Functional Nerve Regeneration Research Articles

The effect of catheter use on vein grafting of a peripheral nerve defect: an experimental study

Since vein grafts have been used in the repair of nerve defects, studies regarding this procedure have accumulated, and after coming into clinical use, it was noticed that there is a problem of collapse in the vein graft. Forty Sprague-Dawley rats were used, divided into five groups. No surgical intervention was performed in the first group. The defect was created in the sciatic nerve in Group 2 and left unrepaired. In Group 3, the defect was repaired with a nerve graft. In Group 4, the defect was repaired with a vein graft, while in Group 5, the repair was performed using a vein graft with an inserted catheter. In order to evaluate functional recovery and nerve regeneration, walking track analysis, electrophysiologic and histomorphometric analyses were done at the end of the 12th week. Although there were no functional differences between Groups 5 and 4, comparisons regarding nerve conduction velocity demonstrated that the results obtained in Group 5 were better than those in Group 4. When the number of axons on the distal part of the sciatic nerve and mid-segment of the repaired area was taken into account, no significant difference was found between Groups 3 and 5, whereas there was a significant difference between Groups 4 and 5. In our study, it was experimentally shown that the problem of collapse of a vein graft occurring after its use in the reconstruction of a nerve defect can be overcome by placing a catheter into the vein graft. Consequently, this method may eliminate the need for the use of a nerve graft in selected cases.

Bridging Peripheral Nerve Defects Using a Single-Fascicle Nerve Graft

The criterion standard of nerve reconstruction is an autologous nerve graft, identical in cross-section to the severed nerve stumps. This study investigates single-fascicle nerve transplantation to bridge nerve defects. Forty female Sprague-Dawley rats, each weighing approximately 225 g, were randomized into five groups. Groups A and B were the positive and negative control groups, respectively. In the experimental groups (groups C, D, and E), a nerve defect of 25 mm was created in the right sciatic nerve and reconstructed with a reversed sciatic nerve graft, a three-fascicle sural nerve graft, or a single-fascicle sural nerve graft, respectively. Functional muscle evaluation of the triceps surae differed significantly among the reverse nerve (group C, n=10; 600±154.16 mN), triple-fascicle (group D, n=10; 411±262 mN), and single-fascicle (group E, n=10; 324±215.8 mN) graft groups (p<0.05). The results of retrograde labeling of the reverse nerve group (group C, 518±2) showed that there were twice as many motor neurons as in the three-fascicle nerve group (group D, 221±19). In the single-fascicle nerve group, motor neurons were scarce and faintly labeled (group E, 180±25). The results showed that single-fascicle nerve grafting has potential for good functional nerve regeneration.

English

The peripheral nervous system has the ability to regenerate after injury. Peripheral nerve injuries are caused by penetrating injury, crush, traction and ischemia compression. However, the availability of various nerve coaptation and other techniques for the attainment of functional nerve regeneration is still inadequate. The objective of this study was to compare the effectiveness of bone marrow stromal cells (BMSCs) implantation and epineural nerve suture on peripheral nerve regeneration in a rabbit model. Ten male New Zealand white rabbits were divided into two groups. In the primary epineural repair group (control group), the left sciatic nerve was skeletonized from the sciatic notch to the point of bifurcation, with the nerve been transected at the mid-shaft of the femoral bone and repaired with six epineural sutures. In the treated group, the epineural repaired nerve was implanted with BMSCs in the proximal and distal segments of the transected sciatic nerve. Assessment of the nerve regeneration was based on functional (motor and sensory), histological and morphometric criteria, including the number of myelinated nerve fibers, nerve fiber diameter, axon diameter, myelin sheath thickness, g ratio and relative gastrocnemius muscle weight. The results of the examination showed that the treated group had the best regeneration and functional recovery. &nbsp; Keyword:&nbsp;Bone marrow stromal cells (BMSCs), histopathology, morphometric, peripheral nerve, regeneration.

Neuroprotective and neuroregenerative effects of low-intensity aerobic exercise on sciatic nerve crush injury in mice

Here, we established a program of low-intensity aerobic exercise and compared the effects of exercise preoperative, postoperative, and a combination of both pre- and postoperative protocols on recovery from sciatic nerve crush injury in mice using behavioral, biochemical, and morphological assays. Sciatic nerve crush was performed in adult male mice. The animals were submitted to preoperative (for 2 weeks), postoperative (for 2 weeks), and a combination of preoperative-postoperative (for 4 weeks) training protocols. During the training period, functional recovery was monitored using the Sciatic Functional Index, the Sciatic Static Index, and mechanical and cold hypersensitivity analyses. Morphological and biochemical alterations were analyzed on the 14th day post-crushing. The functional recovery values of all of the exercised groups were significantly better than the nonexercised group. Biochemically, all of the exercise groups showed a reduction in the increase of interleukin-1β (IL-1β) in the sciatic nerve and in the IL-1β and interleukin-6 receptor (IL-6R) levels in the spinal cord. However, the levels of tumor necrosis factor alpha (TNF-α) decreased only in the postoperative group and in the combination exercise protocols. In the morphological analysis, the combination exercise subjects presented an increase in fiber and axon diameter, in the myelination degree and in the number of myelinated fibers. The present study showed that pre- and postoperative exercise achieved values for functional and morphological sciatic nerve regeneration that were significantly better than either the preoperative or postoperative protocols. This experimental study suggests that physical exercise can restore motor and nerve function to a substantial degree when performed using a prophylactic and therapeutic approach.

Electrical Stimulation Accelerates Axonal and Functional Peripheral Nerve Regeneration across Long Gaps

Short-term low-frequency electrical stimulation (ESTIM) of proximal peripheral nerve stumps prior to end-to-end coaptation or tubular bridging of small distances has been reported to increase preferential motor reinnervation and functional motor recovery in animal models and human patients undergoing carpal tunnel release surgery. We investigated the effects of ESTIM on regeneration across rat sciatic nerve gaps, which exceed distances that allow spontaneous regeneration. Three different reconstruction approaches were combined with ESTIM in the experimental groups. Nerve gaps (13 mm) were bridged using (I) nerve autotransplantation, (II) transplantation of differentially filled silicone tubes, or (III) transplantation of tubular grafts containing fibroblast growth factor-2 overexpressing Schwann cells (SCs) for gene therapy. The regeneration outcome was followed for up to 8 weeks, and functionally as well as histomorphometrically analyzed in comparison to non-stimulated control groups. Combining ESTIM with nerve autotransplantation significantly increased the nerve fiber density in the regenerated nerve, and the grade of functional recovery as detected by electrodiagnostic recordings from the gastrocnemius muscle. The combination of ESTIM with transplantation of naïve SCs increased the regeneration of gap-bridging nerve tissue. Although macroscopic tissue regeneration was not further improved after combining ESTIM with FGF-2(21/23-kD) gene therapy, the latter resulted in a high rate of regenerated nerves that functionally reconnected to the target muscle. Based on our results, brief ESTIM shows high potential to accelerate axonal as well as functional (motor and sensory) outcomes in the clinical setting of peripheral nerve gap reconstruction in human patients.

Adhesion molecule L1 overexpressed under the control of the neuronal Thy-1 promoter improves myelination after peripheral nerve injury in adult mice

L1 is an adhesion molecule favorably influencing the functional and anatomical recoveries after central nervous system (CNS) injuries. Its roles in peripheral nervous system (PNS) regeneration are less well understood. Studies using knockout mice have surprisingly revealed that L1 has a negative impact on functional nerve regeneration by inhibiting Schwann cell proliferation. To further elucidate the roles of L1 in PNS regeneration, here we used a novel transgenic mouse overexpressing L1 in neurons, but not in PNS or CNS glial cells, under the control of a neuron-specific Thy-1 promoter. Without nerve injury, the transgene expression, as compared to wild-type mice, had no effect on femoral nerve function, numbers of quadriceps motoneurons and myelinated axons in the femoral nerve but resulted in slightly reduced myelination in the sensory saphenous nerve and increased neurofilament density in myelinated axons of the quadriceps motor nerve branch. After femoral nerve injury, L1 overexpression had no impact on the time course and degree of functional recovery. Unaffected were also numbers of regenerated quadriceps motoneurons, precision of muscle reinnervation, axon numbers and internodal lengths in the regenerated nerves. Despite the lack of functional effects, myelination in the motor and sensory femoral nerve branches was significantly improved and loss of perisomatic inhibitory terminals on motoneurons was attenuated in the transgenic mice. Our results indicate that L1 is a regulator of myelination in the injured PNS and warrant studies aiming to improve function in demyelinating PNS and CNS disorders using exogenous L1.

Mouse Hind Limb Transplantation: A New Composite Tissue Allotransplantation Model Using Nonsuture Supermicrosurgery

The development of microsurgical techniques has facilitated the establishment of vascularized composite tissue transplant models in small mammals. Because the mouse would be the ideal model to study various composite tissue allotransplantation (CTA)-related problems, we designed two new surgical techniques for orthotopic (ORT) and heterotopic (HET) hind limb transplantation. BALB/c hind limbs were transplanted to BALB/c or C57BL6 recipients using a nonsuture cuff technique. ORT: donor femoral vessels were anastomosed to recipient femoral vessels, the sciatic nerve approximated end-to-end and osteosynthesis was performed using an intramedullary rod. HET/cervical: Donor femoral vessels of a reduced size osteomyocutaneous hind limb CTA were anastomosed to recipient common carotid artery and external jugular vein without nerve approximation. Both procedures could be performed with a high success rate (ORT: 62%; HET: 90%). Donor operation lasted for 100±12 min and recipient operation 114±27 min (ORT) and 54±16 min (HET). Complication rates in terms of bleeding, and thrombosis at the cuff side was slightly higher in the ORT group. All syngeneic grafts survived long term (>100 days). FK506 (2 mg/kg) significantly prolonged graft survival (87±22 days) when compared with untreated controls (6±1 day). Functional evaluation of ORT grafts by means of video gait kinematics and CatWalk analysis revealed specific differences of gait parameters when compared with nontransplanted controls (P<0.05). The ORT hind limb transplant model seems to be best suited to study functional outcome and nerve regeneration in CTA. The technically less demanding HET/cervical model may be used to investigate basic immunology and clinically relevant questions related to acute and chronic rejection, and ischemia reperfusion injury in reconstructive transplantation.

Waterjet Dissection of Peripheral Nerves: An Experimental Study of the Sciatic Nerve of Rats

Although waterjet dissection has been well evaluated in intracranial pathologies, little is known of its qualities in peripheral nerve surgery. Theoretically, the precise dissection qualities could support the separation of nerves from adjacent tissues and improve the preservation of nerve integrity in peripheral nerve surgery. To evaluate the potential of the new waterjet dissector in peripheral nerve surgery. Waterjet dissection with pressures of 20 to 80 bar was applied on the sciatic nerves of 101 rats. The effect of waterjet dissection on the sciatic nerve was evaluated by clinical tests, neurophysiological examinations, and histopathological studies up to 12 weeks after surgery. With waterjet pressures up to 30 bar, the sciatic nerve was preserved in its integrity in all cases. Functional damaging was observed at pressures of 40 bar and higher. However, all but 1 rat in the 80 bar subgroup showed complete functional regeneration at 12 weeks after surgery. Histopathologically, small water bubbles were observed around the nerves. At 40 bar and higher, the sciatic nerves showed signs of direct nerve injury. However, all these animals showed nerve regeneration after 12 weeks, as demonstrated by histological studies. Sciatic nerves were preserved functionally and morphologically at pressures up to 30 bar. Between 40 and 80 bar, reliable functional and morphological nerve regeneration occurred. Waterjet pressures up to 30 bar might be applied safely under clinical conditions. This technique might be well suited to separate intact peripheral nerves from adjacent tumor or scar tissue. Further studies will have to show the clinical relevance of these dissection qualities.

Controlled Neurotrophic Factor Delivery To Promote Functional Peripheral Nerve Regeneration

Controlled Neurotrophic Factor Delivery To Promote Functional Peripheral Nerve Regeneration

Isogenic venous graft supported with bone marrow stromal cells as a natural conduit for bridging a 20 mm nerve gap

In this study, we introduce a technique for bridging large neural gaps, using an isogenic vein graft supported with isogenic bone marrow stromal cells (BMSC). In three groups a nerve defect of 20 mm was bridged with a vein graft. Our first experimental group comprized an empty venous graft, in group II the venous nerve graft was filled with saline where as in group III the venous nerve graft was filled with BMSC. The animals were tested for functional recovery up to 3 months post repair. Our results show that the BMSC filled venous graft resulted in significantly better regeneration of the nerve defect compared to controls, as confirmed by the functional recovery measured by somatosensory evoked potentials, toe spread, pin prick, and gastrocnemius muscle index. Conclusively, the results confirm that the vein graft supported with BMSC is associated with better functional nerve regeneration.

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB-OECs and OM-OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM-OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB-OECs gene profile suggests a prominent role in nervous system development. Hence, OB-OECs and OM-OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application.

Fibrin matrices with affinity‐based delivery systems and neurotrophic factors promote functional nerve regeneration

Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.

Spinal cord injury in vitro: modelling axon growth inhibition

Over the past three decades, tremendous progress has been made in elucidating mechanisms underlying regenerative failure after spinal cord injury and in devising therapeutic approaches to promote functional nerve regeneration. Various in vitro assays have been developed using brain and/or spinal cord neuronal cells to study axon growth in conditions that represent the post-injury environment. This review outlines the current models used to dissect, analyze and manipulate specific aspects of spinal cord injury leading to axon growth inhibition.

Comparison of collagen biomatrix and omentum effectiveness on peripheral nerve regeneration

Despite the presence of various nerve coaptation materials and techniques, achievement of the functional nerve regeneration is still inadequate. This study was aimed to compare the effectiveness of conduit composed of collagen biomatrix and omentum graft on peripheral nerve regeneration. Thirty-five male Wistar rats were divided into four groups. In the control group, the right sciatic nerve was skeletonized from the sciatic notch till the point of bifurcation. In the primary epineural repair group, the nerve was transected 1 cm proximal to the bifurcation with a sharp pair of micro scissors and then repaired with four epineural sutures. In the collagen biomatrix group, the epineural repaired nerve was wrapped with collagen biomatrix. In the collagen group, the epineural repaired nerve was wrapped with the nonpediculated omentum. Assessment of the nerve regeneration was based on functional (Walking Track Analysis, Electrophysiological Measurements), histological, and morphometric criteria. Light and electron microscopic examinations showed that collagen-biomatrix-wrapped specimens have the best regeneration. The electrophysiological study confirmed the recovery of electrical activity in the regenerated axons.

Electrical Stimulation Accelerates Motor Functional Recovery in Autograft-Repaired 10 mm Femoral Nerve Gap in Rats

Electrical stimulation has been shown to enhance peripheral nerve regeneration after nerve injury. However, the impact of electrical stimulation on motor functional recovery after nerve injuries, especially over long nerve gap lesions, has not been investigated in a comprehensive manner. In the present study, we aimed to determine whether electrical stimulation (1 h, 20 Hz) is beneficial for motor functional recovery after a 10 mm femoral nerve gap lesion in rats. The proximal nerve stump was electrically stimulated for 1 h at 20 Hz frequency prior to nerve repair with an autologous graft. The rate of motor functional recovery was evaluated by single frame motion analysis and electrophysiological studies, and the nerve regeneration was investigated by double labeling and histological analysis. We found that brief electrical stimulation significantly accelerated motor functional recovery and nerve regeneration. Although the final outcome, both in functional terms and morphological terms, was not improved by electrical stimulation, the observed acceleration of functional recovery and axon regeneration may be of therapeutic importance in clinical setting.

Effects of local release of hepatocyte growth factor on peripheral nerve regeneration in acellular nerve grafts

Options for reconstructing peripheral nerve gaps after trauma are limited. The acellular nerve is a new kind of biomaterial used to reconstruct the peripheral nerve defect, but its use could be improved upon. We aimed to investigate the effect of adenoviral transfection with hepatocyte growth factor (HGF) on the functional recovery of transected sciatic nerves repaired by acellular nerve grafting. 30 Rats were divided into three groups (10/group) for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF (1 × 10 8 pfu) injected in muscles around the proximal and distal allograft coapation. Sciatic functional index (SFI) was evaluated every 4 weeks to week 16 by measuring rat footprints on walking-track testing. The three groups presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. Weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve grafts were evaluated at week 16. Autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Neovascularization was greater with HGF-treated acellular grafting than with autografting and acellular grafting alone. Axonal regeneration distance of autografting on the 20th postoperative day was the longest in the three groups,while that of acellular grafting alone was the smallest. Acellular nerve grafting may be useful for functional peripheral nerve regeneration, and with human HGF gene transfection may improve on acellular grafting alone in functional recovery.

Experimentelle Untersuchungen zur Verbesserung der Fazialisregeneration

Using a combination of the following, it is possible to investigate procedures to improve the morphological and functional regeneration of the facial nerve in animal models: 1) retrograde fluorescence tracing to analyse collateral axonal sprouting and the selectivity of reinnervation of the mimic musculature, 2) immunohistochemistry to analyse both the terminal axonal sprouting in the muscles and the axon reaction within the nucleus of the facial nerve, the peripheral nerve, and its environment, and 3) digital motion analysis of the muscles. To obtain good functional facial nerve regeneration, a reduction of terminal sprouting in the mimic musculature seems to be more important than a reduction of collateral sprouting at the lesion site. Promising strategies include acceleration of nerve regeneration, forced induced use of the paralysed face, mechanical stimulation of the face, and transplantation of nerve-growth-promoting olfactory epithelium at the lesion site.

Skin‐derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

Human Hair Derived Keratins Mediate Schwann Cell Behavior in vitro and Facilitate Rapid Peripheral Nerve Regeneration in vivo

Nerve defects are a common result of peripheral nerve injury and present a significant challenge clinically. Keratins extracted from human hair fiber are a novel group of biomaterials with superior biocompatibility to many synthetic and naturally derived scaffolds. The specific aims of this study were to investigate the ability of human hair keratins to mediate Schwann cell behavior in vitro and to determine whether keratin scaffolds are capable of accelerating functional nerve regeneration in vivo. Using a variety of in vitro assays we have found that keratins increase Schwann cell proliferation, migration and attachment. In addition, keratins have the ability to self-assemble into fibrous and highly porous scaffolds suitable for cell infiltration and axonal growth. To determine whether a keratin hydrogel scaffold could facilitate functional nerve regeneration in vivo, a tibial nerve axotomy mouse model was employed. After 6 weeks, the extent of regeneration was evaluated using electrophysiology and muscle force testing. Functional testing showed that keratin filled conduits significantly enhanced the nerve regeneration process in comparison to empty conduit controls, and were comparable to autograft. This data demonstrates that keratin hydrogels serve as effective scaffolds for neural tissue engineering and may facilitate functional nerve regeneration by acting on Schwann cells.

Effects of alleviated tension at the nerve repair site using biodegradable tubular conduits: Histological, electrophysiological and functional results in a rat model

BACKGROUND: The effect of mobilization on the functional regeneration of peripheral nerves remains controversial. Functional efficacy of alleviated tension at the nerve repair site in a full range of motion model in the rat was recently published elsewhere. This study sought to determine the effect of alleviated tension at the nerve repair site using biodegradable tubular conduits for segmental nerve splinting to block, thereby blocking variable tension. METHODS: In 60 rats, the right sciatic nerve was transected midthigh and coapted immediately microsurgically. The animals were divided into four groups. Group N, epineural nerve repair (control). For the groups listed below, segmental tubular nerve splinting with alleviated in situ tension at the nerve suture site was performed using tubular conduits of different biodegradable materials. Polyurethane group, biodegradable polyurethane tubular conduits. Neuragen group, collagen I tubular conduits. Neurolac group: DL-lactide-e-caprolactone tubular conduits. Full range of motion of lower limbs was ensured by passive motion of hind limbs once a week after functional testing. Histological and electrophysiological assessment and 12 postoperative weekly function tests (toe spread test and BBB Locomotor Rating Scale) were carried out. RESULTS: Functional results were significantly better in all groups using segmental biodegradable tubular nerve splinting for alleviated in situ tension at the nerve repair site, mainly because of less endoneural scar formation at the nerve suture segment. However, this effect was attenuated when polyurethane tubular conduits were used. Electrophysiologic results showed increased amplitude and compound nerve action potential area in the alleviated tension groups. Nerve conduction velocity was fastest in the Neuragen group. CONCLUSIONS: Complete alleviation of in situ tension at the nerve suture site with biodegradable tubular conduits seems to improve functional peripheral nerve regeneration by significant decrease of endoneural collagenization in the nerve suture segment. Additionally, our findings suggest that alleviated tension in the nerve suture segment using biodegradable conduits depends upon which material is used.