Expression of neuronal and inducible nitric oxide synthase in neuronal and glial cells after transient occlusion of the middle cerebral artery.

We presently investigated the time-course of neuronal nitric oxide synthase and inducible nitric oxide synthase expression and content in the rat striatum up to 6 days after ischemia induced by transient middle cerebral artery occlusion, a condition that potentially allows functional recovery, with the aim to identify the cell types expressing these two enzymes and to correlate neuronal nitric oxide synthase and inducible nitric oxide synthase changes in order to verify whether and how these changes are related to tissue damage, motor-sensory performances and survival. Before and after surgery, the animals underwent neurological evaluation. The results demonstrated that the rats with a score > or = 12 at the neurological evaluation 24 h after ischemia showed a significant increase in neuronal nitric oxide synthase-immunoreactive neurones and absence of inducible nitric oxide synthase-immunoreactive cells and survived up to the sixth day; conversely, the rats with a score < 12 at the neurological evaluation 24 h after ischemia showed a progressive significant decrease in neuronal nitric oxide synthase-immunoreactive neurones and appearance of inducible nitric oxide synthase-immunoreactive cells and none of the rats survived up to the sixth day. Microglia cells were activated in both groups but only in the latter did these cells express inducible nitric oxide synthase. Measurement of the infarct area demonstrated that it occupied a similar territory in both groups of rats but in those with a score < 12 the edema was more extended. In conclusion, we demonstrated that a neurotoxic insult such as ischemia can induce neuronal nitric oxide synthase expression in the neurones and that when neuronal nitric oxide synthase-immunoreactive neurones increase in number, microglia activation is less extended, inducible nitric oxide synthase-immunoreactive cells are absent, tissue damage reduced and the rats survive longer. Conversely, when there is a significant decrease of neuronal nitric oxide synthase-immunoreactive neurones, microglia cells are intensely activated, inducible nitric oxide synthase-immunoreactive cells appear and the animal survival is shortened.

Interstitital cells of Cajal in the human stomach: distribution and relationship with enteric innervation.

Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal muscle coat with a region-specific location, and are considered to be pace-maker and/or mediators of neurotransmission. Little is known about their shape, size, distribution and relationships with excitatory and inhibitory nerves in human stomach. With this aim, we labeled the ICC, using c-Kit immunohistochemistry, followed by a quantitative analysis to evaluate the distribution and area occupied by these cells in the circular and longitudinal muscle layers and at the myenteric plexus level in the human fundus, corpus and antrum. Furthermore, by NADPH-d histochemistry and substance P (SP) immunohistochemistry, we labeled and quantified nitric oxide (NO)-producing and SP-containing nerves and evidenced their relationships with the ICC in these three gastric regions. In the fundus, the ICC appeared as bipolar cells and in the corpus and antrum they mainly appeared as multipolar cells, with highly ramified processes. The networks formed by ICC differed in the three gastric regions. The ICC number was significantly higher and cell area smaller in the fundus compared to the corpus and antrum. The area occupied by the ICC was significantly higher at the myenteric plexus level compared with circular and longitudinal muscle layers. Everywhere, NADPH-d-positive nerves were more numerous than SP-positive ones. Both kinds of fibers were closely apposed to the ICC in the corpus and antrum. In conclusion, in the human stomach, the ICC have region-specific shape, size and distribution and in the corpus and antrum have close contact with both inhibitory and excitatory nerves. Presumably, as suggested for laboratory mammals, these differences are in relationship with the motor activities peculiar to each gastric area.

Alterations of neurokinin receptors and interstitial cells of Cajal during and after jejunal inflammation induced by Nippostrongylus brasiliensis in the rat.

Substance P (SP) and its receptors NK1 and NK2 are widely expressed in the intestinal wall by neurones, interstitial cells of Cajal (ICC) and smooth muscle cells. Changes in SP and/or its NK receptors have been documented during experimental inflammation in animals or inflammatory bowel diseases in humans, but the data concern the acute phase of the inflammatory process. We determined immunohistochemically whether NK receptors and SP were altered in the muscle coat during jejunal inflammation induced by the nematode Nippostrongylus brasiliensis and whether these alterations persisted when inflammation had spontaneously resolved 30 days postinfection. An ultrastructural analysis was also conducted on ICC, nerves and muscle. At day 14, when inflammation peaked, there was a reduction in NK1 receptors in myenteric neurones and in SP-immunoreactive nerve endings. There were also ultrastructural anomalies in synaptic vesicles and NK2 receptor loss in the circular muscle layer. The SP decrease persisted at day 30, whereas neurones and circular muscle cells re-expressed NK1 and NK2 receptors, respectively. The ICC at the deep muscular plexus, located near to the inflammatory site, underwent alterations leading to their complete loss at day 30. These morphological changes are probably associated with impairment in tachykinergic control of jejunal functions leading to the alterations of motility and sensitivity to distension already described in these animals.

Co-distribution of NK2 tachykinin receptors and substance P in nerve endings of guinea-pig ileum.

The distribution of NK2 tachykinin receptors-immunoreactivity (NK2r-IR) in the guinea-pig ileum and the co-distribution of NK2r-IR with substance P-immunoreactivity (SP)-IR were investigated. NK2r-IR was detected in varicose fibers of myenteric and submucous ganglia and nerve strands, in the longitudinal and circular muscle layers and at the deep muscular plexus (DMP). Except for the submucous plexus, some of the NK2r-IR varicose fibers were co-distributed with SP-IR ones and quantitative analysis showed significant regional differences in the percentages of these fibers. These results demonstrate that presynaptic NK2 receptors are located at varicose fibers likely originating from motor neurons projecting to muscle layers and DMP, and from interneurons. Furthermore, the NK2r/SP-IR co-distribution suggests that some of these receptors are autoreceptors on SP nerve endings.

Myenteric neurons and interstitial cells of Cajal of mouse colon express several nitric oxide synthase isoforms.

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.

Substance P immunoreactive nerves and interstitial cells of Cajal in the rat and guinea-pig ileum. A histochemical and quantitative study.

The substance P (SP)-containing nerves at the deep (DMP) and myenteric (MP) plexuses and the related interstitial cells of Cajal (ICC-DMP and ICC-MP) were immunohistochemically studied in rat and guinea-pig ileum. All the ICC expressed SP-preferred receptor NK1r: the ICC-DMP showed an intense and the ICC-MP a faint NK1r-immunoreactivity(IR). c-kit-labeling confirmed that they were ICC. The SP-IR nerves at the DMP were significantly more numerous in the guinea-pig than in the rat, and more numerous than those at the MP in both animal species. All the ICC-DMP in the guinea-pig and half of them in the rat were close to SP-IR nerves. The ICC-MP were rarely near to SP-IR nerves in either species. The SP-innervation shows interspecies differences at the DMP that imply a different tachykinergic control of the local ICC.

Expression of dystrophin in the mouse myenteric neurones.

Dystrophin, a membrane-associated protein, plays relevant roles in cell functions. Its lack or trunkated expression results in Duchenne muscular dystrophy (DMD), a pathology associated with alterations in gastrointestinal motility considered to be neural in origin. No data are available on the presence of dystrophin in myenteric neurones. We labelled mouse myenteric neurones with DYS1-, DYS2-, DYS3-antibodies; staining was located on the perikarya and processes, with no differences in distribution or intensity among the antibodies; the western immunoblot analysis indicated that myenteric neurones express several dystrophin isoforms; anti-dystrophins/anti-neuronal specific enolase double-labeling confirmed that all neurones express dystrophin. Dystrophin in myenteric neurones might play a role in cytoskeletal organization, axonal transport and signal pathways; its lack might cause the intestinal motor abnormalities reported in DMD patients.

Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice.

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic (mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that N(omega)-nitro- L-arginine methyl ester (L-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca(2+)-free solution. In colon from mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca(2+) through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.