Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube

Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination reg-ulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group (epineurium suture). Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-reg-ulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notch1 mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hes1 mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a simi-lar trend as Hes1 mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling.This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.     

Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve

The transforming growth factors beta (TGF-β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-βs, and since TGF-β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF-β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-β 1-3 mRNA were determined for each segment. We demonstrate that TGF-ß1 mRNA levels are higher than those of TGF-ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-ß2 mRNA could not be detected. For TGF-ß1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-β-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-β-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-β-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-β expression. © 1994 Wiley-Liss, Inc.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Axonal regeneration into chronically denervated distal stump

During the first 2 weeks after an injury to peripheral nerve, endoneurial cells proliferate and express integrin 1 and mRNA for collagen types I and III. Clinical results for surgical repair within this time are clearly better than those obtained after delayed (months after original injury) surgery. The question of whether this is due to changes in the proliferative capacity of endoneurial cells or to changes in expression of mRNA for collagen types I and III or integrin 1 was studied using rats. The left common peroneal nerve was transected and allowed to degenerate for 3 and 6 months. After these times, the tibial nerve of the same animals were transected, and the fresh proximal stump of the transected tibial nerve was sutured into the chronically denervated distal stump of the common peroneal nerve. At 3 and 6 weeks after the reoperation, samples were collected from the distal stump for morphometry, immunohistochemistry and in situ hybridization. Proliferating cells and Schwann cells were identified by immunohistochemistry. These cells increased markedly in number during the axonal reinnervation. In situ hybridization revealed that in the epineurium and perineurium, which were fibrotic, especially type I but also type III collagen mRNA were highly expressed. The amount of type I collagen mRNA in the endoneurium seemed to increase with progressing axonal reinnervation. Immunostaining for integrin 1 was negative in these distal stumps. In the present study the proliferation of endoneurial cells and expression of type I collagen mRNA in the endoneurium were similar to those found after immediate regeneration of transected peripheral nerve. However, the failure of endoneurial fibroblasts to express the integrin 1 subunit may indicate an advanced degeneration of the denervated distal stump. Moreover, clear expression of type I and III collagen mRNA in epineurial fibroblasts indicates scarring at the region of the transection and may play a role in prohibitting full structural recovery of the injured peripheral nerve.     

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.     

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.     

Specificity in regenerative outgrowth and target reinnervation by mammalian peripheral axons

There are indications that specific factors are present in the distal stump of transected nerves which preferentially attract axons of the corresponding proximal stump into the distal nerve stumps. However, the impact of these factors is unclear, since there is abundant evidence that numerous regenerating motor and sensory axons are topographically misdirected after nerve transection and repair. Topographic reinnervation is improved after fascicular repair of fasciculated nerves, and quite precise after nerve crush. The latter may not be true, however, for non-myelinated axons, which show a high degree of aberrant growth even after crush. In contrast, regenerative outgrowth appears to be topographically specific after neonatal nerve transection. Reinnervation of muscle fibers appears to be unspecific in adult mammals, but specific after neonatal injury under certain circumstances. Some preference for reinnervation of the appropriate sensory receptors seems to exist although this preference does not preclude reinnervation of receptors by 'foreign' sensory fibers. In conclusion, incorrect topographic and target reinnervation commonly occurs after peripheral regeneration in adult mammals, and most certainly explains some of the functional disturbances after peripheral nerve lesions. Topographic regeneration appears to be better after nerve injury in developing mammals indicating that mechanisms from the developmental period may persist and aid in accurate regenerative outgrowth.     

Temporal and Spatial Expression of Ciliary Neurotrophic Factor after Peripheral Nerve Injury

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions.     

The effects of an autologous transplant on patterns of regeneration in rat sciatic nerve

Autologous transplants are often used in repair of peripheral nerve injury. Quantitative evaluation of the results of such a transplant is obviously desirable. In previous study, we determined numerical and cytologic parameters of the regeneration that followed transection of rat sciatic nerve, but no transplant was used. This work now serves as a basis for evaluating the use of an autologous transplant in the same transection paradigm. Our procedure is to remove 8 mm of sciatic nerve in the thigh. The removed segment is then put into the center of a silicone tube and the proximal and distal stumps of the severed nerve are placed into the ends of the tube. The data show: (1) a high percentage of successful regenerations; (2) a relatively large nerve in the gap; (3) a typical outer perineurium underlying the epineurium; (4) a well-developed fascicular perineurium; and (5) approximately equal numbers of myelinated and unmyelinated axons in the gap and distal stump. If a transplant is not used there are: (1) a greater number of failures of regeneration; (2) a smaller nerve in the gap; (3) a less well-developed fascicular perineurium; (4) unequal numbers of axons in the gap as compared to the distal stump; and (5) no outer perineurium forms. The presence of a typical outer perineurium after a transplant and its absence if a transplant is not used is probably the most striking cytologic difference between the two paradigms. The equal numbers of axons in the gap and distal stump following regeneration after transplantation presumably indicate that all axons in the gap enter the distal stump without branching or ending blindly, a situation that is presumably beneficial and contrasts with the findings when a transplant is not used. Both paradigms show a remarkable increase in the density of blood vessels in the regenerated nerve in the gap between the two stumps. These findings will serve as a basis for further studies on the mechanisms of peripheral nerve regeneration.     

Differences in peripheral nerve degeneration/regeneration between wild-type and neuronal nitric oxide synthase knockout mice

Nitric oxide (NO), a unique biological messenger molecule, is synthesized by three isoforms of the enzyme NO synthase (NOS) and diffuses from the site of production across cellular membranes. A postulated role for NO in degeneration and regeneration of peripheral nerves has been explored in a sciatic nerve model comparing wild-type mice and mice lacking neuronal NOS after transection and microsurgical repair. In NOS knockout mice, regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). In the regenerated nerve, pruning of uncontrolled sprouts was disturbed, leading to an enhanced number of axons, whereas remyelination seemed to be less affected. Delayed regeneration was associated with a delayed recovery of sensor and motor function. In such a context, possible NO targets are neurofilaments and myelin sheaths of the interrupted axon, filopodia of the growth cone, newly formed neuromuscular endplates, and Schwann cells in the distal nerve stump. The results presented suggest that 1) local release of NO following peripheral nerve injury is a crucial factor in degeneration/regeneration, 2) success of fiber regeneration in the peripheral nervous system depends on a regular WD, and 3) manipulation of NO supply may offer interesting therapeutic options for treatment of peripheral nerve lesions.     

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.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons

GAP-43 is a membrane-associated phosphoprotein enriched in elongating axons (Meiri et al., 1986; Skene et al., 1986). After an axon has been interrupted by cutting or crushing a nerve (axotomy), the portion of the axon disconnected from the cell body (distal stump) degenerates and is replaced by the outgrowth (elongation) of regenerating sprouts arising from the proximal stump. Previous studies have shown that increased amounts of pulse-labeled GAP-43 undergo fast axonal transport in regenerating neurons (Benowitz et al., 1981; Skene and Willard, 1981 a, b). Using hybridization with a cloned cDNA probe, I now show that mRNA levels for GAP-43 increase in lumbar sensory neurons of rat after regeneration is initiated by crushing the sciatic nerve; the relatively high levels of GAP-43 mRNA in regenerating neurons are comparable to those in the developing neurons of 5-d-old animals. I further demonstrate that the induction of GAP-43 expression in regenerating neurons coincides temporally with an increase in mRNA levels for class II beta tubulin (Hoffman and Cleveland, 1988), suggesting that the expression of these proteins is closely coordinated during axonal elongation.     

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.     

End-to-side nerve repair: review of the literature

End-to-side (ETS) nerve repair, in which the distal stump of a transected nerve is coapted to the side of an uninjured donor nerve, offers a technique for repair of peripheral nerve injuries where the proximal nerve stump is unavailable or a significant nerve gap exists. Details of animal models are explored including motor and sensory regeneration to further clarify the mechanism of collateral sprouting while eliminating false positive results from contaminating axons. Some experimental studies support the conclusion that sensory or motor reinnervation may be derived from collateral sprouting while others suggest that reinnervation requires an injury to the donor nerve. Clinical experience with ETS neurorrhaphy includes management of upper extremity nerve injury, facial reanimation, reconstruction following tumor ablation, and the prevention of neuroma formation. Our interpretation of the ETS literature suggests that sensory axons may sprout without deliberately attempting to injure them, while motor axons regenerate only in response to a deliberate injury. Experimental and clinical experience with ETS neurorrhaphy has rendered mixed results. Our interpretation of the literature suggests that the success of this technique is dependent upon axonal injury of motor and possibly sensory nerves. While continued clinical and laboratory experimentation with ETS nerve repair is warranted, it should not yet replace more established techniques of nerve repair.     

Functional aspects of the regeneration of unmyelinated axons in the rat saphenous nerve

Electrophysiological experiments have been carried out to investigate aspects of unmyelinated axon regeneration in a transected cutaneous nerve. Some comparisons with regeneration of myelinated axons in the same nerve have also been made.

By 3 months after injury approximately 80% of the unmyelinated axons that had survived in the proximal stump had regenerated into the distal stump. About the same proportion of myelinated axons had regrown into the distal stump by this time. With both groups of axons there was no marked increase in the amount of regeneration across the injury site with longer recovery times. Conduction velocities in the regenerated unmyelinated axons tended to be slower across the injury site than proximally; the proximal conduction velocities did not differ from those in control nerves. The unmyelinated axons seemed to take longer to resupply the skin than did the myelinated ones, but in both cases the extent of skin innervation had reached about 60% of control values by 6 months after the injury.