Spatiotemporal patterns of postsynaptic density (PSD)-95 expression after rat spinal cord injury

Aims: Postsynaptic density (PSD)-95 is a scaffolding protein linking the N-methyl-D-aspartate receptor with neuronal nitric oxide synthase (nNOS), which contributes to many physiological and pathological actions. We here investigated whether PSD-95 was involved in the secondary response following spinal cord injury (SCI). Methods: Spinal cord contusion (SCC) and spinal cord transection (SCT) models at thoracic (T) segment 9 (T₉) were established in adults rats. Real-time polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence were used to detect the temporal profile and spatial distribution of PSD-95 after SCI. The association between PSD-95 and nNOS in the injured cords was also assessed by coimmmunoprecipation and double immunofluorescent staining. Results: The mRNA and protein for PSD-95 expression were significantly increased at 2 h or 8 h, and then gradually declined to the baseline level, ultimately up-regulated again from 5 days to 7 days for its mRNA level and at 7 days or 14 days for its protein level after either SCC or SCT. PSD-95 immunoreactivity was found in neurones, oligodendrocytes and synaptic puncta of spinal cord tissues within 5 mm from the lesion site. Importantly, injury-induced expression of PSD-95 was colabelled by active caspase-3 (apoptotic marker), Tau-1 (the marker for pathological oligodendrocytes) and nNOS. Conclusions: Accompanied by the spatio-temporal changes for PSD-95 expression, the association between PSD-95 and nNOS undergoes substantial alteration after SCI. These two molecules are likely to form a complex on apoptotic neurones and pathological oligodendrocytes, which may in turn be involved in the secondary response after SCI.     

Changes in mRNA for post-synaptic density-95 (PSD-95) and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection

When the axon of motoneurons is transected, the number of synaptic boutons contacting the cell body is decreased, and the recovery of synapses depends on muscle reinnervation. Post-synaptic density-95 (PSD-95) is a protein which is located at the post-synaptic density (PSD) and it plays a pivotal role in regulating synaptic plasticity and synaptogenesis. In addition, PSD-95 binds with neuronal nitric oxide synthase (nNOS), which is competitively inhibited by carboxy-terminal PDZ ligand of nNOS (CAPON) and, thereby, nNOS activity is thought to be regulated by PSD-95 and CAPON. We investigated the changes in mRNA for PSD-95, CAPON and nNOS in the facial motor nucleus of adult rats following axotomy, by in situ hybridization, in combination with the time course of muscle reinnervation, by retrograde tracing and nNOS protein expression, by examining nicotinamide adenine nucleotide phosphate diaphorase (NADPH-d) activity. Signals of mRNA for PSD-95 and CAPON were initially expressed in the facial motoneurons, transiently decreased following axotomy and gradually recovered to the control level. When reinnervation of the axotomized nerve into muscle was observed, mRNA expression of PSD-95 and CAPON started to recover in the facial motoneurons. It was also found that mRNA and protein expression of nNOS started to increase in the axotomized facial motoneurons just prior to the recovery of mRNA expression of PSD-95 and CAPON. These results suggest that PSD-95 and CAPON are involved in synaptogenesis and/or recovery of synaptic function in motoneurons after axotomy.     

Nicotine attenuates iNOS expression and contributes to neuroprotection in a compressive model of spinal cord injury

Primary impact to the spinal cord results in stimulation of secondary processes that potentiate the initial trauma. In the present study, we hypothesized that the altered expression of nitric oxide synthase (NOS) may contribute to these effects. Recent evidence indicates that nicotine can exert potent antioxidant and neuroprotective effects in spinal cord injury (SCI). Therefore, the aim of the present study was to evaluate whether the administration of nicotine can influence expression of inducible NOS (iNOS) and/or neuronal NOS (nNOS) in injured spinal cords. Adult male Long-Evans rats were subjected to a moderate contusion model of SCI and received a single intraperitoneal injection of either saline or nicotine (0.35, 3.5, or 7 mg/kg) 2 hr after trauma. SCI dramatically increased iNOS (but not nNOS) mRNA and protein levels in microglial cells in the thoracic and lumbar regions of spinal cords. iNOS overexpression resulted in increased nitrotyrosine formation, decreased number of NeuN (neuronal nuclei)-immunoreactive cells, and up-regulation of inflammatory genes. Most importantly, these effects were markedly attenuated by nicotine acting via a receptor-mediated mechanism. These data may have significant therapeutic implications for the targeting of nicotine receptors in the treatment of compressive spinal cord trauma.     

Involvement of CAPON and Nitric Oxide Synthases in Rat Muscle Regeneration After Peripheral Nerve Injury

Carboxy-terminal PDZ ligand of nNOS (CAPON) protein, as an adaptor, binds to nNOS via the PDZ domain helping regulate neuronal nitric oxide synthase (nNOS) activity at post-synaptic sites in neurons (Jaffrey et al., Neuron, 20, 115–124, 1998). Recently, it has been reported that CAPON is present in mouse muscle and may be involved in mouse muscle growth, injury, and repair possibly by regulating the stability, activity, or position of nNOS (Segalat et al., Experimental Cell Research, 302, 170–179, 2005). The present study was to explore the expression patterns and roles of CAPON as well as NOS in rat muscle regeneration after nerve injury. Normal Sprague–Dawley rats were subjected to right sciatic nerve crush injury. Walking track analysis, real time polymerase chain reaction, Western blotting, in situ hybridization, immunocytochemistry, and co-immunoprecipitation techniques were used. It revealed that CAPON mRNA increased, which peaked on days 1 and 28, whereas nNOS mRNA underwent a downregulation in the ipsilateral gastrocnemius muscles after sciatic nerve injury. Their proteins approximately paralleled the mRNA expression. CAPON and nNOS were identified in the activated satellite cells or myotubes and their in vivo interaction was verified. However, eNOS and iNOS proteins suffered an upregulation and were detected in activated satellite cells or myotubes. These data suggest that CAPON and all these three isoforms of NOS might be involved in muscle regeneration after nerve injury. Further study is necessary for a better understanding of the potential functional link between CAPON, NOS, and muscle regeneration, with possible application to therapy for skeletal muscle repair from nerve injury. M. Chen and C. Cheng contribute equally to this work.     

Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation

alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.     

Inhibition of the Ca2?-dependent K? channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K? channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca2? signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.     

Tempol reduces injury area in rat model of spinal cord contusion injury through suppression of iNOS and COX-2 expression

The present study focused on the biologic effects of tempol on anti-inflammatory and nitric oxide generation in contusion spinal cord injury (SCI). The animal model of SCI was induced by dropping a 10-g rod (2.0 mm in diameter) at a height of 25 mm. Tempol was injected intraperitoneally a dose of 100 mg/kg at 15 min before SCI. Controls was injected with saline. The contused spinal segments were removed according to time courses, and the expression level of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was analyzed along with the size of irreversibly damaged region. After SCI, the relative amounts of COX-2 and iNOS mRNA were peaked at 8 h after post-injury, and then decreased up to 7 days post-injury, and normal level at 14 days. Expression of COX-2 protein was peaked at 8 h post-injury. With the tempol pre-treatment, the immunoreactivity of COX-2 and nitrotyrosine in paraffin-embedded tissue slices was profoundly decreased. The irreversibly damaged area of the spinal cord was peaked at 3 days after SCI. With tempol pre-treatment, the irreversibly damaged area shows a statistically significant decrease at 3 days after SCI. These evidences indicate that tempol pre-treatment reduces irreversibly damaged area on the contusion SCI in rat. The mechanisms of biologic reactions of tempol might be related to the decreased expressions of COX-2 and iNOS in spinal cord cells, neurons and glia. It is expected that the tempol effect on the SCI is not only antioxidant activity but also anti-inflammatory reaction.     

一氧化氮合酶抑制剂对脊髓损伤后运动功能的影响

目的观察诱导型和神经型一氧化氮合酶(iNOS,nNOS)抑制剂对大鼠脊髓损伤(SCI)后运动功能的影响和机理。方法大鼠脊髓压迫伤后分别给予iNOS和nNOS抑制剂—氨基胍(AG)和7-硝基吲唑(7-NI)进行治疗,24h后用分光光度法测定组织中一氧化氮(NO)含量和一氧化氮合酶(NOS)活性,72h后用流式细胞仪检测神经细胞凋亡情况,4周后用电生理和动物行为学等指标评价运动功能的恢复情况。结果AG和7-NI均可以抑制组织中的NO含量,并使NOS活性下降,同时降低神经细胞的凋亡比率,对运动功能的恢复前者优于后者。结论脊髓损伤后应用NOS抑制剂可以使伤后运动功能得到改善,AG的作用似乎更明显,提示iNOS活性变化可能对脊髓损伤的恢复更具决定作用。     

Expression of DDAH1 in chick and rat embryos

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that metabolizes methylated arginine to citrulline and methylamine, thus working to produce nitric oxide (NO). We isolated a gene encoding chick DDAH1. In situ hybridization analysis revealed characteristic DDAH1 mRNA expression in the embryonic spinal cord, which was especially strong in the ventral horn and dorsal root ganglion (DRG). DDAH1 was also detected in the brain, kidney, digestive tract, and in other tissues. We examined the expression pattern of DDAH1 in developing rats and compared this with the expression pattern in chicks. The expression pattern in the rats was very similar to that in the chicks, but there were some differences between the chicks and rats in the amount of DDAH1 detected in the heart, liver, lung, and DRG. We also investigated neural nitric oxide synthase (nNOS) mRNA expression patterns in rat embryos. The DDAH1 expression patterns were completely different from nNOS expression patterns. Our study suggests that DDAH1 plays an important role in development.     

Neuropathic pain is associated with alterations of nitric oxide synthase immunoreactivity and catalytic activity in dorsal root ganglia and spinal dorsal horn

Previous experiments have suggested that nitric oxide may play an important role in nociceptive transmission in the spinal cord. To assess the possible roles of neuronal nitric oxide synthase (nNOS) in spinal sensitization after nerve injury, we examined the distribution of nNOS immunoreactivity in dorsal root ganglia (DRGs) and dorsal horn of the corresponding spinal segments. NOS catalytic activity was also determined by monitoring the conversion of [3H]arginine to [3H]citrulline in the lumbar (L4-L6) spinal cord segments and DRGs in rats 21 days after unilateral loose ligation of the sciatic nerve. Behavioral signs of tactile and cold allodynia developed in the nerve-ligated rats within 1 week after surgery and lasted up to 21 days. Immunocytochemical staining revealed a significant increase (approximately 6.7-fold) of nNOS-immunoreactive neurons and fibers in the DRGs L4-L6. No significant changes were detected in the number of nNOS-positive neurons in laminae I-II of the spinal segments L4-L6 ipsilateral to nerve ligation. However, an increased number of large stellate or elongated somata in deep laminae III-V of the L5 segment expressed high nNOS immunoreactivity. The alterations of NOS catalytic activity in the spinal segments L4-L6 and corresponding DRGs closely correlated with nNOS distribution detected by immunocytochemistry. No such changes were detected in the contralateral DRGs or spinal cord of sham-operated rats. The results indicate that marked alterations of nNOS in the DRG cells and in the spinal cord may contribute to spinal sensory processing as well as to the development of neuronal plasticity phenomena in the dorsal horn.     

Spatiotemporal Patterns of Dexamethasone-Induced Ras Protein 1 Expression in the Central Nervous System of Rats with Experimental Autoimmune Encephalomyelitis

Dexamethasone-induced Ras protein 1 (Dexras 1), a brain-enriched member of Ras subfamily of guanosine triphosphatases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that could form a ternary complex with carboxy-terminal PDZ ligand of nNOS (CAPON) and nNOS, to specific targets to enhance NO signaling. The present study was to explore the expression pattern of Dexras 1 in the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Western blot and immunochemistry analysis showed that the gene and protein expression of Dexras 1 in the central nervous system (CNS) of rats increased significantly during the process of EAE compared with control groups (p < 0.01) and maintain a high level in the remission period. The protein expressions of nNOS and CAPON in hippocampus were approximately paralleled Dexras 1. Immunofluorescence revealed that both neurons and glial cells expressed the Dexras 1 in EAE CNS. Importantly, the damaged CNS in EAE-affected rats showed the codistribution between Dexras 1 and caspase 3, indicating the role of Dexras 1 played in the apoptotic process in EAE. Furthermore, colocalizations of Dexras 1 were observed in neurons and glial cells in CNS with nNOS or CAPON, supporting the ternary complex in this model. Thus, these findings suggest the postulation that Dexras 1 might participate into CNS neuronal cell death and demyelination in the whole process of EAE through regulating the NO signaling by binding to nNOS and CAPON.     

Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

Activation of the neuronal-glial network in the spinal cord dorsal horn (SCDH) mediates various chronic painful conditions. We studied spinal neuronal-astrocyte signaling interactions involved in the maintenance of painful diabetic neuropathy (PDN) in type 2 diabetes. We used the db/db mouse, an animal model for PDN of type 2 diabetes, which develops mechanical allodynia from 6 to 12 wk of age. In this study, enhanced substance P expression was detected in the presynaptic sensory fibers innervating lamina I-III in the lumbar SCDH (LSCDH) of the db/db mouse at 10 wk of age. This phenomenon is associated with enhanced spinal ERK1/2 phosphorylation in projection sensory neurons and regional astrocyte activation. In addition, peak phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR), along with upregulation of neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression were detected in diabetic mice. Expression of nNOS and iNOS was detected in both interneurons and astrocytes in lamina I-III of the LSCDH. Treatment with MK801, an NMDAR inhibitor, inhibited mechanical allodynia, ERK1/2 phosphorylation, and nNOS and iNOS upregulation in diabetic mice. MK801 also reduced astrocytosis and glial acidic fibrillary protein upregulation in db/db mice. In addition, N(G)-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor, had similar effects on NMDAR signaling and NOS expression. These results suggest that nitric oxide from surrounding interneurons and astrocytes interacts with NMDAR-dependent signaling in the projection neurons of the SCDH during the maintenance of PDN.     

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.     

Amitriptyline attenuates astrocyte activation and morphine tolerance in rats: role of the PSD-95/NR1/nNOS/PKCγ signaling pathway

The tricyclic antidepressant amitriptyline binds with high affinity to N-methyl-d-aspartate receptors (NMDARs) and inhibits NMDAR-mediated events. Activation of the postsynaptic density protein-95 (PSD-95)/NMDAR-mediated downstream signaling cascade, including neuronal nitric oxide synthase (nNOS) and protein kinase gamma (PKCγ), has been shown to be involved in morphine tolerance. The present study examined the potential effect of amitriptyline on chronic morphine infusion-induced spinal PSD-95/NMDAR/nNOS/PKCγ signaling in morphine tolerance. Male Wistar rats were implanted with an intrathecal catheter and received an intrathecal infusion of saline or amitriptyline (15 μg/h), morphine+saline (tolerance induction, 15 μg/h), or morphine+amitriptyline for 5 days. Co-administration of amitriptyline with morphine not only preserved the antinociceptive effect of morphine, but also attenuated astrocyte activation in the rat spinal cord dorsal horn. On day 5 after drug infusion, increased expression and phosphorylation of spinal membrane NMDAR NR1 subunit and expression of PSD-95 were observed following chronic morphine infusion and these effects were attenuated by amitriptyline co-infusion. Upregulation of NMDAR-induced intracellular nNOS expression was also inhibited by amitriptyline co-infusion in chronic morphine-infused rats. Furthermore, amitriptyline co-infusion significantly inhibited morphine-induced PKCγ expression in both the cytosol and membrane of spinal neurons. These findings suggest that the attenuation of morphine tolerance caused by amitriptyline is due to downregulation of NMDAR NR1 subunit expression in the synaptosomal membrane accompanied by decreased expression of the scaffolding protein PSD-95. The effects of amitriptyline in attenuating astrocyte activation and reversing tolerance to morphine may be due, at least in part, to inhibition of the PSD-95/NMDAR NR1/nNOS/PKCγ signaling cascade.     

NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O2*-)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO-O2*- interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91phox and P67phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.     

Nicotine attenuates arachidonic acid-induced overexpression of nitric oxide synthase in cultured spinal cord neurons

Primary spinal cord trauma can initiate a cascade of pathophysiologic events which markedly contribute to the expansion and amplification of the primary insult. The detailed mechanisms of these secondary neurochemical reactions are largely unknown; however, they involve membrane lipid derangements with the release of free fatty acids, in particular, arachidonic acid (AA). AA can induce several injury effects on spinal cord neurons. We hypothesize that upregulation of nitric oxide synthase (NOS) is among the most important mechanisms of arachidonic-acid-induced neuronal dysfunction and that nicotine can attenuate this effect. To study these hypotheses, spinal cord neurons were exposed to AA and/or nicotine, and several markers of neuronal nitric oxide synthase (nNOS) metabolism were measured. In addition, cotreatments with either inhibitors of nicotinic receptors or inhibitors of specific NOS isoforms were employed. Treatment with AA markedly increased activity of nNOS, as well as mRNA and protein levels of this enzyme. Changes in nNOS expression were accompanied by an increase in cellular cGMP and medium nitrite levels. Pretreatment with nicotine decreased AA-induced overexpression of nNOS and elevation of nitrite levels. In addition, it appeared that these nicotine effects could be partially modulated both by the alpha7 nicotinic receptors or by nonreceptor mechanisms. Alternatively, the observed changes could also be mediated by an alternate nicotinic receptor mechanism which is not blocked by alpha-bungarotoxin or mecamylamine. Results of the present study indicate that exposure to AA can lead to induction of nNOS in cultured spinal cord neurons. In addition, nicotine can exert a neuroprotective effect by attenuation of AA-induced upregulation of nNOS metabolism. These data may have therapeutic implications for the treatment of acute spinal cord trauma.     

Effects of MPSS and a potent iNOS inhibitor on traumatic spinal cord injury

ONO-1714, a selective inhibitor of inducible nitric oxide synthetase (iNOS) attenuated the increase of apoptosis and improved the functional outcome of urinary bladder after traumatic spinal cord injury. These findings suggest that iNOS plays a role in the process of SCI. Early treatment with 30 mg/kg methylprednisolone sodium succinate (MPSS) could also inhibit the expression of iNOS gene, apoptosis and the loss of urinary bladder function. We confirmed that early MPSS treatment may prevent injury associated with apoptosis and urinary bladder disability by reducing iNOS mRNA. However, delayed single MPSS treatment 8 h after spinal cord injury was not effective. Early repeated MPSS treatment might allow greater recovery from acute spinal cord injury.     

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the spinal cord of transgenic mice with G93A mutant SOD1 gene

Immunohistochemical and quantitative analyses were used to examine the evolution of neuronal nitric oxide synthase (nNOS) with time in spinal motor neurons of transgenic mice with a G93A mutant Cu/Zn superoxide dismutase (SOD1) gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the controls, the anterior horn including the anterior horn neurons was not immunostained for nNOS. In the transgenic mice, at the age of 24 weeks (early presymptomatic), when no pathological change was observed in the spinal cord, anterior horn neurons were only occasionally immunostained for nNOS (0.3%). At the age of 28 weeks (late presymptomatic), nNOS-positive anterior horn neurons and their neuronal processes were occasionally observed (7.6%), and at the age of 32 weeks (early symptomatic), nNOS-positive anterior horn cells, including degenerated ones showing central chromatolysis, were frequently demonstrated (27.6%) and nNOS-positive cord-like swollen proximal axons were also observed in the anterior horns. nNOS expression in the anterior horn neurons was almost always observed in the somata. At the age of 35 weeks (end stage), neuronal loss of the anterior horn cells was severe, and nNOS-positive anterior horn neurons and cord-like swollen axons in the anterior horns were less prominent compared to those at the age of 32 weeks (33.8%), but many reactive astrocytes were immunostained for nNOS. Thus, nNOS immunoreactivity in the anterior horn neurons is observed as early as the presymptomatic stage and varies with the progression of the disease. The selective localization of positive nNOS immunoreactivity in the anterior horn neurons and degenerated ones in particular, and swollen proximal axons suggests that nNOS immunoreactivity may be involved in the degeneration of anterior horn neurons in this SOD1 transgenic mouse model.     

Intrathecal interleukin-1beta administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord

The effect of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) on the inducible nitric oxide synthase-nitric oxide (iNOS-NO) cascade in nociceptive signal transduction was examined in the intact rat spinal cord. All rats were implanted with an intrathecal (i.t.) catheter; some were also implanted with an i.t. microdialysis probe. The paw withdrawal latency to radiant heat was used to assess thermal hyperalgesia. The iNOS protein expression in the spinal cord dorsal horn was examined by western blot analysis and NOS activity assay. NO production in the CSF dialysate was also measured. IL-1beta i.t. (100 ng) produced thermal hyperalgesia from 4 to 24 h after i.t. injection. The iNOS protein expression was induced at 4 h after i.t. IL-1beta injection, peaked at the 6th hour, and disappeared at 24 h. The iNOS activity showed a similar time-dependent change as the iNOS protein expression. NO release increased by 1.1- to 1.9-fold between 4 and 12 h, also with a peak at the 6th hour, after i.t. IL-1beta administration. Pretreatment with the iNOS inhibitor 1400W (10 microg, i.t.) 1 h before i.t. IL-1beta injection prevented all the responses of IL-1beta. Neither 1400W nor artificial CSF (aCSF) affected the thermal nociceptive threshold and NO production. These results demonstrate that i.t. administration of IL-1beta induced thermal hyperalgesia by activating the iNOS-NO cascade in the rat spinal cord. On the basis of the present findings, we suggest that i.t. administration of iNOS inhibitors may have potential in the treatment of inflammatory and neuropathic pain syndromes.