Electrical Stimulation Promotes Regeneration of Defective Peripheral Nerves after Delayed Repair Intervals Lasting under One Month

Background

Electrical stimulation (ES) has been proven to be an effective means of enhancing the speed and accuracy of nerve regeneration. However, these results were recorded when the procedure was performed almost immediately after nerve injury. In clinical settings, most patients cannot be treated immediately. Some patients with serious trauma or contaminated wounds need to wait for nerve repair surgery. Delays in nerve repair have been shown to be associated with poorer results than immediate surgery. It is not clear whether electrical stimulation still has any effect on nerve regeneration after enough time has elapsed.

Methods

A delayed nerve repair model in which the rats received delayed nerve repair after 1 day, 1 week, 1 month, and 2 months was designed. At each point in time, the nerve stumps of half the rats were bridged with an absorbable conduit and the rats were given 1 h of weak electrical stimulation. The other half was not treated. In order to analyze the morphological and molecular differences among these groups, 6 ES rats and 6 sham ES rats per point in time were killed 5 days after surgery. The other rats in each group were allowed to recover for 6 weeks before the final functional test and tissue observation.

Results

The amounts of myelinated fibers in the distal nerve stumps decreased as the delay in repair increased for both ES rats and sham ES rats. In the 1-day-delay and 1-week-delay groups, there were more fibers in ES rats than in sham ES rats. And the compound muscle action potential (CMAP) and motor nerve conduction velocity (MNCV) results were better for ES rats in these two groups. In order to analyze the mechanisms underlying these differences, Masson staining was performed on the distal nerves and quantitative PCR on the spinal cords. Results showed that, after delays in repair of 1 month and 2 months, there was more collagen tissue hyperplasia in the distal nerve in all rats. The brain-derived neurotrophic factor (BDNF) and trkB expression levels in the spinal cords of ES rats were higher than in sham ES rats. However, these differences decreased as the delay in repair increased.

Conclusions

Electrical stimulation does not continue to promote nerve regeneration after long delays in nerve repair. The effective interval for nerve regeneration after delayed repair was found to be less than 1 month. The mechanism seemed to be related to the expression of nerve growth factors and regeneration environment in the distal nerves.     

Brain-Derived Neurotrophic Factor from Bone Marrow-Derived Cells Promotes Post-Injury Repair of Peripheral Nerve

Brain-derived neurotrophic factor (BDNF) stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs) in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/−) received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT) were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI), and motor nerve conduction velocity (MNCV) simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/− mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/− BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/− mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.     

Altered Neurotrophin mRNA Levels in Peripheral Nerve and Skeletal Muscle of Experimentally Diabetic Rats

Abstract: The levels of neurotrophin mRNA in sensory ganglia, sciatic nerve, and skeletal muscle were measured in the streptozotocin-diabetic rat using northern blotting. Periods of diabetes of 4, 6, and 12 weeks significantly elevated brain-derived neurotrophic factor (BDNF) mRNA levels in soleus muscle compared with age-matched controls, the increase being highest at 6 weeks. At all time periods studied, the levels of nerve growth factor (NGF) mRNA in soleus muscle were decreased by 21–47%. Following 12 weeks of diabetes, BDNF mRNA levels were increased approximately two-to threefold in L4 and L5 dorsal root ganglia (DRG), and in sciatic nerve, NGF mRNA levels were raised 1.65-fold. Intensive insulin treatment of diabetic rats for the final 4 weeks of the 12-week period of diabetes reversed the up-regulation of BDNF mRNA in DRG and muscle and NGF mRNA in sciatic nerve. All diabetes-induced changes in neurotrophin mRNA were not paralleled by similar alterations in the levels of β-actin mRNA in muscle and nerve, or of GAP-43 mRNA in DRG and nerve. It is proposed that the up-regulation of neurotrophin mRNA is an endogenous protective and/or repair mechanism induced by insult and, as such, appears as an early marker of peripheral nerve and muscle damage in experimental diabetes.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

Effect of Delayed Peripheral Nerve Repair on Nerve Regeneration,Schwann Cell Function and Target Muscle Recovery

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2–3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.     

Effect of weak, interrupted sinusoidal low frequency magnetic field on neural regeneration in rats: functional evaluation

A study of the effect of weak, interrupted sinusoidal low frequency magnetic field (ISMF) stimulation on regeneration of the rat sciatic nerve was carried out. In the experiment, 60 Wistar rats were used: 24 rats underwent unilateral sciatic nerve transection injury and immediate surgical nerve repair, 24 rats underwent unilateral sciatic nerve crush injury, and the remaining 12 rats underwent a sham surgery. Half of the animals (n = 12) with either sciatic nerve lesion were randomly chosen and exposed between a pair of Helmholtz coils for 3 weeks post-injury, 4 h/day, to an interrupted (active period to pause ratio = 1.4 s/0.8 s) sinusoidal 50 Hz magnetic field of 0.5 mT. The other half of the animals (n = 12) and six rats with sham surgery were used for two separate controls. Functional recovery was followed for 6 weeks for the crush injuries and 7(1/2) months for the transection injuries by video assisted footprint analysis in static conditions and quantified using a recently revised static sciatic index (SSI) formula. We ascertained that the magnetic field influence was weak, but certainly detectable in both injury models. The accuracy of ISMF influence detection, determined by the one-way repeated measures ANOVA test, was better for the crush injury model: F(1, 198) = 9.0144, P = .003, than for the transection injury model: F(1, 198) = 6.4826, P = .012. The Student-Newman-Keuls range test for each response day yielded significant differences (P < .05) between the exposed and control groups early in the beginning of functional recovery and later on from the points adjacent to the beginning of the plateau, or 95% of functional recovery, and the end of observation. These differences probably reflect the ISMF systemic effect on the neuron cell bodies and increased and more efficient reinnervation of the periphery.     

Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap

A new type of a biodegradable nerve graft conduit material, the amnion tube, has been developed in our laboratory. To test the tube in the peripheral nerve regeneration process, it was initially applied across a 1-cm sciatic nerve gap in rats and was compared with other nerve conduit materials. We used male Sprague-Dawley rats as our animal model. The experiment included 66 rats that were randomly assigned into five groups: autograft (n = 17), amnion tube (n = 19), silicone tube (n = 20), no repair (n = 7), and sham group (n = 3). The process of peripheral nerve regeneration was evaluated at 2, 4, 10, and 17 weeks following injury and repair by using morphologic and functional assessments of the outcome of nerve regeneration in each animal. Nerve regeneration across the amnion tube nerve conduit was comparable with that seen in autograft and superior to that of the silicone group. A uniform nerve tissue was seen filling and crossing the amnion conduit, and the regenerated nerve from the proximal stump reached the distal end and was undifferentiated from the normal nerve tissues. At 4 months, the amnion tube biodegraded and no longer could be identified and differentiated from the nerve tissues. The amnion tube animal group showed a number of axons very close to that in the nerve autograft group (37,157 versus 33,054). Functional recovery at a 2- to 4-week interval was significantly statistically higher only in the amnion tube animal group (p = 0.01). However, the improvement disappeared between 10 and 17 weeks. In conclusion, the amnion tube is a potential ideal nerve conduit material secondary to its unique characteristics: it contains important neurotropic factors, is biodegradable, provokes a very weak immune response, is semiflexible, is readily available, and is easily manufactured into different sizes and diameters.     

A role for free radicals and nitric oxide in delayed recovery in aged rats with chronic constriction nerve injury

Using a reversible chronic constriction injury (CCI) model of neuropathic pain, we previously demonstrated that changes in thermal hyperalgesia correlate with the changes in peripheral microvascular blood flow in the affected paw, and that recovery can be assessed by normalization of both behavioral and vascular responses. Using the same model, this study examined age-related changes in recovery after nerve injury and the involvement of free radicals and nitric oxide (NO) in these changes. Four loose, nonconstrictive ligatures were applied to the sciatic nerve in the right, mid-thigh region of young and old (3 and 24 months) Sprague Dawley rats. All rats were monitored weekly (for 8-10 weeks) for their thermal threshold using a 46 degrees C water bath and some groups were used to examine endothelial and smooth muscle-dependent microvascular responses to substance P (SP) and sodium nitroprusside (SNP), respectively. These substances were perfused over the base of blisters raised on the footpad innervated by the injured nerve. Free radical activity in the sciatic nerve was assessed by measuring the activity of xanthine oxidase (XO) and lipid hydroperoxides (LPO). Young rats showed signs of recovery (reduction in thermal hyperalgesia and improvement of peripheral microvascular blood flow) from the fifth week. No signs of recovery were observed in old rats for 8 weeks, with some reduction in thermal hyperalgesia observed by weeks 9 and 10. XO activity was significantly higher in young injured nerves compared to sham (400%) and was even significantly greater in old injured nerves (680%). Similarly, old injured nerves showed 300% increase in LPO levels compared to sham. The role of reactive oxygen species (ROS) in delayed recovery in old rats was examined using the antioxidant tirilazad mesylate. Tirilazad (20 mg/kg) was injected intramuscularly (im) in the mid-thigh region starting on day 1 post CCI, (early treatment) or day 7 (late treatment). Levels of LPO in the injured sciatic nerves were significantly reduced using either early or late treatment, however tirilazad had opposing effects on recovery, prolonging or alleviating thermal hyperalgesia, respectively. The role of neuronal nitric oxide (nNO) was then examined using the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (3Br-7NI) (10 mg/kg). 3Br-7NI resulted in a significant alleviation of thermal hyperalgesia with improvement in the vascular responses from weeks 5 and 6 onwards. A combination of 3Br-7NI and tirilazad treatment was also used but did not show an additive effect. The results suggest that ROS and nNO contribute to delayed recovery of injured nerves in old rats and to the maintenance of thermal hyperalgesia and the reduction in microvascular blood flow in the area innervated by the injured nerve. The results also raise the notion that possible interaction of free radicals with NO to form peroxynitrite might be responsible for such delayed recovery. Ironically, this study also reveals a positive role for free radicals in tissue repair and raises the notion that early intervention with antioxidants could exert a negative effect on repair of injured nerves.     

Synergistic Effects of Bone Mesenchymal Stem Cells and Chondroitinase ABC on Nerve Regeneration After Acellular Nerve Allograft in Rats

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague–Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.     

碱性成纤维细胞生长因子对大鼠晚期周围神经再生作用的实验研究

目的探讨外源性碱性成纤维细胞生长因子(bFGF)对晚期周围神经再生的作用.方法50只SD大鼠随机分治疗组、对照组各25只,切断右侧坐骨神经,12周后予以修复,修复术后每日分别给予bFGF和生理盐水,行神经电生理和组织学检查.结果治疗组和对照组修复处远段神经均有不同程度再生,4周时已可见到再生轴突,且治疗组多见.计量分析治疗组运动神经传导速度、神经肌肉动作电位幅值、髓鞘厚度、再生轴突直径和截面积明显优于对照组.治疗组与对照组相比,差异有显著性.结论bFGF能促进晚期周围神经再生.     

Pulsed electromagnetic fields induce peripheral nerve regeneration and endplate enzymatic changes

An experimental study was carried out in rats with the purpose of demonstrating the capacity of pulsed electromagnetic fields (PEMFs) to stimulate regeneration of the peripheral nervous system (PNS). Wistar and Brown Norway (BN) rats were used. Direct sciatic nerve anastomoses were performed after section or allograft interposition. Treatment groups then received 4 weeks of PEMFs. Control groups received no stimulation. The evaluation of the results was carried out by quantitative morphometric analysis, demonstrating a statistically significant increase in regeneration indices (P < 0.05) in the stimulated groups (9000 +/- 5000 and 4000 +/- 6000) compared to the non-stimulated groups (2000 +/- 4000 and 700 +/- 200). An increase of NAD specific isocitrate dehydrogenase (IDH) activity was found along with an increase in the activity of acetyl cholinesterase at the motor plate. The present study might lead to the search for new alternatives in the stimulation of axonal regenerative processes in the PNS and other possible clinical applications.     

Enhancement of nerve regeneration and orientation across a gap with a nerve graft within a vein conduit graft: a functional, stereological, and electrophysiological study

In this study, the right sciatic nerves of 40 rats were used to determine whether a nerve graft within a vein graft might accelerate and facilitate axonal regeneration, compared with a nerve graft alone. The animals were separated into four groups, as follows: group 1, sham control; group 2 (control), segmental nerve resection and no repair; group 3, segmental nerve resection and nerve grafting; group 4, segmental nerve resection and reconstruction with a nerve graft within a vein conduit graft. For all groups, sciatic functional indices were calculated before the operation and on postoperative days 7 and 90. On postoperative day 90, the sciatic nerves were reexposed and nerve conduction velocities were recorded. The sciatic nerves were harvested from all groups for counting of the myelinated axons with a stereological method. No statistically significant differences with respect to return of gait function, axon count, or nerve conduction were noted between groups 3 and 4 (p > 0.05). However, functional recovery in group 4 on postoperative day 90 was significant, compared with group 2 (p < 0.05); the recovery difference between groups 2 and 3 was not significant (p > 0.05). This study was not able to demonstrate any functional benefits with the use of a nerve graft within a vein graft, compared with standard nerve grafting.     

Improving Nerve Regeneration of Acellular Nerve Allografts Seeded with SCs Bridging the Sciatic Nerve Defects of Rat

The objective of the paper is to evaluate the effect of acellular nerve allografts (ANA) seeded with Schwann cells to promote nerve regeneration after bridging the sciatic nerve defects of rats and to discuss its acting mechanisms. Schwann cells were isolated from neonatal Wistar rats. In vitro Schwann cells were microinjected into acellular nerve allografts and co-cultured. Twenty-four Wistar rats weighing 180–220 g were randomly divided into three groups with eight rats in each group: ANA seeded with Schwann cells (ANA + SCs), ANA group and autografts group. All the grafts were, respectively, served for bridging a 10-mm long surgically created sciatic nerve gap. Examinations of regeneration nerve were performed after 12 weeks by transmission electron microscope (TEM), scanning electron microscope (SEM), and electrophysiological methods, and then analyzed statistically. The results obtained indicated that in vitro Schwann cells displayed the feature of bipolar morphology with oval nuclei. Compared with ANA group, the conduction velocity of ANA + SCs group and autograft group was faster after 12 weeks, latent period was shorter, and wave amplitude was higher (P < 0.05). The difference between ANA + SCs group and autograft group is not significant (P > 0.05). Regeneration nerve myelinated fiber number, myelin sheath thickness, and myelinated fibers/total nerves (%) in both ANA + SCs group and autograft group are higher than that in ANA group; the difference is significant (P < 0.05). The difference between the former two is not significant (P > 0.05). In conclusion, ANA seeded with SCs could improve nerve regeneration and functional recovery after bridging the sciatic nerve gap of rats, which offers a novel approach for the repair peripheral nerve defect.     

Skeletal muscle reinnervation by reduced axonal numbers results in whole muscle force deficits

Patients sustaining a peripheral nerve injury will frequently experience residual muscle weakness after muscle reinnervation, even if the nerve repair is performed under optimal circumstances to allow rapid muscle reinnervation. The mechanisms responsible for this contractile dysfunction remain unclear. It is hypothesized that after peripheral nerve injury and repair, a reduced number of axons are available for skeletal muscle reinnervation that results in whole muscle force and specific force deficits. A rat model of peroneal nerve injury and repair was designed so that the number of axons available for reinnervation could be systematically reduced. In adult rats, the peroneal nerve to the extensor digitorum longus muscle was either left intact (sham group, n = 8) or divided and repaired with either 50 percent (R50 group, n = 7) or 100 percent (R100 group, n = 8) of the axons in the proximal stump included in the repair. Four months after surgery, maximal tetanic isometric force was measured and specific force was calculated for each animal. Mean tetanic isometric force for extensor digitorum longus muscles from R50 rats (2765.7 +/- 767.6 mN) was significantly lower than sham (4082.8 +/- 196.5 mN) and R100 (3729.0 +/-370.2 mN) rats (p < 0.003). Mean specific force calculations revealed significant deficits in both the R100 (242.1 +/- 30 kN/m2) and R50 (190.6 +/- 51.8 kN/m2) rats compared with the sham animals (295.9 +/- 14 kN/m2) (p < 0.0005). These data support our hypothesis that after peripheral nerve injury and repair, reinnervation of skeletal muscle by a reduced number of axons results in a reduction in tetanic isometric force and specific force. The greater relative reduction in specific force compared with absolute force production after partial nerve repair may indicate that a population of residual denervated muscle fibers is responsible for this deficit.     

Therapeutic Effects of 15 Hz Pulsed Electromagnetic Field on Diabetic Peripheral Neuropathy in Streptozotocin-Treated Rats

Although numerous clinical studies have reported that pulsed electromagnetic fields (PEMF) have a neuroprotective role in patients with diabetic peripheral neuropathy (DPN), the application of PEMF for clinic is still controversial. The present study was designed to investigate whether PEMF has therapeutic potential in relieving peripheral neuropathic symptoms in streptozotocin (STZ)-induced diabetic rats. Adult male Sprague–Dawley rats were randomly divided into three weight-matched groups (eight in each group): the non-diabetic control group (Control), diabetes mellitus with 15 Hz PEMF exposure group (DM+PEMF) which were subjected to daily 8-h PEMF exposure for 7 weeks and diabetes mellitus with sham PEMF exposure group (DM). Signs and symptoms of DPN in STZ-treated rats were investigated by using behavioral assays. Meanwhile, ultrastructural examination and immunohistochemical study for vascular endothelial growth factor (VEGF) of sciatic nerve were also performed. During a 7-week experimental observation, we found that PEMF stimulation did not alter hyperglycemia and weight loss in STZ-treated rats with DPN. However, PEMF stimulation attenuated the development of the abnormalities observed in STZ-treated rats with DPN, which were demonstrated by increased hind paw withdrawal threshold to mechanical and thermal stimuli, slighter demyelination and axon enlargement and less VEGF immunostaining of sciatic nerve compared to those of the DM group. The current study demonstrates that treatment with PEMF might prevent the development of abnormalities observed in animal models for DPN. It is suggested that PEMF might have direct corrective effects on injured nerves and would be a potentially promising non-invasive therapeutic tool for the treatment of DPN.     

Non-thermal plasma application enhances the recovery of transected sciatic nerves in rats

This experimental research aimed to investigate the effects of non-thermal plasma on nerve regeneration after transected nerve damage using the sciatic nerve in Wistar albino (A) rats. The experiments were performed on 27 Wistar A rats. The rats underwent surgery for right sciatic nerve exposure and were divided into three groups (each group, n = 9) according to sciatic nerve transected injury (SNTI) and non-thermal plasma application: a non-nerve damage (non-ND) group, a only nerve damage without non-thermal plasma application (ND) group, and a nerve damage with non-thermal plasma application (ND + NTP) group. Subsequent to SNTI and immediate suture, non-thermal plasma was administered three times per week for eight weeks. Evaluation for functional recovery was performed using the static sciatic index measured over the full treatment period of eight weeks. The sciatic nerve specimens were obtained after euthanasia and third day from the last non-thermal plasma application. The sciatic nerve tissues were subjected to histological analysis. Behavior analysis presented that the ND + NTP group showed improved static sciatic index compared with the nerve damage group. Histopathological findings demonstrated that the ND + NTP group had more dense Schwann cells and well-established continuity of nerve fibers, greater than the nerve damage group. Immunohistochemistry showed that the ND + NTP group had increased levels of markers for microtubule-associated protein 2 (MAP2), tau, S100 calcium-binding protein B, and neurofilament-200 and regulated the overexpression of CD68 and MAP2. These results indicated that non-thermal plasma enhanced the motor function and restored the neuronal structure by accelerating myelination and axonal regeneration. Additionally, non-thermal plasma was confirmed to have a positive effect on the recovery of SNTI in rats.     

Functional assessment in the rat by ground reaction forces

Although the rat sciatic nerve model is used extensively in the investigation of repair techniques, and a variety of evaluation methods utilized to assess the results, a means to measure directly and accurately the return of function in these animals is absent. Histologic, histomorphometric, and electrophysiologic methods can be reliable indicators of nerve regeneration but do not correlate to functional recovery. The purposes of this study were to develop apparatus to continuously measure ground reaction forces (GRF) and use GRF parameters in the assessment of gait parameters in normal rats preoperatively and following peripheral nerve severance and repair. Three neurorrhaphy methods: direct sciatic nerve repair, direct tibial nerve repair and double sciatic nerve repair simulating autograft, as well as a non-repaired tibial nerve transection were evaluated. The testing apparatus was designed to measure the spontaneous and voluntary effort of the rat with objective data. Three orthogonal components - vertical, craniocaudal (braking and propulsion), and mediolateral - of the ground reaction force were measured. Preoperative data showed that vertical forces were comparable among the four limbs but propulsion and braking forces displayed significant differences. At 12 weeks, functional recovery was most evident in the direct tibial nerve repair group and absent in the non-repaired tibial defect group. Direct sciatic nerve repairs and sciatic nerve grafts resulted in lesser degrees of improvement. Results indicated that the propulsive force is the optimal GRF parameter for evaluating recovery of useful function.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

Functional motor recovery is improved due to local placement of GDNF microspheres after delayed nerve repair

The majority of bioengineering strategies to promote peripheral nerve regeneration after injury have focused on therapies to bridge large nerve defects while fewer therapies are being developed to treat other nerve injuries, such as nerve transection. We constructed delivery systems using fibrin gels containing either free GDNF or polylactide–glycolic acid (PLGA) microspheres with GDNF to treat delayed nerve repair, where ELISA verified GDNF release. We determined the formulation of microspheres containing GDNF that optimized nerve regeneration and functional recovery in a rat model of delayed nerve repair. Experimental groups underwent delayed nerve repair and treatment with GDNF microspheres in fibrin glue at the repair site or control treatments (empty microspheres or free GDNF without microspheres). Contractile muscle force, muscle mass, and MUNE were measured 12 weeks following treatment, where GDNF microspheres (2 weeks formulation) were superior compared to either no GDNF or short‐term release of free GDNF to nerve. Nerve histology distal to the repair site demonstrated increased axon counts and fiber diameters due to GDNF microspheres (2 weeks formulation). GDNF microspheres partially reversed the deleterious effects of chronic nerve injury, and recovery was slightly favored with the 2 weeks formulation compared to the 4 weeks formulation. Biotechnol. Bioeng. 2013; 110: 1272–1281. © 2012 Wiley Periodicals, Inc.