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In the present study, we examined whether activation of p-38alpha MAPK modulates mechanical allodynia and neuronal hyperexcitability, and if propentofylline (PPF, a glial modulator) modulates specifically localized activated p-38alpha MAPK expression in caudal regions remote from a low thoracic hemisection injury in rats. T13 spinal hemisection produces bilateral mechanical allodynia in hindpaws with evoked (in response to mechanical stimuli) neuronal hyperexcitability in lumbar spinal wide dynamic range (WDR) neurons compared to sham controls. The mechanical allodynia and the evoked activity of WDR neurons is attenuated by intrathecal and topical administration of SB203580, an inhibitor of p-38 MAPK activation, dose dependently (p<0.05); however, the spontaneous activity showed no significant differences compared to sham controls. After T13 spinal hemisection, significantly increased phosphorylated (activated form) p-38alpha MAPK expression was present in both superficial and deep dorsal horn neurons as well as in microglia, but not in astrocytes, in the lumbar spinal cord compared to sham controls (p<0.05). Intrathecal application of PPF significantly attenuated the expression of phosphorylated p-38alpha MAPK in superficial dorsal horn neurons (10 mM) and in microglia (1 and 10 mM) in the lumbar spinal cord compared to the hemisection group (p<0.05). In conclusion, our present data demonstrate that activated neuronal and microglial, but not astrocytic, p-38alpha MAPK contributes to the maintenance of neuronal hyperexcitability in caudal regions following spinal cord injury.

Several lines of evidence indicate that phosphatidylinositol 3-kinase (PI3K) and PI3K-protein kinase B/Akt (PKB/Akt) signal pathway mediate the pain hypersensitivity induced by intradermal injection of capsaicin or nerve growth factor. However, the role of PI3K and PI3K-PKB/Akt signal pathway activation in neuropathic pain is still unclear. Using L5 spinal nerve ligation (L5 SNL) and immunohistochemistry, we found that the numbers of phospho-PKB/Akt-immunoreactive (p-PKB/Akt IR) positive neurons were significantly increased in ipsilateral L5 dorsal root ganglia (DRG) and adjacent L4 DRG started at 12 h after surgery and maintained to the 3rd day. Meanwhile, L5 SNL also induced an increased expression of p-PKB/Akt in ipsilateral L5 spinal dorsal horn. Double immunofluorescence staining showed that p-PKB/Akt expressed entirely in DRG neurons, especially in IB4-positive neurons. Intrathecal injection of PI3K inhibitor wortmannin or LY294002 and PKB/Akt inhibitor Akt inhibitor IV or (-)-Deguelin, started before L5 SNL, reduced the behavioral signs of neuropathic pain. Intraperitoneal injection of wortmannin or (-)-Deguelin as above also reduced the pain hypersensitivity. Post-treatment with wortmannin, started at the 1st day or the 3rd day after L5 SNL, decreased abnormal pain behaviors. Whereas the inhibitory effect of Akt inhibitor IV on established neuropathic pain was observed only in those rats that received the drug treatment started at the 1st day. Immunohistochemistry revealed that intrathecal injection of wortmannin significantly inhibited the activation of PKB/Akt in L5 DRG and L5 spinal cord. The data suggested that PI3K and PI3K-PKB/Akt signal pathway activation might contribute to the development of neuropathic pain.

Compelling evidence shows that the adjacent uninjured primary afferents play an important role in the development of neuropathic pain after nerve injury. The underlying mechanisms, however, are largely unknown. In the present study, the selective motor fiber injury was performed by L5 ventral root transection (L5 VRT), and p38 activation in dorsal root ganglia (DRG) and L5 spinal dorsal horn was examined. The results showed that phospho-p38 immunoreactivity (p-p38-IR) was increased in both L4 and L5 DRGs, starting on day 1 and persisting for nearly 3 weeks (P<0.05) following L5 VRT and that the activated p38 was confined in neurons, especially in IB4 positive C-type neurons. L5 VRT also induced p38 activation in L5 spinal dorsal horn, occurred at the first day after the lesion and lasted for 2 weeks (P<0.05). The activated p38 is restricted entirely in spinal microglia. In contrast, selective injury of sensory neurons by L5 dorsal root transection (L5 DRT) failed to induce behavioral signs of neuropathic pain and activated p38 only in L5 DRG but not in L4 DRG and L5 spinal dorsal horn. Intraperitoneal injection of thalidomide, an inhibitor of TNF-alpha synthesis, prevented p38 activation in DRG and spinal cord. Intrathecal injection of p38 inhibitor SB203580, starting before L5 VRT, inhibited the abnormal pain behaviors. Post-treatment with SB203580 performed at the 1st day or at the 8th day after surgery also reduced established neuropathic pain. These data suggest that p38 activation in uninjured DRGs neurons and in spinal microglia is necessary for the initiation and maintenance of neuropathic pain induced by L5 VRT.

The role of c-Jun activation for survival and regeneration of sensory neurons is unclear. Here we report that c-Jun N-terminal kinase (JNK)-mediated c-Jun activation is important for axonal outgrowth of sensory neurons in rat nodose and dorsal root ganglia (DRG). Peripheral severance of the vagus or the sciatic nerve resulted in a massive and rapid, but transient increase of the activated JNK (p-JNK) in neuronal nuclei, followed by c-Jun phosphorylation and activating transcription factor-3 (ATF3) induction. JNK inhibition by the selective JNK inhibitors SP600125 and (D)-JNKI1 did not affect neuronal survival in explanted or dissociated ganglia, but dramatically reduced axonal outgrowth, c-Jun activation, and ATF3 induction. Using retrograde labeling, we demonstrated that activated c-Jun (p-c-Jun) and ATF3 were associated with regenerative neurons. Taken together, our results suggest that JNK-mediated c-Jun activation is one of the first cell body reactions in response to nerve injury and that this activation and subsequent ATF3 induction are associated with axonal outgrowth.

Peripheral neuropathy is the most common neurological symptom in patients with acquired immunodeficiency syndrome (AIDS). Here, we examine possible mechanisms of gp120 and nucleoside reverse transcriptase inhibitors (NRTIs) in the pathogenesis of AIDS peripheral neuropathy. Neonatal dorsal root ganglion (DRG) neurons were found to undergo apoptosis in response to chronic treatment with gp120IIIB, an effect enhanced by the co-application of hCD4, as well as upon exposure to the nucleoside reverse transcriptase inhibitor (NRTI), 2',3'-dideoxyinosine (ddI). DRG neurons were rescued from the neurotoxic effects of these agents by CEP-1347, an inhibitor of the mixed lineage kinases (MLKs), upstream activators of the c-Jun N-terminal kinase (JNK) signaling pathway. In addition, gp120- or ddI-mediated toxicity were also inhibited by neuronal expression of dominant negative versions of the MLKs. Our results suggest that both gp120 and the NRTIs cause sensory neuron apoptosis through the activation of the JNK pathway, and that CEP-1347-like compounds may serve as a therapeutic option in patients with AIDS-associated peripheral neuropathy.

The stimulation of C-fiber sensory neurons is known to induce activation of the ERK MAP kinase signaling pathway in the spinal cord dorsal horn. In this study we have elucidated some of the signaling components of C-fiber transmission responsible for ERK activation. Using an in vitro slice preparation of the mouse spinal cord dorsal horn, we compared the release of substance P (SP) and BDNF with the activation of ERK in postsynaptic neurons. We observed that primary afferent stimulation recruiting C-fibers was required for both SP and BDNF release and ERK activation in post-synaptic dorsal horn neurons. Glutamate transmission via NMDA and mGluR1 but not AMPA receptors was critical to this ERK activation. BDNF signaling via TrkB receptors but not SP signaling via NK(1) were also involved in ERK recruitment. In conclusion, glutamate and BDNF are the important C-fiber signaling components for ERK activation in dorsal horn neurons.

MAP1B is a major microtubule-associated phospho-protein in growing axons and growth cones. Recent findings suggest that glycogen synthase kinase 3beta (GSK-3beta) phosphorylation of MAP1B may act as a molecular switch to regulate microtubule stability during axonogenesis. The effects of lithium, an inhibitor of GSK-3beta, on neurons in culture, are consistent with this suggestion. However, lithium is not a specific inhibitor of GSK-3beta. In the experiments reported here we have compared the effects of lithium with SB-216763, a new, potent and specific inhibitor of GSK-3 that has a different mechanism of action from lithium. We examined the effects of inhibition of GSK-3beta on axonogenesis, microtubule distribution, and growth cone behavior in cultured embryonic chick primary sensory neurons. Both compounds reduced axon elongation rates and increased growth cone size. In addition, both compounds slowed growth cone filopodia dynamics. These behavioral changes correlated with a decrease in MAP1B phosphorylation and an increase in the number of stable microtubules in growth cones. These results suggest that a major role of MAP1B in growing axons and growth cones is to regulate microtubule and actin filament stability. Furthermore, this function is regulated by phosphorylation of MAP1B by GSK-3beta.

Several molecules inhibit axonal growth cones and may account for the failure of central nervous system regeneration, including myelin proteins and various chondroitan sulfate proteoglycans expressed at the site of injury. Axonal growth inhibition by myelin and chondroitan sulfate proteoglycans may in part be controlled by Rho-GTPase, which mediates growth cone collapse. Here, we tested in vitro whether pharmacological inhibition of a major downstream effector of Rho, Rho-kinase, promotes axonal outgrowth from dorsal root ganglia grown on aggrecan. Aggrecan substrates stimulated Rho activity and were inhibitory to axonal growth. Y-27632 treatment promoted the growth of axons by 5- to 10-fold and induced "steamlined" growth cones with longer filopodia and smaller lamellipodia. Interestingly, more actin bundles reminiscent of stress fibers in the central domain of the growth cone were observed when grown on aggrecan compared to laminin. In addition, Y-27632 significantly promoted axonal growth on both myelin and adult rat spinal cord cryosections. Our data suggest that suppression of Rho-kinase activity may enhance axonal regeneration in the central nervous system.

Multiple protein tyrosine kinases regulate neurite outgrowth in the developing nervous system. To begin to unravel the complexity of this regulation, we addressed the role of one specific kinase, pp60(c-src), in chick dorsal root ganglion (DRG) neurons grown on laminin-1, a well-characterized system to study neurite outgrowth. Pharmacological inhibition of all tyrosine kinases by genestein treatment of chick DRG neurons significantly increased neurite number and length by approximately 50%. Similar increases in these parameters occurred when src-family kinases were inhibited using PP2. To implicate pp60(c-src) directly in neurite outgrowth, we inactivated it in DRG neuronal growth cones using Chromophore-Assisted Laser Inactivation (CALI). CALI of pp60(c-src) resulted in an 85% inactivation of its kinase activity and a 63% reduction in phosphotyrosine immunofluorescence in neurons. Microscale CALI of pp60(c-src) in DRG growth cones caused a significant and acute two-fold increase in neurite extension rate during irradiation. These findings demonstrate that pp60(c-src) is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons.

Mature dorsal root ganglion cells respond to neurotrophins, and the intracellular signalling pathways activated by neurotrophins have been characterized in vitro. We have now used immunocytochemistry and Western blots to examine the expression and activation of extracellular signal-regulated protein kinase-1/2 (ERK) in rat dorsal root ganglion cells in vivo, using antisera to total (tERK) and phosphorylated (pERK) forms. This has revealed a number of novel findings. tERK immunoreactivity is present in most dorsal root ganglion cells but is expressed most strongly in small (nociceptive) cells and, surprisingly, is absent in a population of large cells that expressed trkB or trkC but mainly lack p75(NTR) immunoreactivity. In contrast pERK is prominent in a few trkA cells and in satellite glial cells, and is further increased by NGF treatment. tERK and pERK both undergo fast anterograde and retrograde axonal transport, indicated by accumulation at a sciatic nerve ligature, and NGF reduces the level of retrograde pERK transport., (Copyright 2001 Academic Press.)

A previous study showed that basic fibroblast growth factor (FGF-2) promotes the effects of brain-derived neurotrophic factor (BDNF) on migration and neurite outgrowth from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage 22 (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes growing into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal outgrowth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor., (Copyright 2000 Academic Press.)

Histochemical detection of NADPH-d activity in rat barrel-field cortex reveals four types of distributions. (i) A transient, diffuse neuropil staining is visible in the cortical plate and in deeper layers until postnatal day (P) 4. Thereafter, until P15, it is segregated in whisker-specific patches in layer IV, then the pattern gradually disappears, becoming virtually indistinct by P21. This transient patterning of diffuse NADPH-d activity in layer IV disappears after cortical injections of kainic acid and is affected by neonatal damage to the contralateral snout. An intense labeling (ii) of scattered cells and (iii) of a plexus of fibers is present. With maturation, the cells become localized mostly in layers II/III, in the lower part of layer V, and in layer VI. They are sparse in layer I, in upper layer V, and in layer IV where their somata are located primarily in the interbarrel septa. (iv) Light staining of cortical neurons is detected mostly in layers II-IV but occasionally also in layers V-VI. Cytochrome c oxidase (CO)-positive patches associated with barrels are first detected in layer IV around P4-P5; their staining density increases with development, then stays high. In the adult, CO activity is moderate in supragranular layers, highest in the barrels in layer IV, low in upper layer V, medium dense in the deeper half of layer V, and low in lamina VI. Thus, NADPH-d and CO activities are not necessarily colocalized in the rodent barrel-field cortex. The varied (transient and long-lasting) distributions of NADPH-d activity indicate that the enzyme and its associated production of NO serve multiple roles in developing and adult barrel-field cortex., (Copyright 1999 Academic Press.)

Oxidative injury has been implicated in the pathophysiology of neuronal injury and neurodegenerative disease. Antioxidant proteins provide an endogenous defense against such oxidative injury and may yield important clues to mechanisms of cytoprotection and neuronal recovery. Axotomy is the simplest model of neuronal injury and lesioning the sciatic nerve allows concurrent study of both motor (spinal cord) and sensory (dorsal root ganglia, DRG) neurons affected by the same injury. This study was designed to examine the expression of superoxide dismutase (SOD), an essential antioxidant protein, in motor and sensory neurons following complete axotomy of peripheral nerve. Immunocytochemical, quantitative immunoblot, and enzymatic activity assay techniques are used. By 12 days after axotomy, immunocytochemical expression of Mn-SOD is markedly increased in affected DRG and spinal cord. A similar increase in Cu/Zn-SOD is not seen in DRG or spinal cord. This immunocytochemical staining is associated with a significant increase in specific activity and Mn-SOD protein content as measured on quantitative immunoblots. This report suggests, for the first time, that Mn-SOD and not Cu/Zn-SOD increases in sensory neurons of the DRG and motor neurons of the spinal cord following distal axotomy of the sciatic nerve. Quantitative measurements of Mn-SOD following axotomy reveals that the increase in immunocytochemical reactivity is associated with an approximately 30% increase in specific activity when comparing lesioned and contralateral spinal cord samples. These data suggest that Mn-SOD may have a more significant role in the pathophysiology of neuronal injury than Cu/Zn-SOD.

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.