Local isoform‐specific NOS inhibition: A promising approach to promote motor function recovery after nerve injury

Physical injury to a nerve is the most frequent cause of acquired peripheral neuropathy, which is responsible for loss of motor, sensory and/or autonomic functions. Injured axons in the peripheral nervous system maintain the capacity to regenerate in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Therefore, the identification of molecules that regulate degenerative and regenerative processes is indispensable in developing therapeutic tools to accelerate and improve functional recovery. Here, I consider the role of nitric oxide (NO) synthesized by the three major isoforms of NO synthases (NOS) in motor neuropathy. Neuronal NOS (nNOS) seems to be the primary source of NO that is detrimental to the survival of injured motoneurons. Endothelial NOS (eNOS) appears to be the major source of NO that interferes with axonal regrowth, at least soon after injury. Finally, NO derived from inducible NOS (iNOS) or nNOS is critical to the process of lipid breakdown for Wallerian degeneration and thereby benefits axonal regrowth. Specific inhibitors of these isoforms can be used to protect injured neurons from degeneration and promote axonal regeneration. A cautious proposal for the treatment of acquired motor neuropathy using therapeutic tools that locally interfere with eNOS/nNOS activities seems to merit consideration. © 2010 Wiley‐Liss, Inc.     

Altered expression of Smad family members in injured motor neurons of rat

We examined changes in the expression of Smad family members, which transduce signals from TGF-beta superfamily ligands, following hypoglossal nerve injury. RT-PCR and in situ hybridization revealed that Smad1, 2, 3 and 4 mRNAs were significantly up-regulated in injured side, whereas Smad8 mRNA was down-regulated. Immunohistochemistry and Western blotting analysis confirmed the alterations of Smad1, 2 and 4 in injured neurons. These results suggest that the Smad signaling may be important for nerve regeneration.     

Reduced synaptic activity precedes synaptic stripping in vagal motoneurons after axotomy

Activated microglia, which spread on the motor neurons following nerve injury, engage in the displacement of detached afferent synaptic boutons from the surface of regenerating motor neurons. This phenomenon is known as "synaptic stripping." The present study attempted to examine whether changes in the synaptic inputs after motor nerve injury correlated with the microglial attachment to the dorsal motor neurons of the vagus (DMV). DMV neurons in Wistar rats could survive after nerve injury, whereas most of injured DMV neurons in the C57BL/6 mice died. At 2 days after nerve injury, a significant decrease was observed in the frequencies of both spontaneous and miniature EPSCs and IPSCs recorded from DMV neurons in the slice preparation but not from the mechanically dissociated neurons in the Wistar rats. At this stage, no direct apposition of microglia on the injured neurons was observed. High-K(+) stimulation restored their frequencies to control levels. Furthermore, PPADS and DPCPX, antagonists of P2 and adenosine receptors, respectively, also stimulated the recovery of their frequencies. In contrast, no significant change was detected in the spontaneous EPSCs frequency recorded from the severely injured DMV neurons in the slice preparation of the C57BL/6 mice. These observations strongly suggest that presynaptic inhibition through glia-derived ATP and adenosine, thus precedes synaptic stripping in regenerating DMV neurons following nerve injury.     

Primary motor neurons fail to up-regulate voltage-gated sodium channel Na(v)1.3/brain type III following axotomy resulting from spinal cord injury

Epilepsy occurs in a small proportion of patients with spinal cord injury (SCI), but whether it is due to concomitant traumatic head injury or to changes in cortical motor neurons secondary to axotomy within the spinal cord is not known. Na(v)1.3/brain type III sodium channel expression is up-regulated following peripheral axotomy of dorsal root ganglion (DRG) and facial motor neurons, but, to date, Na(v)1.3 expression has not been examined in upper (cortical) motor neurons following axotomy associated with SCI. In the present study, we examine Na(v)1.3 expression in upper motor neurons within rat primary motor cortex following midthoracic (T9) dorsal column transection, which severs the axons of those cells. Axotomized pyramidal cells were identified by retrograde transport of fluorogold. Immunolabeled cells were confined to layer V of the primary motor cortex and exhibited low levels of Na(v)1.3 staining. After axotomy, no significant changes were detected in Na(v)1.3 density or distribution in injured or uninjured cells, compared with control brains, in contrast to up-regulation of Na(v)1.3 in ipsilateral DRG neurons after sciatic nerve transection. These results do not preclude a role for voltage-gated sodium channels in post-SCI epilepsy but suggest that up-regulated expression of Na(v)1.3 channel is not involved.     

Expression and role of low-affinity nerve growth factor receptor (p75) in spinal motor neurons of aged rats following axonal injury

Expression of low-affinity nerve growth factor receptor (p75) and its regulation in spinal motor neurons of aged rats following axonal injury were investigated by immunocytochemical staining with antibody against p75. Under normal conditions, approximately 60% of spinal motor neurons expressed p75 in aged rats whereas no p75 expression was observed in spinal motor neurons of young adult rats. We examined the effects of spinal motor neuron injury on aged rats by two approaches, i.e. distal axotomy and spinal nerve root avulsion. A 20% increase in the number of p75-positive motor neurons was observed in aged rats 2 weeks after distal axotomy after which it returned to normal by 8 weeks post-injury and remained constant. Following root avulsion, a transient and slight up-regulation of p75 expression was observed in injured motor neurons. The number of p75-positive motor neurons decreased quickly to below normal levels 1 week after lesion and progressively declined with time post-injury, 40% by 2 weeks, 33% by 4 weeks, 23% by 8 weeks, and 5.8% by 12 weeks compared with the normal controls. This study demonstrates that p75 is re-expressed in aged spinal motor neurons. Following axonal injury in aged rats, up-regulation of p75 seems to coincide with the survival of injured motor neurons. Potential roles of re-expression of p75 in aged motor neurons are discussed.     

Effect of 17 beta-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-alpha and estrogen receptor-beta localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-alpha and estrogen receptor-beta mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-alpha and -beta mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i). axotomized motor neurons increase expression of estrogen receptors-alpha and -beta mRNA, (ii). estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii). estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.     

Regulation of Semaphorin III/Collapsin-1 Gene Expression during Peripheral Nerve Regeneration

The competence of neurons to regenerate depends on their ability to initiate a program of gene expression supporting growth and on the growth-permissive properties of glial cells in the distal stump of the injured nerve. Most studies on intrinsic molecular mechanisms governing peripheral nerve regeneration have focussed on the lesion-induced expression of proteins promoting growth cone motility, neurite extension, and adhesion. However, little is known about the expression of intrinsic chemorepulsive proteins and their receptors, after peripheral nerve injury and during nerve regeneration. Here we report the effect of peripheral nerve injury on the expression of the genes encoding sema III/coll-1 and its receptor neuropilin-1, which are known to be expressed in adult sensory and/or motor neurons. We have shown that peripheral nerve crush or transection results in a decline in sema III/coll-1 mRNA expression in injured spinal and facial motor neurons. This decline was paralleled by an induction in the expression of the growth-associated protein B-50/GAP-43. As sema III/coll-1 returned to normal levels following nerve crush, B-50/GAP-43 returned to precrush levels. Thus, the decline in sema III/coll-1 mRNA coincided with sensory and motor neuron regeneration. A sustained decline in sema III/coll-1 mRNA expression was found when regeneration was blocked by nerve transection and ligation. No changes were observed in neuropilin-1 mRNA levels after injury to sensory and motor neurons, suggesting that regenerating peripheral neurons continue to be sensitive to sema III/coll-1. Therefore we propose that a decreased expression of sema III/coll-1, one of the major ligands for neuropilin-1, during peripheral nerve regeneration is an important molecular event that is part of the adaptive response related to the success of regenerative neurite outgrowth occurring following peripheral nerve injury.     

Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons

Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.     

Induction of the 27-kDa Heat Shock Protein (Hsp27) in the Rat Medulla Oblongata after Vagus Nerve Injury

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in motor and sensory neurons of the brainstem. Hsp27 is also rapidly induced in the nervous system following oxidative and cellular metabolic stress. In this study, we examined the distribution of Hsp27 in the rat medulla oblongata by means of immunohistochemistry after the vagus nerve was cut or crushed. After vagal injury, rats were allowed to survive for 6, 12, 24 h, 2, 4, 7, 10, 14, 30, or 90 days. Vagus nerve lesions resulted in a time-dependent up-regulation of Hsp27 in vagal motor and nodose ganglion sensory neurons that expressed Hsp27 constitutively andde novoinduction in neurons that did not express Hsp27 constitutively. In the dorsal motor nucleus of the vagus nerve (DMV) and nucleus ambiguus, the levels of Hsp27 in motor neurons were elevated within 24 h of injury and persisted for up to 90 days. Vagal afferents to the nucleus of the tractus solitarius (NTS) and area postrema showed increases in Hsp27 levels within 4 days that were still present 90 days postinjury. In addition, increases in Hsp27 staining of axons in the NTS and DMV suggest that vagus nerve injury resulted in sprouting of afferent axons and spread into areas of the dorsal vagal complex not normally innervated by the vagus. Our observations are consistent with the possibility that Hsp27 plays a role in long-term survival of distinct subpopulations of injured vagal motor and sensory neurons.     

Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC(1) and VPAC(2), in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC(1) and VPAC(2) receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC(2) receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC(1) receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses.     

βAPP Gene Expression is Increased in the Rat Brain After Motor Neuron Axotomy

The response of the βAPP gene to neuronal injury was studied in the facial and hypoglossal nerve nuclei of the rat after corresponding nerve axotomy. Increased levels of βAPP 695, 714, 751 and 770 mRNAs were observed after either facial or hypoglossal nerve axotomy in the parent ipsilateral motor neurons. The increase was gradual, with maximal values 7 days after axotomy. βAPP mRNA expression returned to normal values 60 days after the lesion. Increased βAPP immunostaining was also detected in ipsilateral chromatolytic motor neurons. No change in βAPP immunoreactivity was observed in oligodendrocytes, another cell type expressing βAPP under normal conditions. A rapid increase in the expression of the GFAP gene was observed in reactive astrocytes surrounding chromatolytic neurons in the ipsilateral facial or hypoglossal nuclei. Thus, in contrast with other models of neuronal injury, where only the Kunitz protease inhibitor-containing βAPP mRNA isoforms are increased, all βAPP mRNAs are increased in the axotomy model. Furthermore, although βAPP expression has been shown to be increased in reactive astrocytes following neuronal injury, in the present study the increase was essentially found in the motor neurons reacting to axotomy.     

Prolonged induction of neuronal NOS expression and activity following cortical spreading depression (SD): implications for SD- and NO-mediated neuroprotection

Cortical spreading depression (CSD) is associated with various short- and long-term physiological and neurochemical changes and has been shown to confer an increased susceptibility to accompanying ischemic injury or provide protection against a subsequent experimental ischemia. Nitric oxide is involved in the processes of ischemic injury and under certain conditions mediates cellular protection. To investigate the possibility that CSD-induced alterations in nitric oxide synthase (NOS) expression and activity occur and might be associated with the time-dependent enhancement or prevention by CSD of ischemic damage, this study examined the spatiotemporal changes in nNOS expression and activity in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl and were killed at various times thereafter. CSD increased both nNOS mRNA and protein levels throughout layers II-III of cortex. nNOS mRNA in the affected neocortex was significantly increased by 30-90% at 2, 7, and 14 days (P < or = 0.05) compared with contralateral levels, but was not significantly above control values at 1-6 h, 1 day, and 28 days after CSD induction. Levels of [3H]-L-N(G)-nitroarginine binding to NOS were increased by 40-170% 7, 14, and 28 days (P < or = 0.01) after CSD in a similar, but delayed, profile to nNOS mRNA. Levels of nNOS-immunoreactivity were also increased in both neurons and astrocytes of ipsilateral cortex 7 and 14 days after CSD--confirmed by double-immunofluorescence localization. Ex vivo NOS activity in layers I-III of ipsilateral cortex was also increased by 30-50% (P < or = 0.01) at 7 and 14 days after CSD, times coincident with reported maximal ischemic protection. These results demonstrate that nNOS is up-regulated by cellular depolarization/depression occurring during CSD, or by resultant stimuli and suggest that "CSD-conditioned" cortex may be capable of producing appropriate levels of NO to mediate or contribute to protective/adaptive responses to subsequent physical ischemic injury.     

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.     

Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.     

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.     

Nitric oxide-induced neurotoxicity versus neuroprotection; relationship with selective motor neuronal death]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuronal death. In addition to elucidate the "cell death mechanism", we think it is also important to clarify the "cell survival mechanism", to understand the pathogenesis of this intractable disease. Glutamate (Glu) is an excitatory neurotransmitter in the central nervous system, and is implicated in the pathogenesis of ALS. In this report, we presented our current research, investigating the mechanism of Glu-induced selective motor neuronal death, derived from the study of primary culture of rat embryonic spinal cord. In brief, 1) motor neurons are selectively injured by long-term exposure to low-dose Glu through the activation of nNOS to generate NO and ONOO-: 2) nonmotor neurons are protected by cGMP which is formed by NONdependent guanylyl cyclase: 3) chronic exposure of spinal neurons to Glu increases nNOS positive neurons only in nonmotor neurons. These results indicate the cascade of Glu-calcium influx-NO generation is toxic to motor neurons and protective to nonmotor neurons. The different effect of cGMP on motor neurons and nonmotor neurons against Glu-induced excitotoxicity may explain the selective motor neuronal death of ALS. Further investigation might advance the possibility of new therapy against ALS.     

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Tα1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.