Evaluation of safety and early efficacy of AAV gene therapy in mouse models of vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.

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.

Pathologic remodeling in human neuromas: insights from clinical specimens.

BACKGROUND: Neuroma pathology is commonly described as lacking a clear internal structure, but we observed evidence that there are consistent architectural elements. Using human neuroma samples, we sought to identify molecular features that characterize neuroma pathophysiology. METHODS: Thirty specimens-12 neuromas-in-continuity (NICs), 11 stump neuromas, two brachial plexus avulsions, and five controls-were immunohistochemically analyzed with antibodies against various components of normal nerve substructures. RESULTS: There were no substantial histopathologic differences between stump neuromas and NICs, except that NICs had intact fascicle(s) in the specimen. These intact fascicles showed evidence of injury and fibrosis. On immunohistochemical analysis of the neuromas, laminin demonstrated a consistent double-lumen configuration. The outer lumen stained with GLUT1 antibodies, consistent with perineurium and microfascicle formation. Antibodies to NF200 revealed small clusters of small-diameter axons within the inner lumen, and the anti-S100 antibody showed a relatively regular pattern of non-myelinating Schwann cells. CD68+ cells were only seen in a limited temporal window after injury. T-cells were seen in neuroma specimens, with both a temporal evolution as well as persistence long after injury. Avulsion injury specimens had similar architecture to control nerves. Seven pediatric specimens were not qualitatively different from adult specimens. NICs demonstrated intact but abnormal fascicles that may account for the neurologically impoverished outcomes from untreated NICs. CONCLUSIONS: We propose that there is consistent pathophysiologic remodeling after fascicle disruption. Particular features, such as predominance of small caliber axons and persistence of numerous T-cells long after injury, suggest a potential role in chronic pain associated with neuromas.

Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration.

The cellular and molecular underpinnings of Wallerian degeneration have been robustly explored in laboratory models of successful nerve regeneration. In contrast, there is limited interrogation of failed regeneration, which is the challenge facing clinical practice. Specifically, we lack insight on the pathophysiologic mechanisms that lead to the formation of neuromas-in-continuity (NIC). To address this knowledge gap, we have developed and validated a novel basic science model of rapid-stretch nerve injury, which provides a biofidelic injury with NIC development and incomplete neurologic recovery. In this study, we applied next-generation RNA sequencing to elucidate the temporal transcriptional landscape of pathophysiologic nerve regeneration. To corroborate genetic analysis, nerves were subject to immunofluorescent staining for transcripts representative of the prominent biological pathways identified. Pathophysiologic nerve regeneration produces substantially altered genetic profiles both temporally and in the mature neuroma microenvironment, in contrast to the coordinated genetic signatures of Wallerian degeneration and successful regeneration. To our knowledge, this study presents as the first transcriptional study of NIC pathophysiology and has identified cellular death, fibrosis, neurodegeneration, metabolism, and unresolved inflammatory signatures that diverge from pathways elaborated by traditional models of successful nerve regeneration.

Simultaneous Quantification of Anisotropic Microcirculation and Microstructure in Peripheral Nerve.

Peripheral nerve injury is a significant public health challenge, and perfusion in the nerve is a potential biomarker for assessing the injury severity and prognostic outlook. Here, we applied a novel formalism that combined intravoxel incoherent motion (IVIM) and diffusion tensor imaging (DTI) to simultaneously characterize anisotropic microcirculation and microstructure in the rat sciatic nerve. Comparison to postmortem measurements revealed that the in vivo IVIM-DTI signal contained a fast compartment (2.32 ± 0.04 × 10−3 mm2/s mean diffusivity, mean ± sem, n = 6, paired t test p < 0.01) that could be attributed to microcirculation in addition to a slower compartment that had similar mean diffusivity as the postmortem nerve (1.04 ± 0.01 vs. 0.96 ± 0.05 × 10−3 mm2/s, p > 0.05). Although further investigation and technical improvement are warranted, this preliminary study demonstrates both the feasibility and potential for applying the IVIM-DTI methodology to peripheral nerves for quantifying perfusion in the presence of anisotropic tissue microstructure.

Incorporating Blood Flow in Nerve Injury and Regeneration Assessment.

Peripheral nerve injury is a significant public health challenge, with limited treatment options and potential lifelong impact on function. More than just an intrinsic part of nerve anatomy, the vascular network of nerves impact regeneration, including perfusion for metabolic demands, appropriate signaling and growth factors, and structural scaffolding for Schwann cell and axonal migration. However, the established nerve injury classification paradigm proposed by Sydney Sunderland in 1951 is based solely on hierarchical disruption to gross anatomical nerve structures and lacks further information regarding the state of cellular, metabolic, or inflammatory processes that are critical in determining regenerative outcomes. This review covers the anatomical structure of nerve-associated vasculature, and describes the biological processes that makes these vessels critical to successful end-organ reinnervation after severe nerve injuries. We then propose a theoretical framework that incorporates measurements of blood vessel perfusion and inflammation to unify perspectives on all mechanisms of nerve injury.

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.

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.

Does Blast Exposure to the Torso Cause a Blood Surge to the Brain?

The interaction of explosion-induced blast waves with the torso is suspected to contribute to brain injury. In this indirect mechanism, the wave-torso interaction is assumed to generate a blood surge, which ultimately reaches and damages the brain. However, this hypothesis has not been comprehensively and systematically investigated, and the potential role, if any, of the indirect mechanism in causing brain injury remains unclear. In this interdisciplinary study, we performed experiments and developed mathematical models to address this knowledge gap. First, we conducted blast-wave exposures of Sprague-Dawley rats in a shock tube at incident overpressures of 70 and 130 kPa, where we measured carotid-artery and brain pressures while limiting exposure to the torso. Then, we developed three-dimensional (3-D) fluid-structure interaction (FSI) models of the neck and cerebral vasculature and, using the measured carotid-artery pressures, performed simulations to predict mass flow rates and wall shear stresses in the cerebral vasculature. Finally, we developed a 3-D finite element (FE) model of the brain and used the FSI-computed vasculature pressures to drive the FE model to quantify the blast-exposure effects in the brain tissue. The measurements from the torso-only exposure experiments revealed marginal increases in the peak carotid-artery overpressures (from 13.1 to 28.9 kPa). Yet, relative to the blast-free, normotensive condition, the FSI simulations for the blast exposures predicted increases in the peak mass flow rate of up to 255% at the base of the brain and increases in the wall shear stress of up to 289% on the cerebral vasculature. In contrast, our simulations suggest that the effect of the indirect mechanism on the brain-tissue-strain response is negligible (<1%). In summary, our analyses show that the indirect mechanism causes a sudden and abundant stream of blood to rapidly propagate from the torso through the neck to the cerebral vasculature. This blood surge causes a considerable increase in the wall shear stresses in the brain vasculature network, which may lead to functional and structural effects on the cerebral veins and arteries, ultimately leading to vascular pathology. In contrast, our findings do not support the notion of strain-induced brain-tissue damage due to the indirect mechanism.

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.

A 3-D Rat Brain Model for Blast-Wave Exposure: Effects of Brain Vasculature and Material Properties.

Exposure to blast waves is suspected to cause primary traumatic brain injury. However, existing finite-element (FE) models of the rat head lack the necessary fidelity to characterize the biomechanical responses in the brain due to blast exposure. They neglect to represent the cerebral vasculature, which increases brain stiffness, and lack the appropriate brain material properties characteristic of high strain rates observed in blast exposures. To address these limitations, we developed a high-fidelity three-dimensional FE model of a rat head. We explicitly represented the rat's cerebral vasculature and used high-strain-rate material properties of the rat brain. For a range of blast overpressures (100 to 230 kPa) the brain-pressure predictions matched experimental results and largely overlapped with and tracked the incident pressure-time profile. Incorporating the vasculature decreased the average peak strain in the cerebrum, cerebellum, and brainstem by 17, 33, and 18%, respectively. When compared with our model based on rat-brain properties, the use of human-brain properties in the FE model led to a three-fold reduction in the strain predictions. For simulations of blast exposure in rats, our findings suggest that representing cerebral vasculature and species-specific brain properties has a considerable influence in the resulting brain strain but not the pressure predictions.

Distribution of blood-brain barrier disruption in primary blast injury.

Traumatic brain injury (TBI) resulting from explosive-related blast overpressure is a topic at the forefront of neurotrauma research. Compromise of the blood-brain barrier (BBB) and other cerebral blood vessel dysfunction is commonly reported in both experimental and clinical studies on blast injury. This study used a rifle primer-driven shock tube to investigate cerebrovascular injury in rats exposed to low-impulse, pure primary blast at three levels of overpressure (145, 232, and 323 kPa) and with three survival times (acute, 24, and 48 h). BBB disruption was quantified immunohistochemically by measuring immunoglobulin G (IgG) extravasation with image analysis techniques. Pure primary blast generated small lesions scattered throughout the brain. The number and size of lesions increased with peak overpressure level, but no significant difference was seen between survival times. Despite laterally directed blast exposure, equal numbers of lesions were found in each hemisphere of the brain. These observations suggest that cerebrovascular injury due to primary blast is distinct from that associated with conventional TBI.