Nitric oxide is involved in the lesions of the peripheral autonomic neurons observed in the acute phase of experimental Trypanosoma cruzi infection

Our aim was to investigate the possible involvement of nitric oxide (NO) in peripheral denervation during the acute phase of murine experimental Trypanosoma cruzi infection. Wistar male rats were infected with the Y strain of T. cruzi. One group of animals was also treated with the NO synthase inhibitor N-nitro-l-arginine. A group of uninfected animals was the control. At the 18th day of infection the animals were sacrificed. Quantification of neurons in the colon and heart and tissue parasitism in the heart was performed. Serum concentration of nitrate was measured and a histochemical technique for assessing NADPH-diaphorase activity in the colon was also performed. The infected animals presented a statistically significant decrease in the number of peripheral neurons in the colon and heart and a 2-fold increase in serum NO(3) concentration compared with controls. The animals treated with N-nitro-l-arginine showed almost an absence of NO(3) concentration in the serum and did not show loss of neurons compared with controls. These treated animals displayed a 15-fold increase in tissue parasitism compared with nontreated infected animals. The NADPH-diaphorase activity was much more intense in the muscle layers of the colon of the infected animals than in those of the controls. Taken together, these data suggest that NO is involved in the peripheral denervation observed in the acute phase of experimental T. cruzi infection.     

Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons

The submucous layers of human small and large intestines contain at least two separate neuron populations. Besides morphological features, they differ in their immunoreactivities for calretinin (CALR) and somatostatin (SOM), respectively. In this study, submucosal wholemounts of 23 patients or body donors (including all segments of small intestine and colon) were immunohistochemically quadruple stained for CALR and SOM as well as for substance P (SP) and choline acetyltransferase (ChAT). We found that all SOM-positive neurons co-stained for ChAT and the majority for SP [between 50 % in the small intestinal external submucosal plexus (ESP) and 75 % in the colonic ESP]. In contrast, a majority of CALR-neurons contained ChAT (between 77 % in the small intestinal ESP and 92 % in the large intestinal ESP) whereas less than 4 % of CALR-neurons were co-immunoreactive for SP. Another set of wholemounts was co-stained for peripherin, a marker enabling morphological analysis. Where identifiable, both SOM alone- and SOM/SP-neurons displayed a uniaxonal (supposed pseudouniaxonal) morphology. We suggest that the chemical code of SOM-immunoreactive, human submucosal neurons may be “ChAT+/SOM+/SP±”. In additional sections double stained for SOM and SP, we regularly found double-labelled nerve fibres only in the mucosa. In contrast, around submucosal arteries mostly SOM alone- fibres were found and the muscularis propria contained numerous SP-alone fibres. We conclude that the main target of submucosal SOM(/SP)-neurons may be the mucosa. Due to their morpho-chemical similarity to human myenteric type II neurons, we further suggest that one function of human submucosal SOM-neurons may be a primary afferent one.     

Morphometric investigations of the colon mucosa in chronic Trypanosoma cruzi infected rats.

The objective of this investigation was to study the morphometry of the epithelial mucosa in the chronic phase of T. cruzi infection. Nine young female Wistar rats were inoculated with T. cruzi. Ten months after inoculation the animals were sacrificed and the proximal colon was collected for morphometric measurements of the thickness of the muscle layers, the number of neurons in the myenteric plexus, the crypt cell population (CCP), crypt cell production per crypt (CCPC) and turnover time (TT) of the epithelium. There was no muscle layer hypertrophy but there was significant denervation in the group inoculated with T. cruzi, which also showed hyperplasia of the epithelium. The data suggest that denervation of the myenteric plexus did not induce hypertrophy of the propria muscle layer itself but altered the morphometry of the colonic epithelium in T. cruzi-infected animals, with increased development of CCP and TT. It is possible that this epithelial hyperplasia, as a consequence of a longer crypt cell TT, increased the absorption and secretion activities of the colon, which in turn may participate in the genesis of the enteromegalies observed in the chronic phase of Chagas' Disease.     

Diabetic state affects the innervation of gut in an animal model of human type 1 diabetes

Gastrointestinal symptoms in diabetic patients are commonplace, and are believed to be due, at least partly, to neuropathy of the gut. In the present study, therefore, some important neurotransmitters in the myenteric plexus were investigated in non-obese diabetic mice, an animal model of human type 1 diabetes. For this purpose, immunocytochemistry was applied on sections from antrum, duodenum and colon, subsequently quantified by computerized image analysis. Whereas the number of vasoactive intestinal peptide (VIP)-positive neurons was increased in antral myenteric ganglia of diabetic mice, there was a decreased density of nerve fibres in muscularis propria. No difference was seen in the VIP of duodenum and colon. Acetylcholine-containing nerve fibres showed an increased volume density in muscularis propria of antrum and duodenum, but a decreased density in colon of diabetic mice, as compared with controls. There was a decreased number of neurons containing nitric oxide synthase (NOS) in myenteric ganglia of antrum and duodenum. No difference was seen in density of NOS-containing nerve fibres in muscularis propria. There was no difference regarding neuropeptide Y (NPY) and galanin between diabetic and control mice; nor was there any difference between pre-diabetic NOD mice and controls regarding all bioactive substances investigated. It is concluded that the diabetic state affects the innervation of gut in this animal model. The present findings may be of some relevance to the gastrointestinal symptoms seen in patients with diabetes.     

Myenteric plexus of obese diabetic mice (an animal model of human type 2 diabetes)

The myenteric plexus of the gastrointestinal tract was investigated in the obese diabetic mouse, an animal model of human type 2 diabetes. Sections were immunostained by the avidin-biotin complex method, using a general nerve marker, protein gene product 9.5 (PGP 9.5), as well as antibodies to several important neurotransmitters. Computerized image analysis was used for quantification. When diabetic mice were compared with controls, no difference was found in the density of PGP 9.5-immunoreactive (IR) nerve fibres in antrum, duodenum or colon. In antrum and duodenum, diabetic mice showed a decreased number of vasoactive intestinal peptide (VIP)-IR neurons in myenteric ganglia as well a decreased relative volume density in myenteric plexus (though not significantly in antrum, p=0.073). No difference was found regarding VIP-IR nerves in colon. The volume density of nitric oxide synthase (NOS)-IR nerve fibres was decreased in antrum and duodenum of diabetic mice, whereas no difference was found in colon. The density of galanin-IR nerve fibres was decreased in duodenum. Whereas neuropeptide Y (NPY)- and vesicular acetylcholine transporter (VAChT)-IR nerve fibres was increased in density in colon of diabetic mice, no difference was found in antrum and duodenum. Regarding substance P, there was no difference between diabetic and control mice in antrum, duodenum or colon. The present study shows that gut innervation is affected in this animal model of human type 2 diabetes. It is possible that the present findings may have some relevance for the gastrointestinal dysfunctions seen in patients with type 2 diabetes.     

Differences in the chemical coding of nerve fibres supplying major populations of neurons between the caudal mesenteric ganglion and anterior pelvic ganglion in the male pig

The present study was designed to investigate and to compare the chemical coding of nerve fibres supplying major populations of neurons in the caudal mesenteric (CaMG) and anterior pelvic (APG) ganglion in juvenile male pigs (n=5) using double-labelling immunofluorescence. The co-existence patterns of some biologically active substances including tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) as well as vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), Leu5-enkephalin (LENK) and serotonin (5-HT) were analysed under a confocal laser scanning microscope. Profound differences in the neurochemical features of the nerve terminals between the ganglia were observed. Moreover, there were also distinct differences in the chemical coding of nerve fibres associated with the particular populations and subpopulations of neurons within the ganglia. In both ganglia, nearly all adrenergic and cholinergic neurons were supplied with VAChT-positive nerve fibres (putative preganglionic fibres). However, in the CaMG, they were more numerous and, in contrast to the APG, many of them also stained for VIP. In the APG, a great number of nerve terminals expressed immunoreactivity to SP and CGRP (putative collaterals of sensory neurons). Interestingly, they densely supplied almost exclusively adrenergic neurons. SP-positive nerve fibres were moderate in number in the CaMG, but, in addition to VAChT-IR nerve terminals, the most numerous populations of nerve fibres in this ganglion were those expressing highly colocalized immunoreactivities to CGRP and LENK, and those which stained for 5-HT (putative processes of enteric neurons). However, these fibres supplied almost exclusively larger, intensely stained for TH and clustered adrenergic neurons. This diversity of the nerve terminals reflects the complexity of nerve circuits involved in the innervation of structures supplied by neurons in the porcine CaMG and APG. It also demonstrates the importance of nerve inputs for the proper function of autonomic neurons and thus their target tissues.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Projections of nitric oxide synthesizing neurons in the guinea-pig colon

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.     

Trypanosoma cruzi: treatment with the iron chelator desferrioxamine reduces parasitemia and mortality in experimentally infected mice

The effects of prolonged treatment with iron chelator (desferrioxamine) on the development of infection in mice inoculated with Y Trypanosoma cruzi were determined. Infected/treated mice presented lower levels of parasitemia and reduced mortality rate compared with infected/non-treated animals. The five out of twenty infected/treated mice that survived the acute phase of infection showed negative hemoculture and positive ELISA in the acute and chronic phases and positive PCR in the acute phase: in the chronic phase, three of the animals presented negative PCR. The single surviving infected/non-treated animal exhibited positive hemoculture, PCR and ELISA in both phases of infection. Infected groups presented lower levels of iron in the liver compared with treated/non-infected or non-treated/non-infected animals. The serum iron levels of the infected/non-treated group were higher on the 21st day post-infection in comparison with control and infected/treated groups. These results suggest that decrease of iron in the host leads to T. cruzi infection attenuation.     

Distribution of enteric nerve cells projecting to the superior and inferior mesenteric ganglia of the guinea-pig

Retrograde tracing, using Fast Blue dye, was employed to determine the distribution of enteric nerve cells that project to the superior mesenteric and inferior mesenteric ganglia of the guinea-pig. Retrogradely labelled neurons were found in the myenteric but not submucous ganglia. When the superior mesenteric ganglion was injected, labelled neurons were found in low frequencies (less than 5 nerve cell bodies/cm²) in the duodenum, jejunum, ileum, caecum and proximal colon. The distal colon was analysed in five segments of equal length (1–5; oral to anal). Segment 1 had about 4 labelled nerve cells/cm², whereas segments 2 to 5 displayed an average of about 25 nerve cells/cm². The rectum contained about 36 labelled neurons/cm². After injection of the inferior mesenteric ganglia with Fast Blue, no labelled neurons were found in the duodenum, jejunum, ileum or caecum. No labelled cells were observed in the gallbladder. A small number of labelled cells occurred in the proximal colon and in segment 1 of the distal colon. The frequency of labelled cells increased markedly in the more anal regions of the distal colon, and reached a peak in the rectum (138 cells/cm²). Both nerve lesions and immersion of the cut nerve in Fast Blue solution showed that the superior mesenteric nerve carries the axons of neurons located in the middle distal colon to the superior mesenteric ganglion. Almost half of the neurons in the rectum that project to the inferior mesenteric ganglia do so via the hypogastric nerves. Of neurons that projected to the inferior or superior mesenteric ganglia from the colon or rectum, similar proportions (about 75–80%) showed immunoreactivity for calbindin or VIP. For each of the prevertebral ganglia (coeliac, superior mesenteric and inferior mesenteric) the great majority of peripheral inputs arise from the large intestine.     

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.     

Immunohistochemical characterisation of cholinergic neurons in the anterior pelvic ganglion of the male pig

This study investigated immunohistochemical properties of cholinergic neurons in the anterior pelvic ganglion (APG) of juvenile male pigs (n=7). Cholinergic neurons were identified using antibodies against choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). Immunoblotting was applied to verify the specificity of ChAT-immunostaining. Western blotting performed on APG tissue homogenates detected single immunoreactive protein with a molecular weight matching that of ChAT (71.6 kDa). It was found that many APG neurons expressed immunoreactivity to ChAT or VAChT (40% and 39% of the neurons, respectively). The analysis of adjacent sections from the ganglion revealed complete colocalization of ChAT and VAChT in these nerve cells. Furthermore, virtually all the ChAT-positive neurons were tyrosine hydroxylase (TH)-negative (non-adrenergic) but many of them displayed immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or somatostatin (SOM). There were also single nerve cell bodies that stained for neither ChAT nor TH. The comparison of the adjacent sections revealed that NOS, VIP, NPY and SOM were simultaneously co-expressed in the majority of the cholinergic somata. ChAT- or VAChT-positive varicose nerve terminals supplied nearly all neuronal profiles within the ganglion often forming loose basket-like formations surrounding the particular nerve cell bodies. The present study for the first time has revealed that nearly all non-adrenergic neurons in the porcine APG are cholinergic in nature, i.e. express immunoreactivity for ChAT and VAChT. Considering a high coincidence between the chemical coding of non-adrenergic (cholinergic) nerve fibres supplying some porcine male reproductive organs described in earlier papers and that of cholinergic pelvic neurons found in this study it is further concluded that pelvic ganglia are probably the major source of cholinergic innervation for the porcine urogenital system.     

Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract,with particular reference to the mucosa

Summary The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions.VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers.These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Selective survival of calretinin- and vasoactive-intestinal-peptide-containing nerve elements in human chagasic submucosa and mucosa

Chronic Chagas' disease is frequently characterized by massive myenteric neuron loss resulting in megacolon with severely and irreversibly disturbed motility. Here, we focused on two submucosal neuron populations, immunoreactive for calretinin (CALR) or somatostatin (SOM), and their respective mucosal nerve fibres in chagasic megacolon. Surgically removed megacolonic segments of seven chagasic patients were compared with seven age- and region-matched non-chagasic control segments. Evaluation included immunohistochemical triple-staining of cryosections for CALR, SOM and peripherin or for CALR and vasoactive intestinal peptide (VIP) and of submucosal whole-mounts for CALR, SOM and the pan-neuronal marker anti-HuC/D. Submucosal neuron counts in chagasic tissue revealed neuron numbers reduced to 51.2?% of control values. In cryosections, nerve fibre area measurements revealed 8.6?% nerve fibre per mucosal area in control segments, but this value decreased to 1.5?% in megacolonic segments. In both evaluations, a disproportionate decrease of SOM-reactive nerve elements was observed. The proportions of SOM-positive neurons related to the total neuron number declined to 2?% (control 10?%) and the proportion of SOM-reactive mucosal nerve fibres related to the whole mucosal area to 0.014?% (control 1.8?%)in chagasic tissue. The second set of cryosections revealed extensive colocalization of CALR with VIP in both surviving submucosal perikarya and mucosal nerve fibres. We suggest that VIP, a neuroprotective and neuroeffectory peptide typically contained in submucosal neurons, allows both the VIP-containing neurons to endure and the patients to survive by maintaining their mucosal barrier, despite the almost complete loss of colonic motility for decades.     

Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig

Summary The distributions within the coeliac ganglion of different chemically coded subgroups of noradrenaline neurons, and the relationships between these neurons and nerve fibres projecting to the ganglion from the intestine, have been assessed quantitatively by use of an immunohistochemical double-staining method. Noradrenaline (NA) neurons made up 99% of all cell bodies. Of these, 21% were also reactive for somatostatin (NA/SOM neurons), 53% were also reactive for NPY (NA/NPY neurons), and 26% were not reactive for either peptide. NA neurons without reactivity for any of the peptides whose localization was tested have been designated NA/-. A small percentage, about 1%, of neurons were reactive for both NPY and SOM. The three major types of NA neurons were arranged in clumps or ribbons throughout the ganglia, with a tendency for NA/SOM neurons to be medial and NA/NPY neurons to be lateral in the ganglia. A small group of neurons (<1%) encoded with dynorphin, NPY and vasoactive intestinal peptide (VIP) was encountered. VIP-immunoreactive nerve terminals, projecting to the ganglion from cell bodies in the intestine, ended around NA/SOM and NA/neurons but not around NA/NPY neurons. Thus, the VIP axons from the intestine end selectively around neurons that modify intestinal function (NA/SOM and NA/-neurons) but not around neurons, the terminals of which supply blood vessels (NA/NPY neurons).     

Enteric Neuronal Damage,Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11^th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.     

Trypanosoma cruzi reinfections in mice determine the severity of cardiac damage

In two murine models we studied Trypanosoma cruzi reinfection in the acute and chronic phase of experimental Chagas' disease in order to elucidate the relevance of reinfections in determining the variability of cardiac symptoms and the irreversible cardiac damage. They were followed for 120 and 600 days post infection (p.i.) for the acute and chronic model, respectively. Reinfected mice reached higher parasitaemia levels than infected mice. The survival was 33 and 21% in the chronic phase for mice reinfected in the acute phase and 13% for mice reinfected in the chronic stage at the end of the experiments. Sixty-six percent of the infected group presented electrocardiographic abnormalities (heart frequency, prolonged PQ segment or QRS complex) in the chronic stage whereas 100% of the reinfected animals exhibited electric cardiac dysfunction since 90 and 390 days p.i. for the acute and chronic reinfected model, respectively (P<0.01). Heart histopathological studies showed fibrosis and necrosis areas and mononuclear infiltrates supporting the view that parasite persistence is a major factor in continuing the tissue inflammation. This work shows that T. cruzi reinfections could be related to the variability and severity of the clinical course of Chagas' disease and that parasite persistence is involved in exacerbation of the disease.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.