Effect of mesenchymal stem cells and melatonin on experimentally induced peripheral nerve injury in rats.

Injury of a peripheral nerve (PNI) leads to both ischemic and inflammatory alterations. Sciatic nerve injury (SNI) represents the most widely used model for PNI. Mesenchymal stem cell-based therapy (MSCs) has convenient properties on PNI by stimulating the nerve regeneration. Melatonin has cytoprotective activity. The neuroprotective characteristics of MSCs and melatonin separately or in combination remain a knowledge need. In the rats-challenged SNI, therapeutic roles of intralesional MSCs and intraperitoneal melatonin injections were evaluated by functional assessment of peripheral nerve regeneration by walking track analysis involving sciatic function index (SFI) and two electrophysiological tests, electromyography and nerve conduction velocity, as well as measurement of antioxidant markers in serum, total antioxidant capacity (TAC) and malondialdehyde, and mRNA expression of brain derived neurotrophic factor (BDNF) in nerve tissues in addition to the histopathological evaluation of nerve tissue. Both individual and combination therapy with MSCs and melatonin therapies could effectively ameliorate this SNI and promote its regeneration as evidenced by improving the SFI and two electrophysiological tests and remarkable elevation of TAC with decline in lipid peroxidation and upregulation of BDNF levels. All of these led to functional improvement of the damaged nerve tissues and good recovery of the histopathological sections of sciatic nerve tissues suggesting multifactorial synergistic approach of the concurrent usage of melatonin and MSCs in PNI. The combination regimen has the most synergistic neuro-beneficial effects in PNI that should be used as therapeutic option in patients with PNI to boost their quality of life.

Comparative neuroprotective effects of Cerebrolysin, dexamethasone, and ascorbic acid on sciatic nerve injury model: Behavioral and histopathological study.

Background: The majority of the suggested experimental modalities for peripheral nerve injury (PNI) result in varying degrees of recovery in animal models; however, there are not many reliable clinical pharmacological treatment models available. To alleviate PNI complications, research on approaches to accelerate peripheral nerve regeneration is encouraged. Cerebrolysin, dexamethasone, and ascorbic acid (vitamin C) drug models were selected in our study because of their reported curative effects of different mechanisms of action. Methodology: A total of 40 adult male albino rats were used in this study. Sciatic nerve crush injury was induced in 32 rats, which were divided equally into four groups (model, Cerebrolysin, dexamethasone, and vitamin C groups) and compared to the sham group (n = 8). The sciatic nerve sensory and motor function regeneration after crushing together with gastrocnemius muscle histopathological changes were evaluated by the sciatic function index, the hot plate test, gastrocnemius muscle mass ratio, and immune expression of S100 and apoptosis cascade (BAX, BCL2, and BAX/BCL2 ratio). Results: Significant improvement of the behavioral status and histopathological assessment scores occurred after the use of Cerebrolysin (as a neurotrophic factor), dexamethasone (as an anti-inflammatory), and vitamin C (as an antioxidant). Despite these seemingly concomitant, robust behavioral and pathological changes, vitamin C appeared to have the best results among the three main outcome measures. There was a positive correlation between motor and sensory improvement and also between behavioral and histopathological changes, boosting the effectiveness, and implication of the sciatic function index as a mirror for changes occurring on the tissue level. Conclusion: Vitamin C is a promising therapeutic in the treatment of PNI. The sciatic function index (SFI) test is a reliable accurate method for assessing sciatic nerve integrity after both partial disruption and regrowth.

Evaluation of peripheral nerve regeneration in Murphy Roths Large mouse strain following transection injury.

Aim: Murphy Roths Large (MRL/MpJ) mice have demonstrated the ability to heal with minimal or no scar formation in several tissue types. In order to identify a novel animal model, this study sought to evaluate whether this attribute applies to peripheral nerve regeneration. Materials & methods: This was a two-phase study. 6-week-old male mice were divided into two interventional groups: nerve repair and nerve graft. The MRL/MpJ was compared with the C57BL/6J strain for evaluation of both functional and histological outcomes. Results: MRL/MpJ strain demonstrated superior axon myelination and less scar formation, however functional outcomes did not show significant difference between strains. Conclusion: Superior histological outcomes did not translate into superior peripheral nerve regeneration in MRL/MpJ strain.

Potential Assessment of Topical Felbinac-Loaded Cubosomal Gel in Soft Tissue Injury in Albino Rats.

Muscle strain is one of the most common injuries with high intermittence rate. Due to diverseness of strain injuries, different experimental animal models are employed to investigate such injuries with reproducible results. Cubosomes, an emerging nano drug delivery tool, are considered ideal carriers for the topical delivery of lipophilic drugs to treat local inflammations with reduced frequency of application for prolonged periods. This work describes the development of Felbinac-loaded cubosomal gel and investigated the treatment of inflammation and tissue injury in vivo. Sciatic Function Index (SFI) is a simple clinical method to observe hind limb recovery in rats after induced injuries. First, cubosomes were fabricated by high-pressure homogenization process and evaluated for in vitro parameters. The optimized cubosome formulation was chosen to develop cubosomal gel and evaluated for in vitro parameters and also investigated time to recovery of SFI after strain induction in tibialis anterior muscles in rats. The cubosome formulation (F4) exhibited low droplet size (51.04 ± 1.37 nm)and polydispersity index (0.085 ± 1.13), and negative zeta potential (-32.8 ± 0.67 mV). In rats, topical application of cubosomal gel formulation (CGF) exhibited significant improvement in skin permeation (402 ± 6.08 µg) and drug flux (15.71 ± 0.82 µg/cm2 h) compared to plain gel. Also, CGF demonstrated significant difference in SFI from first to seventh day. The histology of rat skin showed significant effect for groups treated with Felbinac-loaded CGF compared to a negative control group.

Sustained delivery of vascular endothelial growth factor mediated by bioactive methacrylic anhydride hydrogel accelerates peripheral nerve regeneration after crush injury.

Neurotrophic factors, currently administered orally or by intravenous drip or intramuscular injection, are the main method for the treatment of peripheral nerve crush injury. However, the low effective drug concentration arriving at the injury site results in unsatisfactory outcomes. Therefore, there is an urgent need for a treatment method that can increase the effective drug concentration in the injured area. In this study, we first fabricated a gelatin modified by methacrylic anhydride hydrogel and loaded it with vascular endothelial growth factor that allowed the controlled release of the neurotrophic factor. This modified gelatin exhibited good physical and chemical properties, biocompatibility and supported the adhesion and proliferation of RSC96 cells and human umbilical vein endothelial cells. When injected into the epineurium of crushed nerves, the composite hydrogel in the rat sciatic nerve crush injury model promoted nerve regeneration, functional recovery and vascularization. The results showed that the modified gelatin gave sustained delivery of vascular endothelial growth factors and accelerated the repair of crushed peripheral nerves.

Comparison of the Effects of Electroacupuncture and Melatonin on Nerve Regeneration in Experimentally Nerve-Damaged Rats.

Background: Development of methods to accelerate nerve regeneration in peripheral nerve damage is important. Electroacupuncture is a new therapeutic method that combines traditional acupuncture with modern electrotherapy. Melatonin has been shown to reduce nerve damage. Objectives: In this study, we aimed to determine and compare the therapeutic effects of electroacupuncture and melatonin on rat sciatic nerve injury. Methods: A total of 56 adult male Wistar Albino rats were divided into four study groups with 14 animals in each group: intact control (group I), subcutaneous saline (group II), subcutaneous melatonin (group III), and electroacupuncture (group IV). Surgical procedure including unilateral (right) sciatic nerve injury was applied to groups II, III, and IV. Saline and melatonin started immediately after surgery for six weeks, while electroacupuncture was given two weeks after surgery for 3 weeks. Functional and histological assessments were used as outcome measurements. Results: Sciatic nerve damage caused a significant decrease in nerve conduction velocity. Both electroacupuncture treatment and melatonin treatment significantly increased the nerve conduction velocity. Both sciatic functional recovery and histological regeneration were faster in these treatment groups compared to the saline. However, no significant difference was observed between the two treatment groups. Conclusion: Electroacupuncture and melatonin are promising alternative treatment strategies for peripheral nerve damage and can be examined in detail in future studies.

Effects of adipose derived stromal vascular fraction on diabetic neuropathy: an experimental study.

The stromal vascular fraction (SVF) obtained from inguinal adipose tissue was injected into the sciatic nerve region in diabetic rats. The effects of the SVF on the sciatic nerve and functional, electrophysiological and histopathological changes were examined in this study. Rats were divided into five groups; a non-diabetic control group, and four diabetic groups. In the first diabetic rat group, the SVF was obtained from inguinal adipose tissue. The remaining diabetic groups included a sham control group, a phosphate-buffered saline (PBS) injection group and an SVF injection group. Injections were made into the sciatic nerve region. Electromyography and walking track analyses were conducted on all groups at the beginning of the experiment. Diabetes was induced via a single dose of streptozotocin. Walking track analysis and electromyography measurements were repeated in week 8. SVF or PBS was injected into the right sciatic nerve region on week 8 of experiment group rats. Walking track analysis and electromyography were repeated in week 12 and all sciatic nerves were examined histopathologically. In the diabetic SVF group, the sciatic functional index calculated from walking track analysis in week 12 was better than week 8. Additionally, the myelin sheaths of the right sciatic nerve were thicker and more uniform and the nerve fibers were thicker than those of the left, untreated sciatic nerve. No statistical differences were detected in electromyographic measurements. The adipose-derived SVF may be beneficial for nerve regeneration in diabetic neuropathy.

Qian-Zheng-San promotes regeneration after sciatic nerve crush injury in rats.

Qian-Zheng-San, a traditional Chinese prescription consisting of Typhonii Rhizoma, Bombyx Batryticatus, Scorpio, has been found to play an active therapeutic role in central nervous system diseases. However, it is unclear whether Qian-Zheng-San has therapeutic value for peripheral nerve injury. Therefore, we used Sprague-Dawley rats to investigate this. A sciatic nerve crush injury model was induced by clamping the right sciatic nerve. Subsequently, rats in the treatment group were administered 2 mL Qian-Zheng-San (1.75 g/mL) daily as systemic therapy for 1, 2, 4, or 8 weeks. Rats in the control group were not administered Qian-Zheng-San. Rats in sham group did not undergo surgery and systemic therapy. Footprint analysis was used to assess nerve motor function. Electrophysiological experiments were used to detect nerve conduction function. Immunofluorescence staining was used to assess axon counts and morphological analysis. Immunohistochemical staining was used to observe myelin regeneration of the sciatic nerve and the number of motoneurons in the anterior horn of the spinal cord. At 2 and 4 weeks postoperatively, the sciatic nerve function index, nerve conduction velocity, the number of distant regenerated axons and the axon diameter of the sciatic nerve increased in the Qian-Zheng-San treatment group compared with the control group. At 2 weeks postoperatively, nerve fiber diameter, myelin thickness, and the number of motor neurons in the lumbar spinal cord anterior horn increased in the Qian-Zheng-San treatment group compared with the control group. These results indicate that Qian-Zheng-San has a positive effect on peripheral nerve regeneration.

Comparative effects of implanted electrodes with differing contact patterns on peripheral nerve regeneration and functional recovery.

Electrical stimulation could enhance nerve regeneration and functional recovery. The objective of this study was to evaluate the regenerative effects of implanted electrodes with different contacts in resected sciatic nerve. Sciatic nerve resection and microsurgical repair models were established and randomly divided into four groups (point contact, 1/4 circle contact; whole-circle contact; no electrodes as control). Electrical stimulation was performed and electrophysiological, morphological and histological exams (of the sciatic nerve and muscle) were conducted at 4 and 10 weeks post-implantation. Point and 1/4 circle contact groups showed significantly higher scores in the sciatic functional index (SFI), increased amplitude of compound muscle action potential (AMP) and motor nerve conduction velocity (MNCV) compared to the control group at both 4 and 10 weeks post-implantation. Point and 1/4 circle contact morphologically promoted sciatic nerve regeneration and reduced muscular atrophy with less mechanical injury to the nerve trunk observed compared with the whole-circle contact group at both 4 and 10 weeks post-implantation. Electrodes with point and 1/4 circle contacts represented an alternatively portable and effective method of electrical stimulation to facilitate injured sciatic nerve regeneration and reduce subsequent muscular atrophy, which might offer a promising approach for treating peripheral nerve injuries.

Validation of a novel animal model for sciatic nerve repair with an adipose-derived stem cell loaded fibrin conduit.

Despite the regenerative capabilities of peripheral nerves, severe injuries or neuronal trauma of critical size impose immense hurdles for proper restoration of neuro-muscular circuitry. Autologous nerve grafts improve re-establishment of connectivity, but also comprise substantial donor site morbidity. We developed a rat model which allows the testing of different cell applications, i.e., mesenchymal stem cells, to improve nerve regeneration in vivo. To mimic inaccurate alignment of autologous nerve grafts with the injured nerve, a 20 mm portion of the sciatic nerve was excised, and sutured back in place in reversed direction. To validate the feasibility of our novel model, a fibrin gel conduit containing autologous undifferentiated adipose-derived stem cells was applied around the coaptation sites and compared to autologous nerve grafts. After evaluating sciatic nerve function for 16 weeks postoperatively, animals were sacrificed, and gastrocnemius muscle weight was determined along with morphological parameters (g-ratio, axon density & diameter) of regenerating axons. Interestingly, the addition of undifferentiated adipose-derived stem cells resulted in a significantly improved re-myelination, axon ingrowth and functional outcome, when compared to animals without a cell seeded conduit. The presented model thus displays several intriguing features: it imitates a certain mismatch in size, distribution and orientation of axons within the nerve coaptation site. The fibrin conduit itself allows for an easy application of cells and, as a true critical-size defect model, any observed improvement relates directly to the performed intervention. Since fibrin and adipose-derived stem cells have been approved for human applications, the technique can theoretically be performed on humans. Thus, we suggest that the model is a powerful tool to investigate cell mediated assistance of peripheral nerve regeneration.

Time to recovery of sciatic function index after induced tibialis anterior strain in rats.

BACKGROUND: Muscle strain is a common injury with a high recurrence rate. Due to the heterogeneity of strain injuries, experimental animals provide controlled and reproducible models to investigate such injuries. Sciatic Function Index (SFI) is a clinically feasible method to assess hind limb recovery in rodents after induced injuries. OBJECTIVES: To investigate time to recovery of SFI after induced-strain in tibialis anterior (TA) muscle in rats. METHODS: Sixteen adult male Wister rats were randomly and equally divided to a normal control group that received no intervention, and TA induced muscle strain group. Muscle strain was induced using an external weight that corresponded to 150% of the animal body weight. SFI was tested only once in the control group. For the muscle strain group, SFI was tested on the 1st, 2nd, 3rd, 4th, 7th, 11th, 20th and 24th days after strain induction. RESULTS: Comparisons between group showed significant difference in SFI on the 1st, 2nd, 3rd and 4th days (p= 0.012, 0.012, 0.012 and 0.028, respectively). CONCLUSIONS: In a rat animal model of TA induced muscle strain, functional recovery measured by SFI is evident on the 7th day post-injury, which corresponds to the sub-acute phase of injury. LEVEL OF EVIDENCE: V.

Sciatic nerve regeneration in KLF7-transfected acellular nerve allografts.

OBJECTIVE: Krüppel-like Factor 7 (KLF7) is a transcription factor that promotes axon regeneration in the central nervous system. Here, we assessed whether KLF7 stimulates regeneration after peripheral nerve injury. METHODS: C57BL/6 mice received an acellular nerve allograft (ANA) injected with either adeno-associated virus 2 (AAV2) vector or AAV2-KLF7 for sciatic nerve gap repair. After 4 weeks, KLF7 was detected by RT-PCR, western blot and immunohistochemistry in regenerated nerves. Axonal regeneration and functional recovery were examined by immunohistochemistry, Fluorogold (FG) and cholera toxin B (CTB) retrograde neural tracing, sciatic function index (SFI), angle of ankle, Hargreaves test and electrophysiological analysis. RESULTS: With AAV2-KLF7 injection, KLF7 expression increased in regenerated nerves, and amplitude, score of SFI, angle of ankle and FG-labelled spinal cord neurons were increased. We observed elevated CTB-labelled neurons in dorsal root ganglia (DRG), neurofilaments, P0 (peripheral myelin) and S100 and decreased latency period and withdrawal latencies in the Hargreaves test. The SFI was significantly correlated with amplitude and regenerated axon number. Tyrosine kinase A (TrkA) and B (TrkB) receptors were also increased in the DRG. CONCLUSIONS: Our findings suggest that KLF7 promoted peripheral nerve axonal regeneration, further supporting a role for KLF7 as a growth-promoting transcription factor in the injured nervous system.

Study of Wide Pulse Wave Electroacupuncture at Zusanli (ST36) for Preventing and Treating Lower Limb Muscle Atrophy / 上海针灸杂志

Objective To sift and optimize pulse width for electroacupuncture prevention and treatment of muscular atrophy. Method A model of lower limb muscle atrophy was made in 40 rats. They were randomized into groupsⅠ (no electroacupuncture),Ⅱ (a pulse width of 0.5 ms and a frequency of 2 Hz), Ⅲ (a pulse width of 100 ms and a frequency of 2 Hz) and Ⅳ (a pulse width of 200 ms and a frequency of 2 Hz). Points Zusanli (ST36) and Chengshan(BL57) were given electroacupuncture. Sciatic function index (SFI), gastrocnemius muscle wet weight ratio (GW), the cross-sectional area of gastrocnemius muscle fibers (GC), gastrocnemius muscle cell diameter (GD) and gastrocnemius muscle cell apoptosis index (AI) were used as indicators of therapeutic effect. Result Compared with group Ⅰ, SFI and GW increased significantly in groups Ⅲ and Ⅳ (P<0.05). Compared with groupsⅠ and Ⅱ, AI on the affected side decreased significantly in groups Ⅲ and Ⅳ (P<0.05). Conclusion The therapeutic effect of electroacupuncture is related to its pulse width. The pulse widths of 100 ms and 200 ms are significantly more effective than a pulse width of 0.5 ms from a conventional electroacupuncture device in preventing and treating muscular atrophy.

Regenerative effect of adipose tissue-derived stem cells transplantation using nerve conduit therapy on sciatic nerve injury in rats.

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT-extracts were non-toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i-ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i-ASCs nerve conduit as a guidance channel in the repair of a 10-mm gap in the sciatic nerve of rats. At eight weeks post-implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P < 0.05) in the GGT/i-ASC group than in the autograft group. Furthermore, the mean recovery index of compound muscle action potential (CMAP) differed significantly between GGT/i-ASCs and autograft groups (P < 0.05), both of which were significantly superior to the GGT group (P < 0.05). No severe inflammatory reaction in the peripheral nerve tissue at the site of implantation was observed in either group. Histological observation and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/i-ASCs nerve conduits were similar to those of the autografts. These promising results achieved through a combination of regenerative cells and GGT nerve conduits suggest the potential value in the future development of clinical applications for the treatment of peripheral nerve injury.

Effects of 660nm red light on crush-induced sciatic nerve injury / 中华物理医学与康复杂志

Objective To explore the therapeutic effects of 660 nm red light on sciatic nerve injury in adult rats.Methods Forty-five adult,male rats were divided into a control group and treatment groups 1,2,3 and 4.Sciatic nerve injury was modeled by crushing the nerve.The treatment groups received irradiation with red light once daily for 21 consecutive days.The power density of red light and irradiation time varied among the groups.The latency and amplitude of compound muscle action potentials (CMAPs) and nerve conductive velocity were examined at different time points.The Sciatic Function Index (SFI) was used to evaluate walking function.Results After 21 days of red light therapy no statistically significant differences were observed between the control group and treatment groups 1 to 4 with regard to the latency or the amplitude of the CMAPs.There was a significant difference between the control group and treatment group 3 in terms of sciatic nerve conduction velocity.The average Sciatic Function Indexes of treatment groups 2,3 were significantly different from that of the control group.Conclusion Red light irradiation can promote recovery after sciatic nerve injury,at least in rats,thereby improving walking function.

Effects of Herbal Bath "HAC" on Functional Recovery and c-Fos Expression in the Ventrolateral Periaqueductal Gray Region after Sciatic Crushed Nerve Injury in Rats

Peripheral nerve injuries are a commonly encountered clinical problem and often result in a chronic pain and severe functional deficits. c-Fos expression is sometimes used as a marker of increased neuronal activity. We have developed herbal bath "HAC" for pain control using the following herbs: Harpagophytum procumbens, Atractylodes japonica, and Corydalis tuber. In the present study, we investigated the effects of herbal bath "HAC" on the recovery rate of the locomotor function and the expression of c-Fos in the ventrolateral periaqueductal gray (vlPAG) region of brain following sciatic crushed nerve injury in rats. Walking track analysis for the evaluation of functional recovery and immunohistochemistry for the c-Fos expression were used for this study. In the present results, characteristic gait change with dropping of the sciatic function index (SFI) was observed and c-Fos expression in the vlPAG was suppressed following sciatic crushed nerve injury in rats. Immersion into herbal bath "HAC" enhanced SFI value and restored c-Fos expression in the vlPAG to the control value. These results suggest that herbal bath "HAC" might activate neurons in the vlPAG, and it facilitates functional recovery from peripheral nerve injury. Here we showed that herbal bath "HAC" could be used as a new therapeutic intervention for pain control and functional recovery from peripheral nerve injury.

Experimental study of peripheral nerve regeneration by using non-tubular natural cellulose membrane nerve conduit

Styela clava, called non-native tunicate or sea squirt, is habitat which include bays and harbors in Korea and several sites in the sea faced world. We fabricate cellulose membrane nerve conduit (CMNC) from this native sea squirt skin, and evaluate the capacity of promoting peripheral nerve regeneration in the rat sciatic nerve defect model. After processing the pure cellulose membrane from the sea squirt skin as we already published before, CMNC was designed as a non-tubular sheet with 14 mm length and 4 mm width. Total eleven male Spraque-Dawley rats (12 weeks, weighing 250 to 300g) were divided into sham group (n=2), silicone tube grafted control group (n=3) and experimental group (n=6). Each CMNC grafted nerve was evaluated after 4, 8 and 12 weeks in the experimental group, and after 12 weeks, sciatic function was evaluated with sciatic function index (SFI) and gait analysis, and histomorphology of nerve conduit and the innervated tissues of sciatic nerve were all examined using image analyzer and electromicroscopic methods in the all groups. The regenerated axon and nerve sheath were found only in the inner surface of the CMNC after 4 weeks and became more thicker after 8 and 12 weeks. In the TEM study, CMNC grafted group showed more abundant organized myelinated nerve fibers with thickened extracellular matrix than silicone conduit grafted group after 12 weeks. The sciatic function index (SFI) and ankle stance angle (ASA) in the functional evaluation were -47.2+/-3.9, 35.5.+/-4.9.in CMNC grafted group (n=2) and -80.4+/-7.4, 29.2.+/-5.3.in silicone conduit grafted group (n=3), respectively. And the myelinated axon was 41.59% in CMNC group and 9.51% in silicone conduit group to the sham group. The development of a bioactive CMNC to replace autogenous nerve grafts offers a potential and available approach to improved peripheral nerve regeneration. As we already published before, small peptide fragment derived from the basement membrane matrix proteins of squirt skin, which is a kind of anchoring protein composed of glycocalyx, induced the effective axonal regeneration with rapid growth of Schwann cells beneath the inner surface of CMNC. So the possibilities of clinical application as a peripheral nerve regeneration will be able to be suggested.