Expression of type I and III collagen and laminin beta1 after rat sciatic nerve crush injury

Extracellular matrix changes are thought to be essential to the regeneration of peripheral nerves. The production of this matrix is believed to be regulated by interactions between axons and their supporting cells. In this study matrix production and cell proliferation were studied during rat sciatic nerve regeneration after a crush injury, and compared to that after rat sciatic nerve transection. Expression of proalpha1(I) and proalpha1(III) collagen and laminin beta1 mRNAs was followed in isolated endoneuria by Northern and in situ hybridization both proximally and distally to the site of either a crush injury or transection of rat sciatic nerve up to 18 weeks. Changes in the Schwann cell and fibroblast populations were monitored by morphometric analysis of endoneurial cross-sections immunostained for S-100 protein. The process of axonal regeneration was followed by Bielschowsky's silver staining. A crush injury initially resulted in increased expression of all mRNAs studied in the endoneurial cells. However, with progressing axonal regeneration the amount of collagen mRNAs returned to control levels, whereas the amount of laminin beta1 mRNA in the distal site of the crush remained elevated throughout the study period. The expression of type I collagen mRNA was enhanced after nerve transection injury compared to that after the crush injury. The epineurial fibroblasts actively expressed both type I and III collagen mRNAs after the injury. The proliferation of Schwann cells and the expression of collagen mRNAs are not, at least directly, related to the axonal regeneration. However, the long-lasting and strong expression of laminin beta1 mRNA after a nerve crush injury may be related to good axonal regeneration. The expression of type I collagen in the epineurium may lead to clinically well-recognized epineurial scarring and thus impede axonal regeneration.     

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

Reelin is transiently expressed in the peripheral nerve during development and is upregulated following nerve crush

Reelin is an extracellular matrix protein which is critical for the positioning of migrating post-mitotic neurons and the laminar organization of several brain structures during development. We investigated the expression and localization of Reelin in the rodent peripheral nerve during postnatal development and following crush injury in the adult stage. As shown with Western blotting, immunocytochemistry and RT-PCR, Schwann cells in the developing peripheral nerve and in primary cultures from neonatal nerves produce and secrete Reelin. While Reelin levels are downregulated in adult stages, they are again induced following sciatic nerve injury. A morphometric analysis of sciatic nerve sections of reeler mice suggests that Reelin is not essential for axonal ensheathment by Schwann cells, however, it influences the caliber of myelinated axons and the absolute number of fibers per unit area. This indicates that Reelin may play a role in peripheral nervous system development and repair by regulating Schwann cell-axon interactions.     

Induction of neuropilins-1 and -2 and their ligands, Sema3A, Sema3F, and VEGF, during Wallerian degeneration in the peripheral nervous system

The neuropilins, NP-1 and NP-2, are coreceptors for Sema3A and Sema3F, respectively, both of which are repulsive axonal guidance molecules. NP-1 and NP-2 are also coreceptors for vascular endothelial growth factor (VEGF). The neuropilins and their ligands are known to play prominent roles in axonal pathfinding, fasciculation, and blood vessel formation during peripheral nervous system (PNS) development. We confirmed a prior report (Exp. Neurol. 172 (2001) 398) that VEGF mRNA levels rise during Wallerian degeneration in the PNS and herein demonstrate that NP-1, NP-2, Sema3A, and Sema3F mRNA levels increase in peripheral nerves distal to a transection or crush injury. In a sciatic nerve crush model, in which axonal regeneration is robust, the highest levels of Sema3F mRNA below the injury site are in the epi- and perineurium. Our results suggest the possibility that the neuropilins and their semaphorin ligands serve to guide, rather than to impede, regenerating axons in the adult PNS.     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Induction of parathyroid hormone-related peptide following peripheral nerve injury: role as a modulator of Schwann cell phenotype

Parathyroid hormone-related peptide (PTHrP) is widely distributed in the rat nervous system, including the peripheral nervous system, where its function is unknown. PTHrP mRNA expression has recently been shown to be significantly elevated following axotomy of sympathetic ganglia, although the role of PTHrP was not investigated. The role of PTHrP in peripheral nerve injury was investigated in this study using the sciatic nerve injury model and dorsal root ganglion (DRG) explant model of nerve regeneration. We find that PTHrP is a constitutively secreted peptide of proliferating Schwann cells and that the PTHrP receptor (PTH1R) mRNA is expressed in isolated DRG and in sciatic nerve. Using the sciatic nerve injury model, we show that PTHrP is significantly upregulated in DRG and in sciatic nerve. In addition, in situ hybridization revealed significant localization of PTHrP mRNA to Schwann cells in the injured sciatic nerve. We also find that PTHrP causes a dramatic increase in the number of Schwann cells that align with and bundle regrowing axons in explants, characteristic of immature, dedifferentiated Schwann cells. In addition to stimulating migration of Schwann cells along the axonal membrane, PTHrP also stimulates migration on a type 1 collagen matrix. Furthermore, treatment of purified Schwann cell cultures with PTHrP results in the rapid phosphorylation of the cAMP response element protein, CREB. We propose that PTHrP acts by promoting the dedifferentiation of Schwann cells, a critical requirement for successful nerve regeneration and an effect consistent with known PTHrP functions in other cellular differentiation programs.     

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.     

Spatiotemporal increases in epidermal growth factor receptors following peripheral nerve injury.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.     

Dorsal root ganglion neurons up-regulate the expression of laminin-associated integrins after peripheral but not central axotomy

The favorable prognosis of regeneration in the peripheral nervous system after axonal lesions is generally regarded as dependent on the Schwann cell basal lamina. Laminins, a heterotrimeric group of basal lamina molecules, have been suggested to be among the factors playing this supportive role. For neurons to utilize laminin as a substrate for growth, an expression of laminin binding receptors, integrins, is necessary. In this study, we have examined the expression of laminin binding integrin subunits in dorsal root ganglion (DRG) neurons after transection to either their peripherally projecting axons, as in the sciatic nerve, followed by regeneration, or the centrally projecting axons in dorsal roots, followed by no or weak regenerative activity. In uninjured DRG, immunohistochemical staining revealed a few neurons expressing integrin subunit alpha6, whereas integrin subunits alpha7 and foremost beta1 were expressed in a majority of neurons. After an injury to the sciatic nerve, mRNAs encoding all three integrins were up-regulated in DRG neurons. By anterograde tracing, immunoreactivity for all studied integrins was also found in association with growing axons after a sciatic nerve crush lesion in vivo. In contrast, mRNA levels remained constant in DRG neurons after a dorsal root injury. Together with previous findings, this suggests that integrin subunits alpha6, alpha7, and beta1 have an important role in the regenerative response following nerve injury and that the lack of regenerative capacity following dorsal root injury could in part be explained by the absence of response in integrin regulation.     

Axons modulate the expression of transforming growth factor-betas in Schwann cells

We have investigated the expression of transforming growth factor (TGF)-β1,-β2, and -β3 in developing, degenerating, and regenerating rat peripheral nerve by immunohistochemistry and Northern blot analysis. In normal adult sciatic nerve, TGF-β1, -β2, and -β3 are detected in the cytoplasm of Schwann cells, and the levels of TGF-β1 and -β3 mRNAs are constant during post-natal development. When sciatic nerves are transected to cause axonal degeneration and prevent axonal regeneration, the level of TGF-β1 mRNA in the distal nerve-stump increases markedly and remains elevated, whereas the level of TGF-β3 mRNA falls modestly and remains depressed. When sciatic nerves are crushed to cause axonal degeneration and allow axonal regeneration, the level of TGF-β1 mRNA initially increases as axons degenerate, and then falls as axons regenerate. TGF-β2 mRNA was not detected in developing or lesioned sciatic nerves at any time. Cultured Schwann cells have high levels of TGF-β1 mRNA, the amount of which is reduced by forskolin, which mimicks the effect of axonal contact. These data demonstrate that Schwann cells express TGF-β1, -β2, and -β3, and that TGF-β1 and -β3 mRNA predominate over TGF-β2 mRNA in peripheral nerve. Axonal contact and forskolin decrease the expression of TGF-β1 in Schwann cells. © 1993 Wiley-Liss, Inc.     

Induction of myelin genes during peripheral nerve remyelination requires a continuous signal from the ingrowing axon

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were (1) down-regulated to basal levels (PO and MBP mRNAs), (2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), (3) upregulated (mRNAs encoding 2′3′-cyclic nucleotide phosphodiesterase and β-actin), or (4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons. © 1993 Wiley-Liss, Inc.     

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.     

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin ( reeler ) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.     

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.     

Expression of the netrin receptor UNC-5 in lamprey brain: modulation by spinal cord transection

The sea lamprey recovers from spinal cord transection by a process that involves directionally specific regeneration of axons. The mechanisms underlying this specificity are not known, but they may involve molecular cues similar to those that guide the growth of spinal cord axons during development, such as netrins and semaphorins. To test the role of guidance cues in regeneration, we cloned netrin and its receptor UNC-5 from lamprey central nervous system (CNS) and studied their expression after spinal cord transection. In situ hybridization showed that (1) mRNA for netrin is expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells; (2) mRNA for UNC-5 is expressed in lamprey reticulospinal neurons; (3) following spinal cord transection, UNC-5 message was dramatically downregulated at two weeks, during the period of axon dieback; (4) UNC-5 message was upregulated at three weeks, when many axons are beginning to regenerate; and (5) axotomy-induced expression of UNC-5 occurred primarily in neurons whose axons regenerate poorly. Because the UNC-5 receptor is thought to mediate the chemorepellent effects of netrins, netrin signaling may play a role in limiting or channeling the regeneration of certain neurons. These data strengthen the rationale for studying the role of developmental guidance molecules in CNS regeneration.     

Nogo-C is sufficient to delay nerve regeneration

Axonal regeneration succeeds in the peripheral but not central nervous system of adult mammals. Peripheral clearance of myelin coupled with selective CNS expression of axon growth inhibitors, such as Nogo, may account for this reparative disparity. To assess the sufficiency of Nogo for limiting axonal regeneration, we generated transgenic mice expressing Nogo-C in peripheral Schwann cells. Nogo-C includes the panisoform inhibitory Nogo-66 domain, but not a second Nogo-A-specific inhibitory domain, allowing a selective consideration of the Nogo-66 region. The oct-6::nogo-c transgenic mice regenerate axons less rapidly than do wild-type mice after mid-thigh sciatic nerve crush. The delayed axonal regeneration is associated with a decreased recovery rate for motor function after sciatic nerve injury. Thus, expression of the Nogo-66 domain by otherwise permissive myelinating cells is sufficient to hinder axonal reextension after trauma.