suctioned into a nerve-recording electrode to monitor the response to stimulation (
All rat experiments were performed following protocols approved by the Institutional Animal Care and Use Committee. Female Sprague-Dawley rats (n=9) weighing 150-200 g (Charles River) were anesthetized with continuously inhaled isoflurane (induction: 3% isoflurane, 2.0 LPM oxygen; maintenance: 2-2.5% isoflurane, 1.5 LPM oxygen). A rectal probe and heating pad (catalog 40-90-8, FHC, Bowdoin, Me.) were used to maintain the rat at a target body temperature of 35-37° C. throughout the experiment. The lateral sides of the animals' back legs were shaved and the sciatic nerve exposed proximal to the knee via an incision in the overlying muscle. The muscular fascia over the nerve was removed while the nerve's epineurial layer was left intact. Saline was added periodically to keep the nerve from dehydrating throughout the experiment. A custom Sylgard platform was anchored to a micromanipulator and placed below the sciatic nerve with minimal added tension to minimize motion of the nerve due to the animal's respiration (
Analysis of hybrid stimulation requires an appropriately defined endpoint. In Aplysia, the endpoint is defined as the visible detection of single and/or compound extracellular nerve spikes in response to stimulation (
Extracellular stimulating electrodes were made from thin-wall borosilicate capillary glass (catalogue 615000; A-M Systems, Everett, Wash.) pulled to resistances of about 0.2 MQ (PP-830; Narishige). For each Aplysia experiment, two electrodes were capillary filled with Aplysia saline and placed on either side of the nerve in contact with the nerve sheath. This created a bipolar stimulus, with the pipettes oriented transverse to the longitudinal axis of the nerve. Pipettes were positioned such that their angle of approach to the nerve was as shallow as was allowed by the edge of the recording dish. For the rat experiments, two glass pipettes were filled with normal saline and placed in contact with the nerve along the nerve's longitudinal axis. The stimulating pipette arrangement for each species was chosen based on consistency of stimulation thresholds and ability to achieve reliable supra-threshold stimulation on each nerve tested. Monophasic currents were supplied by a bipolar stimulus isolator (A365R; WPI) and passed between the two pipettes in each preparation. Electrical stimulation was defined as the minimal current that would yield five consecutive evoked potentials in response to pulsed stimuli.
For the optical stimulation, both a holmium:yttrium-aluminum-garnet (Ho:YAG) solid state laser (SEO Laser 1-2-3, Schwartz Electro-Optics, Orlando, Fla.) and a tunable pulsed diode laser were used (Capella; Lockheed-Martin-Aculight, Bothwell, Wash.). Two different lasers were chosen due to the established performance in peripheral nerves offered by the Ho:YAG and the ease of use and INS-specific design of the Capella. While the Capella was used in our previous demonstration of hybrid nerve stimulation, the Ho:YAG is the laser of choice for much of the INS literature pertaining to peripheral mammalian nerves [46-48, 52, 58, 59]. However, the Capella offers vastly improved ease of use and greatly reduced pulse-to-pulse variability when compared with the Ho:YAG. The Capella is also known to work exceptionally well for INS in a wide array of excitable tissues including the cochlea, somatosensory cortex, embryonic heart, cardiomyocytes and the vestibular system [60-64]. While the Ho:YAG provides pulses of infrared light (λ=2.12 μm) having fixed pulse duration (τp=0.25 ms), the Capella has slightly tunable wavelength (λ=1.855-1.875 μm) and a variable pulse duration. The important parameter for INS is penetration depth in tissue (as pulse duration was shown to have negligible effects [17]); therefore, the Capella is set to have a wavelength of λ=1.875 μm for all experiments to match the absorption (i.e., penetration depth) of the Ho:YAG laser [65].
For the Aplysia experiments, laser output was coupled into either a flat-polished 100 or 200 μm diameter optical fiber (Ocean Optics, Dunedin, Fla.). For each experiment, the tip of the optical fiber was immersed in the Aplysia saline bath and brought into contact with the nerve sheath. The optical fiber was then slowly retracted with a micromanipulator and gently translated back and forth transverse to the nerve until the optical fiber was just out of contact with the nerve sheath. For radiant exposures presented in this study, the laser-irradiated area is assumed to be a circular spot on the incident surface of the nerve sheath having diameter equal to that of the optical fiber (i.e., 0.0314 mm2 for a 200 μm fiber and 0.00785 mm2 for a 100 μm fiber). For simplicity, as the optical fiber is just out of contact with the nerve sheath, this assumes no divergence of the beam from the tip of the optical fiber to incident surface of the nerve sheath.
For the rat experiments, laser output was coupled into a flat-polished 400 μm diameter optical fiber (Ocean Optics, Dunedin, Fla.). The fiber diameter for rat experiments was chosen to match the 400-600 μm optical fibers used in mammalian peripheral nerve studies, while smaller fibers were used in Aplysia studies to scale with the size of the Aplysia buccal nerves [49, 58, 59]. The optical fiber was positioned 500 μm from the incident surface of the nerve at an angle just off of vertical with a layer of saline just covering the surface of the nerve. The laser-spot size was measured using the knife-edge technique where two perpendicular measurements were taken along the axes of the presumed circularly shaped laser spot, yielding an irradiated area of 0.19 mm2 [66]. Pyroelectric energy detectors were used to measure pulse energies from the tip of the optical fiber for the Ho:YAG laser (J25, Coherent-Molectron Inc., Santa Clara, Calif.) and Capella laser (PE50BB-SH-V2, Ophir Optronics Ltd).
For INS alone, an optical stimulation threshold was defined as the minimum radiant exposure that would yield five consecutive evoked potentials in response to pulsed stimuli. In the Aplysia buccal nerve, using the Capella laser coupled to a 200 μm optical fiber that was retracted just out of contact with the nerve, threshold radiant exposures averaged 8.93 J/cm2 with a 95% confidence interval of 8.72-9.14 J/cm2 (25 measurements from 7 nerves). In the rat sciatic nerve, using the Ho:YAG laser coupled to a 400 μm optical fiber, threshold radiant exposures averaged 1.12 J/cm2 with a 95% confidence interval of 0.92-1.32 J/cm2 (12 measurements from 8 nerves).
Previous published studies found threshold radiant exposures in mammalian peripheral nerves ranging from 0.32 to 1.77 J/cm2 [46-49, 52, 58, 59]. However, directly comparing these values with published data is difficult. Ongoing studies in our lab show stimulation thresholds in the rat sciatic nerve from 0.7 to 1.3 J/cm2 (unpublished). In the cochlea, stimulation thresholds are on the order of mJ/cm2 [67]. To make direct comparisons, it is imperative that certain factors be controlled; in particular, spot-size determination and measures of threshold must be the same. Radiant exposures are highly dependent on the spot-size. Differences in the way spot-sizes are calculated or measured between studies propagate into large differences in reported radiant exposures (due to the squared term in the denominator). In addition to variations in experimental preparations (i.e., neural model system, in vivo, ex vivo or in situ), thresholds may vary based on the definition of the endpoint for a given study, for example, whether the threshold is defined by the appearance of muscle or nerve action potentials, or by a visibly identified muscle twitch [47, 49, 67]. A noteworthy aspect of this study is that no visible damage or loss of function (as indicated by the response to electrical stimulation) was noted as a result of stimulation with the radiant exposures used. This is particularly relevant to Aplysia, where optical- and hybrid-evoked potentials remained steady over several hours of stimulation (not shown).
All nerve stimulation was coordinated through computer software (AxoGraph X; AxoGraph Scientific, Sydney, Australia) and applied at a repetition rate of 2 Hz. In both preparations, electrical pulses of 100 μs were used. Optical pulse durations were 250 μs for the Ho:YAG and 2-3 ms for the Capella lasers, respectively. This is due to the fixed pulse duration of the Ho:YAG and the minimum pulse duration of the Capella required to achieve optical energies for stimulation. Since the underlying mechanism of INS has been shown to be thermally mediated and dependent on a temperature gradient [52], as long as the pulse duration is significantly shorter than the thermal diffusion time (about 100 ms), the laser pulse can be considered as an input delta function to the system. For hybrid stimulation, pulses were synchronized such that they ended concurrently. This allowed for the total charge and total thermal deposition to occur simultaneously. Nerve recordings were triggered and acquired for 10 ms prior to stimulation through 140 ms post stimulation.
To investigate spatial factors contributing to the reproducibility of hybrid stimulation, sub-threshold pulses of electrical current (90% of electrical stimulation threshold) were applied simultaneously with optical pulses of a set magnitude. During hybrid stimulation, the optical fiber was translated across the nerve between the stimulating pipettes using a micromanipulator. A CMOS color USB camera and accompanying software (catalog 59-367; Edmund Optics, Barrington, N.J.) were used to record the position of the optical fiber. A LED was triggered by computer software to flash synchronously with the laser pulse so that we could reconstruct the exact position of the optical fiber at the time of stimulation. The center of the tip of the optical fiber was plotted and correlated with the presence or absence of stimulation as indicated by an evoked potential on the nerve recording.
Temporal factors were examined by investigating how fluctuations in the electrical stimulation threshold over time affect the optical component of hybrid stimulation. Threshold currents were measured every 2-3 min for 1-3 hr to monitor underlying changes in electrical stimulation with time and to assure that hybrid stimulation was not inducing alterations in threshold currents. One hour of each trial was an experimental period where radiant exposures eliciting hybrid stimulation were measured along with electrical stimulation threshold currents. Every 2-3 min during this experimental period, electrical stimulation threshold currents were first measured and then the stimulus current was reduced to 90% of electrical stimulation threshold. For the Aplysia experiments, five pulses of five different radiant exposures were then systematically applied with the sub-threshold current pulses. For the rat experiments, eight pulses of five different radiant exposures were applied. The order in which the radiant exposures were applied was determined by a random sequence generator so as to limit any conditioning effects or bias. Each hybrid stimulus pulse was recorded as either a 1 or 0 as determined by the presence (1) or absence (0) of a visibly identified nerve (Aplysia) or muscle (rat) action potential. This process was repeated every 2-3 min for the duration of the experimental period.
For spatial data, movie files were analyzed with custom software (Matlab r2010b; Mathworks, Natick, Mass.). Locations of successful stimulation were compared using non-parametric statistical tests. The two-sample Kolmogorov-Smirnov test compares two empirical distributions and responds to both the overall shape and location of the distributions. While this test indicates if the distributions are statistically different, it does not tell whether it is due to the relative size or location of the distributions. To distinguish whether differences are due to changes in size or location of the region of excitability (ROE), the Mann-Whitney test was also performed, which is a non-parametric test that determines if the median of one data set is greater than another. The interquartile range was used as a measure of the size of the ROE.
Temporal data were aggregated using Matlab with statistical analysis performed in Microsoft Excel (Microsoft Office Professional Plus 2010) and Slide Write Plus Version 6 (Advanced Graphics Software, Inc., Encinitas, Calif.). For each radiant exposure, the number of ones was divided by the sum of ones and zeros to achieve a probability of firing. The cumulative distribution function (CDF) of the standard normal distribution,
where x is a random variable with mean μ and variance σ2, was then fitted to the data to determine the radiant exposure yielding 50% probability of firing (RE50). While the RE50 is not practically useful for stimulation, we use this approach as a generally well-accepted model for making comparisons and identifying thresholds [64, 68-71]. One of the objectives of the invention is to establish a methodology and identify pertinent considerations for successful hybrid stimulation rather than prescribe optimal conditions for stimulation.
When translating the optical fiber back and forth across the nerve, it was determined that there exists a finite region between the cathode and anode where hybrid stimulation is possible (
After identifying the existence of a finite ROE, how the strength of the optical stimulus altered its size was investigated. With electrical current at 90% of electrical stimulation threshold, the ROE size for optical stimuli of 1.78 and 4.71 J/cm2 using the Capella in Aplysia and 0.29-1.18 J/cm2 was compared with both the Ho:YAG and Capella lasers in the rat. These values were chosen to cover a range of optical radiant exposures that, in the absence of the electrical stimulus, are sub-threshold for stimulation in their respective neural systems. Locations of hybrid stimulation were binned and plotted as a probability histogram by dividing the number of stimuli evoking a response by the total number of attempts for each bin (
In Aplysia, a total of 28 trials were acquired from 3 nerves (3 different animals). In the rat, a total of 26 trials were acquired from 4 nerves (4 different animals). Equal radiant exposures from the same nerve and animal were combined into one data set. In Aplysia, a statistically significant increase (p<0.05) in the ROE size with increasing radiant exposure was observed for all nerve tested (
It was hypothesized that the polarity of the electrical stimulus would shift the location of the ROE. To test this, the ROE was identified as before, and then the polarity was reversed (while keeping the electrodes in place) and the new ROE was found. In Aplysia, this experiment was repeated using both the Capella and Ho:YAG lasers with a constant optical stimulus (2.42-4.71 J/cm2) across a total of 8 nerves from 7 animals yielding 11 polarity pairs. The Mann-Whitney test was used to evaluate whether a shift in the ROE median occurred with a change in polarity. For all polarity pairs, a reversal in polarity showed a statistically significant shift (p<0.05) in the ROE median such that the ROE was located adjacent to the cathode (
Electrical stimulation threshold currents as well as the RE50 for hybrid stimulation were monitored in the same nerve to determine if fluctuations in the former affect the latter. The RE50 for hybrid stimulation was determined by first generating probabilities of firing at a given radiant exposure for each time point (by dividing the number of stimulation attempts evoking a response by the number of total attempts) and then fitting those probabilities to a CDF (Equation (1)). The RE50 was defined as the radiant exposure providing a 50% probability of firing as indicated by the CDF fit.
For the Aplysia, 5 pulses of 5 radiant exposures (using the Capella laser) yielded 25 total data points every 2 min. These data were not sufficient for a reliable CDF fit at each time point, so a sliding window was applied to fit a CDF to 6 min windows of data.
To evaluate the consistency over time of the RE50 for hybrid stimulation, all of the data acquired from a given nerve were compiled and each radiant exposure was converted to a probability of firing. The probability of firing as a function of radiant exposure was then fit to a CDF. In Aplysia, a total of four nerves from four animals (n=610 data points at each radiant exposure) yielded a 50% probability of firing at 1.34 J/cm2 with a 95% confidence interval between 1.13 and 1.55 J/cm2 (
In the course of evaluating temporal factors affecting the RE50 for hybrid stimulation in Aplysia, it was discovered that at higher radiant exposures, the probability of firing began to decrease rather than asymptotically approach 100% as expected. To further investigate this phenomenon, the electrical stimulus was set to 90% of electrical stimulation threshold every 2 min and five pulses of five radiant exposures were applied in the manner described above. However, for this experiment the radiant exposures were higher than those used for identifying the RE50. The results from four nerves from two animals (n=600 data points per radiant exposure) are shown in
Reducing the optical energy required to stimulate excitable tissues may facilitate clinical translation of infrared neural interfaces due to the reduced likelihood of thermal tissue damage, and by making the design criteria for laser sources less restrictive. The purpose of this study was to assess potential factors that might contribute to variability in hybrid electro-optical stimulation, as well as to create a methodology for reliable and reproducible hybrid stimulation. This task was approached by comparing trends seen in two different neurobiological systems—the tractable and well-characterized Aplysia californica buccal ganglion and the myelinated and more clinically relevant rat sciatic nerve. Given the variability and lack of reproducibility as previously experienced, this approach allowed for identification of factors in the more experimentally tractable system that could subsequently be applied to the more clinically relevant preparation. Some concern may arise as to the translation of hybrid stimulation between an unmyelinated, invertebrate nerve and a myelinated, mammalian nerve. However, this study shows that the information gathered from experiments in Aplysia directly led to improved understanding and performance of hybrid stimulation in the rat sciatic nerve. Although some aspects of the experimental protocol differ between the two preparations (i.e., orientation of stimulating pipettes, source of optical stimulation, endpoint definition), overarching trends were clearly evident across both species. Prior to both adopting the methods used in this study and controlling for the spatial and temporal factors we have assessed, our efficacy for hybrid stimulation in the Aplysia buccal nerve and the rat sciatic nerve was 35% and 23%, respectively (unpublished data). In this paper, we define efficacy as a nerve demonstrating a hybrid stimulation event where a sub-threshold electrical stimulus and sub-threshold optical stimulus are combined to achieve an evoked response. We attempt to determine whether or not sub-threshold electrical and optical stimuli were combined to achieve supra-threshold stimulation. At the conclusion of this study, we now have an efficacy of 93% ( 42/45 nerves) in the Aplysia buccal nerve and 76% ( 13/17 nerves) in the rat sciatic nerve.
Relative mechanical stability between the target neural tissue, optical fiber and electrodes was imperative to achieving reliable and reproducible hybrid stimulation. This allowed for consistent location of the stimuli throughout a given experiment by minimizing nerve movement due to optical fiber movement, fluid flow (Aplysia) or animal respiration (rat). Stabilization challenges are likely to be alleviated as hybrid stimulation progresses to multi-modality nerve cuff stimulators where microfabricated cuffs will be able to adapt to changes in nerve shape and movement.
The orientation of the stimulating glass pipettes is also an important part of the physical setup that must be taken into account. In the rat, electrical stimulation was more reliable with the pipettes oriented along the longitudinal axis of the nerve than in a transverse configuration. For electrical stimulation of myelinated nerves, it is necessary to induce longitudinal axonal currents, which may explain the reason that pipettes oriented longitudinally to the nerve were most effective. Recent models of intrafascicular stimulation support these observations. As a function of position relative to nodes of Ranvier, bipolar stimulation with a longitudinal configuration was shown to have less variability in threshold currents as compared to a transverse configuration [37]. While Aplysia nerves are unmyelinated, and thus do not possess nodes of Ranvier, they do exhibit clustering of voltage-gated sodium channels that may aid in the conduction of action potentials along the nerves [38]. However, it was found in Aplysia nerves that electrical stimulation was more reliable with the pipettes oriented transverse to the nerve. Due to the thick outer sheath protecting the nerve, placing the glass pipettes along the longitudinal axis of the nerve may result in electrical current dissipating into the bath rather than penetrating to the axons. When placing the pipettes transverse with respect to the midline of the nerve, the current may take a more direct path through the axonal tissue.
The choice of laser is also a contributor to the reproducibility of hybrid stimulation. The two lasers used in this study differ in many respects, but are expected to perform equally from the point of view of thermal laser-tissue interaction. However, the Ho:YAG laser yielded greater reproducibility in the rat than did the Capella. To understand how this may have occurred, the two laser sources were examined. The Capella used for this study is a diode laser, which is chopped to produce square pulses having tunable pulse duration at a center wavelength of 1.875 μm. The Ho:YAG laser is a pulsed solid-state laser at 2.12 μm, which produces a 250 μs pulse (full width at half maximum), exhibiting an initial rising phase followed by a decay, with spikes in output energy throughout the pulse duration. The mechanism by which pulsed infrared light produces neural activation is known to be thermally mediated, and directly associated with the absorption of infrared light by water in tissue [52]. Which attribute of the laser contributes most significantly to the thermal gradient is the most relevant issue. A comparison of the absorption coefficient as a function of wavelength for pure water reveals that 1.875 μm and 2.12 μm have similar absorption coefficients (μa=26.9 cm−1 and μa=24.01 cm−1, respectively) [65]. Although tissue is predominantly water, these values may differ slightly in our preparation and are known to be temperature dependent. However, it is unlikely that the differing wavelengths of the lasers is the source of the Ho:YAG laser's superior reproducibility in myelinated peripheral nerves. A second obvious difference is the pulse durations of the two lasers. However, there is conflicting evidence as to whether pulse duration plays a role in optical stimulation thresholds [52, 67]. A third possibility is that the broad spectral width of the Capella (15-20 nm, FWHM) causes much of the laser's output to occur at wavelengths that are not optimal for optical stimulation of peripheral, myelinated nerves. In applications with more direct access to the target neural tissue, the effects of spectral width are minimized due to all of the light being absorbed at the site of neuronal activation. However, in peripheral nerves, where the optical energy must penetrate through connective tissue and myelin surrounding the axons, longer wavelengths emitted by the Capella may be absorbed before they ever reach the axons. Thus, stimulation thresholds would be higher and quickly approach damage thresholds. The differing temporal pulse structure has not been investigated, but may also contribute to the relative effectiveness of the lasers. Whereas the Capella is a chopped diode laser exhibiting a square pulse, the Ho:YAG laser has a temporal structure in which the optical energy varies and includes numerous energy spikes throughout the pulse duration [74]. This could result in higher peak power and peak irradiance for the Ho:YAG laser.
There are two broad categories of factors that affect the reproducibility of hybrid stimulation related to the interaction of the optical and electrical stimuli. In the first category are spatial factors, where the relative location of the two stimuli determines the efficacy of stimulation. The initial working hypothesis was that for a given sub-threshold radiant exposure, hybrid stimulation would be possible for all locations between the cathode and anode of a bipolar stimulus. The results of this study have shown that hypothesis to be false. In
This raises the question of where the ROE is located. This answer is clearer in Aplysia, where the ROE was consistently located adjacent to the cathode. Within a single nerve, the location of the ROE was effectively ‘steered’ by reversing the polarity of the electrical stimulus. In the rat sciatic nerve, half of the nerves showed a statistically significant shift in ROE location upon polarity reversal, though the effect was not as dramatic as in Aplysia. In the other trials, the ROE location either did not shift, or hybrid stimulation was ineffective when the polarity was reversed. However, in cases of successful hybrid stimulation, different evoked potentials were recruited for each stimulus polarity. This suggests that hybrid stimulation offers two forms of selectivity, as both the position of the optical stimulus and the polarity of the electrical stimulus dictate the units recruited. The results also imply that the ROE location in the rat sciatic nerve is influenced more by whether or not optical stimulation is possible rather than by the direction of current flow. Anecdotal evidence reveals that there are ‘sweet spots’ on the sciatic nerve where optical stimulation is most effective; in particular, these spots are found just proximal to the branch point of the fascicles, but also at some additional locations along the nerve trunk. This could potentially be due to thinning of the epineurium, proximity of fascicles to the irradiated surface or to increased concentration of nodes of Ranvier in these locations.
The existence of a finite ROE with the potential for shifting location in response to polarity reversal must be taken into account for reproducible hybrid stimulation. Much of the previously observed variability is also likely to be due to the relationship between ROE size and applied radiant exposure. The results indicate an approximately linear increase in ROE size over the range of radiant exposures tested (
A second category of factors contributing to the reproducibility of hybrid stimulation is temporal factors. These factors include how the electrical stimulation threshold and the hybrid stimulation RE50 change with time and relative to one another. It was initially expected that the excitability of a nerve to the combination of electrical and hybrid optical stimuli would follow a similar temporal pattern. However,
The underlying electrical stimulation threshold must be taken into account to reduce variability and enhance the reproducibility of hybrid stimulation. Whenever short-term fluctuations (minutes) in threshold radiant exposures are present, controlling for these fluctuations yields overall long-term (1 h) threshold radiant exposures that are consistent (
In the course of investigating temporal factors affecting hybrid stimulation, it was discovered that elevated radiant exposures (although still below threshold radiant exposures for optical stimulation alone) resulted in a decline in the probability of firing (
We previously showed the proof-of-concept potential for combined optical and electrical stimulation of neural tissue [49]. This study extends that work by outlining some potential sources of variability that may be controlled to provide reproducible hybrid stimulation. The results presented here also demonstrate the potential of combining optical and electrical stimulation techniques by providing further evidence for selectivity as well as the ability to inhibit neuronal firing. Finally, the study demonstrates the translational value of parallel studies in invertebrates and vertebrates. The key aspects of the methodology to capitalize on the potential of hybrid electro-optical stimulation are summarized as follows.
Having taken these points into account, the efficacy is improved by threefold in both the Aplysia californica buccal nerve and the rat sciatic nerve. There are other potential sources of variability that could be controlled to bring the current efficacy up to 100%. In Aplysia, the three nerves that did not show hybrid stimulation were from animals with questionable health, but were included in the success rate calculations for completeness. In myelinated peripheral nerves, the efficacy of optical stimulation is crucial to the success of hybrid stimulation. Elucidating the mechanism of INS will provide a priori knowledge of where on the nerves to stimulate (e.g., near the nodes of Ranvier). Improving the efficacy of optical stimulation in turn improves the efficacy and reduce variability of hybrid stimulation. Knowing the mechanism of INS also provides a clearer understanding of the interaction between electrical and optical stimuli that drives hybrid stimulation. In this study, it was demonstrated that mechanical stabilization of the nerve, electrodes and optical fiber is of utmost importance. Even with the efforts taken to stabilize the system, there is potentially still movement-inducing variability. To address this issue, we envision a hybrid stimulation cuff that moves with the nerve and is thus able to hold the stimuli in place relative to the nerve. However, the results thus far have provided the ability to begin assessing the clinical utility of hybrid neural stimulation. It is believed that the concepts and techniques presented in this study will facilitate the application of spatially selective neural interfaces where thermal tissue damage and/or laser design constraints are currently of concern.
The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
This application claims priority to and the benefit of, pursuant to 35 U.S.C. §119(e), U.S. provisional patent application Ser. No. 61/699,735, filed Sep. 11, 2012, entitled “OPTICAL INHIBITION OF EXCITABLE TISSUES,” by Austin Robert Duke et al., the disclosure of which is incorporated herein in its entirety by reference. Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference. In terms of notation, hereinafter, “[n]” represents the nth reference cited in the reference list. For example, [14] represents the 14th reference cited in the reference list, namely, A. R. Duke, H. Lu, M. W. Jenkins, H. J. Chiel, E. D. Jansen, Spatial and temporal variability in response to hybrid electro-optical stimulation. J Neural Eng 9, 036003 (Apr. 16, 2012).
This invention was made with government support under grant number CiPHER—HR0011-10-1-0074 awarded by the Department of Defense, and under grant number R01NS052407-01/05 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61699735 | Sep 2012 | US |
