Letizia Corsani

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.

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.

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.

Inducible nitric oxide synthase appears and is co-expressed with the neuronal isoform in interneurons of the rat hippocampus after transient ischemia induced by middle cerebral artery occlusion.

The hippocampus (dentate gyrus DG plus Cornu Ammonis, CA) is vulnerable to neuropathological events such as ischemia. The DG is a region where neurogenesis takes place and it has been demonstrated that ischemia stimulates neurogenesis. Nitric oxide (NO) plays a major role in ischemic damage evolution and increases in rat hippocampus after ischemia. No information is available on the presence of nNOS-immunoreactive (IR) neurons in the hippocampus of ischemic animals; whereas, the presence of the iNOS protein has been reported in the DG after focal ischemia. We evaluated, immunohistochemically, the cell types expressing nNOS and iNOS in the rat hippocampus by 24 up to 144 h after transient middle cerebral artery occlusion to ascertain whether ischemia induces changes in nNOS or iNOS expression and whether a relationship exists between these changes and the animal survival. nNOS-IR interneurons were detected in control and ischemic rats; in the latter, their number was significantly decreased at all time points. iNOS-IR interneurons appeared in the hippocampus of ischemic rats at 24 h; their number was significantly higher in the animals with longer survival and did not change at later time points. More than 50% of the nNOS-IR interneurons co-expressed iNOS-IR. All these changes were seen both in the ipsilateral and contralateral hippocampus. In conclusion, the focal ischemia affects the hippocampus which responds bilaterally to the injury. We hypothesize that the decrease in the nNOS-IR neurons is likely due to either a neuronal loss or a switching towards the iNOS production which, by inducing neurogenesis, might compensate the neuronal loss.

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.

Synaptic vesicle morphology and recycling are altered in myenteric neurons of mice lacking dystrophin (mdx mice)

Several dystrophin isoforms are known. The full‐length isoform is present in striated and smooth muscles and neurons and its lack causes Duchenne Muscular Dystrophy, a progressive myopathy accompanied by mild cognitive deficits and gastrointestinal dismotility. An ultrastructural study was undertaken in the colon of mice lacking full‐length dystrophin and maintaining shorter isoforms (mdx mice) to ascertain whether myenteric neurons have an altered morphology. Results showed a significant increase in the size of synaptic vesicle and in the number of recycling vesicles. An enlargement of endoplasmic reticulum cisternae in a subpopulation of neurons was also seen. Immunohistochemistry confirmed that the shorter isoforms were expressed in mdx mice myenteric neurons. These findings indicate the presence of a neuropathy at the myenteric plexus which might justify the defective neuronal control of gastrointestinal motility reported for these animals and which might be correlated with full‐length dystrophin loss, since the shorter isoforms are present. J. Cell. Physiol. 197: 232–242, 2003.

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.

Functional activity and expression of inducible nitric oxide synthase (iNOS) in muscle of the isolated distal colon of mdx mice.

The inducible isoform of nitric oxide (NO) synthase (iNOS), expressed in endothelium, epithelium, and inflammatory cells, produces a large amount of NO. Previous studies on mouse intestine indicate that a muscular iNOS may have a role in the storage of intraluminal content. In this study we investigated the presence and function of iNOS in the colonic smooth muscle cells of 2‐ and 12‐month‐old dystrophic (mdx) mice. By using an in vitro isovolumic technique, and immunohistochemical and Western blot analysis, we demonstrated that iNOS is expressed and active in the smooth muscle cells of normal mouse and defective in young adult (2‐month‐old) mdx mice. Therefore, an altered activity of the muscle iNOS might explain the motility disorders observed in the colon of mdx mice and, from a clinical point of view, the impairment of intestinal function in dystrophic patients. Muscle Nerve 29: 795–803, 2004

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.