Melatonin prevents dopaminergic cell loss induced by lentiviral vectors expressing A30P mutant alpha-synuclein.

Two hallmarks of Parkinson's disease (PD) are dopaminergic cell loss and the presence of cytoplasmic inclusions (Lewy bodies). Different point mutations in alpha-synuclein, the main constituent of Lewy bodies, have been identified in familial PD. Alpha-synuclein also constitutes one of the main components of Lewy bodies in sporadic cases of PD. Moreover, oxidant stress and generation of free radicals from both mitochondrial impairment and dopamine metabolism are considered to play critical roles in PD etiopathogenesis. Melatonin, a known potent antioxidant secreted by the pineal gland, may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage. However, the neuroprotective effect of melatonin has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. Lentiviral vectors encoding A30P mutant human alpha-synuclein (lenti-A30P) were stereotactically injected into the right substantia nigra of adult male Sprague-Dawley rats and neuroprotection was examined by administration of melatonin or vehicle from two days before nigral administration of lenti-A30P until eight weeks after injection. It was found that lenti-A30P induced a significant TH⁺ cell-loss both in the medial and lateral substantia nigra versus the contrallateral side injected with lenti-eGFP. However, melatonin administration showed a total neuroprotective effect in both regions of the substantia nigra. In conclusion, the data here show that melatonin is neuroprotective against mutant alpha-synuclein-induced injury in the substantia nigra.

Molecular and cellular events in alcohol-induced muscle disease.

Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms.

Tetrahydrobiopterin stimulates L-DOPA release from striatal tissue.

In the present study we have analyzed the effect of tetrahydrobiopterin (BH4) essential cofactor for tyrosine hydroxylase and nitric oxide synthase, on the 3,4-dihydroxyphenylalanine (L-DOPA) release from in vitro incubated striatal tissue. dl-6-methyl-5,6,7,8 tetrahydropterine (6-MPH4)-stimulated L-DOPA release in a concentration-dependent manner in the range from 25 to 100 microM. At these concentrations 6-MPH4 did not have any effect on dopamine release. Presence of Nomega-Nitro-L-arginine methyl ester (L-NAME, 200 microM), a nitric oxide synthase inhibitor, but not of alpha-methyl-rho-tyrosine (alpha-MPT, 100 microM), a tyrosine hydroxylase inhibitor, blocked L-DOPA release induced by 6-MPH4 (200 microM). Also, the addition to the incubation medium of melatonin (MEL, 300 microM), which is a scavenger of NO and other free radicals, blocked the L-DOPA release induced by 6-MPH4 (200 microM) but this effect did not occur with the addition of the peroxynitrite scavenger uric acid (UA, 300 microM). Sodium nitroprusside (SNP, 100 muM), a NO generator and l-DOPA releaser as previously reported, potentiated the L-DOPA releasing effect of 6-MPH4 (200 microM) which was also blocked by melatonin. In summary 6-MPH4 stimulates L-DOPA release from striatal fragments incubated in vitro by a mechanism which involves NO or other free radicals derived from NO but not peroxynitrite.

Consequences of unilateral nigrostriatal denervation on the thalamostriatal pathway in rats.

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

Alcoholic myopathy: lack of effect of zinc supplementation.

A chronic form of myopathy has been described in alcoholics, characterized by atrophy of type II fibers, due both to reduced protein synthesis and increased protein breakdown. Increased production of reactive oxygen species could probably play a role in increased protein breakdown. In addition, treatment with zinc might be beneficial, since it acts as a cofactor of several enzymes involved in the synthesis of proteins and antioxidants as copper-zinc-superoxidedismutase (SOD) and selenium dependent glutathione peroxidase (GPX). Based on these facts, we analyze the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in form of zinc sulphate, on muscle changes in 8 groups of adult Sprague-Dawley rats fed following the Lieber-de Carli model during 5 weeks. We also study the association between muscle histological changes and the activity of GPX, SOD and lipid peroxidation products (MDA), with hormones such as IGF-1, and with trace elements involved in antioxidant systems and/or in lipid peroxidation, such as selenium, copper, zinc, and iron. We found type IIa and IIb fiber atrophy in the alcoholic animals, especially in the low-protein fed ones. This effect was mainly due to protein deficiency. Zinc played no role at all. Muscle iron increased in ethanol, low protein fed rats, either with or without zinc, and was directly related with muscle MDA levels, which in turn were related with muscle atrophy, as was also found for serum IGF-1 levels. Ethanol was the main responsible for all these changes, although protein undernutrition also played an independent role in MDA levels. A positive interaction between ethanol and protein deficiency on serum IGF-1 was also detected. These results suggest that both protein deficiency and ethanol contribute to muscle atrophy observed in alcoholized rats; this atrophy is associated with increased lipid peroxidation and muscle iron overload. Treatment with zinc sulphate confers no benefit.

Effect of intracerebroventricular injection of lipopolysaccharide on the tuberoinfundibular dopaminergic system of the rat.

Numerous studies indicate that monoaminergic systems are sensitive to both peripheral and central inflammatory stimuli, and in particular dopaminergic neurons in the nigrostriatal system degenerate after local injection of lipopolysaccharide (LPS). However, data about the response of other dopaminergic groups to local inflammation are very sparse. In this study, we have examined the effect of i.c.v. injection of LPS on the tuberoinfundibular dopaminergic (TIDA) system by using biochemical and morphological parameters. Our results show that 6 h after i.c.v. injection of LPS, in parallel to a transient and intense immunoreaction to interleukin-1beta in arcuate microglial cells, there is a decrease in tyrosine hydroxylase (TH) activity in the median eminence and in the number of TH- and TH mRNA-positive cells in the arcuate nucleus, and at 12 h, an increase of prolactin levels in serum. Posterior changes were found in the TH mRNA labeling pattern, mostly in the ventrolateral region of the arcuate nucleus, but they were not accompanied by any changes in TH activity and immunoreactivity and TH-cell count. This suggests that the TIDA system is functionally susceptible to local inflammation, but the effects are transient and do not induce neurodegeneration.

Protein deficiency and muscle damage in carbon tetrachloride induced liver cirrhosis.

Protein undernutrition, alterations of hormones such as IGF-1, testosterone and cortisol, and increased lipid peroxidation-which may be related with deranged metabolism of some elements such as iron (Fe), zinc (Zn), manganese (Mn), selenium (Se) or copper (Cu)-may contribute to muscle damage in non alcoholic cirrhosis. Here, we analyse the effect of protein deficiency on muscle Cu, Fe, Zn, Mn and Se in carbon-tetrachloride (CCl(4)) induced liver cirrhosis. We also study the association between protein undernutrition and these trace elements with the activity of glutathione peroxidase (GPX), superoxide dismutase (SOD) and lipid peroxidation products, and how all these are related with muscle morphological changes in 40 male adult Sprague-Dawley rats. Liver cirrhosis was induced by intraperitoneal injection of CCl(4) to 10 rats fed a 2% protein diet, and to another 10 fed a 18% protein control diet. Two further groups included rats without cirrhosis fed the 2% protein and the 18% protein diets. After sacrifice (6 weeks later), we found type IIa fibre atrophy in the cirrhotic animals, especially in the low-protein fed ones and this was due to protein deficiency. Muscle Fe increased in low protein fed cirrhotic rats. No relationship was found between muscle changes and any of the hormones, enzymes and trace elements analysed, or with liver fibrosis. These results suggest that muscle atrophy observed in CCl(4)-induced cirrhosis is related with protein deficiency, but not with cirrhosis itself.

[Neuroimmune epidemiological value of reibergram in the first echovirus 16 meningoencephalitis outbreak in Cuba]. / Valor neuroinmunoepidemiologico del reibergrama en la primera epidemia de meningoencefalitis por echovirus 16 en Cuba.

INTRODUCTION: During the third quarter of 2000, an outbreak of echovirus 16 meningoencephalitis was firstly occurred in Cuba and produced vomiting, headache, fever and exanthem that differentiate it from other enterovirus epidemies. OBJECTIVE: To describe the intrathecal immunoglobulin synthesis pattern of the epidemie from the reibergram. PATIENTS AND METHODS: Diagnostic serum and cerebrospinal fluid from 18 children during the acute phase by nephelometrie assay, besides cytochemical and virological study. RESULTS: A predominant absence of intrathecal immunoglobulin synthesis and two patients with IgA and IgM synthesis was produced. 66% of cerebrospinal blood barrier dysfunction, 6.26 10 3 mean albumin ratio, lactate below 2.1 mmol/L and glucose concentration in cerebrospinal fluid was 50% over glucose blood content and 168 cells 10 6 L mainly lymphocytes were observed. CONCLUSIONS: The intratecal immunoglobulin synthesis pattern differs from other enterovirus outbreaks that have affected this population and it seems the one found for the diagnostic period in adults. This finding alerts the possibility of genetic changes in echovirus 16 strain, interesting from the neuroimmunoepidemiological point of view.

Response of GABAergic cells in the deep mesencephalic nucleus to dopaminergic cell degeneration: an electrophysiological and in situ hybridization study.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region located between the substantia nigra compacta and the superior colliculus. It contains GABAergic cells that share striatal afferents, thalamic and collicular efferents, as well as neurochemical and electrophysiological similarities, with those of the substantia nigra reticulata. In the present paper we used electrophysiological (firing rate and firing pattern) and morphological (densitometric analysis of in situ hybridization histochemical labeling for glutamic acid decarboxylase (GAD)65 and GAD67 mRNA) techniques, to study the response of DMN GABAergic cells to the degeneration of nigral dopaminergic cells. Our results showed that unilateral dopaminergic cell loss (after injection of 6-hydroxydopamine in the medial forebrain bundle) induces a bilateral and symmetrical increase in both firing rate and GAD67 mRNA levels and a decrease in GAD65 mRNA levels. These findings support the involvement of DMN GABAergic cells in the basal ganglia modifications that follow dopaminergic cell loss, also suggesting its participation in the pathophysiology of Parkinson's disease. The symmetry of effects, together with its recently reported bilateral projections to the thalamus and superior colliculus, suggest that unlike substantia nigra reticulata, DMN is involved in the interhemispheric regulation of basal ganglia, probably keeping their functional symmetry even after asymmetric lesions.

The deep mesencephalic nucleus as an output center of basal ganglia: morphological and electrophysiological similarities with the substantia nigra.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region between the superior colliculus, the substantia nigra compacta, the periaqueductal gray, and the medial geniculate body. Although some data suggest that it is involved in nociception and visceral control, its functions remain unclear. In the present study, by using morphological (combination of anterograde and retrograde tracers with immunocytochemistry and in situ hibrydization) and electrophysiological (firing activity and transynaptic response to striatal stimulation) methods, we show that a subpopulation of DMN cells shares many morphological and electrophysiological characteristics with those of the substantia nigra reticulata (SNR). These similarities include the following: 1) firing rate, firing pattern, and conduction velocity; 2) expression of GAD65, GAD67, and PV; 3) excitatory and inhibitory inputs from the striatum; and 4) projections to the ventral thalamus, superior colliculus, and pedunculopontine tegmental nucleus. Some differences were also found. In comparison with SN, DMN cells and striatal afferents are more sparsely distributed and they show conspicuous contralateral projections to the thalamus and superior colliculus. This suggests that, similarly to the SNR, the DMN acts as an output center of basal ganglia and probably facilitates the inter-hemispheric regulation of these centers.

Motor behavioural changes after intracerebroventricular injection of 6-hydroxydopamine in the rat: an animal model of Parkinson's disease.

At the beginning of the 1970s, different studies reported behavioural disturbances after the intracerebroventricular (icv) administration of 6-hydroxydopamine (6-OHDA) in the rat. Despite the fact that this neurotoxic agent degenerates brain dopaminergic (DA-) cells, its potential utility to produce a rat model of Parkinson's disease (PD) was never systematically studied because the aphagia and adipsia were often observed. In the present study, a procedure that induces a marked DA-cell degeneration that bypasses these and other undesirable complications of icv injection of 6-OHDA is reported. Lesioned animals (50-500 microg of 6-OHDA) showed a persistent motor syndrome composed of hypokinesia, purposeless chewing and catalepsy. The intensity of motor signs was dose-dependent, and recovered partially after administration of DA-receptor agonists, exposure to sensorial stimuli and stress, three procedures that reduce motor dysfunctions in Parkinson's disease (PD). Lesioned animals showed bilateral and symmetrical midbrain DA-cell degeneration with the highest cell-loss in A9 group (substantia nigra), followed by A8 (retrorubral field) and A10 (ventral tegmental area) groups. The similarity between the behavioural syndrome and the topographical profile of cell-loss after icv injection of 6-OHDA in rats and the clinical and neuropathological features of PD indicates that this may be a convenient animal model of PD particularly useful for checking in rats the possible efficacy of new anti-parkinsonian drugs on specific parameters of motor dysfunctions.

Dopamine cell degeneration induced by intraventricular administration of 6-hydroxydopamine in the rat: similarities with cell loss in parkinson's disease.

In an attempt to find a convenient rat model to study cell vulnerability in Parkinson's disease, we have investigated the cell-loss profile in different midbrain dopaminergic nuclei and subnuclei of rats injected with 6-hydroxydopamine (6-OHDA) in the third ventricle. Following administration of different doses (5-1000 microgram) of 6-OHDA, motor behavior was evaluated and tyrosine hydroxylase-immunostained neurons were counted in the A8 group and different subdivisions of A9 and A10 groups. Animals developed hypokinesia, repetitive chewing movements, and catalepsia. Signs of cell degeneration were evident from the first day after injection, reaching the definitive pattern at the end of the first week. There was a similar degeneration in both brain sides, the A9 group showing the highest degree of cell-loss, followed by A8 and A10 groups. In the A9 group, the degeneration mostly affected those subgroups located in its ventral, lateral, and posterior regions. In the A10 group the degeneration mainly affected the parabrachial pigmented nucleus, the paranigral nucleus and the ventral tegmental area. This topographic pattern of degeneration is very similar to that previously described in Parkinson's disease, suggesting that this model may be a useful tool in the study of the cell vulnerability mechanisms in this neurodegenerative disorder. In addition, our results also showed that small dopaminergic neurons are more resistant to degeneration than the large ones. In some DA subgroups, the cells that contained calbindin but not calretinin were less vulnerable to the neurotoxic effect of 6-OHDA.

Colocalization of tyrosine hydroxylase and GAD65 mRNA in mesostriatal neurons.

Although dopamine has been considered as the only neurotransmitter in the nigrostriatal pathway, studies carried out in the last two decades have suggested the existence of a nondopaminergic nigrostriatal projection, and more recently, gamma-aminobutyric acid (GABA) has been identified as its neurotransmitter. In this study, we used the combination of immunocytochemistry for tyrosine hydroxylase (TH; a marker of dopaminergic neurons), in situ hybridization (ISH) for two different isoforms of glutamic acid decarboxylase (GAD65 and GAD67, the rate-limiting enzyme in GABA synthesis) and retrograde tracing techniques to investigate the possible existence of nigrostriatal neurons containing both neurotransmitters (dopamine and GABA) in the rat. Our results revealed that approximately 10% of mesostriatal dopaminergic neurons, most of them lying in the medial region of the substantia nigra pars compacta (SNC) and neighbouring A10 region, contain GAD65 mRNA. These findings reveal a third nigrostriatal pathway formed by dopaminergic/GABAergic neurons. Contrasting with the idea that in the basal ganglia, dopamine and GABA are released from different cell populations, the results suggest a more complex dopamine/GABA interaction than previously assumed, probably including cotransmission.

Gender differences and the effect of different endocrine situations on the NOS expression pattern in the anterior pituitary gland.

The presence of neuronal nitric oxide synthase (nNOS) in two populations of pituitary cells, gonadotrophs (LH) and folliculostellate (FS) cells, suggests that pituitary nitric oxide (NO) is involved in the control of hormone secretion. We have used single and double immunostaining and quantitative procedures to investigate possible gender-related differences in the nNOS expression pattern in the anterior pituitary lobe and its possible alterations in different endocrine situations. Our results reveal a sexual dimorphism in the pattern of nNOS expression. In males, nNOS is mainly found in FS cells, whereas only a few LH cells express nNOS. Conversely, in females, nNOS is mainly found in LH cells. After gonadectomy, paralleling an increase in LH cell size and serum luteinizing hormone (LH) levels, there is nNOS upregulation in LH cells and nNOS downregulation in FS cells. After testoterone replacement, LH cells become nNOS-immunonegative again. In lactating rats, LH cells overexpress nNOS, but LH cell size and serum LH levels are low. This suggests that, depending on its cellular source, pituitary NO can exert either an inhibitory or a stimulatory effect on hormone secretion. When released from FS cells, NO exerts a paracrine inhibitory effect, and when released from gonadotrophs it exerts an autocrine or paracrine stimulatory effect on LH or prolactin secretion, respectively.

Nitric oxide synthase expression in the cerebral cortex of patients with epilepsy.

PURPOSE: Nitric oxide (NO), a short-lived radical synthesized from L-arginine by activation of the enzyme nitric oxide synthase (NOS), has been implicated in the pathophysiology of epilepsy by some investigators. However, the current data about NO and NOS in epilepsy are controversial and are derived only from animal models of epilepsy. In this study we investigated possible changes in NOS expression in the cerebral cortex of patients with epilepsy. METHODS: Qualitative and quantitative parameters of the immunolabeling pattern of the neuronal, endothelial, and inducible isoforms of NOS were analyzed in biopsy material obtained from patients with short and long seizure history and from patients without epilepsy. RESULTS: The comparative study showed that in the cerebral cortex of patients with epilepsy, particularly in those with a long seizure history, the number and labeling intensity of NOS-positive neurons increased, and that a subpopulation of nonpyramidal GABAergic neurons (type II NOS neurons) was responsible for this phenomenon. CONCLUSIONS: The fact that NOS upregulation is more evident in patients with a long seizure history suggests that this is a consequence of seizures, acting probably as an adaptative response to the sustained release of excitatory amino acids.

Compartmental organization and chemical profile of dopaminergic and GABAergic neurons in the substantia nigra of the rat.

The substantia nigra (SN) is a midbrain center composed of dopaminergic (DA-) and gamma aminobutyric acid (GABA)ergic (GABA-) neurons. In this study, we investigated the topographical relationship between both cell populations and their chemical profile by using single and double immunostaining for tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), cholecystokinin (CCK), calretinin (CR), calbindin (CB), parvalbumin (PV), and nitric oxide synthase (NOS). Our results showed that DA-cells are arranged in two bands, one rostrodorsal that corresponds to the SN pars compacta (SNC), and another caudoventral that corresponds to the SN pars reticulata (SNR) and emits cell bridges that make contact with the rostrodorsal one. In the SNR, GABA-cells are arranged in dorsoventrally elongated clusters that occupy DA-cell free regions. According to cytoarchitectural, topographical, and chemical criteria, we identified ten different cell groups: five dopaminergic ones, and five GABAergic ones. Within DA-cells, we found a cell group in the dorsomedial portion of the SNC which contains CCK, CR, and CB (dmSNC); DA-cells in the SN pars lateralis (SNL) which also contain CCK, CR and CB; DA-cells in the rostral half of the SNC containing CCK and CR (rSNC); DA-cells in the SNR and the caudal half of the SNC which only express CR (cSNC-SNR), and a DA-cell group in the lateral part of the SNC that contains none of the markers studied (lSNC). Within GABA-cells, we distinguished: large GABA-cells in the SNL that contain PV; large GABA-cells in the rostrolateral part of the SNR containing PV and NOS (rlSNR), small GABA-cells in the caudomedial part of the SNR containing PV (cmSNR), and two groups of small GABA-cells in the rostromedial portion of the SNR, one of them containing CR (rmcSNR), and the other containing NOS (rmnSNR). These data suggest that over a compartmental and complementary organization, DA- and GABA-nigral cells form a mosaic of neurochemically different subnuclei which probably differ in their physiological and pharmacological properties and vulnerability to aggression.

Electrophysiological and morphological evidence for a GABAergic nigrostriatal pathway.

The electrophysiological and neurochemical characteristics of the nondopaminergic nigrostriatal (NO-DA) cells and their functional response to the degeneration of dopaminergic nigrostriatal (DA) cells were studied. Three different criteria were used to identify NO-DA cells: (1) antidromic response to striatal stimulation with an electrophysiological behavior (firing rate, interspike interval variability, and conduction velocity) different from that of DA cells; (2) retrograde labeling after striatal injection of HRP but showing immunonegativity for DA cell markers (tyrosine hydroxylase, calretinin, calbindin-D28k, and cholecystokinin); and (3) resistance to neurotoxic effect of 6-hydroxydomine (6-OHDA). Our results showed that under normal conditions, 5-8% of nigrostriatal neurons are immunoreactive for GABA, glutamic acid decarboxylase, and parvalbumin, markers of GABAergic neurons, a percentage that reached 81-84% after 6-OHDA injection. Electrophysiologically, NO-DA cells showed a behavior similar to that found in other nigral GABAergic (nigrothalamic) cells. In addition, the 6-OHDA degeneration of DA cells induced a modification of their electrophysiological pattern similar to that found in GABAergic nigrothalamic neurons. Taken together, the present data indicate the existence of a small GABAergic nigrostriatal pathway and suggest their involvement in the pathophysiology of Parkinson's disease.

Expression of three forms of nitric oxide synthase in peripheral nerve regeneration.

Nitric oxide (NO) is a short-lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis, exists in three different forms: the neuronal (nNOS), present in discrete neuronal populations; the endothelial (eNOS), present in vascular endotheliun, and the inducible isoform (iNOS), expressed in various cell types when activated, including macrophages and glial cells. In this study, we have investigated the possible involvement of NO in Wallerian degeneration and the subsequent regeneration occurring after sciatic nerve ligature, using histochemistry and immunocytochemistry for the three NOS isoforms, at different postinjury periods. Two days after lesion, the three NOS isoforms are overexpressed, reaching their greatest expression during the second week. nNOS is upregulated in dorsal root ganglion neurons, centrifugally transported and accumulated in growing axons. eNOS is overexpressed in vasa nervorum of the distal stump and around ligature, and iNOS is induced in recruited macrophages. These findings indicate that different cellular sources contribute to maintain high levels of NO at the lesion site. The parallelism between NOS inductions and well-known repair phenomena suggests that NO, acting in different ways, may exert a beneficial effect on nerve regeneration.

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury.

A possible role for nitric oxide in growth and regeneration of dorsal root ganglion (DRG) afferents has been explored in lesion experiments by comparing immunocytochemistry for nitric oxide synthase (NOS) with that for the growth-associated phosphoprotein 43 (GAP-43). Sciatic nerve ligature induced a progressive increase in the number of small DRG cell profiles immunopositive for NOS between 2 days and 4 weeks of survival. In the proximal stump of the ligature, NOS-immunopositive fibers began to appear 2 days after injury and their growth cones were especially evident after 7 days. NOS-immunopositive fibers appeared past (i.e., distal to) the ligature at 14 days of survival and extended for at least 6 mm in either direction 4 weeks after the lesion. Dorsal root ligature alone at L4-L5 did not result in expression of NOS in DRG neurons or in the appearance of NOS-immunopositive fibers. In rats with dorsal root ligature and nerve ligature, the results were similar to those with nerve ligature only. DRG cell profiles immunopositive for GAP-43 kept increasing from 2 days to 4 weeks after sciatic nerve ligature and included small neurons initially and large neurons subsequently. Numerous axons became GAP-43 immunopositive on both sides of the ligature from 2 days after injury. In double-labeled material, about 80% of DRG cell profiles immunopositive for NOS were also immunopositive for GAP-43. The two antigens co-occurred in peripheral nerve axons proximal to the ligature starting at about 7 days and distal to it at about 2 weeks after ligature. Thus, in response to nerve lesion, nitric oxide may not only provide an injury signal to the central nervous system but may also contribute to the growth and regeneration of injured axons.