Retrograde labeling correlates with motor unit number estimation in rapid-stretch nerve injury.

INTRODUCTION/AIMS: Rapid-stretch nerve injuries represent a substantial treatment challenge. No study has examined motor neuron connection after rapid-stretch injury. Our objective in this study was to characterize the electrophysiological properties of graded rapid-stretch nerve injury and assess motor neuron health using retrograde labeling and muscle adenosine triphosphatase (ATPase) histology. METHODS: Male C57BL/6 mice (n = 6 per group) were rapid-stretch injured at four levels of severity: sham injury, stretch within elastic modulus, inelastic deformation, and stretch rupture. Serial compound muscle action potential (CMAP) and motor unit number estimation (MUNE) measurements were made for 48 days, followed by retrograde labeling and muscle ATPase histology. RESULTS: Elastic injuries showed no durable abnormalities. Inelastic injury demonstrated profound initial reduction in CMAP and MUNE (P < .036) on day 2, with partial recovery by day 14 after injury (CMAP: 40% baseline, P = .003; MUNE: 55% baseline, P = .033). However, at the experimental endpoint, CMAP had recovered to baseline with only limited improvement in MUNE. Inelastic injury led to reduced retrograde-labeled neurons and grouped fiber type histology. Rupture injury had severe and nonrecovering electrophysiological impairment, dramatically reducing labeled neurons (P = .005), and atrophic or type 1 muscle fibers. There was an excellent correlation between MUNE and retrograde-labeled tibial motor neurons across injury severities (R2  = 0.96). DISCUSSION: There was no significant electrophysiological derangement in low-severity injuries but there was recoverable conduction block in inelastic injury with slow recovery, potentially due to collateral sprouting. Rupture injuries yielded permanent failure of injured axons to reinnervate. These results provide insight into the pathophysiology of clinical injuries and recovery.

A novel anatomo-physiologic high-grade spondylolisthesis model to evaluate L5 nerve stretch injury after spondylolisthesis reduction.

L5 nerve palsy is a well-known complication following reduction of high-grade spondylolisthesis. While several mechanisms for its occurrence have been proposed, the hypothesis of L5 nerve root strain or displacement secondary to mechanical reduction remains poorly studied. The aim of this cadaveric study is to determine changes in morphologic parameters of the L5 nerve root during simulated intraoperative reduction of high-grade spondylolisthesis. A standard posterior approach to the lumbosacral junction was performed in eight fresh-frozen cadavers with lumbosacral or lumbopelvic screw fixation. Wide decompressions of the spinal canal and L5 nerve roots with complete facetectomies were accomplished with full exposure of the L5 nerve roots. A 100% translational slip was provoked by release of the iliolumbar ligaments and cutting the disc with the attached anterior longitudinal ligament. To evaluate the path of the L5 nerves during reduction maneuvers, metal bars were inserted bilaterally at the inferomedial aspects of the L5 pedicle at a distance of 10 mm from the midpoint of the L5 pedicle screws. There was no measurable change in length of the L5 nerve roots after 50% and 100% reduction of spondylolisthesis. Mechanical strain or displacement during reduction is an unlikely cause of L5 nerve root injury. Further anatomical or physiological studies are necessary to explore alternative mechanisms of L5 nerve palsy in the setting of high-grade spondylolisthesis correction, and surgeons should favor extensive surgical decompression of the L5 nerve roots when feasible.

Does the L5 spinal nerve move? Anatomical evaluation with implications for postoperative L5 nerve palsy.

PURPOSE: While palsy of the L5 nerve root due to stretch injury is a known complication in complex lumbosacral spine surgery, the underlying pathophysiology remains unclear. The goal of this cadaveric study was to quantify movement of the L5 nerve root during flexion/extension of the hip and lower lumbar spine. METHODS: Five fresh-frozen human cadavers were dissected on both sides to expose the lumbar vertebral bodies and the L5 nerve roots. Movement of the L5 nerve root was tested during flexion and extension of the hip and lower lumbar spine. Four steps were undertaken to characterize these movements: (1) removal of the bilateral psoas muscles, (2) removal of the lumbar vertebral bodies including the transforaminal ligaments from L3 to L5, (3) opening and removing the dura mater laterally to visualize the rootlets, and (4) removal of remaining soft tissue surrounding the L5 nerve root. Two metal bars were inserted into the sacral body at the level of S1 as fixed landmarks. The tips of these bars were connected to make a line for the ruler that was used to measure movement of the L5 nerve roots. Movement was regarded as measurable when there was an L5 nerve excursion of at least 1 mm. RESULTS: The mean age at death was 86.6 years (range 68-89 years). None of the four steps revealed any measurable movement after flexion/extension of the hip and lower lumbar spine on either side (< 1 mm). Flexion of the hip and lower lumbar spine revealed lax L5 nerve roots. Extension of the hip and lower lumbar spine showed taut ones. CONCLUSION: Significant movement or displacement of the L5 nerve root could not be quantified in this study. No mechanical cause for L5 nerve palsy could be identified so the etiology of the condition remains unclear.

Sesamin protects SH-SY5Y cells against mechanical stretch injury and promoting cell survival.

BACKGROUND: Sesamin is a well-known antioxidant extracted from sesame seeds that exhibits various curative effects. The present study investigated whether sesamin would protect neuroblastoma SH-SY5Y cells against mechanical stretch injury-induced increases in reactive oxygen species (ROS) and apoptosis. Additionally, the mechanisms underlying these actives were investigated. Following exposure to mechanical stretch injury, cells were incubated for further investigations. Lactate dehydrogenase and Cell Counting Kit-8 assays were used to assess cell viability, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometric analysis were performed to evaluate changes in mitochondrial membrane potential (ΔΨm). Furthermore, intracellular levels of ROS production were measured by 20, 70-dichlorofluorescein diacetate staining, the mRNA levels of matrix metallopeptidase 9 (MMP-9) were evaluated using real-time polymerase chain reaction analysis, and the determinations had also been made on related proteins by Western blot analysis. RESULTS: Exposure to mechanical stretch injury significantly decreased cell viability but this decrease was attenuated by pretreatment with sesamin (50 µM). Sesamin also significantly inhibited mechanical stretch injury-induced increases in intracellular ROS production, attenuated declines in ΔΨm, diminished the expressions of pro-apoptotic proteins, and decreased cell apoptosis. Stretch injury increased Bax and cleaved caspase 3 levels, enhanced the gene expression of MMP-9, increased the phosphorylation levels of Akt, p38, and JNK and decreased Bcl-2 levels in the cells. However, pretreatment with sesamin reduced the mechanical stretch injury-induced overexpression of MMP-9. CONCLUSIONS: Sesamin protected SH-SY5Y cells against stretch injury by attenuating increases in ROS levels and suppressing apoptosis. Accordingly, sesamin seems to be a potentially therapeutic agent in the treatment of traumatic brain injury.

Axillary nerve palsy consequent to a guided manual stretch of the upper extremities: a case report.

Reflexive breathing therapy and complementary manual therapy aim to improve breathing by mobilizing the chest, reducing the muscle tone of the breath supporting muscles, and improving secretion and chest wall compliance. We describe an uncommon case of axillary nerve (AN) palsy in association with a treatment session of reflexive breathing therapy combined with manual therapy. After a therapist's guided intensive movement of crossed arms in front of the body, upward, as high as possible, a sudden ache in the left shoulder that radiated to the lateral upper arm occurred along with loss of elevation and abduction of the shoulder. An electrophysiological examination demonstrated a diminished and delayed compound muscle action potential that indicated AN injury. Following conservative treatment, the nerve dysfunction resolved completely. Stretching of the nerve may have resulted in tearing some nerve fibers and is discussed as a possible pathophysiological mechanism.

Biomechanical characterization of cadaveric brachial plexus regions using uniaxial tensile tests.

INTRODUCTION: The proximal regions of the brachial plexus (roots, trunks) are more susceptible to permanent damage due to stretch injuries than the distal regions (cords, terminal branches). A better description of brachial plexus mechanical behavior is necessary to better understand deformation mechanisms in stretch injury. The purpose of this study was to model the biomechanical behavior of each portion of the brachial plexus (roots, trunks, cords, peripheral nerves) in a cadaveric model and report differences in elastic modulus, maximum stress and maximum strain. METHODS: Eight cadaveric plexi, divided into 47 segments according to regions of interest, underwent cyclical uniaxial tensile tests, using a BOSE® Electroforce® 3330 and INSTRON® 5969 material testing machines, to obtain the stress and strain histories of each specimen. Maximum stress, maximum strain and elastic modulus were extracted from the load-displacement and stress-strain curves. Statistical analyses used 1-way ANOVA with post-hoc Tukey HSD (Honestly Significant Difference) and Mann-Whitney tests. RESULTS: Mean elastic modulus was 8.65 MPa for roots, 8.82 MPa for trunks, 22.44 MPa for cords, and 26.43 MPa for peripheral nerves. Differences in elastic modulus and in maximum stress were statistically significant (p < 0.001) between proximal (roots, trunks) and distal (cords, peripheral nerves) specimens. CONCLUSIONS: Proximal structures demonstrated significantly smaller elastic modulus and maximum stress than distal structures. These data confirm the greater fragility of proximal regions of the brachial plexus.

Biomechanical characterization of cadaveric brachial plexus microstructure.

INTRODUCTION: Peripheral nerves consist of axons and connective tissue. The amount of connective tissue in peripheral nerves such as the brachial plexus varies proximally to distally. The proximal regions of the brachial plexus are more susceptible to stretch injuries than the distal regions. A description of the mechanical behavior of the peripheral nerve components is necessary to better understand the deformation mechanisms during stretch injuries. The purpose of this study was to model the biomechanical behavior of each component of the peripheral nerves (fascicles, connective tissue) in a cadaveric model and report differences in elastic modulus, maximum stress and maximum strain. METHODS: Forty-six specimens of fascicles and epi-perineurium were subjected to cyclical uniaxial tensile tests to obtain the stress and strain histories of each specimen, using a BOSE® Electroforce® 3330 and INSTRON® 5969 materials testing machines. Maximum stress, maximum strain and elastic modulus were extracted from the load-displacement and stress-strain curves, and analyzed using Mann-Whitney tests. RESULTS: Mean elastic modulus was 6.34 MPa for fascicles, and 32.1 MPa for connective tissue. The differences in elastic modulus and maximum stress between fascicles and connective tissue were statistically significant (p < 0.001). CONCLUSIONS: Peripheral nerve connective tissue showed significantly higher elastic modulus and maximum stress than fascicles. These data confirm the greater fragility of axons compared to connective tissue, suggesting that the greater susceptibility to stretch injury in proximal regions of the brachial plexus might be related to the smaller amount of connective tissue.

Transplantation of astrocyte-derived mitochondria into injured astrocytes has a protective effect following stretch injury.

Traumatic brain injury (TBI) is a global public-health problem. Astrocytes, and their mitochondria, are important factors in the pathogenesis of TBI-induced secondary injury. Mitochondria extracted from healthy tissues and then transplanted have shown promise in models of a variety of diseases. However, the effect on recipient astrocytes is unclear. Here, we isolated primary astrocytes from newborn C57BL/6 mice, one portion of which was used to isolate mitochondria, and another was subjected to stretch injury (SI) followed by transplantation of the isolated mitochondria. After incubation for 12 h, cell viability, mitochondrial dysfunction, calcium overload, redox stress, inflammatory response, and apoptosis were improved. Live-cell imaging showed that the transplanted mitochondria were incorporated into injured astrocytes and fused with their mitochondrial networks, which was in accordance with the changes in the expression levels of markers of mitochondrial dynamics. The astrocytic IKK/NF-κB pathway was decelerated whereas the AMPK/PGC-1α pathway was accelerated by transplantation. Together, these results indicate that exogenous mitochondria from untreated astrocytes can be incorporated into injured astrocytes and fuse with their mitochondrial networks, improving cell viability by ameliorating mitochondrial dysfunction, redox stress, calcium overload, and inflammation.

Dorsal root ganglion neurons recapitulate the traumatic axonal injury of CNS neurons in response to a rapid stretch in vitro.

Introduction: In vitro models of traumatic brain injury (TBI) commonly use neurons isolated from the central nervous system. Limitations with primary cortical cultures, however, can pose challenges to replicating some aspects of neuronal injury associated with closed head TBI. The known mechanisms of axonal degeneration from mechanical injury in TBI are in many ways similar to degenerative disease, ischemia, and spinal cord injury. It is therefore possible that the mechanisms that result in axonal degeneration in isolated cortical axons after in vitro stretch injury are shared with injured axons from different neuronal types. Dorsal root ganglia neurons (DRGN) are another neuronal source that may overcome some current limitations including remaining healthy in culture for long periods of time, ability to be isolated from adult sources, and myelinated in vitro. Methods: The current study sought to characterize the differential responses between cortical and DRGN axons to mechanical stretch injury associated with TBI. Using an in vitro model of traumatic axonal stretch injury, cortical and DRGN neurons were injured at a moderate (40% strain) and severe stretch (60% strain) and acute alterations in axonal morphology and calcium homeostasis were measured. Results: DRGN and cortical axons immediately form undulations in response to severe injury, experience similar elongation and recovery within 20 min after the initial injury, and had a similar pattern of degeneration over the first 24 h after injury. Additionally, both types of axons experienced comparable degrees of calcium influx after both moderate and severe injury that was prevented through pre-treatment with tetrodotoxin in cortical neurons and lidocaine in DRGNs. Similar to cortical axons, stretch injury also causes calcium activated proteolysis of sodium channel in DRGN axons that is prevented by treatment with lidocaine or protease inhibitors. Discussion: These findings suggest that DRGN axons share the early response of cortical neurons to a rapid stretch injury and the associated secondary injury mechanisms. The utility of a DRGN in vitro TBI model may allow future studies to explore TBI injury progression in myelinated and adult neurons.

The Integrin Pathway Partially Mediates Stretch-Induced Deficits in Primary Rat Microglia.

Stretch-injured microglia display significantly altered morphology, function and inflammatory-associated gene expression when cultured on a synthetic fibronectin substrate. However, the mechanism by which stretch induces these changes is unknown. Integrins, such as α5ß1, mediate microglial attachment to fibronectin via the RGD binding peptide; following integrin ligation the integrin-associated signaling enzyme, focal adhesion kinase (FAK), autophosphorylates tyrosine residue 397 and mediates multiple downstream cellular processes. We therefore hypothesize that blocking the RGD binding/integrin pathway with a commercially available RGD peptide will mimic the stretch-induced morphological alterations and functional deficits in microglia. Further, we hypothesize that upregulation of stretch-inhibited downstream integrin signaling will reverse these effects. Using primary rat microglia, we tested the effects of RGD binding peptide and a FAK activator on cellular function and structure and response to stretch-injury. Similar to injured cells, RGD peptide administration significantly decreases media nitric oxide (NO) levels and iNOS expression and induced morphological alterations and migratory deficits. While stretch-injury and RGD peptide administration decreased phosphorylation of the tyrosine 397 residue on FAK, 20 nM of ZINC 40099027, an activator specific to the tyrosine 397 residue, rescued the stretch-induced decrease in FAK phosphorylation and ameliorated the injury-induced decrease in media NO levels, iNOS expression and inflammatory associated gene expression. Additionally, treatment alleviated morphological changes observed after stretch-injury and restored normal migratory behavior to control levels. Taken together, these data suggest that the integrin/FAK pathway partially mediates the stretch-injured phenotype in microglia, and may serve as a pathway to modulate microglial responses.

Stretch on the L5 nerve root in high-grade spondylolisthesis reduction.

OBJECTIVE: L5 nerve root (L5-NR) injury after surgery for high-grade spondylolisthesis (HGS) was considered a nerve stretch associated with reduction. Currently, however, no study has directly measured the stretch on the L5-NR during HGS reduction procedures. METHODS: CT data of 4 patients with mild lumbar degeneration (control group [CG]) and 4 patients with HGS (spondylolisthesis group [SG]) were used for 3D printing to develop L5 vertebrae and sacrum models. These models were mounted on a self-designed reduction apparatus, which performed vertical translation (disc heights of 0, 5, and 10 mm), anterior-posterior translation (reduction, 0%-100%), and slip-angle correction (0° to -30°). The L5-NR was simulated by using a rabbit sciatic nerve. The cephalic side of the nerve was fixed at the upper base of the L5 pedicle, while the caudal side was connected to a high-precision sensor and an indicator to measure the tension (stretch) on the nerve during the reduction procedures in real time. RESULTS: The SG had shorter L5-NRs than the CG. At a 0-mm disc height, the peak tension on the L5-NR changed from 0 N (reduction 0%) to 1.81 ± 0.54 N (reduction 100%) in the SG and to 1.78 ± 0.71 N in the CG. At a 10-mm disc height, the tension changed from 1.50 ± 0.67 N to 4.97 ± 1.04 N in the SG and from 0.92 ± 0.45 N to 3.26 ± 0.88 N in the CG. In both the CG and SG, at the same disc height, all values from the complete reduction process were statistically significant. Furthermore, at the same degree of reduction, the comparisons between different disc heights were almost all statistically significant. Intergroup comparisons showed that an increased disc height would cause more tension on the L5-NR in the SG than in the CG. At a 10-mm disc height, all results between the groups demonstrated statistical significance. The slip-angle correction produced a slight increase in the tension on the L5-NR in both groups. CONCLUSIONS: Increased disc height and reduction significantly increased the tension on the L5-NR, which demonstrated a nonlinear curve. The slip-angle correction from 0° to -30° slightly increased the tension on the L5-NR. Under the same degree of reduction and restored disc height, the SG had more tension on the L5-NR than the CG.

Assessment of the Effects of Stretch-Injury on Primary Rat Microglia.

Mechanical stretch-injury is a prominent force involved in the etiology of traumatic brain injury (TBI). It is known to directly cause damage and dysfunction in neurons, astrocytes, and endothelial cells. However, the deleterious effects of stretch-injury on microglia, the brain's primary immunocompetent cell, are currently unknown. The Cell Injury Controller II (CICII), a validated cellular neurotrauma model, was used to induce a mechanical stretch-injury in primary rat microglia. Statistical analysis utilized Student's t test and one- and two-way ANOVAs with Tukey's and Sidak's multiple comparisons, respectively. Cells exposed to stretch-injury showed no signs of membrane permeability, necrosis, or apoptosis, as measured by media-derived lactate dehydrogenase (LDH) and cleaved-caspase 3 immunocytochemistry, respectively. Interestingly, injured cells displayed a functional deficit in nitric oxide production (NO), identified by media assay and immunocytochemistry, at 6, 12, 18, and 48 h post-injury. Furthermore, gene expression analysis revealed the expression of inflammatory cytokines IL-6 and IL-10, and enzyme arginase-1 was significantly downregulated at 12 h post-injury. Time course evaluation of migration, using a cell exclusion zone assay, showed stretch-injured cells display decreased migration into the exclusion zone at 48- and 72-h post-stretch. Lastly, coinciding with the functional immune deficits was a significant change in morphology, with process length decreasing and cell diameter increasing following an injury at 12 h. Taken together, the data demonstrate that stretch-injury produces significant alterations in microglial function, which may have a marked impact on their response to injury or their interaction with other cells.

Rapid-Stretch Injury to Peripheral Nerves: Histologic Results.

BACKGROUND: Although most severe peripheral nerve injuries result from high-speed mechanisms, there is no laboratory model to replicate this clinical condition. OBJECTIVE: To qualitatively and quantitatively describe microanatomical injury of rapid stretch. METHODS: The sciatic nerves of 36 Sprague-Dawley rats were subjected to rapid-stretch nerve injury, using fixed-direction strain produced via constrained weight drop applied to an intact nerve. Nerve injury severity was categorized by biomechanical parameters. Injury to nerve microarchitecture was quantified with serial longitudinal sectioning, with specific focus on the endoneurium, perineurium, and epineurium. RESULTS: Four grades of stretch injury severity were determined by mathematical cluster analysis: sham, elastic stretch, inelastic stretch, and stretch rupture. Two patterns of injury to endoneurial architecture were quantified: loss of fiber undulation (straightened fibers) and rupturing of individual fibers ("microruptures"). Straightening of nerve fibers was the earliest accommodation to stretch injury and accounted for elongation during elastic stretch. Microruptures were distributed along the length of the nerve and were more severe and involved greater volume of the nerve at higher biomechanical severity. Epineurium and perineurium disruption increased in frequency with progressive injury severity, yet did not predict transition from one injury grade to another (P = .3), nor was it a hallmark of severe injury. Conversely, accumulation of microruptures provided strong correlation to nerve injury severity (Pearson's R = .9897) and progression to mechanical failure. CONCLUSION: Nerve architecture is injured in a graded fashion during stretch injury, which likely reflects tissue biomechanics. This study suggests new considerations in the theoretical framework of nerve stretch trauma.

Current ex Vivo and in Vitro Approaches to Uncovering Mechanisms of Neurological Dysfunction after Traumatic Brain Injury.

Traumatic brain injury often leads to progressive alterations at the molecular to circuit levels resulting in epilepsy and memory impairments. Ex vivo and in vitro models have provided a powerful platform for investigating the multimodal alteration after trauma. Recent ex vivo analyses using voltage sensitive dye imaging, optogenetics, and glutamate uncaging have revealed circuit abnormalities following in vivo brain injury. In vitro injury models have enabled examination of early and progressive changes in activity while development of three-dimensional organoids derived from human induced pluripotent stem cells have opened novel avenues for injury research. Here, we highlight recent advances in ex vivo and in vitro systems, focusing on their potential for advancing mechanistic understandings, possible limitations, and implications for therapeutics.

Rapid-stretch injury to peripheral nerves: comparison of injury models.

OBJECTIVE: Traditional animal models of nerve injury use controlled crush or transection injuries to investigate nerve regeneration; however, a more common and challenging clinical problem involves closed traction nerve injuries. The authors have produced a precise traction injury model and sought to examine how the pathophysiology of stretch injuries compares with that of crush and transection injuries. METHODS: Ninety-five late-adolescent (8-week-old) male mice underwent 1 of 7 injury grades or a sham injury (n > 10 per group): elastic stretch, inelastic stretch, stretch rupture, crush, primary coaptation, secondary coaptation, and critical gap. Animals underwent serial neurological assessment with sciatic function index, tapered beam, and von Frey monofilament testing for 48 days after injury, followed by trichrome and immunofluorescent nerve histology and muscle weight evaluation. RESULTS: The in-continuity injuries, crush and elastic stretch, demonstrated different recovery profiles, with more severe functional deficits after crush injury than after elastic stretch immediately following injury (p < 0.05). However, animals with either injury type returned to baseline performance in all neurological assessments, accompanied by minimal change in nerve histology. Inelastic stretch, a partial discontinuity injury, produced more severe neurological deficits, incomplete return of function, 47% ± 9.1% (mean ± SD) reduction of axon counts (p < 0.001), and partial neuroma formation within the nerve. Discontinuity injuries, including immediate and delayed nerve repair, stretch rupture, and critical gap, manifested severe, long-term neurological deficits and profound axonal loss, coupled with intraneural scar formation. Although repaired nerves demonstrated axon regeneration across the gap, rupture and critical gap injuries demonstrated negligible axon crossing, despite rupture injuries having healed into continuity. CONCLUSIONS: Stretch-injured nerves present unique pathology and functional deficits compared with traditional nerve injury models. Because of the profound neuroma formation, stretch injuries represent an opportunity to study the pathophysiology associated with clinical injury mechanisms. Further validation for comparison with human injuries will require evaluation in a large-animal model.

Nerve stretching: a history of tension.

Stretch injuries are among the most devastating forms of peripheral nerve injury; unfortunately, the scientific understanding of nerve biomechanics is widely and impressively conflicting. Experimental models are unique and disparate, victim to different testing conditions, and thus yield gulfs between conclusions. The details of the divergent reports on nerve biomechanics are essential for critical appraisal as we try to understand clinical stretch injuries in light of research evidence. These conflicts preclude broad conclusion, but they highlight a duality in thought on nerve stretch and, within the details, some agreement exists. To synthesize trends in nerve stretch understanding, the author describes the literature since its introduction in the 19th century. Research has paralleled clinical inquiry, so nerve research can be divided into epochs based largely on clinical or scientific technique. The first epoch revolves around therapeutic nerve stretching-a procedure known as neurectasy-in the late 19th century. The second epoch involves studies of nerves repaired under tension in the early 20th century, often the result of war. The third epoch occurs later in the 20th century and is notable for increasing scientific refinement and disagreement. A fourth epoch of research from the 21st century is just dawning. More than 150 years of research has demonstrated a stable and inherent duality: the terribly destructive impact of stretch injuries, as well as the therapeutic benefits from nerve stretching. Yet, despite significant study, the precise border between safe and damaging stretch remains an enigma.

Rapid Stretch Injury to Peripheral Nerves: Biomechanical Results.

BACKGROUND: Although most adult brachial plexus injuries result from high-speed mechanisms, no laboratory model has been created to mimic rapid-stretch nerve injuries. Understanding the biomechanical response of nerves to rapid stretch is essential to understanding clinical injury patterns and developing models that mimic the clinical scenario. OBJECTIVE: To assess the influence of rate, loading direction, and excursion of stretch injuries on the biomechanical properties of peripheral nerves. METHODS: The sciatic nerves of 138 Sprague-Dawley rats were dissected and subjected to rapid- and slow-stretch methods. Maximal nerve strain, persistent deformation, regional strain variation, and location of nerve failure were recorded. RESULTS: Nerve rupture was primarily determined by weight-drop momentum >1 N/sec (odds ratio = 27.8, P < .0001), suggesting a threshold condition. Loading direction strongly determined maximal strain at rupture (P = .028); pull along the nerve axis resulted in nerve rupture at lower strain than orthogonal loading. Regional variations in nerve compliance and rupture location correlated with anatomic zones. Nerve branch anatomy was the largest contributing factor on maximum strain and rupture location. Rapidly stretched nerves are characterized by a zone of elastic recovery, followed by inelastic response at increasing strain, and finally rupture. CONCLUSION: The large variation in previous results for nerve strain at rupture can be attributed to different testing conditions and is largely due to loading direction or segment of nerve tested, which has significant clinical implications. Nerve stretch injuries do not reflect a continuous variability to applied force but instead fall into biomechanical patterns of elastic, inelastic, and rupture injuries.

Rapid-stretch injury to peripheral nerves: implications from an animal model.

OBJECTIVE: Rapid-stretch nerve injuries are among the most devastating lesions to peripheral nerves, yielding unsatisfactory functional outcomes. No animal model has yet been developed that uses only stretch injury for investigation of the pathophysiology of clinical traction injuries. The authors' objective was to define the behavioral and histopathological recovery after graded rapid-stretch nerve injury. METHODS: Four groups of male B6.Cg-Tg(Thy1-YFP)HJrs/J mice were tested: sham injury (n = 11); stretch within elastic limits (elastic group, n = 14); stretch beyond elastic limits but before nerve rupture (inelastic group, n = 14); and stretch-ruptured nerves placed in continuity (rupture group, n = 16). Mice were injured at 8 weeks of age, comparable with human late adolescence. Behavioral outcomes were assessed using the sciatic functional index (SFI), tapered-beam dexterity, Von Frey monofilament testing, and the Hargreaves method. Nerve regeneration outcomes were assessed by wet muscle weight and detailed nerve histology after 48 days. RESULTS: Post hoc biomechanical assessment of strain and deformation confirmed that the differences between the elastic and inelastic cohorts were statistically significant. After elastic injury, there was a temporary increase in foot faults on the tapered beam (p < 0.01) and mild reduction in monofilament sensitivity, but no meaningful change in SFI, muscle weight, or nerve histology. For inelastic injuries, there was a profound and maintained decrease in SFI (p < 0.001), but recovery of impairment was observed in tapered-beam and monofilament testing by days 15 and 9, respectively. Histologically, axon counts were reduced (p = 0.04), muscle atrophy was present (p < 0.01), and there was moderate neuroma formation on trichrome and immunofluorescent imaging. Stretch-ruptured nerves healed in continuity but without evidence of regeneration. Substantial and continuous impairment was observed in SFI (p < 0.001), tapered beam (p < 0.01), and monofilament (p < 0.01 until day 48). Axon counts (p < 0.001) and muscle weight (p < 0.0001) were significantly reduced, with little evidence of axonal or myelin regeneration concurrent with neuroma formation on immunofluorescent imaging. CONCLUSIONS: The 3 biomechanical grades of rapid-stretch nerve injuries displayed consistent and distinct behavioral and histopathological outcomes. Stretch within elastic limits resembled neurapraxic injuries, whereas injuries beyond elastic limits demonstrated axonotmesis coupled with impoverished regeneration and recovery. Rupture injuries uniquely failed to regenerate, despite physical continuity of the nerve. This is the first experimental evidence to correlate stretch severity with functional and histological outcomes. Future studies should focus on the pathophysiological mechanisms that reduce regenerative capacity after stretch injury.

BrainPhys® increases neurofilament levels in CNS cultures, and facilitates investigation of axonal damage after a mechanical stretch-injury in vitro.

Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. METHODS/RESULTS: Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as "BrainPhys® maintained neurons"). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau. CONCLUSION: BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro.

An In Vitro Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a significant problem causing high mortality globally. Methods to increase possibilities for treatment and prevention of secondary injuries resulting from the initial physical insult are thus much needed. TBI affects the central nervous system (CNS) and the neurovascular unit as a whole in numerous ways but one of the primarily compromised components is the blood-brain barrier (BBB).In this chapter, we present a detailed procedure on how stretch injury and oxygen-glucose deprivation (OGD) are applied to brain microvascular endothelial cells of the BBB in order to replicate the actual impact they receive during TBI.