Peripheral Nerve Injury In Mice Research Articles

Denervation‑induced NRG3 aggravates muscle heterotopic ossification via the ErbB4/PI3K/Akt signaling pathway.

Peripheral nerve injury exacerbates progression of muscle heterotopic ossification (HO) and induces changes in expression of local cytokines in muscle tissue. The objective of the present study was to assess the impact of peripheral nerve injury on muscle HO development and the mechanism of cytokine modulation. A mouse model of gastrocnemius muscle HO was established and the sciatic nerve cut to simulate peripheral nerve injury. To evaluate the underlying factors contributing to the exacerbation of muscle HO resulting from denervation, fresh muscle tissue was collected and micro‑computed tomography, histochemical staining, RNA‑sequencing, reverse transcription‑quantitative PCR, Western blot, muscle tissue chip array were performed to analyze the molecular mechanisms. Sciatic nerve injury exacerbated HO in the gastrocnemius muscle of mice. Moreover the osteogenic differentiation of nerve‑injured muscle tissue‑derived fibro‑adipogenic progenitors (FAPs) increased in vitro. The expression of neuregulin 3 (NRG3) was demonstrated to be increased after nerve injury by muscle tissue chip array. Subsequent transcriptome sequencing analysis of muscle tissue revealed an enrichment of the PI3K/Akt pathway following nerve injury and an inhibitor of the PI3K/Akt pathway reduced the osteogenic differentiation of FAPs. Mechanistically, in vitro, peripheral nerve injury increased secretion of NRG3, which, following binding to ErbB4 on the cell surface of FAPs, promoted expression of osteogenesis‑associated genes via the PI3K/Akt signaling pathway, thus contributing to osteogenic differentiation of FAPs. In vivo, inhibition of the PI3K/Akt pathway effectively protected against muscle HO induced by peripheral nerve injury in mice. The present study demonstrated that the regulatory roles of NRG3 and the PI3K/Akt pathway in peripheral nerve injury exacerbated muscle HO and highlights a potential therapeutic intervention for treatment of peripheral nerve injury‑induced muscle HO.

Improved Gut Microbiota Escalates Muscle Function Rehabilitation and Ameliorates Oxidative Stress Following Mechanically Induced Peripheral Nerve Injury in Mice

Improved Gut Microbiota Escalates Muscle Function Rehabilitation and Ameliorates Oxidative Stress Following Mechanically Induced Peripheral Nerve Injury in Mice

Functional Gait Assessment Using Manual, Semi-Automated and Deep Learning Approaches Following Standardized Models of Peripheral Nerve Injury in Mice.

Objective: To develop a standardized model of stretch–crush sciatic nerve injury in mice, and to compare outcomes of crush and novel stretch–crush injuries using standard manual gait and sensory assays, and compare them to both semi-automated as well as deep-learning gait analysis methods. Methods: Initial studies in C57/Bl6 mice were used to develop crush and stretch–crush injury models followed by histologic analysis. In total, 12 eight-week-old 129S6/SvEvTac mice were used in a six-week behavioural study. Behavioral assessments using the von Frey monofilament test and gait analysis recorded on a DigiGait platform and analyzed through both Visual Gait Lab (VGL) deep learning and standardized sciatic functional index (SFI) measurements were evaluated weekly. At the termination of the study, neurophysiological nerve conduction velocities were recorded, calf muscle weight ratios measured and histological analyses performed. Results: Histological evidence confirmed more severe histomorphological injury in the stretch–crush injured group compared to the crush-only injured group at one week post-injury. Von Frey monofilament paw withdrawal was significant for both groups at week one compared to baseline (p < 0.05), but not between groups with return to baseline at week five. SFI showed hindered gait at week one and two for both groups, compared to baseline (p < 0.0001), with return to baseline at week five. Hind stance width (HSW) showed similar trends as von Frey monofilament test as well as SFI measurements, yet hind paw angle (HPA) peaked at week two. Nerve conduction velocity (NCV), measured six weeks post-injury, at the termination of the study, did not show any significant difference between the two groups; yet, calf muscle weight measurements were significantly different between the two, with the stretch–crush group demonstrating a lower (poorer) weight ratio relative to uninjured contralateral legs (p < 0.05). Conclusion: Stretch–crush injury achieved a more reproducible and constant injury compared to crush-only injuries, with at least a Sunderland grade 3 injury (perineurial interruption) in histological samples one week post-injury in the former. However, serial behavioral outcomes were comparable between the two crush groups, with similar kinetics of recovery by von Frey testing, SFI and certain VGL parameters, the latter reported for the first time in rodent peripheral nerve injury. Semi-automated and deep learning-based approaches for gait analysis are promising, but require further validation for evaluation in murine hind-limb nerve injuries.

Traumatic Peripheral Nerve Injury in Mice

Traumatic Peripheral Nerve Injury in Mice

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.

Single-cell RNA sequencing reveals time- and sex-specific responses of mouse spinal cord microglia to peripheral nerve injury and links ApoE to chronic pain

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unclear whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. Using single-cell RNA-sequencing, we show that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in male as compared to female mice. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following peripheral nerve injury. Apolipoprotein E (Apoe) is the top upregulated gene in spinal cord microglia at chronic time points after peripheral nerve injury in mice. Furthermore, polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell RNA sequencing analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a link between ApoE and chronic pain in humans.

Characterizing Sex Differences in Depressive-Like Behavior and Glial Brain Cell Changes Following Peripheral Nerve Injury in Mice.

Chronic pain and depression are intimately linked; the combination of the two leads to higher health care costs, lower quality of life, and worse treatment outcomes with both conditions exhibiting higher prevalence among women. In the current study, we examined the development of depressive-like behavior in male and female mice using the spared nerve injury (SNI) model of neuropathic pain. Males displayed increased immobility on the forced-swim test – a measure of depressive-like behavior – 2 weeks following injury, while females developed depressive-like behavior at 3-week. Since the pathogenesis of chronic pain and depression may involve overlapping mechanisms including the activation of microglial cells, we explored glial cell changes in brain regions associated with pain processing and affect. Immunohistochemical analyses revealed that microglial cells were more numerous in female SNI mice in the contralateral ventral anterior cingulate cortex (ACC), a brain region important for pain processing and affect behavior, 2-week following surgery. Microglial cell activation was not different between any of the groups for the dorsal ACC or nucleus accumbens. Analysis of astrocyte density did not reveal any significant changes in glial fibrillary acidic protein (GFAP) staining in the ACC or nucleus accumbens. Overall, the current study characterized peripheral nerve injury induced depression-like behavior in male and female mice, which may be associated with different patterns of glial cell activation in regions important for pain processing and affect.

Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice

BackgroundPlenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury.MethodsImmunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the LyzCre+/−; RhoAflox/flox (cKO) mice or Lyz2Cre+/− (Cre) mice, regardless of sex. Macrophages’ migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages.Results1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages.ConclusionsOur findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

Up-Regulation of CD146 in Schwann Cells Following Peripheral Nerve Injury Modulates Schwann Cell Function in Regeneration.

CD146 is cell adhesion molecule and is implicated in a variety of physiological and pathological processes. However, the involvement of CD146 in peripheral nerve regeneration has not been studied yet. Here, we examine the spatial and temporal expression pattern of CD146 in injured mouse sciatic nerve via high-throughput data analysis, RT-PCR and immunostaining. By microarray data analysis and RT-PCR validation, we show that CD146 mRNA is significantly up-regulated in the nerve bridge and in the distal nerve stump following mouse sciatic nerve transection injury. By single cell sequencing data analysis and immunostaining, we demonstrate that CD146 is up-regulated in Schwann cells and cells associated with blood vessels following mouse peripheral nerve injury. Bioinformatic analysis revealed that CD146 not only has a key role in promoting of blood vessel regeneration but also regulates cell migration. The biological function of CD146 in Schwann cells was further investigated by knockdown of CD146 in rat primary Schwann cells. Functional assessments showed that knockdown of CD146 decreases viability and proliferation of Schwann cells but increases Schwann cell migration. Collectively, our findings imply that CD146 could be a key cell adhesion molecule that is up-regulated in injured peripheral nerves to regulate peripheral nerve regeneration.

The Potential Repurposing Effect 4‐Aminopyridine in Nerve and Muscle Injury‐induced Bone Loss

Muscle injuries are one of the most common injuries occurring in physical and sporting activities, and account for 10-55% of all injuries. Peripheral nerve injury (PNI) itself accounts for 3-5% of all injuries in trauma patients, and muscle injuries can be concurrent with PNI because of their close proximity. Importantly, the peripheral nervous system is critically involved in bone metabolism, bone mineralization, and bone remodeling, and PNI results in substantial bone loss. While first aid for muscle injuries follows the conservative RICE (Rest, Ice, Compression and Elevation) principle, there is no medical treatment available after the acute phase except anti-inflammatory medications and rehabilitation. The poor healing process in damaged muscle, prolonged muscle disuse, and the lack of therapeutic strategies affect the performance of daily activities and predispose to further risk of muscle injury and bone loss. Therefore, there is an unmet need to develop therapeutic strategies that can help the recovery of damaged muscle and nerve, and limit bone loss. Recently we demonstrated the potential repurposing effect of 4-aminopyridine (4-AP), a potassium channel blocker, on neurogenic muscle atrophy and functional recovery after PNI in mice. However, it is unknown whether these beneficial effects of 4-AP are present in traumatic muscle injury and muscle injury-induced bone loss. This study was thus designed to evaluate the potential therapeutic effect of 4-AP in traumatic muscle healing and PNI-induced bone loss. Standardized crush injury was performed on right sciatic nerve, biceps femoris (BF) muscle (innervated by sciatic nerve), and quadriceps femoris (QF) muscle (innervated by femoral nerve) of adult male mice (6 animals/group). Post-surgical animals were randomly assigned to normal saline and 4-AP. 4-AP (40 µg, intraperitoneal) was given daily for 21 days, and the functional recovery after injury was assessed by sciatic function index (SFI), sensation by von Frey test (VFT), and grip strength test. After 21 days, the muscles at the injury/peri-injury site were processed for histomorphometry and tibial bone density was analyzed using DEXA scanning. We found that 4-AP significantly enhanced SFI, VFT, and hind limb paw grip strength from post-injury day 7, improved BF muscle morphology, cross-sectional area, and minimum ferret diameter, and totally reversed trauma-induced muscle fiber type composition changes (type-1 vs. type-2). 4-AP-induced muscle effects were also associated with a significantly increased number of regenerating stem cells (Pax7+) and proliferating cells (Ki67+) compared with saline group. Of note, 4-AP-induced muscle effects were either less pronounced or nonsignificant in QF muscle. Most importantly, 4-AP treatment significantly reduced PNI-induced bone loss by enhancing bone mineral density and bone mineral content. In conclusion, these interesting findings, for the first time, demonstrate the potential therapeutic effect of 4-AP on the recovery of traumatic muscle and bone loss after mixed muscle and nerve injury.

Local (4‐Aminopyridine)‐PLGA‐PEG Treatment Improves Functional Recovery and Muscle Morphology after Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) represents a major medical problem resulting in loss of both motor and sensory function, and in severe cases, permanent disability. Inadequate axonal regeneration and delayed muscle reinnervation contribute to muscle atrophy and poor functional recovery. To date, there is no effective pharmacologic treatment which can promote neuromuscular outcomes after TPNI. In repurposing studies, we have shown that daily systemic 4-aminopyridine (4-AP), an FDA-approved potassium channel blocker for multiple sclerosis, enhances functional recovery, improves myelination, and attenuates muscle atrophy after TPNI. However, 4-AP at higher doses cause adverse effects such as seizures. Thus, we sought to develop a novel locally injectable formulation of 4-AP which could be administered to a nerve injury site and deliver 4-AP at a sustained rate for several weeks. We developed (4-AP)-Poly(lactide-co-glycolide)-b-Poly(ethylene glycol)-b-Poly(lactide-co-glycolide) (PLGA-PEG-PLGA), consisting of two FDA-approved, biodegradable, and biocompatible hydrogels. It is unknown whether this locally injectable formulation of 4-AP would have an impact on TPNI-induced functional recovery, as well as on muscle atrophy. In this study, an established mouse model of right sciatic nerve crush injury was used and mice received one of 4 treatments: (4-AP)-PLGA-PEG, PLGA-PEG vehicle, systemic 4-AP, or saline control (n = 5/group). Functional recovery was assessed using sciatic function index, grip strength, and von Frey filament testing. At post-injury day 28, gastrocnemius (GN) and tibialis anterior (TA) muscles were harvested from both injured (innervated by crushed nerve) and uninjured (contralateral healthy) hind limbs for histomorphological analysis. Our results show that a single local administration of (4-AP)-PLGA-PEG remarkably enhanced motor and sensory functional recovery on post-sciatic nerve crush injury days 1, 3, 7, 14, and 21. Histomorphometric analysis of the muscles revealed that (4-AP)-PLGA-PEG treatment significantly increased right TA cross-sectional area (CSA) (1488.2 µm2 vs. 438.4 µm2, p<0.01) and minimum Feret's diameter (MFD) (53.9 µm vs. 30.3 µm, p<0.01) compared with the other groups. Similarly, (4-AP)-PLGA-PEG treatment significantly increased right GN CSA (1213.8 µm2 vs. 471.9 µm2, p<0.001) and MFD (48.5 µm vs. 32.6 µm, p<0.01) compared with the other groups. These preliminary findings on global functional and muscle fiber morphological parameters demonstrate that (4-AP)-PLGA-PEG can be used locally for neuromuscular outcomes after TPNI. Further cellular, molecular, and biochemical studies will define the mechanistic insights for this promising long-acting local therapeutic agent in TPNI.

Sexually Dimorphic Immune and Neuroimmune Changes Following Peripheral Nerve Injury in Mice: Novel Insights for Gender Medicine.

Neuropathic pain (NeP) in humans is often a life-long condition with no effective therapy available. The higher incidence of female gender in NeP onset is worldwide reported, and although the cause is generally attributed to sex hormones, the actual mechanisms and the players involved are still unclear. Glial and immune cells take part in NeP development, and orchestrate the neuroimmune and inflammatory response, releasing pro-inflammatory factors with chemoattractant properties that activate resident immune cells and recruit immune cells from circulation. The neuro-immune crosstalk is a key contributor to pain hypersensitivity following peripheral nervous system injury. Our previous works showed that in spite of the fact that female mice had an earlier analgesic response than males following nerve lesion, the recovery from NeP was never complete, suggesting that this difference could occur in the very early stages after injury. To further investigate gender differences in immune and neuroimmune responses to NeP, we studied the main immune cells and mediators elicited both in plasma and sciatic nerves by peripheral nerve lesion. After injury, we found a different pattern of distribution of immune cell populations showing either a higher infiltration of T cells in nerves from females or a higher infiltration of macrophages in nerves from males. Moreover, in comparison to male mice, the levels of cytokines and chemokines were differently up- and down-regulated in blood and nerve lysates from female mice. Our study provides some novel insights for the understanding of gender-associated differences in the generation and perseveration of NeP as well as for the isolation of specific neurodegenerative mechanisms underlying NeP. The identification of gender-associated inflammatory profiles in neuropathy is of key importance for the development of differential biomarkers and gender-specific personalized medicine.

Upregulation of Nucleotide-Binding Oligomerization Domain-, LRR- and Pyrin Domain-Containing Protein 3 in Motoneurons Following Peripheral Nerve Injury in Mice

Neuronal injuries are accompanied by release and accumulation of damage-associated molecules, which in turn may contribute to activation of the immune system. Since a wide range of danger signals (including endogenous ones) are detected by the nucleotide-binding oligomerization domain-, LRR- and pyrin domain-containing protein 3 (NLRP3) pattern recognition receptor, we hypothesized that NLRP3 may become activated in response to motor neuron injury. Here we show that peripheral injury of the oculomotor and the hypoglossal nerves results in upregulation of NLRP3 in corresponding motor nuclei in the brainstem of mice. Although basal expression of NLRP3 was observed in microglia, astroglia and neurons as well, its upregulation and co-localization with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, suggesting inflammasome activation, was only detected in neurons. Consequently, increased production of active pro-inflammatory cytokines interleukin-1β and interleukin-18 were detected after hypoglossal nerve axotomy. Injury-sensitive hypoglossal neurons responded with a more pronounced NLRP3 upregulation than injury-resistant motor neurons of the oculomotor nucleus. We further demonstrated that the mitochondrial protector diazoxide was able to reduce NLRP3 upregulation in a post-operative treatment paradigm. Our results indicate that NLRP3 is activated in motoneurons following acute nerve injury. Blockade of NLRP3 activation might contribute to the previously observed anti-inflammatory and neuroprotective effects of diazoxide.

Functional recovery with histomorphometric analysis of nerves and muscles after combination treatment with erythropoietin and dexamethasone in acute peripheral nerve injury.

IntroductionPeripheral nerve injury (PNI) often leads to significant functional loss in patients and poses a challenge to physicians since treatment options for improving functional outcomes are limited. Recent studies suggest that erythropoietin and glucocoticoids have beneficial effects as mediators of neuro-regenerative processes. We hypothesized that combination treatment with erythropoietin and glucocoticoids would have a synergistic effect on functional outcome after PNI.Materials and methodsSciatic nerve crush injury was simulated in ten-week-old male C57BL/6 mice. The mice were divided into four groups according to the type of drugs administered (control, erythropoietin, dexamethasone, and erythropoietin with dexamethasone). Motor functional recovery was monitored by walking track analysis at serial time points up to 28 days after injury. Morphological analysis of the nerve was performed by immunofluorescent staining for neurofilament (NF) heavy chain and myelin protein zero (P0) in cross-sectional and whole-mount nerve preparations. Additionally, morphological analysis of the muscle was performed by Hematoxylin and eosin staining.ResultsCombination treatment with erythropoietin and dexamethasone significantly improved the sciatic functional index at 3, 7, 14, and 28 days after injury. Fluorescence microscopy of cross sectional nerve revealed that the combination treatment increased the ratio of P0/NF-expressing axons. Furthermore, confocal microscopy of the whole-mount nerve revealed that the combination treatment increased the fluorescence intensity of P0 expression. The cross-sectional area and minimum Feret’s diameter of the muscle fibers were significantly larger in the mice which received combination treatment than those in the controls.ConclusionOur results demonstrated that combination treatment with erythropoietin and dexamethasone accelerates functional recovery and reduces neurogenic muscle atrophy caused by PNI in mice, which may be attributed to the preservation of myelin and Schwann cell re-myelination. These findings may provide practical therapeutic options for patients with acute PNI.

Neuropathic and cAMP-induced pain behavior is ameliorated in mice lacking CNGB1

Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1−/−) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.

Human induced pluripotent stem cell-derived GABAergic interneuron transplants attenuate neuropathic pain

Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here, we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated human induced pluripotent stem cell-derived GABAergic (iGABAergic) neurons provide significant, long-term, and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.

Human equivalent dose of oral 4-aminopyridine differentiates nerve crush injury from transection injury and improves post-injury function in mice.

4-Aminopyridine (4-AP), an FDA-approved drug for the symptomatic treatment of multiple sclerosis, is used to improve neuromuscular function in patients with diverse demyelinating disorders. We recently demonstrated that local, transdermal or injectable forms of 4-AP improve myelination, nerve conduction velocity, muscle atrophy, and motor function after traumatic peripheral nerve injury in mice. While oral 4-AP is most commonly used in the clinic, it is unknown whether human equivalent oral doses of 4-AP have effects on traumatic peripheral nerve injury differentiation, myelination, muscle atrophy, functional recovery, and post-injury inflammatory processes in animals. Mice with sciatic nerve crush or denervation injury received oral or intraperitoneal 4-AP (10 μg) or vehicle alone and were examined for pharmacokinetics, motor function, muscle mass, intrinsic muscle force, nerve morphological and gene expression profiles. 4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to 4-AP dose. Acute single dose of oral 4-AP administration induced a rapid transient improvement in motor function that was different in traumatic peripheral nerve injury with or without nerve continuity, chronic daily oral 4-AP treatment significantly enhanced post crush injury motor function recovery and this effect was associated with improved myelination, muscle mass, and ex vivo muscle force. Polymerase chain reaction array analysis with crushed nerve revealed significant alterations in gene involved in axonal inflammation and regeneration. These findings provide convincing evidence that regardless of the route of administration, 4-AP can acutely differentiate traumatic peripheral nerve injury with or without nerve continuity and can enhance in vivo functional recovery with better preservation of myelin sheaths, muscle mass, and muscle force. The animal experiments were approved by the University Committee on Animal Research (UCAR) at the University of Rochester (UCAR-2009-019) on March 31, 2017.

Regenerative activity of Hericium erinaceus on axonal injury model using in vitro laser microdissection technique

ABSTRACTObjective: Peripheral nerve injury (PNI) is an important global health problem. Nerve Growth Factor (NGF) plays crucial role in the survival, growth, and maintenance of various neurons in the mammalian nervous system, human included. Hericium erinaceus (HE), an edible and medicinal mushroom, has been extensively studied for its neuroprotective properties. In this study, the neuroprotective and neurotogenic effects of HE and NGF were compared on mouse PNI model by using a laser microdissection technique.Methods: Neuronal cultures were prepared from dorsal root ganglia (DRG) of 6–8 week aged mice, pretreated them with phosphate-buffered saline (PBS), NGF, HE, or the combination of NGF and HE. To model axonal injury in vitro, axons were cut (axotomy) with a microscope-controlled laser beam. Axotomized neurons were imaged under the microscope. Axotomized neurons’ survival ratios were calculated using the propidium iodide (PI), which is a red-fluorescent nuclear dye. Their axon lengths were measured using the AxioVision 4.8 software.Results: Although both HE and NGF have neuroprotective and regenerative effects on axotomized peripheral sensory neurons, HE exhibits a higher neuroprotective activity compared to the NGF. The combination of HE and NGF maximizes axonal regeneration ability of axotomized neurons.Conclusion: HE has capabilities of preventing the death of neurons and regenerating their axons in the experimental axonal injury model. Our findings provide experimental evidence that HE may serve as a neuroprotective and regenerative candidate for treating peripheral nerve injuries. Present study warrants further investigation of HE as a potential natural compound to remedy PNI.

Effect of ulinastatin on neuronal apoptosis in spinal cord after peripheral nerve injury in mice

Objective To evaluate the effect of ulinastatin on neuronal apoptosis in spinal cord after peripheral nerve injury in mice. Methods A total of 225 healthy male SPF C57BL/6J mice, aged 6-8 weeks, weighing 18-22 g, were divided into 3 groups (n=75 each) using a random number table method: sham operation group (group Sham), peripheral nerve injury group (group PNI) and ulinastatin group (group UTI). The model of unilateral sciatic nerve transection was established in PNI and UTI groups.Ulinastatin 10 000 U/kg was injected intraperitoneally once a day for 3 consecutive days in group UTI, while the equal volume of normal saline was given instead in Sham and PNI groups.The mice were sacrificed at 1, 3, 7, 14 and 28 days after administration (T0-4), and the L4-6 segments of the spinal cord were removed for examination of the pathological changes (under the light microscope) and for determination of neuronal apoptosis (by TUNEL) and expression of caspase-12, Bcl-2, Bax and activated caspase-3 (by Western blot) and expression of caspase-12 mRNA (by real-time polymerase chain reaction). Apoptosis index (AI) and Bcl-2/Bax ratio were calculated. Results Compared with group Sham, AI was significantly increased, the expression of Bcl-2 was down-regulated, the expression of caspase-12, activated caspase-3 and Bax was up-regulated, and Bcl-2/Bax ratio was decreased in PNI and UTI groups (P<0.05). Compared with group PNI, AI was significantly decreased, the expression of Bcl-2 was up-regulated, the expression of caspase-12, activated caspase-3 and Bax was down-regulated, and Bcl-2/Bax ratio was increased in group UTI (P<0.05). The pathological changes of the spinal cord were significantly attenuated in group UTI than in group PNI. Conclusion Ulinastatin can inhibit neuronal apoptosis in the spinal cord after peripheral nerve injury in mice. Key words: Trypsin inhibitors; Apoptosis; Peripheral nerves; Spinal cord

Thymoquinone protects DRG neurons from axotomy-induced cell death

ABSTRACTObjective: Peripheral nerve injury (PNI) is a significant health problem that is linked to sensory, motor, and autonomic deficits. This pathological condition leads to a reduced quality of life in most affected individuals. Schwann cells (SCs) play a crucial role in the repair of PNI. Effective agents that promote SC activation may facilitate and accelerate peripheral nerve repair. Thymoquinone (TQ), a bioactive component of Nigella sativa seeds, has an antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective properties. In the present study, the neuroprotective efficacy of TQ was investigated by using a laser microdissection technique in a mouse PNI model.Methods: Single cells were isolated from dorsal root ganglions (DRGs) of 6–8-week-old mice, maintained in defined culture conditions and treated with or without TQ at different concentrations. Axons were cut (axotomy) using a controllable laser microbeam to model axonal injury in vitro. Under fluorescence microscopy, cell viability was evaluated using the fluorescent dyes. The behavior of the cells was continuously monitored with time-lapse video microscopy.Results: TQ significantly increased neuronal survival by promoting the survival and proliferation of SCs and fibroblasts, as well as the migration of SCs. Furthermore, TQ improved the ability to extend neurites of axotomized neurons. The regenerative effect of TQ was dose-dependent suggesting a target specificity. Our studies warrant further preclinical and clinical investigations of TQ as a potential regenerative agent to treat peripheral nerve injuries.Conclusion: TQ exhibits a regenerative potential for the treatment of damaged peripheral nerves.