Neuronal anomalies and normal muscle morphology at the hypomotile ileocecocolonic region of patients affected by idiopathic chronic constipation.

Patients suffering from idiopathic slow-transit chronic constipation have a delayed colonic transit referable to a decrease or loss of propagating contractions. Myogenic and/or neural mechanisms have been implicated in the pathophysiology of this dysfunction and neuronal abnormalities have been described at the ascending, descending and sigmoid colon. The morphology and motile behaviour of the ileocecocolonic region, which in healthy subjects regulates cecum filling and emptying, have never been investigated in such disease. Therefore, we endoscopically ascertained whether a motility impairment was present at these junctional areas and neither spontaneous nor provoked occlusive contractions were found at the cecocolonic junction. Light and electron microscope examination of the entire colon revealed apparently normal features of neurons, smooth muscle cells and interstitial cells of Cajal, while immunohistochemistry and quantitative analysis demonstrated neuronal anomalies at the junctional areas. These anomalies consisted of low total neuron density and significantly few VIP-immunoreactive neurons at the two enteric plexuses, significantly few NOS-immunoreactive neurons at the myenteric plexus and significantly more NOS-immunoreactive neurons at the submucous plexus. These findings exclude a myopathy and demonstrate the existence of a neuropathy. In particular, the presence at the ileocecocolonic region of few VIP- and NO-producing neurons suggests that there might be a reduced VIP and NO production which may result in a compromised relaxation and/or onset of propagating contractions, slowing down bolus transit. The presence at the proximal colon of such an abnormality might explain why left colectomy and/or cecorectal anastomosis are unsuccessful in patients with this disease.

The cytoskeleton of the myenteric neurons during murine embryonic life.

The organization of the cytoskeleton has been studied during mouse differentiation in cells of the myenteric neuronal lineage. The entire gut was examined starting from day 12.5 of embryonic life (E12.5) until birth (P0). Immunocytochemistry was performed to evaluate the expression of five of the most represented neurofilaments proteins (the low, NF-L, medium, NF-M, and heavy, NF-H, molecular weight subunits, alpha-internexin and peripherin) and of two of the microtubule-associated proteins (MAPI and MAP2a+2b). In parallel, the appearance in the differentiating myenteric neurons of filamentous and microtubular structures and their intracytoplasmatic distribution were observed under the electron microscope. A differential immunohistochemical expression of the structural proteins was found. Immature cells expressed alpha-internexin, peripherin, NF-M and MAP1 by day E12.5; alpha-internexin expression was strong in these cells, but gradually decreased with age and was practically absent in adulthood. Conversely, the expression of the other three proteins increased with cell differentiation and was still present in adulthood. NF-L and NF-H expression appeared later, by day E16.5, and was weak for the entire pre- and postnatal life. MAP2a+2b was never expressed. Under the electron microscope, at day E12.5 the cytoskeleton was already organized in filamentous and microtubular structures. At this age neurofilaments were few and mainly located in the cell processes, and microtubules were numerous and mainly assembled in the neuritic growth cones, together with synaptic vesicles. With ageing, neurofilaments and microtubules were ubiquitous in the neuron. Data obtained demonstrate that cytoskeletal proteins gradually accumulate in the cells of the neuronal lineage in parallel with the organization of the cytoskeletal structures, which in turn mediate important neural events by the earliest stages of murine embryonic life, including growth of nerve processes and initiation of axonal transport.

Ultrastructural and biochemical studies on the neuroprotective effects of excitatory amino acid antagonists in the ischemic rat retina.

The effects of glutamate receptor agonists were evaluated, by utilizing the electron microscope, in a photothrombotic occlusion model of rat retinal vessels in order to study the ischemic damage and its antagonism in each morphologically identified population of retinal neurons. Rats were systemically injected with rose bengal fluorescein dye and one of their eyes was then exposed to bright light. This treatment caused neuronal damage and reduced the activities of the neuronal marker enzymes, choline acetyltransferase and glutamate decarboxylase, by approximately 75%. A single intravitreal injection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX, 10-50 nmol), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, or of thiokynurenate (100-400 nmol), which also antagonizes N-methyl-D-aspartate (NMDA) receptors, performed immediately after the lesion, significantly reduced this loss. The electron microscope examination showed major damage in each type of retinal neuron, the pigment epithelium, and the microvessels. NBQX or thiokynurenic acid reduced, in a comparable manner, the effects of ischemia on the pigment epithelium, the photoreceptors, and the bipolar and the horizontal cells. NBQX was particularly efficient in reducing the damage to the amacrine cells located in the inner nuclear layer. The displaced amacrine and ganglion cells were not protected by NBQX but were almost completely spared in animals treated with thiokynurenate. These results show that antagonism of AMPA receptors is sufficient to reduce ischemic damage in a large number of retinal neurons, but that neuroprotection in the ganglion cell layer may be obtained only with agents which also antagonize NMDA receptors.

Ultrastructural localization of neuronal nitric oxide synthase-immunoreactivity in the rat ileum.

The location of neuronal nitric oxide synthase-immunoreactivity (NOS-IR) in whole mount preparations of muscularis externa of rat ileum was determined by using pre-embedding electron microscope immunocytochemistry. Several neurons, nerve fibers and nerve endings in the myenteric plexus (MP) and nerve endings within the muscle layers were found to be NOS-IR. These nerve endings were especially numerous in the deep muscular plexus (DMP) and much closer to interstitial cells of Cajal (ICC) than to smooth muscle cells. Some of the ICC-MP were NOS-IR. These findings indicate that ICC-MP are apparently able to produce NO and ICC-DMP are the ileal ICC type very richly innervated by the NO releasing nerves.

Neurochemical differentiation of rat enteric neurons during pre- and postnatal life.

The presence of nitric oxide synthase (the enzyme which synthesizes nitric oxide), vasoactive intestinal peptide (VIP) and pituitary adenylyl-cyclase-activating peptide (PACAP) in the rat enteric nervous system between term (E18) and 90 days postpartum (P90) was investigated by immunohistochemistry. Neuronal maturity was ascertained by emanining for the presence of two novel neuron intermediate filament proteins: alpha-internexin, which is transiently expressed in developing neurons, and peripherin, which is expressed in differentiating as well as in mature neurons. The alimentary canal of the foetus at E18, unfed newborn, suckling, weaning and adult rats was studied. Throughout the rat gut the myenteric neurons were recognized by the time the foetal stage was reached and the submucous neurons by the suckling period. alpha-Internexin was present at E18; its levels increased during ageing and were markedly reduced at P30. Peripherin was first detected at birth; its levels increased with ageing and practically almost all of the neurons and nerve fibres were labelled from P30. Nitric oxide synthase was present at E18 and the number of labelled neurons gradually increased with ageing, while both VIP and PACAP could be detected by the end of the suckling period in an equal number of neurons to that in adulthood. These data indicate that in the rat gut the onset of the mature neuronal phenotype is not yet achieved at birth and that the neurochemical differentiation is accomplished during the first month of postnatal life.

Constitutive and inducible nitric oxide synthases in mouse uterus and their changes following treatment with ovarian steroids. Immunocytochemical and histoenzymological studies.

Previous studies showed that different nitric oxide synthase isoforms are present in the uterus of laboratory mammals and that their expression is influenced by ovarian steroids. However, the results of these studies are not univocal, probably owing to the different hormonal treatments and techniques applied to reveal nitric oxide synthases. In this study we investigated the distribution and expression of constitutive and inducible nitric oxide synthase isoforms by immunocytochemistry and their changes following treatment of the mice with 17 beta-estradiol alone or in combination with medroxyprogesterone. Moreover, we compared the immunoreactivities for nitric oxide synthases with the histochemical reaction for NADPH-diaphorase, an enzyme that may be associated with nitric oxide synthase. The results obtained show that the two nitric oxide synthase isoforms are differently expressed in surface epithelium, glands, stromal cells and myometrium and that, as compared with the uteri from mice treated with estrogen alone, those from mice treated with estrogen plus progestin showed enhanced expression of constitutive nitric oxide synthase in the myometrium and of the inducible isoform in surface epithelium, glands, stromal cells and myometrium. The results obtained with NADPH-diaphorase reaction show that there is not a colocalization of nitric oxide synthase isoforms and NADPH-diaphorase, apart from a partial colocalization in part of the stromal cell population and myometrium. This provides evidence that NADPH-diaphorase histochemistry is not a valid technique to localize the sites of nitric oxide synthesis in the mouse uterus and that the use of this technique may generate misleading in the interpretation of the effect of ovarian steroids in regulating nitric oxide production by the different components of the uterine wall.

Differentiation of enteric plexuses and interstitial cells of Cajal in the rat gut during pre- and postnatal life.

The stomach, small and large intestine of fetuses at term, of unfed newborns, of suckling, weaning and of adult rats were studied by a combined light (LM) and electron microscope (EM) examination. Neuron-specific enolase was used as a neuronal marker under LM. Zinc-iodide-osmium (ZIO) impregnation was used for a selective staining of neurons and interstitial cells of Cajal under both LM and EM. A routine EM procedure made it possible to identify the nerve elements and ICC and to evaluate their degree of differentiation. The differentiation of enteric plexuses and ICC was poor at birth and was accomplished during the weaning period. The myenteric plexus differentiation preceded the submucous plexus differentiation; in particular, under both LM and EM myenteric neurons were already recognizable in the fetus, while the submucous neurons by day 0 under EM and by day 7 under LM. The ICC were poorly differentiated at birth and acquired the adult morphology during the suckling period. Nerve endings contacting ICC were differentiated before ICC differentiation. The ZIO uptake by both nerve elements and ICC correlated with the establishment of their differentiated features. In conclusion, the present findings confirmed that differentiation of ICC and enteric plexuses is microenvironment dependent, since their differentiative steps are interrelated and correlated with diet changes. ZIO impregnation under EM enabled a distinction to be made between a "morphological' and a "functional' differentiation, and revealed that the former is achieved during the suckling period and the latter by the weaning period. It can be suggested that during the postnatal developmental stages ICC and neuronal functions might be different from those in adulthood.

Morphological changes in the smooth muscle cells of the mouse lower oviduct during pregnancy and post-partum.

The muscle coat of the lower region of the mouse oviduct undergoes morphological changes during pregnancy and post-partum. Ultrastructural examination and morphometrical findings show that, during pregnancy, the smooth muscle cells undergo a significant increase in both the number of mitochondria and caveolae and in the extension of the rough endoplasmic reticulum and Golgi apparatus, suggesting an enhancement of metabolic activities, especially protein synthesis. Within two days after delivery, the number of mitochondria and caveolae is similar to that of non-pregnant mice, whereas the extension of the rough endoplasmic reticulum and Golgi apparatus further increases significantly. The cytological signs of enhanced protein synthesis in the smooth muscle cells of the lower oviduct during pregnancy and, especially, in the post-partum period are probably related to a remodelling of the intercellular connective tissue matrix.

Vasoactive intestinal polypeptide and nitric oxide synthase distribution in the enteric plexuses of the human colon: an histochemical study and quantitative analysis.

UNLABELLED: Vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) positive innervation patterns were immunohistochemically and statistically evaluated in the human colon. Specimens from the right colon (cecum, ascending and right transverse colon) and left colon (left transverse and descending colon) were obtained surgically, fixed either in paraformaldehyde or in Carnoy's or in Bouin's and paraffin embedded. Sections were stained with hematoxylin-eosin, toluidine blue, cresyl violet, neuron-specific enolase, anti-VIP, and anti-NOS. The same results were obtained regardless of the fixative used. Enolase-positive, VIP-positive, and NOS-positive cells were occasionally found within the circular muscle and interpreted as neurons. VIP-positive nerve fibers were evenly distributed within the circular muscle while NOS-positive ones were lacking in its inner portion. The left colon was richer in neurons than the right colon, at both plexuses. VIP- and NOS-positive neuron densities were higher at the left than at the right colon, whereas at all colonic levels VIP-positive neuron percentages at both plexuses and NOS-positive ones at the myenteric plexus were similar. At the submucous plexus the NOS-positive neuron percentage was lower than that of the VIP-positive one. IN CONCLUSION: (a) the right colon contains a lower number of neurons and of VIP- and NOS-positive ones than the left colon, and (b) VIP- and NOS-positive fibers are differently distributed in the inner and outer portions of the circular muscle.

Nitric oxide producing neurons in the human colon: an immunohistochemical and histoenzymatical study.

The nitric oxide producing neurons of the human colonic myenteric plexus have been studied by using antibodies against cerebellar NO synthase type I (NOS-IR) and NADPH-diaphorase (NAPDH-d) histoenzymatic reaction. The majority of the stained neurons were both NOS-IR and NADPH-d-positive, while a few others were either NADPH-d-positive or NOS-IR only. Among the co-stained neurons, four subpopulations sharing various degrees of staining intensities have been identified. These findings indicate that in the human colon a one-to-one correlation between NOS-IR and NADPH-d positivity does not exist and thus the NADPH-d reaction does not delineate with certainty all NO-producing neurons. The degree of staining intensity might account for different intracellular amounts of these two enzymes.

The interstitial cells of Cajal of the rat stomach. A light and electron microscope study.

Interstitial cells of Cajal (ICC), presumed to have a smooth muscle-like nature and to play a pacemaker role, are usually identified for their peculiar ultrastructural features and specific location throughout the gut muscle wall. A Zinc-Iodide-Osmium (ZIO) impregnation for ICC identification under the light microscope has been proposed. However, controversies as to certain ICC identification under both light and electron microscopes are still present, due to their ultrastructural features somewhat similar to the fibroblast ones and the low specificity of the ZIO-staining. The rat stomach has been studied. Some specimens have been routinely processed for electron microscopy, some others have been ZIO-impregnated and further routinely processed for both light and electron microscopy, in order to assure that all the cells presumed to be ICC for their ZIO-staining affinity are the same cells identified as ICC with routine electron microscope procedures, and not fibroblasts. The routine electron microscope examination made it possible to identify within the rat gastric muscle coat two cell populations, one with the same location and morphology as those reported in literature for the gastric ICC, and a second one with a similar location, but showing undoubted fibroblastic features. ZIO-staining, under both light and electron microscopes, revealed ZIO-stained cells distributed within the muscle coat in a manner identical to that of the ultrastructurally identified ICC. Under electron microscope examination, this cell type only was fully impregnated by the zinciodide deposits, whereas all other cell types, including fibroblast-like cells, were devoid of them. These data confirm that ICC can be electively ZIO-stained and that these cells and fibroblasts are two distinct cell types, as ultrastructural and physiological reports had previously suggested.