Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice: effects of 4-aminopyridine.

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine (4-AP), a potassium channel blocker, on functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4-AP is unknown in nerve transection, gap, and grafting models. To evaluate and compare the functional recovery, nerve morphology, and muscle atrophy, we used a novel stepwise nerve transection with gluing (STG), as well as 7-mm irreparable nerve gap (G-7/0) and 7-mm isografting in 5-mm gap (G-5/7) models in the absence and presence of 4-AP treatment. Following surgery, sciatic functional index was determined weekly to evaluate the direct in vivo global motor functional recovery. After 12 weeks, nerves were processed for whole-mount immunofluorescence imaging, and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber cross-sectional area and minimal Feret's diameter. Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model. 4-AP did not affect the sciatic functional index recovery in any model. Compared to STG, nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting. While muscle weight, cross-sectional area, and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7, 4-AP treatment significantly increased right TA muscle mass, cross-sectional area, and minimal Feret's diameter in G-7/0 model. These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap, and 4-AP exerts differential effects on functional recovery and muscle atrophy.

A Novel Standardized Peripheral Nerve Transection Method and a Novel Digital Pressure Sensor Device Construction for Peripheral Nerve Crush Injury.

Peripheral nerve injury (PNI) is common in all walks of life, and the most common PNIs are nerve crush and nerve transection. While optimal functional recovery after crush injury occurs over weeks, functional recovery after nerve transection with microsurgical repair and grafting is poor, and associated with permanent disability. The gold-standard treatment for nerve transection injury is microsurgical tensionless end-to-end suture repair. Since it is unethical to do experimental PNI studies in humans, it is therefore indispensable to have a simple, reliable, and reproducible pre-clinical animal model for successful evaluation of the efficacy of a novel treatment strategy. The objective of this article is two-fold: (A) To present a novel standardized peripheral nerve transection method in mice, using fibrin glue for modeling peripheral nerve transection injury, with reproducible gap distance between the severed nerve ends, and (B) to document the step-wise description of constructing a pressure sensor device for crush injury pressure measurements. We have successfully established a novel nerve transection model in mice using fibrin glue, and demonstrated that this transection method decreases surgical difficulties and variability by avoiding microsurgical manipulations on the nerve, ensuring the reproducibility and reliability of this animal model. Although it is quite impossible to exactly mimic the pathophysiological changes seen in nerve transection with sutures, we hope that the close resemblance of our novel pre-clinical model with gold-standard suturing can be easily reproduced by any lab, and that the data generated by this method significantly contributes to better understanding of nerve pathophysiology, molecular mechanisms of nerve regeneration, and the development of novel strategies for optimal functional recovery. In case of peripheral nerve crush injury, current methods rely on inter-device and operator precision to limit the variation with applied pressure. While the inability to accurately quantify the crush pressure may result in reduced reproducibility between animals and studies, there is no documentation of a pressure monitoring device that can be readily used for real-time pressure measurements. To address this deficit, we constructed a novel portable device comprised of an Arduino UNO microcontroller board and force sensitive resistor (FSR) capable of reporting the real-time pressure applied to a nerve. This novel digital pressure sensor device is cheap, easy to construct and assemble, and we believe that this device will be useful for any lab performing nerve crush injury in rodents.

Traumatic Peripheral Nerve Injury in Mice.

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

Altered gut microbiota composition with antibiotic treatment impairs functional recovery after traumatic peripheral nerve crush injury in mice: effects of probiotics with butyrate producing bacteria.

OBJECTIVE: Antibiotics (ABX) are widely used for life-threatening infections and also for routine surgical operations. Compelling evidence suggests that ABX-induced alterations of gut microbiota composition, termed dysbiosis, are linked with diverse disease states including neurological and neurodegenerative conditions. To combat the consequences of dysbiosis, probiotics (PBX) are widely used. ABX-induced dysbiosis is reported to impair neurological function after spinal cord injury. Traumatic peripheral nerve injury (TPNI) results in profound neurologic impairment and permanent disability. It is unknown whether ABX treatment-induced dysbiosis has any impact on TPNI-induced functional recovery, and if so, what role medical-grade PBX could have on TPNI recovery. RESULTS: In this study, ABX-induced dysbiosis and PBX-induced microbiota enrichment models were used to explore the potential role of gut microbiome in TPNI. Stool analysis with 16S ribosomal RNA (rRNA) gene sequencing confirmed ABX-induced dysbiosis and revealed that ABX-induced changes could be partially restored by PBX administration with an abundance of butyrate producing bacteria. Pre-injury ABX significantly impaired, but pre-injury PBX significantly improved post-TPNI functional recovery. Importantly, post-injury PBX protected against pre-injury ABX-induced functional impairment. These findings demonstrate that reestablishment of gut microbiota composition with butyrate producing PBX during ABX-induced dysbiosis could be a useful adjuvant therapy for TPNI.

Novel Real-time Digital Pressure Sensor Reveals Wide Variations in Current Nerve Crush Injury Models.

INTRODUCTION: Peripheral nerve crush injury (PNCI) models are commonly used to study nerve damage and the potential beneficial effects of novel therapeutic strategies. Current models of PNCI rely on inter-device and operator precision to limit the variation with applied pressure. Although the inability to accurately quantify the PNCI pressure may result in reduced reproducibility between animals and studies, there is very limited information on the standardization and quantification of applied pressure with PNCI. To address this deficit, we constructed a novel device comprised of an Arduino UNO microcontroller board and Force Sensitive Resistor capable of reporting the real-time pressure applied to a nerve. METHODS: Two forceps and two needle drivers were used to perform 30-second PNCIs to the sciatic nerves of mice (n = 5/group). Needle drivers were set to the first notch, and a jig was used to hold the forceps pinch at a reproducible pressure. The Force Sensitive Resistor was interposed in-series between the nerve and instrument during PNCI. RESULTS: Data collected from these procedures displayed average needle driver pressures an order of multitude greater than forceps pressures. Additionally, needle driver inter- and intra-procedure pressure remained more consistent than forceps pressure, with needle driver coefficient of variation equal to 14.5% vs. a forceps coefficient of variation equal to 45.4%. CONCLUSIONS: This is the first demonstration of real-time pressure measurements in PNCI models and it reveals that the applied pressures are dependent on the types of device used. The large disparity in pressure represents an inability to apply graded accurate and consistent intermediate pressure gradients in PNCI. These findings indicate a need for documentation of pressure severity as a screening for PNCI in animals, and the real-time pressure sensor could be a useful tool in monitoring and applying consistent pressure, reducing the outcome variability within the same experimental model of PNCI.

Obligatory role of Schwann cell-specific erythropoietin receptors in erythropoietin-induced functional recovery and neurogenic muscle atrophy after nerve injury.

BACKGROUND: Erythropoietin (EPO) promotes myelination and functional recovery in rodent peripheral nerve injury (PNI). While EPO receptors (EpoR) are present in Schwann cells, the role of EpoR in PNI recovery is unknown because of the lack of EpoR antagonists or Schwann cell-specific EpoR knockout animals. METHODS: Using the Cre-loxP system, we developed a myelin protein zero (Mpz) promoter-driven knockout mouse model of Schwann cell EpoR (MpzCre-EpoRflox/flox , Mpz-EpoR-KO). Mpz-EpoR-KO and control mice were assigned to sciatic nerve crush injury followed by EPO treatment. RESULTS: EPO treatment significantly accelerated functional recovery in control mice in contrast to significantly reduced functional recovery in Mpz-EpoR-KO mice. Significant muscle atrophy was found in the injured hindlimb of EPO-treated Mpz-EpoR-KO mice but not in EPO-treated control mice. CONCLUSIONS: These preliminary findings provide direct evidence for an obligatory role of Schwann-cell specific EpoR for EPO-induced functional recovery and muscle atrophy following PNI.