Lesion-induced changes in the expression of ciliary neurotrophic factor and its receptor in rat optic nerve

There is evidence that ciliary neurotrophic factor (CNTF) is involved in reactive changes following lesions of the nervous system. To investigate, whether differences in the regulation of CNTF and CNTF receptor α (CNTFRα) contribute to the differences in PNS and CNS responses to injury, we have studied their expression on the mRNA and protein level in the rat optic nerve following a crush lesion to compare them with the situation in peripheral nerve. Seven days after the lesion, CNTF mRNA and protein levels were markedly decreased at the lesion site, concommitant with the disappearance of GFAP- and CNTF-immunopositive astrocytes. CNTF levels in proximal and distal parts were less affected. This was in contrast to the situation in the PNS, where CNTF was downregulated at and distal to the lesion site. Different from other CNS regions, optic nerve astrocytes expressed CNTFRα mRNA under normal conditions. Following lesion, CNTFRα was reduced substantially only in distal and proximal parts of the optic nerve but continued to be expressed at high levels at the lesion site, suggesting that GFAP-negative, CNTF-responsive cells are present there. Our results suggest that differences in lesion-induced changes in the optic and sciatic nerve reflect differences in the response to injury of astrocytes and Schwann cells. In the light of the known actions of CNTF in inducing astrogliosis, the expression pattern observed in the optic nerve indicates that CNTF and CNTFRα are involved in glial scar formation in the lesion area. GLIA 23:239–248, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves.

Ciliary neurotrophic factor (CNTF) is a 200-amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942-8947, 1990). To better understand CNTF's role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low-affinity NGF receptor (p75NGFR), which increases following crush. Thus, the changes in CNTF levels do not reflect an overall down-regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.     

The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.     

Cytokine regulation of MMP-9 in peripheral glia: implications for pathological processes and pain in injured nerve

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.     

Altered Connexin Expression after Peripheral Nerve Injury

The identification of connexin32 (Cx32) in myelinating Schwann cells and the association of Cx32 mutations with peripheral neuropathies suggest a functional role for gap junction proteins in the nerve. However, after nerve crush injury, Cx32 expression dramatically decreases in Schwann cells in the degenerating region, returning to control levels at newly formed nodes of Ranvier and Schmidt–Lantermann incisures by 30 days. The present study examined increases in expression of other connexins that occur after peripheral nerve injury. A 56/58-kDa connexin46 (Cx46) protein species was detected in adult rat sciatic nerve, along with very low levels of Cx46 mRNA. However, by 3 days after crush injury, coincident with changes in Schwann cell phenotype, Cx46 mRNA rapidly increased in the degenerating regions. Additionally, the 56/58-kDa Cx46 protein species present in adult nerve decreased and a 53-kDa Cx46 species, which was also present in cultured Schwann cells, became apparent. Connexin43 (Cx43) mRNA and protein, which was localized to perineurial cells in adult nerve, dramatically increased in endoneurial fibroblasts in the crush and distal regions by 3 days, coincident with macrophage infiltration. By 12 days after injury, Cx43 decreased and was comparable to normal nerve. These results suggest that enhanced expression of Cx46 and Cx43, by nonneuronal cells, may be important for the injury and regenerative responses of peripheral nerves.     

Axonal modulation of myelin gene expression in the peripheral nerve.

Myelin gene expression (P0, MBP, P2, and MAG) was investigated during Wallerian degeneration and in the presence or absence of subsequent axonal regeneration and remyelination. The steady state levels of mRNA and protein were assessed in the crushed or permanently transected rat sciatic nerve at 0, 1, 4, 7, 10, 12, 14, 21, and 35 days after injury. The mRNA and protein steady state levels of the myelin specific genes, P0 and the MBPs, decreased to low yet detectable levels during Wallerian degeneration and returned to normal levels with subsequent axonal regeneration. The steady state level of P2 protein also followed a similar pattern of expression. The steady state level of MAG mRNA decreased to undetectable levels by 4 days of injury in the permanently transected nerve. After crush injury, re-expression of MAG to levels comparable to those of normal nerves preceded that of P2 by 2 days and that of P0 and the MBPs by 3 weeks during axonal regeneration and remyelination. These results support the proposed roles for MAG in the formation of initial Schwann cell-axonal contact required for myelin assembly, for P2 in fatty acid transport during myelination, and for P0 and the MBPs in the maintenance of the integrity and compactness of the myelin sheath. In addition, these results indicate that the expression of the myelin specific genes, P0 and MBP, is constitutive and that the level of myelin specific mRNAs is modulated by axonal contact and myelin assembly.     

Expression of myelin protein gene transcripts by Schwann cells of regenerating nerve.

The expression of many myelin-specific molecules in Schwann cells is profoundly decreased following denervation. This study examines the early reexpression of myelin protein genes associated with reinnervation. Following sciatic nerve crush, the distal, regenerated nerve was divided into appropriate (2.5 or 5 mm) consecutive lengths in which gene expression was monitored using Northern blotting, in situ hybridization, and immunostaining. The spatial separation of the distal axon tip and the more proximally located Schwann cells showing initial upregulation of P0 mRNA was constant over the period of 5-13 days after crush at approximately 3-4 mm in fixed, processed material. Axons associated with Schwann cells showing the initial upregulation were completely or partially enveloped in Schwann cell cytoplasm, with very few having any degree of ensheathment. It is probable that only a limited axon-Schwann cell contact is required for induction of the myelin protein genes. Myelin-associated glycoprotein mRNA was upregulated prior to those for P0 and myelin basic protein which had similar time courses. Reexpression of galactocerebroside also preceded that for P0 mRNA. Signal abundance for all myelin proteins decreased in a proximal to distal direction from the crush site, and with time the "wave" of upregulation moved distally down the nerve. In the more proximal, remyelinating zones, the signal intensity exceeded that of the contralateral normal nerve. Signal intensity also varied considerably between adjacent, expressing Schwann cells. The data provide further evidence of the strong temporospatial relationship between axons and the regulation of myelin protein genes in Schwann cells.     

Growth-associated protein-43 (GAP-43) in the regenerating periodontal Ruffini endings of the rat incisor following injury to the inferior alveolar nerve

Alterations in the levels of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) were examined in the lingual periodontal ligament of the rat incisor following two types of injury (resection and crush) to the inferior alveolar nerve (IAN). In normal animals, GAP-43-like immunoreactive (IR) structures were observed as tree-like ramifications in the alveolar half of the lingual periodontal ligament of incisors. Under immunoelectron microscopy, GAP-43-LI appeared in the Schwann sheaths associated with periodontal Ruffini endings; neither cell bodies of the terminal Schwann cells nor axonal profiles showed GAP-43-LI. During regeneration of the periodontal Ruffini endings following resection of the IAN, GAP-43-LI appeared in the cytoplasm of the terminal Schwann cell bodies and axoplasm of the terminals. The distribution of GAP-43-LI in the Ruffini endings returned to almost normal levels on days 28 and 56 following the injury. The changes in the distribution of GAP-43-LI following the crush injury were similar to those following resection; however, expression of GAP-43-LI was slightly higher for the entire experimental period compared with the resection. The transient expression of GAP-43 in the terminal Schwann cells and axonal profiles of the periodontal Ruffini endings following nerve injury suggests that GAP-43 is closely associated with axon–Schwann cells interactions during regeneration.     

Netrin-1 and peripheral nerve regeneration in the adult rat

Axonal guidance during development of the nervous system is thought to be highly regulated through interactions of axons with attractive, repulsive, and trophic cues. Similar mechanisms regulate axonal regeneration after injury. The netrins have been shown to influence the guidance of several classes of developing axons. Although netrins have been implicated as axonal guidance cues in the developing peripheral nervous system, there has been no direct evidence of netrin-1 expression in either developing or adult peripheral nerve. The present study utilized competitive PCR and immunohistochemistry to demonstrate the localization of netrin-1 within adult rat sciatic nerve. The expression of netrin-1 mRNA and protein was compared for normal or regenerated sciatic nerve 2 weeks following either a crush or a transection and repair injury. The PCR data show that netrin-1 mRNA is normally expressed at low levels in peripheral nerve, and similar low levels are found 2 weeks following a crush injury. However, 2 weeks following nerve transection and repair there is approximately a 40-fold increase in netrin-1 mRNA levels. Immunohistochemistry data show that Schwann cells are the major source of netrin-1 protein in peripheral nerve. Our results suggest that netrin-1 mRNA levels are profoundly affected during peripheral nerve injury and regeneration. The localization of netrin-1 to Schwann cells suggests that this protein is strategically situated to influence axon regeneration in adult peripheral nerve.     

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination fol-lowing peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.     

Axonal signals regulate expression of glia maturation factor-beta in Schwann cells: an immunohistochemical study of injured sciatic nerves and cultured Schwann cells

Glia maturation factor-beta (GMF-beta) is a 17 kDa protein purified and sequenced from bovine brains. Using the monoclonal antibody G2-09 directed against GMF-beta, we previously demonstrated endogenous GMF-beta in astroblasts, Schwann cells, and their tumors in culture. In the present study, we have used indirect immunofluorescence microscopy with G2-09 to examine the effects of transection, crush, and regeneration of sciatic nerve on the expression of GMF-beta in Schwann cells in situ and to study the time course of GMF-beta induction in Schwann cells in vitro. For comparison, a parallel study was carried out with monoclonal antibodies directed against nerve growth factor (NGF) receptor. We found that (1) neither GMF-beta nor NGF receptor was detectable in intact sciatic nerves, (2) all Schwann cells of the distal segment of the transected nerve expressed GMF-beta as early as 3 d after axotomy that persisted up to 3 weeks, (3) axonal regeneration repressed the Schwann cell expression of GMF-beta, (4) isolated Schwann cells derived from rat sciatic and adult human sural nerves developed intracellular GMF-beta in culture following an initial lag period, and (5) the induction of Schwann cell NGF receptor coincided temporally with that of GMF-beta in the transected nerve and in culture. These results show that the expression of GMF-beta in Schwann cells, as is the case with the NGF receptor, is induced by the loss of the normal axon-Schwann cell contact. We propose that the induction of GMF-beta, as well as NGF receptor, in Schwann cells after nerve injury plays a role in axonal regeneration.     

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.     

Immunohistochemical localization of CNTFRalpha in adult mouse retina and optic nerve following intraorbital nerve crush: evidence for the axonal loss of a trophic factor receptor after injury

Ciliary neurotrophic factor (CNTF) is important for the survival and outgrowth of retinal ganglion cells (RGCs) in vitro. However, in vivo adult RGCs fail to regenerate and subsequently die following axotomy, even though there are high levels of CNTF in the optic nerve. To address this discrepancy, we used immunohistochemistry to analyze the expression of CNTF receptor alpha (CNTFRalpha) in mouse retina and optic nerve following intraorbital nerve crush. In normal mice, RGC perikarya and axons were intensely labeled for CNTFRalpha. At 24 hours after crush, the immunoreactivity normally seen on axons in the nerve was lost near the lesion. This loss radiated from the crush site with time. At 2 days postlesion, labeled axons were not detected in the proximal nerve, and at 2 weeks were barely detectable in the retina. In the distal nerve, loss of axonal staining progressed to the optic chiasm by 7 days and remained undetectable at 2 weeks. Interfascicular glia in the normal optic nerve were faintly labeled, but by 24 hours after crush they became intensely labeled near the lesion. Double labeling showed these to be both astrocytes and oligodendrocytes. At 7 days postlesion, darkly labeled glia were seen throughout the optic nerve, but at 14 days labeling returned to normal. It is suggested that the loss of CNTFRalpha from axons renders RGCs unresponsive to CNTF, thereby contributing to regenerative failure and death, while its appearance on glia may promote glial scarring.     

Distribution of albumin-like immunoreactivity in rat sciatic nerve after nerve crush injury

To understand better the role of local factors in the response of peripheral nerve to crush injury, we studied the distribution of albumin-like immunoreactivity (A-LI) in the rat sciatic nerve from one day to eight weeks (wk) after a crushing injury; we used electron microscopic immunocytochemistry. In the nerve distal to the crush degenerating axons demonstrated intra-axonal A-LI, and by one wk most of the Schwann cells also showed A-LI. As regenerating sprouts entered the distal nerve, those Schwann cells in contact with sprouts lost their A-LI, while those cells not in contact with axons retained immunoreactivity up to eight wk after injury. Proximal to the nerve crush many axons showed intra-axonal A-LI from one to two wk after injury, despite appearing normal ultrastructurally. This immunoreactivity diminished as the distance from the crush site increased. Many Schwann cells proximal to the crush also showed A-LI from one to four wk after injury. These findings suggest that an albumin-like protein may play a role in the response of Schwann cells and axons to injury.     

Prosaposin: a myelinotrophic protein that promotes expression of myelin constituents and is secreted after nerve injury.

Recently, we demonstrated that prosaposin and prosaptides (peptides encompassing the neurotrophic sequence in prosaposin) prevent cell death and increase extracellular regulated kinase (ERK) phosphorylation and sulfatide content in primary Schwann cells or oligodendrocytes (Hiraiwa et al., 1997a). Here, we examine the effect of prosaptide on other myelin constituents, on Schwann cell morphology and proliferation, and characterize the time course of expression of prosaposin protein after sciatic nerve injury. After 24 h of treatment with 10 nM TX14(A), a 14-mer prosaptide, the specific activity of UDP-galactose:ceramide galactosyltransferase (GalT) in primary Schwann cells was increased by 150% over controls. Under the same conditions, the maximum content of sulfatide increased 3-fold over controls after 48 h of treatment. Northern blot analysis, probed with oligonucleotide sequences from the GalT and P0 cDNAs, revealed that the mRNA levels of GalT and P0 protein were elevated about 30 and 200%, respectively, over controls after 24 h of treatment with TX14(A). Treatment of primary Schwann cells with TX14(A) also induced a morphological change at 10 nM; the peptide-treated cells had a bipolar (spindle-shaped) appearance after 48 h of treatment, compared to control cells which were irregular and multipolar. TX14(A) did not induce cell proliferation, indicating that TX14(A), unlike IGF-I, is not mitogenic. After sciatic nerve transection, Western blot analysis demonstrated the presence of intact prosaposin in tubular fluid in a silicon chamber into which the proximal and distal nerve stumps were sutured. The concentration of prosaposin in the fluid was maximum after 9 days post-surgery and returned to normal after 28 days post-surgery. In uninjured and injured nerve, prosaposin immunolocalized to the smooth muscle of epineurial and endoneurial vessels. These findings indicated that sciatic nerve secreted prosaposin after injury and that prosaposin is a naturally occurring injury-repair protein which acts to prevent degeneration and to promote regeneration of peripheral nerves.     

N-myc downstream-regulated gene 1 expression in injured sciatic nerves

N-myc downstream-regulated gene 1 (NDRG1)/RTP/Drg1/Cap43/rit42/TDD5/Ndr1 is expressed ubiquitously and has been proposed to play a role in growth arrest and cell differentiation. A recent study showed that mutation of this gene is responsible for hereditary motor and sensory neuropathy-Lom. However, the role of this gene in the peripheral nervous system is not fully understood. In our study, rabbit polyclonal antibodies were raised against this gene product and were used to examine changes in its expression over the time course of Wallerian degeneration and ensuing regeneration after crush injury of mouse sciatic nerves. Fluorescent immunohistochemistry showed that NDRG1 was expressed over the intact nerve fibers. Double labeling with a Schwann cell (SC) marker, S-100 protein (S-100), revealed that NDRG1 was localized in the cytoplasm of S-100-positive Schwann cells (SCs). NDRG1 expression was maintained in the early stage of myelin degradation but was then markedly depleted at the end stage of myelin degradation when frequent occurrence of BrdU-labeled SCs was observed (at 7-9 days). The depletion of NDRG1 at this time point was also confirmed by Western blotting analysis. NDRG1 expression finally recovered at the stage of remyelination, with immunoreactivity stronger than that in intact nerves. These findings suggest that NDRG1 may play an important role in the terminal differentiation of SCs during nerve regeneration.     

Delayed Immediate Early Gene Responses Precede Delayed Initiation Of Regeneration In Diabetic Nerve

Nerve fiber regeneration is impaired in diabetic nerve and contributes to the relentless nerve fiber loss characterizing this disorder. Immediate early gene responses constitute the initial response to nerve injury and include upregulation of NGF and IGF-1 primarily by Schwann cells. These responses are believed to initiate macrophage recruitment necessary for initiation of axonal regeneration. We examined NGF, IGF-1 and CNTF mRNA in sciatic nerve at 10 timepoints (0.5hr to 24d) following sciatic nerve crush in diabetic BB/W-rats. The peak of the immediate upregulation of IGF-1 and NGF occurred at 0.5 and 6 hrs respectively in control nerves and was delayed to 24 hrs and 2d for IGF-1 and NGF respectively in diabetic nerve. Also the expression of NGF p75 receptor was significantly attenuated in diabetic nerve. CNTF mRNA showed an immediate downregulation following nerve crush with no significant differences between control and diabetic rats. These findings suggest that attenuations of the immediate gene responses of para- and autocrine IGF-1 and NGF in diabetic nerve may be responsible for the earlier reported defect in macrophage recruitment and delayed initiation of nerve fiber regeneration.