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WO2019173813A1 - Évaluation de microscopie multiphotonique de tissu neurologique - Google Patents

Évaluation de microscopie multiphotonique de tissu neurologique Download PDF

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WO2019173813A1
WO2019173813A1 PCT/US2019/021530 US2019021530W WO2019173813A1 WO 2019173813 A1 WO2019173813 A1 WO 2019173813A1 US 2019021530 W US2019021530 W US 2019021530W WO 2019173813 A1 WO2019173813 A1 WO 2019173813A1
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nerve
injury
nerves
shg
neural
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Paul J. CAGLE
Michael Hausman
Matthew GLUCK
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Icahn School of Medicine at Mount Sinai
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Icahn School of Medicine at Mount Sinai
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0033—Features or image-related aspects of imaging apparatus, e.g. for MRI, optical tomography or impedance tomography apparatus; Arrangements of imaging apparatus in a room
    • A61B5/004—Features or image-related aspects of imaging apparatus, e.g. for MRI, optical tomography or impedance tomography apparatus; Arrangements of imaging apparatus in a room adapted for image acquisition of a particular organ or body part
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4029—Detecting, measuring or recording for evaluating the nervous system for evaluating the peripheral nervous systems
    • A61B5/4041—Evaluating nerves condition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
    • A61B5/7235—Details of waveform analysis
    • A61B5/7264—Classification of physiological signals or data, e.g. using neural networks, statistical classifiers, expert systems or fuzzy systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
    • A61B5/7271—Specific aspects of physiological measurement analysis
    • A61B5/7275—Determining trends in physiological measurement data; Predicting development of a medical condition based on physiological measurements, e.g. determining a risk factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2576/00—Medical imaging apparatus involving image processing or analysis
    • A61B2576/02—Medical imaging apparatus involving image processing or analysis specially adapted for a particular organ or body part
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4887—Locating particular structures in or on the body
    • A61B5/4893—Nerves
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H30/00—ICT specially adapted for the handling or processing of medical images
    • G16H30/40—ICT specially adapted for the handling or processing of medical images for processing medical images, e.g. editing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems

Definitions

  • Peripheral Nerve injury is highly correlated with musculoskeletal trauma which is one of the most common reasons for emergent care presentation.
  • PNI represents a spectrum of events ranging from little to no clinical impact to complete loss of sensory and/or motor function. The degree of loss is highly correlated to the severity of nerve injury, but the nerve is rarely completely ruptured and urgently repaired. More commonly, a nerve injury presents secondary to an adjacent musculoskeletal injury. A macroscopically intact nerve which has undergone a strain or crush injury cannot be assessed for the capacity to spontaneously recover. Patients undergo a period of observation lasting several months. This creates a momentous amount of pain, anxiety and suffering. In addition, the costs of observation are substantial including medical costs, lost wages and loss of production. Therefore, there is a need for a reliable quantitative diagnostic tool for nerve injury assessment allowing for rapid decision making to proceed with immediate surgical care methods or utilize conservative observation.
  • This disclosure provides a method for assessing a nerve injury in vivo.
  • the method may include focusing a source of penetrating energy at neurologic tissue or at least one nerve, receiving the resulting signal as an image, identifying, in the image, at least one neural characteristic selected from a degree of alignment, the amount of residual structures in inter-fiber spaces, internal disorganization or internal disruption; quantifying the image results; and generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic.
  • Nerve characteristics may be used to describe all aspects of the nerve including, but not limited to, collagen, epineurium, endoneurium, perineurium, axons, myelin, fascicles or individual neurons.
  • Also provided herein is a method of assessing a nerve injury in vivo including: generating an image of neural tissue using a handheld multi-photon microscope or other penetrating energy source; identifying, in the image, at least one neural characteristic selected from a degree of fiber, the amount of residual structures in inter-fiber spaces, internal disorganization or internal disruption; quantifying the image results; and generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic.
  • a method of categorizing a nerve injury in vivo may include generating an image of neural collagen surrounding at least one nerve using a handheld multi-photon microscope or other penetrating energy source; identifying, in the image, at least one neural characteristic selected from a degree of fiber alignment, the amount of residual structures in inter-fiber spaces, internal disorganization or internal disruption; quantifying the image results; generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic; and categorizing the injury to predict complete, partial or no recovery of nerve sensory and/or motor function.
  • FIGS. 1A-F are images (40x magnification) of rat median nerve paraffin sections immediately after injury (DayO) - SHG (1 A, C, E) and H&E (1 B, D, F) images. Structural integrity is intact in SC nerves (1 A, B), while mild myelin fragmentation (1 D - arrows) and collagen fiber undulations are observed in LS nerves (1 C, D). Extreme disruption and fiber shredding is exhibited in HS nerves (1 E, F)
  • FIGS. 2A-B are images (40x magnification) of contiguous rat median nerve paraffin embedded sections 1 -week after LS injury. Indicators of regeneration (digestion chambers and Schwann cells) visible in SHG (2A) images can be validated by comparison with H&E (2B) images
  • FIGS. 3A-F are images (40x magnification) of rat median nerve paraffin embedded sections 12-weeks after injury (DayO) - SHG (3A, C, E) and H&E (3B, D, F) images.
  • SCs (3A, B) are linear and intact with evidence of homeostasis and regular maintenance.
  • LS (3C, D) largely regain structural composure with debris completely cleared up however some larger inter-fiber spacing was observed (annotated with black and red boxes).
  • HS (3E, F) also show improved structural integrity but sustain tears (annotated with red and black circles) and increased cellularity.
  • Contiguous H&E sections validate neural structure seen by SHG.
  • FIGS. 4A-D are in vivo SHG Images of collagen (green): (4A) and (4C) Illustrate a linear pattern of Collagen at Day 0 and Week 12; (4B) Shows the disruption of a linear pattern after HS with restoration of linear pattern appreciate at 12 weeks (4D).
  • FIG. 5 is a graph of nerve stretch disorganization. The peaks represent quantification.
  • FIGS. 6A-D are images (40x magnification) of rat median nerve paraffin sections at the site corresponding to clamp placement: SHG (left- 6A&C) and H&E (right- 6B&D). Collagen structure linearity and integrity is observed in the NCs (6A&B), compared with mild waviness and non-linearity in the un-stretched SCs (6C&D).
  • FIG. 7 is SHG images (40x magnification) of rat median nerve paraffin sections that experienced HS injury. Images immediately after injury (DayO) appear on the left progressing through later time points (1wks through 12wks after injury). Regions of interest are displayed through the length of the nerve with the proximal stump appearing at the top and progressing down through the mid-region and distal stump. Loss of structural integrity is consistently greater in the end regions (proximal and distal) than the mid-region.
  • FIGS. 8A-F are images (40x magnification) of rat median nerve paraffin sections 3 weeks after injury (DayO) - SHG (8A, C, E) and H&E (8B, D, F) images.
  • SC nerves (8A, B) are linear and intact with signs of homeostasis and regular maintenance (as annotated by normal vasculature, as well as normal linear collagen appearance).
  • LS nerves (8C, D) show sustained elastic undulations (red and black lines) with signs of reparative activity (digestion chambers).
  • HS nerves (8E, F) show intense waviness (red and black lines) and collagen fiber non-linearity with an intense increase in cellular proliferation.
  • Neural structure seen in SHG can be validated in contiguous H&E sections (circled and labeled). Markings in images such as demonstration of linear vs. undulating or wavy fibers as well as Nodes of Ranvier and digestion chambers seek to show continuity between artifacts observed in H&E images as well as those observed in SHG images.
  • FIGS. 9A-F are images (40x magnification) of rat median nerve paraffin embedded sections 8-weeks after injury (DayO) - SHG (9A, C, E) and H&E (9B, D, F) images.
  • SCs (9A, B) are linear and intact with evidence of homeostasis and regular maintenance.
  • LS (9C, D) regain structural composure with remnant cellular traces of reparative activity.
  • HS (9E, F) also exhibit improved structural integrity but sustain shredding, cellularity, and digestion chambers. Contiguous H&E sections validate neural structure seen by SHG.
  • FIGS. 10A-H are images (40x magnification) of rat median nerve paraffin embedded sections that were immunohistochemically stained for myelin protein zero (P0).
  • SC nerves (10A & B) where (10B) is a region of interest at the site of clamping, nerve 1 -week after LS injury (10C) and HS injury (1 OD & E) where (1 OE) is a sample that appears to have experienced lasting myelin damage, nerves 3-weeks after LS (10F) and HS (10G), nerve images 8-weeks after LS (10H) and HS (101). All insets are images of the proximal region; larger sections are images of the distal region.
  • FIGS. 11A-D are images (40x magnification) of rat median nerve paraffin embedded sections that underwent LS injury and immunohistochemically stained for neurofilament activity (NFM).
  • SCs at DayO (1 1 A) show fully composed structure, while LS nerves, 1 -week after injury, (11 B) exhibit intense neurofilament loss and shredding.
  • NFM is recovered 3-weeks after the injury (11 B), and regain axon density and integrity by 8- weeks (1 1 D).
  • FIGS. 12A-I are images (40x magnification) of rat median nerve paraffin embedded sections that underwent HS injury and immunohistochemically stained for Schwann cells (S100), marked in blue.
  • SCs exhibit organized fiber structure as well as minimal Schwann cell levels related with routine homeostasis at 1 , 3, and 8-weeks after procedure (12A, D, G).
  • LS images show moderately organized collagen structure with sharp increase in Schwann cells 1 -week after injury (12B), with gradually decreasing Schwann cells and increasing linearity 3-weeks (12E) and 8-weeks (12H) after injury.
  • HS images exhibit complete disruption of fiber structure with no Schwann cell response 1 -week after injury (12C), followed by a drastic increase of Schwann cells 3-weeks after injury (12F), sustained even 8-weeks afterwards (121) with limited return to fiber integrity.
  • FIGS. 13A-D are images (40x magnification) of rat median nerve paraffin embedded sections that underwent HS injury and immunohistochemically stained for macrophage activity (ED1 ), marked in brown. Schwann cells are marked by dark blue contrast. 1 -week after injury macrophage activity can be observed (13A), which continues even 3-weeks afterwards alongside Schwann cell proliferation (13B) despite apparent fiber linearity. By 8-weeks post-injury, macrophage-based attempts at repair persist though at lesser numbers (13C). 12-weeks post-injury, macrophages remain alongside nonlinear, unrecovered collagen and abnormal Schwann cell population (13D).
  • FIG. 14 is a comparison between injured and healthy nerves at day 0, 6 weeks, and 12 weeks using in vivo SHG imaging.
  • FIG. 15 is an in vivo SHG image with auto fluorescence included.
  • FIGS. 16A and 16B are images with auto fluorescence removed.
  • FIG. 16A shows the green channel indicates collagen from SHG imaging and FIG. 16B shows the red channel shows what appears to be myelinated axons with nodes of Ranvier (indicated by the arrows).
  • the red channel is not a SHG image, and
  • FIGS. 17A and 17B are illustrations of a vertical multi-photon microscopy objective in one embodiment including an attachment to raise the nerve out of the surgical plane.
  • FIGS. 18A and 18B are illustrations of a horizontal multi-photon
  • microscopy objective in one embodiment consisting of an attachment to raise the nerve out of the surgical plane providing both forward and backward scatter signal collection.
  • FIG. 19 is an illustration of a multi-photon microscopy system in a vertical orientation for acquiring backward scatter including an attachment to raise the nerve out of the surgical field in one embodiment.
  • FIG. 20 is an illustration of a multi-photon microscopy system in a vertical orientation for acquiring forward and backward scatter in one embodiment.
  • FIG. 21 is an illustration of a handheld multi-photon imaging system in one embodiment.
  • references to one or an embodiment in the present disclosure can be references to the same embodiment or any embodiment; and, such references mean at least one of the embodiments.
  • Traumatic peripheral nerve injury can have devastating consequences including substantial pain, disability, loss of motor function and loss of sensory function.
  • the degree of internal nerve injury can lead to full recovery, partial recovery or no recovery.
  • customary assessment modalities such magnetic resonance imaging, electromyography, ultrasound and nerve conduction studies do not provide a definitive assessment. Treating clinicians are left to classify the injuries based physical examination in many cases, and with no predictive method to estimate recovery, patients are observed for periods of months to greater than one year.
  • peripheral nerve injuries undergo a well-characterized series of events often culminating in both myelin sheath breakdown and axonal degradation as well as a potential increase in collagen infiltration which can lead to internal scarring.
  • This process is known as Wallerian degeneration and follows a very consistent timeline from the onset of injury through the degenerative phase. Because Wallerian degeneration typically begins as early as 10 days following an injury, late treatment for these injuries is limited to nerve grafting or nerve transfer which results in improved but varying levels of functional recovery that can be partially associated with the significant delay associated with the time needed to observe for spontaneous nerve recovery.
  • a method to assess internal neurologic damage after peripheral nerve injury would revolutionize the way these peripheral nerve injuries are treated by allowing for early surgical intervention in patients who would not experience neurologic recovery. In addition to early intervention leading to improved pain, function and quality of life, a predictive measure would alleviate the significant cost burden associated with the follow up and therapy required during the post injury observation period.
  • Multi-photon microscopy is a valuable tool for imaging the microanatomy of nerves that holds clinical value and relevance due to its capacity to non-invasively image tissue anatomy-independent of dyes and probes.
  • the connective tissue of peripheral nerves provides strength, structure, and support to the nerve during physiological movement, and thus protects internal neural elements that are usually unable to withstand the multiaxial forces experienced by epineurial and endoneurial collagen. Injuries to the nerve are common clinical occurrences, and they are often difficult to accurately diagnose due to the inability to determine the severity of underlying internal damage.
  • Second Harmonic Generation (SHG) imaging is an application of nonlinear multi-photon microscopy that generates high resolution images of live tissue in a non-destructive manner.
  • SHG is based upon the distinct birefringence of specific tissue types highly enriched in collagen, myosin, or microtubules.
  • SHG is an optical phenomenon in which two near infrared photons of equal energy are incident on a tissue. The result is a single photon excited to a higher energy state (double the energy), which is emitted as visible light. While this event is relatively“rare”, using a pulsed laser allows for optimal timing to take advantage of this phenomenon in a controlled setting.
  • the essential hallmark of SHG is defined by the ability of the live tissue to emit light at exactly half the wavelength of the excitation wavelength.
  • the images generated using SHG are solely based upon fiber resonance and orientation, thus fluorescent probes and extrinsic dyes are not required to visualize the tissue being studied.
  • tissues highly enriched in type I collagen emit a strong SHG signal upon excitation.
  • An excitation wavelength of about 840 nm may be used for collagen, a near infrared wavelength indicating that images are generated at even higher resolutions and with greater depth penetration.
  • the emitted wavelength of 420 nm is consequently higher in energy than the excitation wavelength, meaning no energy from the excitation wavelength is lost to the surrounding tissue. This not only prevents photobleaching and phototoxicity, but also ultimately provides the capacity for non destructive in vivo imaging.
  • a method of assessing a nerve injury in vivo includes focusing a penetrating energy source at a neural tissue; receiving an optical signal from the neural tissue; generating an image of the neural tissue from the optical signal;
  • the neural network identifying, in the image, at least one neural characteristic; quantifying the at least one neural characteristic; and generating a nerve damage index that represents damage to the neural tissue based on the at least one neural characteristic.
  • the penetrating energy source may be a multi-photon
  • the neural tissue assessed by the method may be collagen, epineurium, endoneurium, perineurium, axons, myelin, fascicles, individual neurons, or combinations thereof.
  • multi-photon microscopy may be used for visualizing the collagenous structure of peripheral nerves. Assessing and quantifying collagen continuity and damage after a stretch injury may provide inferential insight into the level of axonal damage present. Multi-photon microscopy can be used to specifically image neural tissue, neural collagen, and other neural structures after trauma to detect initial signs of structural damage within the internal compartments of the nerve without causing further harm. The degree of internal collagen damage may be an important measure of the severity of axonal damage and of how limited the capacity for successful axonal regrowth may be.
  • Multi-photon microscopy may be used to identify the structural and inflammatory response after an in vivo nerve injury.
  • a reliable method for inducing calibrated levels of strain was developed on rat median nerves.
  • SHG images from early time points reveal a clear gradient of damage from low strain to high strain, indicated mostly by the loss of organized and linear fiber alignment as well as the appearance of residual structures in inter-fiber spaces.
  • the delineation of damage provided by SHG images suggests, not only that it can be used for specifically imaging neural collagen, but also that it assists in predicting the amount of structural injury incurred.
  • Later time points may show notable repair and structural reconstitution, though the overall quality of restoration largely correlates with the extent of damage. For example, when an injury is below its plastic threshold or low strain (LS), endoneurial collagen may regain the linearity and continuity seen in control (SC) images, indicating that intraneural repair mechanisms had likely occurred.
  • LS plastic threshold or low strain
  • Multi-photon imaging may reveal substantial structural defects throughout damaged nerves.
  • the images may correlate with signs of myelin and neurofilament degeneration in endoneurial collagen apart from cellularity. This may be especially evident after a high strain (HS) injury.
  • H&E staining of sectioned nerves confirm severe myelin degeneration and internal structural collapse immediately after injury, indicating that the structural effects seen in multi-photon or SHG microscopy mirror the level of internal neuronal pathology.
  • nerves exposed to high strain experience partial structural and myelin regeneration, however the continuity, linearity, and density of myelin may not fully recover to pre-stretch levels.
  • Immunohistochemical staining highlights the level of myelin and axonal collapse and their subsequent regenerative capacity, as characterized by timely Schwann cell proliferation and macrophagial activity in debris cleanup. This suggests that functional defects likely accompanied the induced injuries well into the latest time point post high strain injury, compared to significant reconstitution after low strain injury.
  • the association between SHG imaging, histological, and immunohistochemical staining indicates that damage to internal collagen caused by supraphysiologic strain may result in injury to neighboring internal structures that can drastically reduce proper function shortly after injury, continuing well into late time points.
  • immunohistochemical sections may provide an association between observable patterns of collagen damage and the resulting cellular damage, specifically regarding Schwann cell myelin.
  • H&E sections may validate and correlate with the observations made in SHG images.
  • IHC may allow for the analysis of how internal structural collapse affected critical neuronal elements. Both LS and HS injuries may have notable effects on myelin structure at early and late time points after injury. Myelin fragmentation may be consistently evident in either the extremes or throughout the full section of nerve tissue.
  • SHG images many of the residual structures observed in regions not occupied by endoneurial collagen were often present in H&E images as fragmented myelin sheaths. The same can be said for the Schwann cell and
  • H&E images of nerves subjected to LS and HS appear significantly different from one another, reflecting the trends observed from the SHG images.
  • FIGS. 8B, 8D, and 8F Structural reconstitution is much more evident, regardless of the severity of injury. Endoneurial and perineurial continuity suggests that the capacity for recovery may not be fully compromised in the nerves subjected to HS. For example, rupture or plastic deformation of individual collagen fibers may have occurred during the injury, yet the Schwann cell basal lamina tubes may not be drastically harmed, allowing for at least partial axonal guidance during regeneration.
  • conduction velocity may be used in combination with multi-photon images to confirm an assessment of nerve damage from multi-photon imaging or may be used as a nerve characteristic in quantifying nerve damage.
  • the myelin sheath is a crucial element of the nerve fiber as it allows for saltatory conduction of action potentials down the length of the nerve to and from the central nervous system. Complete or partial loss of the myelin sheath will drastically reduce the sensory-motor information carried by a nerve fiber. Thus, a substantial collection of affected fibers will ultimately affect the normal functioning of the innervated target tissue.
  • Nerve injuries often pose a significant clinical dilemma since the degree of injury and capacity for recovery are difficult to predict at the onset of injury. A loss of structural integrity within the nerve’s interior may immediately lead to axonal injury and may drastically affect the quality and timing of later neural regeneration. SHG
  • microscopy can non-destructively image internal neural elements after injury, and the level of damage corresponds with the severity of myelin injury, axonal damage, and cellular response. Further, multi-photon imaging clearly demarcates between structural response to low strain injuries that peripheral nerves can spontaneously recover from, versus supra-physiologic high strains that can lead to permanent functional losses.
  • the power used during microscopy for all in vivo imaging should be at a level that induces no additional damage to the nerve. Thus, the energy source should use a maximum level of power that can safely be utilized to achieve the highest resolution and the deepest level of penetration that can achieved without compromising safety.
  • the non destructive nature of multi-photon microscopy may be used to determine the severity of internal nerve damage and thus guide crucial diagnostic decisions.
  • the neurologic collagen changes secondary to strain injury may be analyzed using a rat model which can provide the biomechanical basis for a scale up to human tissue.
  • SHG microscopy can be utilized after a calibrated in vivo stretch injury of rat median nerves to evaluate collagen continuity at several time points throughout the recovery process.
  • Endoneurial collagen may be qualitatively assessed in nerves that were subjected to low strain and high strain injuries using SHG microscopy, conventional histology, and immunohistochemistry.
  • both low strain and high strain damaged nerves exhibit signs of structural collagen damage in comparison with sham control nerves.
  • low strain nerves may exhibit signs of full regeneration while high strain nerves may exhibit signs of only partial regeneration with lasting damage and intra-neural scar formation.
  • the method of assessing a nerve injury in vivo may include focusing a light or penetrating energy source at neural tissue; receiving an optical signal; generating an image of the neural tissue; identifying, in the image, at least one neural characteristic selected from a degree of fiber alignment, the amount of residual structures in inter-fiber spaces, internal disorganization or internal disruption; quantifying the image results; generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic; and categorizing the injury to predict complete, partial or no recovery of nerve sensory and/or motor function.
  • neural tissue include collagen, epineurium,
  • the optical signal may be received in at least one detector.
  • the detector may be a photomultiplier tube.
  • the penetrating energy source may be from a multi photon microscopy system.
  • a multi-photon system may further include first and second light detectors set to receive wavelengths of about 430-540nm and 575-630nm, such that one detector receives the desired SFIG collagen signal in a backward or forward scatter manner while the other detector detects any auto
  • the first detector may be set to receive wavelengths of about 495-540nm.
  • the auto fluorescence can be separated from the SFIG signal in order to be further analyzed in a wavelength specific manner as this may provide information regarding other types of non-collagenous tissue within the nerve.
  • FIG. 15 is an in vivo image with auto fluorescence included. After separating the auto fluorescence, the green channel indicates collagen from SFIG, as seen in FIG. 16A, and the red channel shows what appears to be myelinated axons with nodes of Ranvier (indicated by the arrows in FIG. 16B). The red channel is not an SHG image, and corresponds to a wavelength different from the received SHG signal.
  • the energy source may include visual and non visual spectrum.
  • the energy source may be a laser or a pulsed laser.
  • the energy source used in the multi-photon may have a wavelength sufficient for
  • the energy source is non
  • the energy source may have a wavelength ranging from about 350 nm to about 1 100 nm.
  • the energy source used in an SHG microscopy system may have a near-infrared wavelength.
  • the near-infrared wavelength may range from about 750 nm to about 1400 nm, from about 750 nm to about 850 nm, from about 800 nm to about 900 nm, from about 850 nm to about 950 nm, from about 900 nm to about 1000 nm, from about 1000 nm to about 1 100 nm, from about 750 nm to about 1 100 nm, and from about 1000 nm to about 1400 nm.
  • the wavelength of the received second harmonic generation signal may be half the wavelength of the emitted laser wavelength.
  • the wavelength of the laser may be about 860 nm and the wavelength of the generated SHG signal may be collected about 430 nm.
  • the wavelength of the laser may be about 910 nm, and the wavelength of the SHG signal may be about 455 nm.
  • the received optical signal may be a backward scatter signal, a forward scatter, as well as scatter received from various arrangements of detectors not limited to forward or backward, or a combination thereof.
  • the ratio of forward to backward scattered photons depends on ion content and fiber thickness.
  • the thickness of the tissue at the desired location and the depth of the target nerve may determine whether backward scatter or forward scatter is acquired. Forward scatter is much higher resolution, but not all tissues can reliably produce sufficient forward scatter due to thickness.
  • the deeper into tissue the more photon scattering and the less resolution.
  • Increasing numerical aperture and objective magnification decreases working distance. Therefore, resolution is sacrificed for deeper tissue. Ion content and concentration affects protein aggregation and solubility, and thus affects SHG signal emission.
  • FIGS. 17A and 17B are a side and front view of a vertical multi-photon microscopy objective 102 with an attachment 104 or adaptation which may be attached or configured to an objective or objectives to optimize nerve position in vivo for and during imaging.
  • FIGS. 17A and 17B illustrate an attachment 104 to a backward scatter multi-photon objective 102 allowing for the operator to raise the nerve from the surgical field while keeping the distance to the objective lenses consistent and controlling for micro-motion generated from the system.
  • FIGS. 18A and 18B are examples of horizontal multi-photon microscopy objectives 201 , 202 with an attachment 204 or adaptation which may be attached or configured to an objective or objectives to optimize nerve position in vivo for and during imaging.
  • FIG. 18 illustrates how an attachment 204 may be used with objectives 201 , 202 that allow for forward scatter, backward scatter, or a combination thereof.
  • FIG. 19 is a multi-photon microscopy system 100 with an attachment 104 to the objective 102 in a vertical orientation for acquiring backward scatter in one embodiment. Additionally, this demonstrates an arrangement of the multi-photon microscopy system 100 so as to provide a mobile arm 106 for use in a surgical setting.
  • the multi-photon microscopy system may also include a photomultiplier tube 108, a dichroic mirror 110, scanning mirrors 112, a quarter waveplate 114, a polarizer 116, and a penetrating energy source 118, such as a laser.
  • FIG. 19 is a multi-photon microscopy system 100 with an attachment 104 to the objective 102 in a vertical orientation for acquiring backward scatter in one embodiment. Additionally, this demonstrates an arrangement of the multi-photon microscopy system 100 so as to provide a mobile arm
  • the objective attachment 304 or modification in FIG. 20 can be used to optimize the position of the nerve for in vivo imaging.
  • the generated optical signals may then be analyzed for various neural characteristics to assess the damage to the nerve.
  • neural characteristics include the degree of collagen fiber alignment, the degree of fiber undulations, fiber spacing, gross tears in collagen structure, myelin content, synaptic terminal integrity, synaptic terminal count, and the amount of residual structures.
  • more than one optical signals/events may allow for visualization of more than one characteristic.
  • multiple optical events may be combined for evaluation or quantification.
  • Nerve damage may be quantifiable with multi-photon optical events, such as SHG, and the extent of damage as quantified with SHG may correlate with myelin damage and the extent of functional deficit.
  • the differences in nerve integrity may be quantified following increasing nerve strain exposure and the quantified values may be correlated to a histologic evaluation of both epineurium and myelin and to changes in function assessed through nerve stimulation.
  • the SHG or multi-photon generated image may be used to quantify the extent of neural strain or damage. Using image analysis, these characteristics may correlate to a categorical or numerical value designation indicative of the neurological damage.
  • a categorical classification can differentiate nerves which would completely recover, partially recover, or not recover motor and/or sensory function.
  • a numerical classification may provide surgeons with a relative amount of nerve disruption in comparison to images generated from a healthy section of the same nerve.
  • a high degree of organization and linearity in the fibers and a low amount of residual structures may correlate to nerves with a high likelihood of recovery and a low degree of organization or linearity in the fibers and a high number of residual structures may correlate to nerves with a lower likelihood of recovery and therefore would benefit from surgical
  • image analysis software may be used to find the distribution of angles throughout a region of interest in the obtained image(s). Fiber orientation and angle distribution may be assessed in one example as a comparison to the mean direction of angles, the nearest neighbor fiber, an arbitrary 0 value, or a combination thereof.
  • increased or decreased SHG signal may be used to quantify nerve damage, provided the imaging parameters remain constant. The increased or decreased SHG signal corresponds to increased or decreased collagen content. 3D representations may also be generated utilizing the entire stack of images.
  • the region of interest represents an image taken from the image stack with auto-fluorescence tissue identified and separated in standard post imaging processing with bandwidth filters. This ensures the collagen tissue of interest can be examined independent of other tissue that may be present in channels reflecting auto fluorescence.
  • a Gaussian Gradient may be applied to the image.
  • the region of interest may be assessed based on its distribution. For example, the region of interest may be evaluated for a measure of disorganization represented from a deviation from a linear pattern based on a pixel analysis. The resulting data may then be normalized to account for discrepancies in the number of fibers between specimens, and a distribution curve of the number of different angles in the image may be
  • the unit-less value may be a nerve damage index.
  • the nerve damage index will provide threshold values at which complete, partial or absent nerve recovery will be expected.
  • a threshold level of internal nerve damage which correlates with the return of function and a threshold above which recovery does not occur.
  • Quantified SHG nerve collagen images may be correlated with functional and sensory recovery over time. This may allow for identification of the quantified level of collagen disorder where nerve sensation and function begins to improve.
  • the method may allow for identification of a quantified value which represents complete recovery and may identify a quantified value at which neurologic recovery does not occur or occurs incompletely.
  • the nerve damage index may be used to determine the extent of nerve damage or diagnose the nerve damage. A physician or surgeon may then use the nerve damage index to inform patient treatment. In an embodiment, if the nerve damage index is below a threshold value, the patient may choose observation. In another embodiment, if the nerve damage index is above the threshold value, the patient would be considered to have a nerve injury which will not achieve full motor and/or sensory functional recovery. Thus, treatment would be necessary.
  • the nerve damage index may demonstrate a clear difference between normal and damaged nerves.
  • the nerve damage index may provide a numeric comparison of the structural collagens
  • the method may include generating an image of neural tissue surrounding at least one nerve using a handheld multi-photon microscope; identifying, in the image, at least one neural characteristic selected from a degree of fiber alignment and an amount of residual structures in inter-fiber spaces; and
  • the multi-photon device used in vivo to acquire the image from either SHG signals or other optical phenomena, or a combination thereof may be handheld in one embodiment.
  • a multi-photon microscope may be miniaturized such that it may be used in vivo by a doctor or surgeon.
  • FIG. 21 illustrates an example of a handheld multi-photon imaging system 400 in one embodiment.
  • This handheld multi-photon imaging system 400 may be both capable of being placed directly onto the exterior of the nerve or into damaged nerve fibers as is clinically applicable.
  • the handheld multi-photon imaging system 400 uses a penetrating energy source 402 and dichroic mirror 406 to generate a signal which may then be detected by a photomultiplier tube (PMT) 404, interpreted, and quantified.
  • PMT photomultiplier tube
  • the method of treatment or diagnosis may include generating at least one optical event of at least one neural element using a penetrating energy source microscope; generating an image using the at least one optical event or combinations of optical events; identifying, in the image, at least one neural characteristic selected from a degree of fiber alignment, the amount of residual structures in inter-fiber spaces, and internal disorganization or internal disruption; quantifying the at least one neural characteristic; and generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic.
  • the method may further include categorizing the injury by way of the nerve damage index to predict complete, partial or no recovery of nerve sensory and/or motor function.
  • the penetrating energy source microscope may be a multi-photon microscope and, in some examples, the multi-photon microscope is handheld.
  • the at least one neural element is collagen surrounding nerve tissue.
  • the at least one optical event is an SHG signal.
  • the method may include generating an image of a neural tissue from an optical event created from a handheld multi-photon microscope; identifying, in the image, at least one neural characteristic selected from a degree of alignment, an amount of residual structures in inter-fiber spaces, and an internal disorganization or internal disruption; quantifying the at least one neural characteristic; generating a nerve damage index that represents damage to the nerve based on the at least one neural characteristic; and categorizing the nerve damage index to predict complete, partial or no recovery of nerve sensory and/or motor function.
  • the method may include using multi-photon imaging to determine the degree of internal nerve damage and correlate the findings with the capacity to predict functional clinical recovery.
  • imaging and quantifying changes in internal nerve collagen or other aspects of the tissue after strain injury may lead to in vivo human applications and potentially decrease the pain, suffering and cost burden associated with clinical observation. This may also allow for improved decision making regarding immediate surgical procedures in nerves which will not spontaneously recover leading to vastly improved clinical outcomes.
  • SFIG images and histological sections of nerves exposed to low strain revealed structural disorganization and signs of Wallerian degeneration 1 week post-injury, and subsequent cessation. Nerves exposed to high strain show severe structural defects 1 week post-injury that were sustained 3 weeks post-injury, albeit to a lesser degree, and remained all the way until 12 weeks post-injury. Structural defects include disorganized undulating patterns in fibers, crossing fibers, gross tears, as well as fiber shredding and general disorganization.
  • the application of multi-photon imaging, including SFIG imaging, as a method to assess nerve injury in vivo represents a potentially revolutionary non- damaging method to assess an injured structure with no other currently assessment methods.
  • the novel development of a method to quantify nerve injury changes in vivo utilizing multi-photon imaging signifies a technological step towards understanding the internal changes that occur after a strain injury.
  • a comparison of functional and histological results to SHG image quantification provides the unknown link between the internal damaged incurred during nerve strain injury and the capacity to recovery.
  • the application of multi-photon imaging to assess and quantify human nerve tissue in a controlled in vitro strain injury model would represent the fundamental step necessary for in vivo human assessment.
  • the in vivo application of multi-photon microscopy may be a means for real-time, intra-operative, quantitative assessment of nerve damage. Furthermore, a correlation of the SHG nerve injury quantification, functional correlations and histological data obtained in the rat model to in vitro human nerve assessments may provide a daunting step forward in advancing the understanding of human nerve recovery.
  • multi-photon microscopy imaging and quantification may be used with human nerve tissue.
  • Collagen damage in human nerves will increase with the extent of stretch injury and exhibit similar damage patterns as in the rat model.
  • Human nerves may generate different ranges of nerve damage indices and threshold values than rat nerves, but the same quantification measured may be used.
  • Micro dissection tools were used to separate the flexor digitorum superficialis (FDS) from the flexor carpi radialis (FCR) in order to expose approximately 1.5cm of the median nerve.
  • FDS flexor digitorum superficialis
  • FCR flexor carpi radialis
  • a strain device was created.
  • the median nerve was then placed in a specially modified microvascular clip (soldered to radial Vernier calipers for calibrated stretching) the two arms 1 cm apart.
  • the ends of this 1 cm segment, corresponding to the regions of contact with the clamps, were marked with a tissue-marking pen.
  • the distal clamp was controlled via the Vernier to a distance corresponding to a predetermined percentage of elongation.
  • the final distance between the marks on the tissue was then measured to eliminate the possibility of slippage and ensure the desired percentage of elongation was achieved.
  • the nerve remained in the stretch applicator for 5 minutes before the tension was released; at which point the nerves were free to retract to their nominal position.
  • Sham control (SC) operations involved identical clamp placement on the median nerves of the left, contralateral limb. Sham control nerves were clamped and held in the forelimb without inducing any strain for the same 5-minute time period.
  • the median nerves of six Sprague-Dawley rats were utilized. Each animal was anesthetized and the median nerve was microscopically dissected on both forelimbs.
  • the device used in Example 2 allowed for a controlled strain to be applied to one of the median nerve while the contralateral side served as a SC. Nerves were exposed to a HS injury for five minutes and the contralateral side had the device applied for five minutes with no strain.
  • Each nerve was assessed with a graded intraoperative nerve stimulator with capacity to measure between zero and 400 nanocoulombs (nC), the Checkpoint Nerve Stimulator. The nerves were assessed for the lowest current required to generate a paw flicker of movement.
  • nC nanocoulombs
  • Control nerves demonstrated a flicker of paw motion at an average of 25nC. Whereas, nerves exposed to a HS required an average increase of 153.8nC to generate a flicker of paw motion.
  • the mean difference was 128.8nC with a standard error of 10.4nC.
  • the comparison represents a statistically significant difference, and the data set was very closely grouped. The lack of data point outliers and the significant change in the amount of current required to stimulate muscle movement validates the reproducibility of the strain injury utilized in this protocol.
  • SHG imaging was performed on an Olympus FV1000MPE Fluoview multi photon laser scanning microscope controlled by Olympus Fluoview software (version 3.1 b) and fitted with a Coherent Chameleon Vision II Ti:S laser. Images were obtained at 860nm, the optimal wavelength for imaging collagenous nerve tissue based on previous studies observing the SHG signal in type I collagen. A 40x (0.8 numerical aperture) water immersion objective lens was used to focus the excitation beam. 12-bit gray scale resolution images were acquired at a 508.4pm field of view and with an image size of 1024x1024 pixels.
  • Reflected backward scatter SHG signal was collected using a 420- 460nm bandpass filter, a 485 dichroic mirror (GR/XR filter cube, Olympus), and a non-descanned (external) detector.
  • Two additional bandpass filters at 495-540 and 575-630 were utilized to capture and remove auto fluorescence by removing those two channels from the image during processing and analysis. This resulted in images containing just the channel in which the SHG signal was acquired.
  • Immunohistochemistry was also used to assess for neurofilament and myelin markers, indicative of axonal damage (in addition to observed collagen damage). Histological sections were also prepared from every third contiguous section for immunohistochemical assays using antibodies against Schwann cells and
  • macrophages S100 calcium-binding protein staining to mark Schwann cells, and ED1 for macrophages (using mouse anti-rat monocytes/ macrophages [CD68] monoclonal antibody. Some specimens provided additional sections that were assayed using anti chicken antibodies against Myelin Protein Zero and Neurofilament-200kDa.
  • Tissue sections were blocked for non-specific binding with a blocking agent: Blokhen II at an optimal dilution of 1 :10ml_ in PBS). Immunohistochemical staining was performed using optimal dilutions of 1 :100 for both aforementioned antibodies. Green fluorescein-labeled goat anti-chicken IgY secondary antibodies were used at an optimal dilution of 1 :800 to detect signal. Slide staining was carried out using an automated immunohistochemistry slide staining system.
  • Blokhen II at an optimal dilution of 1 :10ml_ PBS. Subsequent immunohistochemical staining was performed according to a previously developed protocol using optimal dilutions of 1 :100 for both antibodies tested. Green fluorescein-labeled goat anti-chicken IgY secondary antibodies were used at an optimal dilution of 1 :800.
  • Example 8 SHG imaging can identify damage that is validated on histology
  • Sectioned SHG images of the paraffin embedded ex vivo nerves demonstrated increased linear disorganization when comparing the SC to the LS and then the HS group (FIGS. 1A-1 F). A comparison of the proximal, mid and distal points did not demonstrate a significant difference in gross visual evaluation.
  • Nerves were then prepared using a H&E stain. Histologic comparisons were quantitatively assessed through histological classification (1 st -5 th degree),
  • SHG images of SCs exhibit organized, linear endoneurial collagen fibers between axons (FIG. 1A).
  • H&E images of SCs also show the similar patterns of congruity and linearity (FIG. 1 B), verified by the nerve interiors, which appear as continuous, parallel white lines.
  • SHG images reveal a notable increase in collagen fiber spacing, suggestive of Wallerian
  • FIG. 1C H&E images of the same nerve show substantial myelin degradation in distinct regions, explaining the increased fiber spacing in SHG images (FIG. 1 D, arrows). Nerves exposed to HS show increased fiber spacing apart from a distinct region of wavy, nonlinear internal fibers, and fiber shredding, which suggests potentially plastic structural damage to these fibers (FIG. 1 E). Corresponding H&E images reveal more severe and diffuse myelin degradation than observed in LS group (FIG. 1 F). H&E images also show complete degeneration and disorganization in the distal region.
  • myelin protein zero (P0) staining allows its direct visualization.
  • Composed myelin fibers in SC sections were consistent with observations made from SFIG as well as FI&E images (FIGS. 10A and 10B).
  • the LS altered this ideal conformation.
  • Myelin appeared globular and fragmented in the distal portion nerve (FIG. 10C), while proximal regions were less altered (FIG. 10C, inset). Nerves exposed to FIS were similar (FIG. 10D), apart from partial regeneration in the proximal segment (FIG. 10D, inset). Though proximal regions suggest partial regeneration (FIG. 10E, inset), the severity of distal myelin collapse
  • FIG. 10E brings into question the potential for successful axonal regeneration.
  • PO stained sections resonate with the themes observed from SHG and H&E images: (a) continued fiber alignment and globules symptomatic of damage clearance in LS nerves (FIG. 10H), and (b) HS nerves (FIG. 101) indicating damage beyond the intrinsic tolerance threshold.
  • HS injury resulted in axonal discontinuity (axonotmesis). A subsequent loss of axon and Schwann cell contact, raised
  • SHG imaging was performed with an Olympus FV1000MPE Fluoview multi-photon laser scanning microscope controlled by Olympus Fluoview software (version 3.1 b) and fitted with a Coherent Chameleon Vision II Ti:S laser. 12-bit gray scale resolution images were acquired at a 508.4pm field of view and with an image size of 1024x1024 pixels. Subgroups for 1 -week, 3-week, 8-week or 12-week time point analysis underwent repeat dissection and imaging.
  • SC nerve at Day 0 demonstrates relatively linear collagen tissue with some auto fluorescence localized around the nerve (red) (FIG. 4A).
  • This same SC nerve imaged 12 weeks also demonstrated linear, organized and linear collagen (FIG. 4C).
  • a 20% elongation injury in HS nerves resulted in gross disorganization, kinking fibers, and crossing fibers at Day 0.
  • Improvement in the linear organization of collagen with ability to again detect auto-fluorescence tissue was demonstrated at 12 weeks (FIG. 4B) which illustrates an improved linear pattern by 12 weeks (FIG. 4D). This data demonstrates the critical ability to image nerves in vivo with repeated measurements, and the ability to detect and separate out collagen and non-collagen tissue.
  • FIG. 5 demonstrates a graphical depiction of the difference between SC and HS nerve for one specimen at Day 0.
  • the figure illustrates a clear difference in the area under the curve between intact SC nerves (green) and damaged HS nerves (red).
  • Nerves were pre-stretched for 5 minutes using 15-gram fishing weights, which was determined to be within the toeing region for all nerves. The nerves were marked and fastened to the device ensuring that they were held at 4% strain (within normal physiological tension). Nerves were then imaged using SHG in the“intact” condition. Following this, the device was tuned to 20% strain and held for 5 minutes. The nerve was again subsequently imaged with SHG microscopy. This initial pilot data
  • Example 13 Identify and quantify specific damage patterns in epineurial collagen and myelin in an in vivo rat model of peripheral nerve injury acutely after injury.
  • SC sham control
  • a nerve stimulator will be utilized to measure paw flicker before and after application of strain device in both strain and SC limbs. All nerves will be imaged immediately in vivo with SHG microscopy. To ensure safety, nerve exposure will be measured with an attached power meter for assessment of photobleaching and cell toxicity.
  • Example 14 Identify and quantify specific damage patterns in epineurial collagen and myelin and correlate with function in an in vivo rat model of peripheral nerve injury following healing after injury
  • Example 15 Develop a method to identify and quantify damage patterns in epineurial collagen and myelin in an in vitro human peripheral nerve model
  • Fresh human cadaveric nerves will be obtained and controlled for diameter.
  • the tibial and sural nerve will be harvested and controlled for size by measurement of gross outer diameter.
  • Full length nerves will be sectioned into segments.
  • Ten tibial and sural nerves segments will be utilized to create a load- deformation curve.
  • the load-deformation curve model will be created to quantify a LS, MS and HS nerve injury.
  • the toeing region of the curve corresponding to the normal elastic threshold of the nerves will be identified to demonstrate a value
  • Nerves will then undergo SHG image assessment and subsequently histologic qualitative and quantitative evaluation. Each nerve will undergo Histologic quantitative and qualitative evaluation as described in Example 7. SHG collagen patterns will be identified and quantified and correlations will be made with findings in the rat model. Demonstrating both the capacity to image and quantify human nerve tissue with SHG microscopy represents a critical step in moving towards an in vivo human application, and the ability to correlate with rat model data would provide insight into potential similar function results in human subjects.

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Abstract

L'invention concerne des procédés d'imagerie, de quantification et de catégorisation d'une lésion nerveuse in vivo. Les procédés comprennent l'utilisation d'une microscopie multiphotonique ou d'autres aspects du spectre visuel et non visuel pour focaliser une source d'énergie pénétrante ou de lumière au niveau d'un tissu neurologique (épineurium, endoeurium, perineurium, axons, myéline, fascicles ou neurones individuels). Les procédés peuvent en outre comprendre la réception d'un signal optique en tant qu'image ; l'identification, dans l'image, d'au moins une caractéristique neuronale sélectionnée parmi un degré d'alignement, la quantité de structures résiduelles dans des espaces inter-fibres, une désorganisation interne ou une interruption interne ; la quantification des résultats d'image ; et la génération d'un indice de lésion nerveuse qui représente un dommage au nerf sur la base de ladite caractéristique neuronale. L'indice peut ensuite être utilisé pour catégoriser la lésion afin de prévoir une récupération complète, partielle ou nulle de la fonction sensorielle et/ou motrice nerveuse.
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