BIOMECHANICS OF RAPID-STRETCH NERVE INJURY
Traumatic injury to the peripheral nervous system leads to loss of function, sensation, and quality of life for thousands each year, with limited options for treatment and repair. A primary focus of our lab is to understand the biomechanics of nerve stretch injury, the most common injury mechanism for peripheral nerves. Determination of injury thresholds, and linking mechanical measurements such as strain to functional and histological changes in model systems will enable characterization of stretch injury, and allow testing of new therapies and techniques to improve outcomes for patients. We use rapid-stretch in order to replicate real life injury conditions with respect to viscoelastic properties of nerves, and measure deformation with high speed video tracking. Our injury model creates a foundation for therapeutic assessment through microstructural changes to tissue, motor and sensory function, and electrodiagnostic measurement.
RAPID-STRETCH INJURY TO PERIPHERAL NERVES: HISTOLOGIC RESULTS OF ACUTE INJURY
Rapid-stretch injury predominates clinical peripheral nerve trauma, yet there exists no laboratory model to recapitulate this injury mechanism. To remedy this situation, we have created a testing apparatus permitting in vivo rapid-stretch nerve injury in the laboratory setting and have determined a gradation of injury severity defined by specific biomechanical properties. Assessment of histological and immunofluorescent stains revealed distinct fragmentation to the internal nerve fibers which is exacerbated with increasing biomechanical injury severity. This is a novel finding within the narrative of nerve injury, which we hypothesize to precipitate the formation of neuroma-in-continuity---the signature of clinical pathology. Collectively, biomechanical injury grade determines degree structural damage to the nerve in rapid-stretch injury; increasing biomechanical injury severity generates more devastating structural damage.
EVALUATION OF CHRONIC RAPID-STRETCH NERVE INJURY
Our model mimics the mechanism of injury most germane to clinic, rapid-stretch, and stratifies grades of stretch injury to establish that the degree of biomechanical strain determines functional outcomes. This injury gradation has also revealed structural damage consistent with human injury histology, with pathological remodeling of extracellular matrix, myelin, and perineurium corresponding to increasing injury severity. Importantly, failed regeneration has led to the pathological formation of neuroma-in-continuity—a novel product in the laboratory setting which recapitulates clinical histology. Neuroma formation is hallmarked by hypercellularity which we hypothesize is the product of chronic inflammation—a persistent, immune hostile environment drives pathological remodeling and prevents the favorable outcome of regeneration.
Future studies will include the addition of the clinically relevant EMG assessment and retrograde labeling to better discern conduction disparities and confirm the fidelity of reinnervation in the rapid-stretch model. Additionally, the use of the Thy1-YFP mouse will permit in-vivo 2-photon imaging of the sciatic nerve at serial end-points and provide further insights into the progressive regeneration of nerve architecture.
COMPARISON OF CHRONIC RAPID-STRETCH NERVE INJURY WITH TRADITIONAL MODELS OF PERIPHERAL NERVE INJURY
Although rapid-stretch injury is the most common cause of a segmental loss of nerve, existing animal models of nerve injury (crush or surgical transection) fail to mimic stretch induced damage. This study aims to compare grades of rapid-stretch injury to traditional models of nerve injuries: crush, primary repair, secondary repair, and no repair. Examination of neurologic deficits, functional outcomes, and histology will be used to assess the consistency of recovery profiles. Further, examination against the common clinical scenarios of acute repair after injury (primary repair) and latency to repair (secondary repair) will provide insights into surgical efficacy and histological examination of post-surgical nerve recovery---an assessment not readily available with clinical populations.
PARTICIPATION OF NEUROINFLAMMATION IN THE PATHOLOGICAL REMODELING OF NEUROMA FORMATION
Functional and histological assessments have determined increased deficits aligning with progressive rapid-stretch injury severity. Qualitatively, histology has revealed neuroma formation hallmarked by hypercellularity, despite sample extraction occurring 6 weeks after injury. As phagocytosis and a supportive regenerative environment has been demonstrated to cease around 2-weeks post-injury, to what cell types do these nuclei belong, and more importantly are they frustrating regeneration efforts?
Much investigation has been devoted to understanding the coordinated cellular and genetic program resulting in successful regeneration, however, the mechanisms of neuroma pathology are not understood. Yet considering the capacity for regeneration, we ask: what drives the dichotomy between successful regeneration and pathological remodeling? Studies in the domains of skeletal muscle regeneration, myocardial injury, and liver injury repair demonstrate the necessity of macrophages for remodeling and recovery----sentinels of inflammation. Macrophages have been implicated in multiple roles: tissue debridement, structural remodeling, and fibrosis, while further governing the recruitment of other inflammatory species such as t-cells, fibroblasts, and myofibroblasts. Yet in the context of peripheral nerve injury, few studies examine the contribution of macrophages to the inflammatory milieu, thus the interplay with other immune cells driving pathology also remains poorly understood. Elucidating the neuro-immune axis of pathological remodeling is cardinal to driving purposeful efforts to circumvent failed regeneration.
In this study, we first aim to define the timeline and participation of inflammatory cells in neuroma formation after rapid-stretch nerve injury with immunofluorescent histology (IHC). Structural visualization through IHC will permit a qualitative visualization of cellular distribution and an appreciable foundation for comparison against RNA-seq libraries. We will next determine the inflammatory transcription program participating in neuroma formation through RNA-seq. Elucidating transcript divergences across injury grades and at serial timepoints will reveal when the immune response frustrates regenerative efforts and which cell types drive this corruption. Collectively, better understanding the immune microenvironment under pathological duress will set the scaffold for future studies of therapeutic intervention.