Transplantation of embryonic spinal cord neurons to the injured distal nerve promotes axonal regeneration after delayed nerve repair

Peripheral nerve injury (PNI) usually results in poor functional recovery. Nerve repair is the common clinical treatment for PNI but is always obstructed by the chronic degeneration of the distal stump and muscle. Cell transplantation can alleviate the muscle atrophy after PNI, but the subsequent recovery of the locomotive function is seldom described. In this study, we combined cell transplantation and nerve repair to investigate whether the transplantation of embryonic spinal cord cells could benefit the delayed nerve repair. The experiment consisted of 3 stages: transection of the tibial nerve to induce ‘pre‐degeneration’, a second surgery performed 2 weeks later for transplantation of E14 embryonic spinal cord cells or vehicle (culture medium) at the distal end of the injured nerve, and, 3 months later, the removal of the grafted cells and the cross‐suturing of the residual distal end to the proximal end of a freshly cut ipsilateral common peroneal (CP) nerve. Cell survival and fate after the transplantation were investigated, and the functional recovery after the cross‐suturing was compared between the groups. The grafted cells could survive and generate motor neurons, extending axons that were subsequently myelinated and forming synapses with the muscle. After the cross‐suturing, the axonal regeneration from the proximal stump of the injured CP nerve and the functional recovery of the denervated gastrocnemius muscle were significantly promoted in the group receiving the cells. Our study presents a new perspective indicating that the transplantation of embryonic spinal cord neurons may be a valuable therapeutic strategy for PNI.     

Chemogenetic enhancement of functional recovery after a sciatic nerve injury

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools used to modulate neuronal excitability. We hypothesized that activation of excitatory (Gq) DREADD by its designer ligand, clozapine‐N‐oxide (CNO), would increase the excitability of neurons whose axons have been transected following peripheral nerve injury, and that this increase will lead to an enhanced functional recovery. The lateral gastrocnemius (LG) muscle of adult female Lewis rats was injected unilaterally with AAV9‐ hsyn‐ hM3Dq‐mCherry (7.6 × 10⁹ viral genomes/μL) to transduce Gq‐DREADD expression in LG neurons. The contralateral LG muscle served as an uninjected control. No significant changes in either spontaneous EMG activity or electrically evoked direct muscle (M) responses were found in either muscle after injection of CNO (1 mg/kg, i.p.). The amplitude of monosynaptic H‐reflexes in LG was increased after CNO treatment exclusively in muscles previously injected with virus, suggesting that Gq‐DREADD activation increased neuronal excitability. After bilateral sciatic nerve transection and repair, additional rats were treated similarly with CNO for up to three days after injury. Electrophysiological data were collected at 2, 4 and 6 weeks after injury. Evoked EMG responses were observed as early as 2 weeks after injury only in Gq‐DREADD expressing virus injected LG muscle. By 4 weeks after injury, both M‐response and H‐reflex amplitudes were significantly greater in muscles previously injected with viral vector than contralateral, uninjected muscles. Increases in the excitability of injured neurons produced by this novel use of Gq‐DREADD were sufficient to promote an enhancement in functional recovery after a sciatic nerve injury.     

One hour electrical stimulation accelerates functional recovery after femoral nerve repair

The clinical outcome of peripheral nerve injuries requiring surgical repair is usually poor and efficient therapies do not exist. Recent work has suggested that low-frequency electrical stimulation of the severed nerve which produces repeated discharges of the parent motoneuron perikarya positively influences axonal regeneration, even if applied once for a period of only 1 h. Here we provide the first evidence for locomotor functional benefits of such stimulation. We transected the femoral nerve of adult C57BL/6J mice proximal to the bifurcation of the quadriceps and saphenous branches and electrically stimulated the proximal nerve stump for 1 h at 20-Hz frequency prior to nerve repair with a silicone cuff. Three months later, the ability of the quadriceps muscle to extend the knee in sham-stimulated mice had recovered to 63% of the preoperative values as estimated by single-frame motion analysis. After electrical stimulation, the outcome was only slightly better (73%) but the rate of functional recovery was considerably accelerated. Near-maximum recovery was achieved 6 weeks earlier than in the control group. The beneficial effects were associated with larger motoneuron cell bodies and increased diameters of regenerated axons in the quadriceps nerve branch, but not with enhanced preferential reinnervation by motoneurons of muscle as opposed to skin. The observed acceleration of functional restoration and the positive effects on motoneurons and regenerated axons indicate the potential of a clinically feasible approach for improvement of nerve repair outcome in human patients in which delayed target reinnervation is a factor limiting recovery.     

Specificity in mammalian peripheral nerve regeneration at the level of the nerve trunk

Previous studies indicate that distal stumps of transected rat peripheral nerves secrete 'tropic' factors which can attract/support axonal regeneration over distances of several mm in vivo. The present study was undertaken in order to determine if there is specificity of neurotropic interaction at the level of the nerve trunk. Proximal stumps of transected peroneal or tibial nerves were inserted into the single inlet end of Y-shaped Silastic implants and offered alternative 'lures' at the paired outlet ends (specifically, grafts of peroneal vs tibial distal stump tissue). Several weeks later, the overwhelming majority of preparations showed exclusive growth of nerve fibers in implant forks attached to 'native' (originally associated) nerve stumps. Inversion of the distal stump grafts (such that the proximal stump was facing an analogous native distal stump, but a different region of it) diminished the frequency and extent of native preference. Taken together, data suggest the possibility that there can be a specificity of nerve regeneration at the level of the nerve trunk.     

Motoneuron survival is not affected by the proximo-distal level of axotomy but by the possibility of regenerating axons to gain access to the distal nerve stump

The aim of this study was to examine whether axotomy-induced motoneuron death in adult mammals differ: (1) with the distance between the site of injury and the nerve cell body, and (2) if contact between the transected nerve stumps is established after the injury, compared with cases where contact is prevented. The hypoglossal nerve of adult rats was transected either proximally in the neck (proximal injury) or close to the tongue (distal injury). The nerve stumps were then either deflected from each other in order to prevent axon regeneration into the distal nerve stump, or sutured. Three months later, the extent of nerve cell loss was examined bilaterally in cresyl violet-stained sections of the hypoglossal nucleus. In addition, we examined hypoglossal neuron survival twelve months after a proximal nerve transection with prevented regeneration. Our results show that there was no significant difference in neuronal survival after a proximal nerve transection compared with a distal one, neither if contact between the nerve stumps was established nor if it was prevented. However, contact between the transected nerve stumps increased the likelihood of neuronal survival significantly after both proximally and distally located injury compared to nerve injury with prevented regeneration. There was no significant decrease in nerve cell survival after twelve months with prevented reinnervation compared with survival after three months. These observations indicate that the extent of axotomy-induced motoneuron death in adult mammals does not correlate with the proximo-distal level of peripheral injury. Furthermore, early contact with the distal stump and/or target musculature is a significant factor for the survival of axotomized motoneurns. However, more than 50% of the original nerve cell population survives a considerable time even after permanent disconnection from the target. Copyright © 1994 Wiley-Liss, Inc.     

Repair of the musculocutaneous nerve using the vagus nerve as donor by helicoid end‐to‐side technique: an experimental study in rats

Although several donor nerves can be chosen to repair avulsed brachial plexus nerve injury, available nerves are still limited. The purpose of this study is to validate whether the vagus nerve (VN) can be used as a donor. Eighteen Sprague‐Dawley male rats were divided into three groups (n = 6). The right musculocutaneous nerve (McN) was transected with differing subsequent repair. (1) HS‐VN group: a saphenous nerve (SN) graft‐end was helicoidally wrapped round the VN side (epi‐and perineurium was opened) with a 30 ° angle, distal SN end was coapted to the McN with end‐to‐end repair. (2) EE‐PN group: a SN was interpositionally grafted between the transected phrenic nerve (PN) and the McN by end‐to‐end coaptation. (3) Sham control group: McN was transected and not repaired and postoperative vital signs were checked daily. At three months, electrophysiology, tetanic force, wet biceps muscle weight, and histology were evaluated. Every tested mean value in HS‐VN group was significantly greater than the EE‐PN or the sham control groups (p < 0.05 or p < 0.005). The mean recovery ratio of regenerated nerve fibers was 96% and, in HS‐VN group, the mean recovery ratio of CMAP was 79%. No vital signs changed in any group. There was no statistical difference (p > 0.5) between the mean VN nerve‐fiber numbers of the segments proximal (2237 ± 134) and distal (2150 ± 156) to the VN graft‐attachment site. Histological analysis revealed no axon injury or intraneural scarring at any point along the VN. This study demonstrated that VN is a practical and reliable donor nerve for end‐to‐side nerve transfer. © 2017 Wiley Periodicals, Inc.     

Simultaneous gradual lengthening of both proximal and distal nerve stumps for repair of peripheral nerve defect in rats

We investigated nerve regeneration following the repair of a segmental nerve defect induced by direct end-to-end neurorrhaphy after simultaneous gradual lengthening of both proximal and distal nerve stumps in rats. A 15-mm-long nerve segment was resected from the sciatic nerve of each rat. The proximal and distal nerve stumps, respectively, were directly lengthened at a rate of 1 mm/day using a custom-made external nerve-lengthening device. After being lengthened for 14 days, both nerve stumps were refreshed, and direct end-to-end neurorrhaphy was performed. For a control, 15-mm nerve grafting was performed immediately after nerve resection. Nerve regeneration was evaluated by motor nerve conduction velocity, muscle contraction force, and histological studies at 6, 8, and 14 weeks after initial nerve resection in both groups. As a result, at 8 and 14 weeks, the motor nerve conduction velocity was significantly higher in the nerve-lengthening group than in the autografting group. In addition, at 14 weeks, the tetanic force and wet weight of the gastrocnemius muscle were significantly higher in the nerve-lengthening group than in the autografting group. Histologically, the mean axonal diameter of myelinated nerve fibers and the total number of myelinated nerve fibers were also significantly higher in the nerve-lengthening group than in the autografting group for each evaluation period. It appears that the simultaneous gradual lengthening of both proximal and distal nerve stumps might have potential application in the repair of peripheral nerve defects.     

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 × 10⁸ 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.     

Electrical stimulation impairs early functional recovery and accentuates skeletal muscle atrophy after sciatic nerve crush injury in rats

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re‐establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N‐CAM) expression, and muscle fiber cross‐sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N‐CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post‐injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury. Muscle Nerve, 2010     

Calcitonin pump improves nerve regeneration after transection injury and repair

Introduction: After nerve injury, excessive calcium impedes nerve regeneration. We previously showed that calcitonin improved nerve regeneration in crush injury. We aimed to validate the direct effect of calcitonin on transected and repaired nerve. Methods: Two rat groups (n = 8) underwent sciatic nerve transection followed by direct repair. In the calcitonin group, a calcitonin‐filled mini‐osmotic pump was implanted subcutaneously, with a catheter parallel to the repaired nerve. The control group underwent repair only, without a pump. Evaluation and comparison between the groups included: (1) compound muscle action potential recording of the extensor digitorum longus (EDL) muscle; (2) tetanic muscle force test of EDL; (3) nerve calcium concentration; and (4) nerve fiber count and calcified spot count. Results: The calcitonin pump group showed superior recovery. Conclusions: Calcitonin affects injured and repaired peripheral nerve directly. The calcitonin‐filled mini‐osmotic pump improved nerve functional recovery by accelerating calcium absorption from the repaired nerve. This finding has potential clinical applications. Muscle Nerve 51 : 229–234, 2015     

Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers

Rat sciatic nerves can be transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers. Anatomical regeneration has been demonstrated across 10 mm long chambers containing both stumps, although little or no axonal outgrowth occurs in chambers omitting the distal stump or exceeding the 10 mm length. We have previously shown that chambers containing both proximal and distal stumps accumulate within days of implantation a clear fluid containing neuronotrophic factors (NTFs) directed to neurons from neonatal mouse dorsal root ganglia. We report here that these chamber fluids also have considerable neuronotrophic activity for chick embryo neurons from embryologic day 4 (E4) lumbar spinal cord, E12 sympathetic ganglia, E12 (but not E8) dorsal root ganglia and E8 ciliary ganglia. Thus, the neuronal types supported by trophic factors of these fluids include all those which contribute axons to the sciatic nerve, namely sensory, spinal motor, and sympathetic. In fluid collected 1 week after implantation, NTF levels directed to different neurons varied independently from one another in chambers with different nerve insertions, suggesting that these activities reside in separate factors. Fluid collected from chamber arrangements allowing little proximal fiber regrowth did not always contain correspondingly lower titers of NTFs. However, generally higher titers of all NTFs were found in chambers containing either or both nerve stumps that in nerve-free chambers. Fluids collected from nerve-containing chambers were subjected to heat, dialysis or trypsin treatments. The behavior of their neuronotrophic activities suggests their association with proteins.     

BDNF/TrkB signaling regulates HNK-1 carbohydrate expression in regenerating motor nerves and promotes functional recovery after peripheral nerve repair

Functional recovery after peripheral nerve injury is often poor despite high regenerative capacity of peripheral neurons. In search for novel treatments, brief electrical stimulation of the acutely lesioned nerve has recently been identified as a clinically feasible approach increasing precision of axonal regrowth. The effects of this stimulation appear to be mediated by BDNF and its receptor, TrkB, but the down-stream effectors are unknown. A potential candidate is the HNK-1 carbohydrate known to be selectively reexpressed in motor but not sensory nerve branches of the mouse femoral nerve and to enhance growth of motor but not sensory axons in vitro. Here, we show that short-term low-frequency electrical stimulation (1 h, 20 Hz) of the lesioned and surgically repaired femoral nerve in wild-type mice causes a motor nerve-specific enhancement of HNK-1 expression correlating with previously reported acceleration of muscle reinnervation. Such enhanced HNK-1 expression was not observed after electrical stimulation in heterozygous BDNF or TrkB-deficient mice. Accordingly, the degree of proper reinnervation of the quadriceps muscle, as indicated by retrograde labeling of motoneurons, was reduced in TrkB+/- mice compared to wild-type littermates. Also, recovery of quadriceps muscle function, evaluated by a novel single-frame motion analysis approach, and axonal regrowth into the distal nerve stump, assessed morphologically, were considerably delayed in TrkB+/- mice. These findings indicate that BDNF/TrkB signaling is important for functional recovery after nerve repair and suggest that up-regulation of the HNK-1 glycan is linked to this phenomenon.     

A collagen-based nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates

When a peripheral nerve is severed and left untreated, the most likely result is the formation of an endbulb neuroma; this tangled mass of disorganized nerve fibers blocks functional recovery following nerve injury. Although there are several different approaches for promoting nerve repair, which have been greatly refined over recent years, the clinical results of peripheral nerve repair remain very disappointing. In this paper we compare the results of a collagen nerve guide conduit to the more standard clinical procedure of nerve autografting to promote repair of transected peripheral nerves in rats and nonhuman primates. In rats, we tested recovery from sciatic nerve transection and repair by 1) direct microsurgical suture, 2) 4 mm autograft, or 3) entubulation repair with collagen-based nerve guide conduits. Evoked muscle action potentials (MAP) were recorded from the gastrocnemius muscle at 4 and 12 weeks following sciatic nerve transection. At 4 weeks the repair group of direct suture demonstrated a significantly greater MAP, compared to the other surgical repair groups. However, at 12 weeks all four surgical repair groups displayed similar levels of recovery of the motor response. In six adult male Macaca fascicularis monkeys the median nerve was transected 2 cm above the wrist and repaired by either a 4 mm nerve autograft or a collagen-based nerve guide conduit leaving a 4 mm gap between nerve ends. Serial studies of motor and sensory fibers were performed by recording the evoked MAP from the abductor pollicis brevis muscle (APB) and the sensory action potential (SAP) evoked by stimulation of digital nerves (digit II), respectively, up to 760 days following surgery. Evoked muscle responses returned to normal baseline levels in all cases. Statistical analysis of the motor responses, as judged by the slope of the recovery curves, indicated a significantly more rapid rate of recovery for the nerve guide repair group. The final level of recovery of the MAP amplitudes was not significantly different between the groups. In contrast, the SAP amplitude only recovered to the low normal range and there were no statistically significant differences between the two groups in terms of sensory recovery rates. The rodent and primate studies suggest that in terms of recovery of physiological responses from target muscle and sensory nerves, entubulation repair of peripheral nerves with a collagen-based nerve guide conduit over a short nerve gap (4 mm) is as effective as a standard nerve autograft.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delayed peripheral nerve repair: methods,including surgical ‘cross-bridging' to promote nerve regeneration

Despite the capacity of Schwann cells to support peripheral nerve regeneration, functional recovery after nerve injuries is frequently poor, especially for proximal injuries that require regenerating axons to grow over long distances to reinnervate distal targets. Nerve transfers, where small fascicles from an adjacent intact nerve are coapted to the nerve stump of a nearby denervated muscle, allow for functional return but at the expense of reduced numbers of innervating nerves. A 1-hour period of 20 Hz electrical nerve stimulation via electrodes proximal to an injury site accelerates axon outgrowth to hasten target reinnervation in rats and humans, even after delayed surgery. A novel strategy of enticing donor axons from an otherwise intact nerve to grow through small nerve grafts(cross-bridges) into a denervated nerve stump, promotes improved axon regeneration after delayed nerve repair. The efficacy of this technique has been demonstrated in a rat model and is now in clinical use in patients undergoing cross-face nerve grafting for facial paralysis. In conclusion, brief electrical stimulation, combined with the surgical technique of promoting the regeneration of some donor axons to ‘protect' chronically denervated Schwa nn cells, improves nerve regeneration and, in turn, functional outcomes in the management of peripheral nerve injuries.     

Electromyographic evaluation of a novel surgical preparation to enhance nerve-muscle specificity that follows mammalian peripheral nerve trunk transection

Previous studies indicated that axons from proximal stumps of transected peripheral nerves "prefer" to grow through Silastic tubes attached to their native (originally associated) rather than foreign (not originally associated) distal stumps. We determined whether or not this specificity is expressed at the level of the neuromuscular junction. Proximal stumps of transected rat sciatic nerves (peroneal and tibial branches) were attached to single inlet ends of 6-mm-long, Y-shape Silastic implants. One outlet was attached to the distal peroneal and the other to the distal tibial stump. Ten weeks later, innervation of the anterior tibialis and interosseous muscles (normally innervated predominantly by peroneal and tibial nerve fibers, respectively) was assessed by measuring compound muscle action potential amplitudes and latencies that follow supramaximal peroneal and tibial nerve stimulation. Results showed higher amplitudes in anterior tibialis muscle, induced by "native" peroneal (vs. tibial) stimulation in four of five animals, and higher amplitudes in interosseous muscles after "native" tibial (vs. peroneal) stimulation in all cases examined. Preparations in which bridges between proximal and distal nerve stumps were bridged with unbranched tubes showed random patterns of muscle innervation. The results suggest that if allowed to express "specificity" at the level of nerve trunk transection, regenerating mammalian peripheral axons can grow into, and form functional connection with, native (vs. foreign) muscle groups. This finding has possible clinical significance.     

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats.After sciatic axotomy, a 10 mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively.Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells.The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery.The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.     

Peripheral nerve lengthening as a regenerative strategy

Peripheral nerve injury impairs motor, sensory, and autonomic function, incurring substantial financial costs and diminished quality of life. For large nerve gaps, proximal lesions, or chronic nerve injury, the prognosis for recovery is particularly poor, even with autografts, the current gold standard for treating small to moderate nerve gaps. In vivo elongation of intact proximal stumps towards the injured distal stumps of severed peripheral nerves may offer a promising new strategy to treat nerve injury. This review describes several nerve lengthening strategies, including a novel internal fixator device that enables rapid and distal reconnection of proximal and distal nerve stumps.     

Axonal growth in mesothelial chambers: Effects of a proximal preconditioning lesion and/or predegeneration of the distal nerve stump

Preformed, autologous mesothelial chambers were utilized to study axonal growth following selective predegeneration of the distal nerve stump and/or preconditioning of the proximal nerve stump. The left and/or right sciatic nerve of rats was exposed and transected in the thigh. Two weeks after transection, the left proximal nerve stump was cross-anastomosed with the right distal nerve stump by using a mesothelial chamber leaving a 15-mm gap between the two nerve stumps. Previous studies have shown that axonal overgrowth normally does not occur over this gap distance to the distal stump. Three months after cross-anastomosing, regeneration across the 15-mm gap was evaluated by muscle action potential recordings and light microscopical examination. In experiments in which a distal nerve stump was selectively degenerated and the proximal segment was freshly cut, axons had bridged the 15-mm gap in six of seven rats. When a proximal preconditioned nerve stump was matched with a freshly cut distal stump, axonal overgrowth occurred in only 4 of 10 experiments. In experiments including a proximal preconditioned nerve stump and a distal predegenerated stump, axons bridged the gap in 6 of 8 experiments. We concluded that a priming lesion, including manipulation with proximal and/or distal stump, enhances axonal growth in mesothelial chambers.