Regeneration of the rat sciatic nerve in the silicone chamber model

The silicone nerve regeneration chamber is a useful model to investigate the cellular and molecular events underlying successful regeneration in the peripheral nervous system. In this model a transected rat sciatic nerve with a 10-mm interstump gap, is repaired with a silicone chamber. The spatial-temporal sequence of regeneration in the silicone chamber has been examined in detail. The chamber rapidly becomes filled with fluid which contains neurotrophic activity for neurons in vitro. The second event to occur is the formation of a fibrin matrix connecting the two nerve stumps. This matrix is then invaded by cellular elements in the following order: perineurial-like cells, vasculature, Schwann cells, and axons. The silicone chamber model also allows manipulation of the regeneration process. Prefilling the chamber at the time of implantation with phosphate-buffered saline or dialyzed plasma stimulates nerve regeneration. Multiple injections into the chamber of a mixture containing laminin, testosterone, and ganglioside GM1 increase the size and the vascularization of the regenerate. Specially designed chambers divided into two compartments by a longitudinally inserted nitrocellulose strip have been used to examine the effects of substrate-bound trophic factors on nerve regeneration. Fibroblast growth factor containing chambers have an improved regeneration and vascularization as compared to controls.     

A two-compartment modification of the silicone chamber model for nerve regeneration

In the nerve regeneration silicone chamber model, the regenerate which forms across a 10-mm gap between proximal and distal nerve stumps is a monofascicular structure with an outer perineurial-like cell sheath. Recent work has provided indications that the geometry of the regenerate within a silicone chamber can be altered by experimental modifications of the chamber matrix. In the present study we modified the standard silicone chamber into a two-compartment chamber by inserting a 6- or 10-mm-long siliconized nitrocellulose strip in order to obtain two separate regenerates. Light microscopy 16 days after implantation revealed that two separate nerve structures had formed, one on each side of the nitrocellulose partition and adjacent to it, and each with its own perineurial-like cell sheath. In chambers with 6-mm-long strips a monofascicular regenerate started from the proximal stump and divided into two separate structures as it approached the proximal end of the strip: the two fascicles joined again into a monofascicular structure in the distal portion of the chambers. The new two-compartment silicone chamber model appears suitable for future examinations of experimental fasciculation. In addition, the nitrocellulose partition should allow one to study specific effects of growth factors on axonal regeneration in vivo, as growth factors bind strongly to untreated nitrocellulose while retaining their biological activity.     

The effects of delayed nerve repair on nerve regeneration in a silicone chamber model

The silicone chamber model for nerve regeneration is suitable to test the effects of exogenous agents or surgical manipulations on nerve regeneration. The total 16-day regeneration period used in this model makes it possible to analyze the effects of certain manipulations on the sequential advancement of the individual cellular components (circumferential perineurial-like cells, vessels, Schwann cells, axons, and myelin) into the chamber fibrin matrix. In the present study we compared the effects on cellular migration of a 7 day delayed chamber repair vs. chamber repair immediately after transection (control chambers) of the rat sciatic nerve. Regeneration was evaluated with light and electron microscopic techniques. Chambers implanted after a delay of 7 days had a statistically significant more advanced migration of vessels, Schwann cells, and axons from the proximal nerve stump and also a significantly increased vascular density as compared to control chambers. We conclude that a 7 day delayed nerve repair stimulates nerve regeneration in this specific silicone chamber model.     

The formation of a 'pseudo-nerve' in silicone chambers in the absence of regenerating axons.

The formation of a regenerate between sciatic nerve segments or stumps inserted into Y-tunnelled silicone chambers was studied under conditions where regenerating axons were prevented from entering the chamber. This was accomplished by using an isolated segment of the nerve as a proximal insert. After one week, a cellular regenerate spanned the proximal and distal inserts. The size of the regenerate increased if circulation was preserved in the distal inserts. At four weeks, a perineurium-like sheath surrounded the regenerate and longitudinally oriented Schwann cell columns could be observed throughout the regenerate. A similar 'pseudo-nerve' formed towards a piece of distally inserted tendon. Thus, the information required for the formation of a nerve-like structure is inherent to the non-neuronal cells entering the chamber. Schwann cells, in contrast to regenerating axons, do not exhibit preferential growth towards nervous tissue.     

The formation of a ‘pseudo-nerve’ in silicone chambers in the absence of regenerating axons

The formation of a regenerate between sciatic nerve segments or stumps inserted into Y-tunnelled silicone chambers was studied under conditions where regenerating axons were prevented from entering the chamber. This was accomplished by using an isolated segment of the nerve as a proximal insert. After one week, a cellular regenerate spanned the proximal and distal inserts. The size of the regenerate increased if circulation was preserved in the distal inserts. At four weeks, a perineurium-like sheath surrounded the regenerate and longitudinally oriented Schwann cell columns could be observed throughout the regenerate. A similar ‘pseudo-nerve’ formed towards a piece of distally inserted tendon. Thus, the information required for the formation of a nerve-like structure is inherent to the non-neuronal cells entering the chamber. Schwann cells, in contrast to regenerating axons, do not exhibit preferential growth towards nervous tissue.     

Nerve regeneration chamber: evaluation of exogenous agents applied by multiple injections

In this laboratory, a silicone chamber model for peripheral nerve regeneration in adult rats has been developed and used to define basic principles of the regenerative events, such as the sequential stages being followed during 'spontaneous' regeneration in vivo and the role of neuronotrophic- and neurite-promoting factors as well as extracellular matrix molecules. Each of the defined stages seems amenable to experimental modulation. Previous attempts to enhance regeneration included increasing the volume of the nerve chambers along with the modification of fibrin matrix formation by prefilling with saline (PBS) or matrix precursors. We present here the results of a series of experiments on the effects of exogenous biochemical agents applied by multiple injections into these in vivo chambers. Out of a variety of agents screened, a mixture of laminin (L), testosterone (T), ganglioside GM 1 (G), and catalase (C) was shown to advance substantially the progress of regeneration in 16 day chambers, as compared to PBS-prefilled and PBS-injected controls. LTGC-treatment at day 0, 6, and 10 postimplantation caused an increasingly frequent occurrence of cellular elements in cross-sections obtained from the middle (S5) of the chambers (i.e. 5 mm from the proximal stump), which was 2-fold for vessels, 3-fold for Schwann cells, and 10-fold for axons. When only sections containing axons 3 mm from the proximal stump (S3) were compared in experimental and control groups, computerized area measurements also revealed an average 2-fold difference for the cross-sectional size of the whole regenerate, the endoneurium and the space occupied by blood vessels.     

Spatial-Temporal progress of peripheral nerve regeneration within a silicone chamber: Parameters for a bioassay

The spatial-temporal progress of peripheral nerve regeneration across a 10-mmgap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7–14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1–5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.     

Regeneration of dorsal root axons is related to specific non-neuronal cells lining NGF-treated intraspinal nitrocellulose implants.

The regeneration of sensory axons from severed dorsal roots can be enhanced by the presence of nerve growth factor (NGF)-treated nitrocellulose strips implanted into an intraspinal lesion cavity. Rather than being directly apposed to the transplant, most regenerating axons are separated from the nitrocellulose by several layers of non-neuronal cells, suggesting that these cells may have a role in the promotion of axonal regrowth. The cellular layers associated with untreated nitrocellulose strips or NGF-treated implants were examined in this study to determine if there were differences in their arrangement or orientation along the implant which might explain some of the possible effects of substrate-bound NGF on axonal regrowth. Into a hemisection lesion cavity created in the adult rat lumbar spinal cord NGF-treated or untreated strips of nitrocellulose were placed vertically, with intact pieces of fetal spinal cord (FSC) tissue transplanted along each side. The distal ends of cut dorsal rootlets were apposed to the fetal tissue. Immunocytochemical and electron microscopic examination 30-60 days post-transplantation revealed a distinct layering of cell types along the NGF-treated strips. Closest to the nitrocellulose was a single layer of macrophages, followed by a separate layer of fibroblasts with dense collagen bundles, then a layer of astroglial cells, before reaching the neuropil of the fetal spinal cord tissue. A thickened basal lamina formed between the fibroblast and astrocytic cell layers and bundles of regenerated sensory axons extended along the interface between these two layers. In contrast, non-neuronal cells along untreated nitrocellulose strips were not as well organized, with an intermixing of fibroblasts and astroglial cells and only scattered macrophage-like cells. Axons rarely were found in conjunction with this mixed population of cells and, overall, fewer regenerated axons extended into transplants with untreated nitrocellulose. The results demonstrate consistent differences in the composition and organization of non-neuronal cells adjacent to NGF-treated nitrocellulose implants, compared to untreated implants. This suggests that the presence of bound NGF influences the recruitment of various cells from the surrounding transplant tissue as well as from the previously injured dorsal rootlets. The capacity for NGF to promote the regeneration of sensory axons may be an indirect effect that is mediated or potentiated by the non-neuronal cell population that gathers in response to the presence of bound NGF.     

Cooperative roles of BDNF expression in neurons and Schwann cells are modulated by exercise to facilitate nerve regeneration

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.     

Mesenchymal stem cell treatment for peripheral nerve injury: a narrative review

Peripheral nerve injuries occur as the result of sudden trauma and lead to reduced quality of life.The peripheral nervous system has an inherent capability to regenerate axons.However,peripheral nerve regeneration following injury is generally slow and incomplete that results in poor functional outcomes such as muscle atrophy.Although conventional surgical procedures for peripheral nerve injuries present many benefits,there are still several limitations including scarring,difficult accessibility to donor nerve,neuroma formation and a need to sacrifice the autologous nerve.For many years,other therapeutic approaches for peripheral nerve injuries have been explored,the most notable being the replacement of Schwann cells,the glial cells responsible for clearing out debris from the site of injury.Introducing cultured Schwann cells to the injured sites showed great benefits in promoting axonal regeneration and functional recovery.However,there are limited sources of Schwann cells for extraction and difficulties in culturing Schwann cells in vitro.Therefore,novel therapeutic avenues that offer maximum benefits for the treatment of peripheral nerve injuries should be investigated.This review focused on strategies using mesenchymal stem cells to promote peripheral nerve regeneration including exosomes of mesenchymal stem cells,nerve engineering using the nerve guidance conduits containing mesenchymal stem cells,and genetically engineered mesenchymal stem cells.We present the current progress of mesenchymal stem cell treatment of peripheral nerve injuries.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

自体神经片段串联再生室修复神经缺损

背景：周围神经缺损在现代生产生活中极为常见,自体神经移植被公认为是修复神经缺损的“金标准”;但是自体神经来源受限,且会导致供区感觉功能障碍,感觉神经细小,无法满足较粗神经缺损的需要,限制了其在临床上的广泛应用。因此,人们一直在努力寻找一种自体神经替代物来桥接神经缺损,同种异体神经﹑自体非神经组织、高分子人工合成材料以及二十世纪80年代兴起的组织工程学人工神经研究,用于修复周围神经缺损,但效果都不理想,临床应用还有很大距离距。 目的：本研究即不是风靡世界的人工神经研究,也不是各种神经细胞的培养,而是从另外一个新角度,使神经缺损很简便易行的转化成多个再生室用自体神经片段串联起来,完成修复。探讨对长距离神经缺损修复的一种新方法。 设计、时间及地点：市场购买成品壳聚糖粉、胶原、CNTF、成年SD大耳白兔。随机分组。在辽宁医学院组织胚胎实验室,附属第一医院动物实验室于2009年6月至2010年1月实验完成。 材料：日本大耳白兔28只,辽宁医学院实验动物中心提供。成品壳聚糖粉、胶原、CNTF、Sigma公司产品。 方法：健康日本大耳白兔28只（4周龄）, 雌雄不限,随机分为A、B、C、D四组,每组7只。麻醉下解剖大耳白兔坐骨神经,分别在梨状肌下缘6mm以远,造成右侧坐骨神经12mm、16 mm、30mm的缺损。A组采用30mm自体神经切取后,翻转180度进行桥接;B、C、D组分别采用10mm自体神经片段串联两个等长(6mm、8mm、10mm)壳聚糖-胶原-CNTF复合再生室桥接坐骨神经缺损。 主要观察指标：四通道肌电图仪、BH-2型光学显微镜及摄像系统、日本日立H/7500电镜检测神经传导速度和再生神经有髓纤维数目。 结果：术后24周A、B、C、D四组坐骨神经传导速度（41.99±2.10）m/s （39.79±2.20 ）m/s（27.94±1.67）m/s（19.89±1.57）m/s;最远端所取标本神经纤维数目（612.8±7.63）.（604.5±7.18）.（341.8±7.19）.（276.2±7.52）。以上指标A、B组比较差异无统计学意义(P >0.05);A组与D、 C组比较差异有统计学意义（ P<0.05）。 结论：1、壳聚糖-胶原-CNTF再生室具有良好的组织相容性,对大白兔坐骨神经缺损具有良好的桥梁作用和促进神经生长作用。 2、串联两个6mm壳聚糖-胶原-CNTF复合再生室修复坐骨神经12mm缺损的效果,近似自体神经移植效果。 3、串联再生室自体神经片段的雪旺细胞是可以分泌多种神经活性物质,在有效趋化距离内,神经活性物质可充分发挥其作用。     

Modification of fibrin matrix formation in situ enhances nerve regeneration in silicone chambers

The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 microliter. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefilling affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2.6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.     

Regenerative sprouting of retinal ganglion cells of adult hamsters induced by the epineurium of a peripheral nerve

Although it is known that transplantation of a peripheral nerve (PN) to the damaged central nervous system (CNS) promotes axonal regeneration, the interactions of cellular components of the PN with CNS neurons are still not well defined. Schwann cells in the PN are thought to be the major element involved in supporting CNS regeneration, but very little information exists with regard to whether other PN components also play an active role. Using our previously established model of transplanting a PN segment into the vitreous to stimulate regenerative sprouting of retinal ganglion cells (RGCs), we found that the epineurium isolated from a PN which had been pre-injured by transection was able to induce RGC sprouting when implanted intravitreally. Since the epineurium is composed mainly of connective tissue components and is devoid of Schwann cells, our results suggest that other cellular elements of the PN besides Schwann cells may have the potential to support CNS regeneration.     

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

A diffusion-reaction model of nerve regeneration

The process of peripheral nerve regeneration has been modeled using 5 populations of mathematical variables to represent the biological activities of Wallerian degeneration, fibrin matrix development, Schwann cell activity, elongating neurites, and neovascularization. The mathematical model provided simulations of nerve regeneration following transection and crush injuries that correspond with growth behaviors quantified in biological experiments. Neovascularization was spatiotemporally quantified in nerve regeneration chambers and following nerve crush injury in order to test the simulations of the mathematical model. The vasculature in both the chamber and following nerve crush responded as predicted by the model, increasing beyond normal levels to a peak only to decrease back to normal. This behavior appeared as a traveling wave in the proximal-distal direction preceding the major thrust of neuritic outgrowth suggesting that development of the vasculature is a rate-limiting step in nerve regeneration.     

Peripheral nerve regeneration through silicone chambers in streptozocin-induced diabetic rats

The silicone chamber model was used to evaluate peripheral nerve regeneration (PNR) in streptozocin (STZ)-induced diabetic rats. Diabetic and control animals underwent sciatic nerve transection and silicone chamber implantation establishing gaps of various lengths between the transected nerve ends. In animals with 5 and 10 mm gaps, diabetes was induced in experimental rats 1 week before surgery, and the animals were sacrificed 3 weeks after surgery. In animals with 8 mm gaps, diabetes induction occurred 3 days after surgery, and they were sacrificed after 7 weeks. Diabetic rats with 10 mm gaps demonstrated an impaired ability to form bridging cables, the initial step of regeneration through chambers. Morphometric studies of bridging cables between transected nerve ends demonstrated a significant reduction in the mean endoneurial area in diabetic animals with 5 and 8 mm gaps compared to controls. The number of regenerated myelinated axons in the chamber was significantly decreased in diabetic rats with 8 and 10 mm gaps. The mean myelinated fiber area in the regenerated cables of the diabetic group was significantly decreased with 5 mm gaps and significantly increased with 8 mm gaps compared to controls. Size-frequency histograms of regenerated myelinated fiber areas suggest a delay in the maturation of small caliber axons. Schwann cell migration across 5 mm gaps was examined with S-100 immunohistochemistry. The total distance of Schwann cell migration into cables from both proximal and distal ends was significantly reduced in diabetic animals. Characterization of PNR across gaps through silicone chambers in diabetic rats showed impairment in multiple aspects of the regenerative process, including cable formation, Schwann cell migration, and axonal regeneration.     

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.     

Axon and Schwann cell partnership during nerve regrowth

Regeneration of peripheral nerve involves an essential contribution by Schwann cells (SCs) in collaboration with regrowing axons. We examined such collaboration between new axons and Schwann cells destined to reform peripheral nerve trucks in a regeneration chamber bridging transected rat sciatic nerves. There was a highly intimate "dance" between axons that followed outgrowing and proliferating SCs. Axons without SCs only grew short distances and almost all axon processes had associated SC processes. When regeneration chambers were infused through an external access port with local mitomycin, a mitosis inhibitor, SC proliferation, migration and subsequent axon regrowth were dramatically reduced. Adding laminin to mitomycin did not reverse this regenerative lag and indicated that SCs provide more than laminin synthesis alone. Laminin infused alone supplemented endogenous laminin and facilitated first SC then axon regrowth. "Wrong way" misdirected axons were associated with misdirected SC processes and were more numerous in bridges exposed to mitomycin, but were fewer in laminin supplemented bridges. Later, by 21 days, there was myelinated axon repopulation of regenerative bridges but those exposed to mitomycin alone at early time points had substantial impairments in axon investment. Reforming peripheral nerve trucks involves a very close and intimate relationship between axons and SCs that must proliferate and migrate, facilitated by laminin.     

The role of extracellular matrix in peripheral nerve regeneration: a wound chamber study

Summary A wound chamber model was used for the study of the interaction between axon, Schwann cell and extracellular matrix during peripheral nerve regeneration. Impermeable silicone tubes, 8 mm long and 1.4 mm in internal diameter were sutured to transected rat sciatic nerve and the contents of the tubes were removed at intervals for chemical, histological, immunocytochemical and electron microscopic studies. There was an initial phase of fluid accumulation and the formation of a fibrin/fibronectin clot or cable which connected the cut ends of the nerve. The chamber fluid was shown to have a protein profile similar to that of rat serum. Schwann cells, endothelial cells and fibroblasts migrated first into the cable, apparently mediated by cell-fibrin interaction. Axons buried within the Schwann cell cytoplasm were led into the cable but an axon-fibrin interaction was not observed. After 1 week, the fibrin matrix underwent dissolution, with replacement by collagen. This marked the onset of myelination and the organization of nerve fibers into fascicles. The findings from the present study suggest that the interactions between axon and Schwann cell and between Schwann cell and a changing extracellular matrix are the essential driving force in nerve growth and differentiation during peripheral nerve regeneration.Supported by a grant from the National Science Council of R. O. C. (NSC 80-0412-B075-67)