Chu Cheng

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.

μ Opioid receptors and analgesia at the site of a peripheral nerve injury

Opioid ligands may exert antinociception through receptors expressed on peripheral afferent axons. Whether local opioid receptors might attenuate neuropathic pain is uncertain. In this work, we examined the function and expression of local μ opioid receptors (MORs) associated with the chronic constriction injury (CCI) model of sciatic neuropathic pain in rats. Low‐dose morphine or its carrier were percutaneously superfused over the CCI site with the injector blinded to the identity of the injectate. Morphine, but not its carrier, and not equimolar systemic doses of morphine reversed thermal hyperalgesia in a dose‐related, naloxone‐sensitive fashion. Moreover, analgesia was conferred at both 48 hours and 14 days after CCI, times associated with very different stages of nerve repair. Equimolar local DAGO ([D‐Ala2, N‐Me‐Phe4, Gly5‐(ol)] enkephalin), a selective MOR ligand, provided similar analgesia. Local morphine also attenuated mechanical allodynia. MOR protein was expressed in axonal endbulbs of Cajal just proximal to the injury site, in aberrantly regenerating small axons in the epineurial sheath around the CCI site and in residual small axons distal to the CCI lesion. Sensory neurons ipsilateral to CCI had an increase in the proportion of neurons expressing MOR. We suggest that local MOR expressed in axons may be exploited to modulate some forms of neuropathic pain. Ann Neurol 2003

Expression of glial cell line-derived neurotrophic factor family of growth factors in peripheral nerve injury in rats.

The glial cell line-derived neurotrophic factor (GDNF) family of growth factors may be involved in the regenerative support of neurons in the peripheral nervous system. In order to study the role of these growth factors and their receptors following rat peripheral nerve injury we examined the changes in their mRNA levels in the spinal cord, the dorsal root ganglia and the peripheral nerve trunk. Following transaction of the sciatic nerve GDNF mRNA was up-regulated rapidly in the denervated nerve distal to the cut along with the mRNA for one of its receptors, GFRα-1. GFRα-1 mRNA was also increased in the DRG ipsilateral to the nerve injury suggesting that GDNF may be involved in the trophic support of DRG sensory neurons. In contrast there were no analogous changes in the mRNA levels of neurturin, persephin and artemin following injury.

Near nerve local insulin prevents conduction slowing in experimental diabetes

Insulin may have direct effects on axons through its actions on insulin receptors or through cross occupancy of insulin-like growth factor-1 receptors. We tested the hypothesis that insulin itself influences conduction of myelinated fibers independent of hyperglycemia in experimental diabetes. Low dose intermittent (0.2 units thrice weekly) Toronto (regular) insulin was injected at the sciatic notch and knee near the left sciatic nerve of rats rendered diabetic with citrate buffered streptozotocin or nondiabetic rats given citrate only. Identical volumes of normal saline were injected near the contralateral right sciatic nerve. The diabetic rats developed hyperglycemia, elevated glycosylated hemoglobin levels and had slowing of right (saline treated) sciatic tibial motor and caudal sensory conduction velocity. In contrast, local insulin treatment on the left side prevented conduction slowing, unilaterally increasing conduction velocity. In nondiabetic rats, conduction velocities were slightly higher on the insulin treated side, but the influence of insulin was less robust than in diabetics. The insulin treated sural branches of the sciatic nerves in diabetics had a higher percentage of small (< or = 9.0 microm diameter) myelinated fibers than the saline treated nerves. Local insulin has a trophic influence on myelinated fibers that is prominent in diabetic nerves and is independent of hyperglycemia.

Distal Degenerative Sensory Neuropathy in a Long-Term Type 2 Diabetes Rat Model

OBJECTIVE—Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats. RESEARCH DESIGN AND METHODS—We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons. RESULTS—ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IRβ protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the α2δ1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-κB (NF-κB; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-κB, and RAGE protein. CONCLUSIONS—ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.

Insulin as an in vivo growth factor.

Insulin peptide has been identified to promote regeneration of axons in culture and in some in vivo model systems. Such actions have been linked to direct actions of insulin, or to cross occupation of closely linked IGF-1 receptors. In this work, we examined insulin support of peripheral nerve regenerative events in mice. Systemic insulin administration accelerated the reinnervation of foot interosseous endplates by motor axons after sciatic nerve transection and enhanced recovery of functional mouse hindpaw function. Similarly, insulin accelerated the regeneration-related maturation of myelinated fibers regrowing beyond a sciatic nerve crush injury. That such benefits might occur through direct signaling on axons was supported by immunohistochemical studies of expression with an antibody directed to the beta insulin receptor (IR) subunit. The proportion of sensory neurons expressing IRbeta increased ipsilateral to a similar sciatic crush injury in the L4 and L5 dorsal root ganglia. Insulin receptors, although widely expressed in axons, were also preferentially and intensely expressed on axons regrowing just beyond a peripheral nerve crush injury zone. The findings indicate that insulin imparts a substantial impact on regenerating peripheral nerve axons through upregulation of its expression following injury. Although the findings do not exclude insulin coactivating IGF-1 receptors during regeneration, its own receptors are present and available for action on injured nerves.

Evidence for nitric oxide and nitric oxide synthase activity in proximal stumps of transected peripheral nerves

Nitric oxide may be liberated as an inflammatory mediator within injured peripheral nerve trunks. We evaluated the proximal stumps of injured peripheral nerve stumps that later form neuromas or regenerative nerve sprouts, for evidence of local nitric oxide elaboration and activity. Proximal stumps were created in male Sprague-Dawley rats by sectioning of the sciatic nerve and resection of its distal portions and branches. There was striking physiological evidence of nitric oxide activity at the tips of 48-h and 14-day-old proximal nerve stumps. We detected local nitric oxide-mediated hyperemia of both extrinsic plexus and endoneurial microvessels that was reversible, in a dose-dependent stereospecific fashion, by the broad-spectrum nitric oxide synthase inhibitors, Nomega-nitro-L-arginine-methyl ester or Nomega-nitro-L-arginine, but not by 7-nitroindazole, an inhibitor with relative selectivity for neuronal nitric oxide. Immunohistochemical studies provided evidence for the localization of nitric oxide generators at the same sites. In 48-h but not 14-day stumps increased expression of two isoforms of nitric oxide synthase was detected: endothelial nitric oxide and to a much lesser extent neuronal nitric oxide synthase. Both isoforms appeared in axonal endbulb-like profiles that co-localized with neurofilament immunostaining. Western immunoblots identified a band consistent with endothelial nitric oxide synthase expression. In 14-day stumps with early neuroma formation, but not 48-h stumps, there was staining for immunological nitric oxide synthase in some endoneurial and epineurial macrophages. Total nitric oxide synthase biochemical enzymatic activity, measured by labelled arginine to citrulline conversion, was increased in 14-day but not 48-h stumps. Injured peripheral nerves have evidence of early nitric oxide action, nitric oxide synthase expression and nitric oxide activity in proximal nerve stumps. Nitric oxide may have an important impact on the regenerative milieu.

Activated RHOA and peripheral axon regeneration

The regeneration of adult peripheral neurons after transection is slow, incomplete and encumbered by severe barriers to proper regrowth. The role of RHOA GTPase has not been examined in this context. We examined the expression, activity and functional role of RHOA GTPase and its ROK effector, inhibitors of regeneration, during peripheral axon outgrowth. We used qRT-PCR, quantitative immunohistochemistry, and assays of RHOA activation to examine expression in sensory neurons of rats with sciatic transection injuries. In vitro, we exposed dissociated adult sensory neurons, not grown on inhibitory substrates, to a RHOA-ROK inhibitor HA-1077 and measured neurite initiation and outgrowth. In vivo, we exposed early regenerating axons and Schwann cells directly to HA-1077 in a conduit connecting the proximal and distal stumps of transected sciatic nerves. Intact adult dorsal root ganglia sensory neurons expressed RHOA and ROK 1 mRNAs and protein and there were rises in RHOA after injury. Activated GTP-bound RHOA, undetectable in intact ganglia, was dramatically upregulated in both neurons and axons after injury. Adult rat sensory neurons in vitro demonstrated a dose-related increase in the initiation of neurite outgrowth, and in the proportion with long neurites when they were exposed to a ROK antagonist. Regenerative bridges that were directly exposed to the ROK inhibitor had a dose-related rise in the extent and distance of in vivo axon and partnered Schwann cell regrowth within them. RHOA activation and signaling are features of adult peripheral axon regeneration within its own milieu, independent of myelin. Inhibition of its activation may benefit peripheral axon lesions.

Inhibition of nitric oxide synthase enhances peripheral nerve regeneration in mice

We tested the hypothesis that inhibition of nitric oxide synthase (NOS) following transection of the sciatic nerve in the mouse would adversely influence regeneration of myelinated fibers from the proximal stump. NOS was inhibited by N(omega)-nitro-L-arginine-methyl ester (L-NAME; 10 mg/kg i.p.), a broad spectrum NOS inhibitor given twice daily for the first 10 days following nerve transection in Swiss mice. Controls received the inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME). Regeneration was assessed by serial recordings of the M potential from interosseous muscles of the foot innervated by sciatic-tibial motor fibers and morphometric analysis of myelinated fibers distal to the injury site. Contrary to expectation, M potentials reappeared earlier in the mice treated with L-NAME and were higher in amplitude (reflecting the number of reinnervating motor fibers) at 10 weeks after the injury. In the L-NAME treated mice, the mean axonal diameter of regenerating tibial myelinated fibers was larger and the fiber size histogram was shifted to larger fibers. Inhibition of NOS in a transected peripheral nerve is associated with enhanced regeneration of myelinated fibers. Local elaboration of NO may be toxic to regenerating axons.

Neurotrophins and other growth factors in the regenerative milieu of proximal nerve stump tips

Classic ideas on mechanisms for axon sprouting and nerve regeneration from peripheral nerves suggest that there is a prominent role for neurotrophin support. There has been comparatively less attention towards features of the regenerative process that develop from the proximal nerve trunk without the support of target tissues or the denervated trunk of a peripheral nerve. We studied early (2–14 d) expression of local growth factors in proximal nerve stump tips of transected sciatic nerves in rats. Immunohistochemical labelling was used to address specific deposition of BDNF, NGF, NT‐3, bFGF, CNTF and IGF‐1. We observed a unique localisation of BDNF, and to a much lesser extent, NGF in mast cells of injured nerve trunks but they were also observed in intact uninjured nerves. Macrophages did not express either BDNF or NGF. CNTF and IGF‐1 were expressed in Schwann cells of intact nerves and stumps. We did not observe bFGF or NT‐3 expression in any of the samples we studied. Mast cells may represent an important reservoir of BDNF in peripheral nerves.