Perineuronal net in the extrinsic innervation of the distal colon of mice and its remodeling in ulcerative colitis.

The perineuronal net (PNN) is a well-described highly specialized extracellular matrix structure found in the central nervous system. Thus far, no reports of its presence or connection to pathological processes have been described in the peripheral nervous system. Our study demonstrates the presence of a PNN in the spinal afferent innervation of the distal colon of mice and characterizes structural and morphological alterations induced in an ulcerative colitis (UC) model. C57Bl/6 mice were given 3% dextran sulfate sodium (DSS) to induce acute or chronic UC. L6/S1 dorsal root ganglia (DRG) were collected. PNNs were labeled using fluorescein-conjugated Wisteria Floribunda (WFA) l lectin, and calcitonin gene-related peptide (CGRP) immunofluorescence was used to detect DRG neurons. Most DRG cell bodies and their extensions toward peripheral nerves were found surrounded by the PNN-like structure (WFA+), labeling neurons' cytoplasm and the pericellular surfaces. The amount of WFA+ neuronal cell bodies was increased in both acute and chronic UC, and the PNN-like structure around cell bodies was thicker in UC groups. In conclusion, a PNN-like structure around DRG neuronal cell bodies was described and found modulated by UC, as changes in quantity, morphology, and expression profile of the PNN were detected, suggesting a potential role in sensory neuron peripheral sensitization, possibly modulating the pain profile of ulcerative colitis.

Colonic motility adjustments in acute and chronic DSS-induced colitis.

AIMS: Inflammatory bowel disease is recurrent inflammation that affects the gastrointestinal tract causing changes in intestinal motility. The evolution of these changes is not completely understood. The aim of this study was to evaluate anatomical and functional changes in the colon during the development of acute and chronic DSS-induced ulcerative colitis (UC) in C57Bl/6 mice. MATERIALS AND METHODS: Mice were relocated into 5 groups: control (GC) and groups exposed to DSS 3 % for 2 (DSS2d), 5 (DSS5d) and 7 DSS7d) days (acute UC) or 3 cycles (DSS3C; Chronic UC). Mice were monitored daily. After euthanasia, colonic tissue was assessed with histological, immunofluorescence and colon manometry methods. KEY FINDINGS: Ulcerative Colitis is a chronic disease characterized by overt inflammation of the colon. Here we investigate whether the morphological changes caused by UC in the colonic wall, in tuft cells and in enteric neurons also promote any alteration in colonic motility patterns. UC Promotes thickening in the colonic wall, fibrosis, reduction in the number of tuft cells and consequently goblet cells also, without promoting neuronal death however there is a change in the chemical code of myenteric neurons. All of these morphological changes were responsible for causing a change in colonic contractions, colonic migration motor complex, total time of gastrointestinal transit and therefore promoting dysmotility. Further studies stimulating a hyperplasia of tuft cells may be the way to try to keep the colonic epithelium healthy, reducing the damage caused by UC. SIGNIFICANCE: Increasing disease pathology of DSS-induced UC induces structural and neuroanatomical changes and driven damage to cholinergic neurons causes colonic dysmotility, including increase of cholinergic myenteric neurons, followed by variations in the motility pattern of different regions of the colon that taking together characterize colonic dysmotility.

Combination Therapy Using Benznidazole and Aspirin during the Acute Phase of Experimental Chagas Disease Prevents Cardiovascular Dysfunction and Decreases Typical Cardiac Lesions in the Chronic Phase.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and heart failure in Latin American countries. The treatment of Chagas disease is directed at eliminating the parasite, decreasing the probability of cardiomyopathy and disrupting the disease transmission cycle. Benznidazole (BZ) and nifurtimox (Nfx) are recognized as effective drugs for the treatment of Chagas disease by the World Health Organization, but both have high toxicity and limited efficacy, especially in the chronic disease phase. At low doses, aspirin (ASA) has been reported to protect against T. cruzi infection. We evaluated the effectiveness of BZ in combination with ASA at low doses during the acute disease phase and evaluated cardiovascular aspects and cardiac lesions in the chronic phase. ASA treatment prevented the cardiovascular dysfunction (hypertension and tachycardia) and typical cardiac lesions. Moreover, BZ+ASA-treated mice had a smaller cardiac fibrotic area than BZ-treated mice. These results were associated with an increase in numbers of eosinophils and reticulocytes and levels of nitric oxide in the plasma and cardiac tissue of ASA-treated mice relative to respective controls. These effects of ASA and BZ+ASA in chronically infected mice were inhibited by pretreatment with the lipoxin A4 (LXA4) receptor antagonist Boc-2, indicating that the protective effects of ASA are mediated by ASA-triggered lipoxin. These results emphasize the importance of exploring new drug combinations for treatments of the acute phase of Chagas disease that are beneficial for patients with chronic disease.

Metabolic syndrome agravates cardiovascular, oxidative and inflammatory dysfunction during the acute phase of Trypanosoma cruzi infection in mice.

We evaluated the influence of metabolic syndrome (MS) on acute Trypanosoma cruzi infection. Obese Swiss mice, 70 days of age, were subjected to intraperitoneal infection with 5 × 102 trypomastigotes of the Y strain. Cardiovascular, oxidative, inflammatory, and metabolic parameters were evaluated in infected and non-infected mice. We observed higher parasitaemia in the infected obese group (IOG) than in the infected control group (ICG) 13 and 15 days post-infection. All IOG animals died by 19 days post-infection (dpi), whereas 87.5% of the ICG survived to 30 days. Increased plasma nitrite levels in adipose tissue and the aorta were observed in the IOG. Higher INF-γ and MCP-1 concentrations and lower IL-10 concentrations were observed in the IOG compared to those in the ICG. Decreased insulin sensitivity was observed in obese animals, which was accentuated after infection. Higher parasitic loads were found in adipose and hepatic tissue, and increases in oxidative stress in cardiac, hepatic, and adipose tissues were characteristics of the IOG group. Thus, MS exacerbates experimental Chagas disease, resulting in greater damage and decreased survival in infected animals, and might be a warning sign that MS can influence other pathologies.

Assemblages A and B of Giardia duodenalis reduce enteric glial cells in the small intestine in mice.

Infection of Giardia duodenalis is one of the most common human parasitic disease worldwide. This infection may be related to important changes in the enteric nervous system. The objective of this study was to evaluate the myenteric and submucosal plexuses, the intestinal muscle layer, and gastrointestinal transit in mice infected with assemblages A and B of G. duodenalis. Swiss albino mice (Mus musculus) were infected with assemblages A and B of G. duodenalis for 15 days. Gastrointestinal transit time was evaluated before euthanasia. Duodenum and jejunum were removed for histological and immunohistochemical analyses. It was observed a reduction in the enteric glial cell count and a decrease in the ratio of enteric glial cells to neurons. The number of neurons did not change, but morphological changes were observed in the duodenum and jejunum in both plexuses, including an increase in the nuclear area and a reduction of cell bodies in the myenteric plexus and a decrease in the nuclear area in the submucosal plexus. A reduction of the thickness of the muscle layer was observed in the duodenum, with no significant differences in the gastrointestinal transit times. Assemblages A and B of G. duodenalis decrease the number of enteric glial cells in the myenteric and submucosal plexuses, decrease the thickness of the muscle layer, and change the morphology of neurons. Graphical abstract ᅟ.

Comparative study of effects of assemblages AII and BIV of Giardia duodenalis on mucosa and microbiota of the small intestine in mice.

AIMS: Giardiasis is one of the major causes of diarrhea worldwide and its symptoms vary in intensity, which can be attributed to different parasite assemblages. The goal of the present study was to compare the effects of infection caused by assemblages AII and BIV ofGiardia duodenalis on the response of the small intestine, microbiota, and behavioral parameters in mice. MAIN METHODS: Swiss mice were infected with assemblages AII and BIV of G. duodenalis for 15 days. Leucometry, pain, intestinal microbiota and histological parameters of the duodenum and jejunum were evaluated in the experimental groups. KEY FINDINGS: Both assemblages modified the composition of the intestinal microbiota. Infection with assemblage AII promoted leukocytosis, reflected by increasing number of polymorphonuclear cells, intraepithelial lymphocytes and pain-related behavior, indicating that this was the more aggressive assemblage with regard to its effects on the intestinal mucosa and duodenum. SIGNIFICANCE: The specific assemblage of the parasite is an important parameter that affects symptomatology in the host.

Acute infection with an avirulent strain of Toxoplasma gondii causes decreasing and atrophy of nitrergic myenteric neurons of rats.

In the enteric nervous system (ENS), nitrergic neurons produce and use nitric oxide (NO) as an inhibitory motor neurotransmitter in response to parasitic infections, including those caused by Toxoplasma gondii. However, damage to the host caused by NO has been reported by various authors, and the role of NO in protection or cytotoxicity continues to be extensively studied. In this study, nitrergic neurons were investigated in the myenteric plexus of the jejunum and the distal colon of rats infected with 500 oocysts of the M7741 strain of T. gondii. Ten rats were randomly assigned into a control group (CG) and infected group (IG; received 500 sporulated oocysts of T. gondii orally). After 24h, the rats were euthanized, and samples of the jejunum and distal colon were obtained and processed for NADPH-diaphorase histochemical analysis. Quantitative and morphometric analysis of the nitrergic neurons in whole mounts containing the myenteric plexus was performed. There was a numeric reduction of nitrergic neurons per mm2 in both jejunum and distal colon. The remaining nitrergic neurons suffered atrophy in the areas of the cell body and nucleus, which resulted in a decrease in cytoplasm. Thus, we conclude that an avirulent strain of T. gondii in a short time causes neuroplastic changes in the small and large intestine of rats.

Treatment with low doses of aspirin during chronic phase of experimental Chagas' disease increases oesophageal nitrergic neuronal subpopulation in mice.

Patients with Chagas' disease may develop dysfunctions of oesophageal and colonic motility resulting from the degeneration or loss of the myenteric neurons of the enteric nervous system. Studies have shown that the use of aspirin, also known as acetylsalicylic acid (ASA), influences the pathogenesis of the disease. However, this remains controversial. The aim of this study was to evaluate the consequences of treatment with low doses of aspirin during the chronic phase of Chagas' disease on oesophageal function. Twenty male Swiss mice, 60 days of age, were used. The animals were infected with Y strain of Trypanosoma cruzi, injected intraperitoneally. Aspirin was given at a dose of 50 mg/kg to some of the infected animals, from the 55th to 63rd day after inoculation on consecutive days, and from the 65th to 75th day on alternate days. We investigated food passage of time, wall structure and nitrergic neuronal population of the distal oesophagus. Our data revealed that the use of low doses of aspirin in chronic Chagas' disease caused an increase in the number of nitrergic neurons and partially prevented hypertrophy of the oesophagus. In addition, the aspirin administration impeded Chagas' diseases associated changes in intestinal transit time. Thus treatment with aspirin in the chronic phase of Chagas' disease changes the natural history of the disease and raises the possibility of using it as a new therapeutic approach to the treatment of this aspect of Chagas' disease pathology.

Immunomodulation over the course of experimental Arthrographis kalrae infection in mice.

Arthrographis kalrae is occasionally described as an opportunistic human pathogen. This study investigated the immune response to A. kalrae during murine experimental infection (7, 14, 28 and 56 days post infection). The fungal load was higher in the early phase and mice presented with neurological syndrome over the course of the infection. There was a gradual increase in the level of anti-A. kalrae IgG and increased levels of DTH at 14 days. There was decreased IFN-γ (14-56 days) and an increase in IL-4 (7 and 56 days). Decreased levels of cytokines (IFN-γ, IL-4, IL-10 and IL-17) were observed in the brain at 56 days p.i. The results suggest that the immune response during murine A. kalrae infection modulates to the pattern of Th2 response. This study shows for the first time the cytokines and cellular immunomodulation that occur in response to an experimental infection with A. kalrae in mice.

Chronic infection with Toxoplasma gondii induces death of submucosal enteric neurons and damage in the colonic mucosa of rats.

Intestinal epithelial secretion is coordinated by the submucosal plexus (SMP). Chemical mediators from SMP regulate the immunobiological response and direct actions against infectious agents. Toxoplasma gondii is a worldwide parasite that causes toxoplasmosis. This study aimed to determine the effects of chronic infection with T. gondii on the morphometry of the mucosa and the submucosal enteric neurons in the proximal colon of rats. Male adult rats were distributed into a control group (n = 10) and an infected group (n = 10). Infected rats received orally 500 oocysts of T. gondii (ME-49). After 36 days, the rats were euthanized and samples of the proximal colon were processed for histology to evaluate mucosal thickness in sections. Whole mounts were stained with methylene blue and subjected to immunohistochemistry to detect vasoactive intestinal polypeptide. The total number of submucosal neurons decreased by 16.20%. Vasoactive intestinal polypeptide-immunoreactive neurons increased by 26.95%. Intraepithelial lymphocytes increased by 62.86% and sulfomucin-producing goblet cells decreased by 22.87%. Crypt depth was greater by 43.02%. It was concluded that chronic infection with T. gondii induced death and hypertrophy in the remaining submucosal enteric neurons and damage to the colonic mucosa of rats.

Aspirin treatment exacerbates oral infections by Trypanosoma cruzi.

Oral transmission of the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease, has been documented in Latin American countries. The reported cases of infection were due to the ingestion of contaminated fresh fruit, juices, or sugar cane juice. There have been few studies on the physiopathology of the disease in oral transmission cases. Gastritis is a common ailment that can be caused by poor dietary habits, intake of alcohol or other gastric irritants, bacterial infection, or by the widespread use of non-steroidal anti-inflammatory drugs (NSAIDs). This study investigated in a mouse model whether gastric mucosal injury, induced by aspirin, would affect the course of disease in animals infected with T. cruzi by the oral route. The CL14 and G strains of T. cruzi, both of low infectivity, were used. To this end, groups of BALB/c mice were treated during 5 days with aspirin (100 mg kg(-1)) before oral infection with T. cruzi metacyclic forms (4 × 10(5) or 5 × 10(7) parasites/mouse). Histological analysis and determination of nitric oxide and TNF-α were performed in gastric samples obtained 5 days after infection. Parasitemia was monitored from the thirteenth day after infection. The results indicate that aspirin treatment of mice injured their gastric mucosa and facilitated invasion by both CL14 and G strains of T. cruzi. Strain CL14 caused more severe infection compared to the G strain, as larger numbers of amastigote nests were found in the stomach and parasitemia levels were higher. Our study is novel in that it shows that gastric mucosal damage caused by aspirin, a commonly used NSAID, facilitates T. cruzi infection by the oral route.

Immunological and histopathological characterization of cutaneous candidiasis.

Chronic mucocutaneous candidiasis constitutes a heterogeneous group of syndromes, characterized by non-invasive infection of the skin, nails and mucosal membranes by the fungus Candida spp. Although symptoms are heterogeneous, in all cases there is a reduction in protective cytokines, favouring the development of disease. The normal role of cytokines in skin lesions is not well understood. The present study aimed to investigate the progression of disease, understand specific cellular and molecular components involved in immunity to Candida albicans and determine the balance between pro- and anti-inflammatory cytokines over the course of cutaneous infection in immunocompetent mice. BALB/c mice (five per group) were inoculated with 5 × 10(6)C. albicans pseudohyphae in the deep dermis of the paw and analysed over 1-14  days post-infection. The contralateral paws were used for negative controls. Haematoxylin and eosin staining of skin sections during C. albicans infection was performed to analyse structural modifications to the epidermis such as hyperplasia, and infiltration of neutrophils and fibroblasts in the dermis. The cytokine populations were determined by capture ELISA using popliteal lymph node tissue. Pro-inflammatory cytokines (IL-6, TNF-α, IL-12, IFN-γ and IL-17) were detected at significant levels during the initial phase of cutaneous infection and correlated with the rapid elimination of C. albicans. Anti-inflammatory cytokines (IL-13, IL-4, IL-10 and transforming growth factor-ß) were detected on day 4 post-infection, and prevented exacerbation of inflammation and participated in healing of lesions. Thus, a balance between pro- and anti-inflammatory cytokines was fundamental for the resolution of infection. Importantly, these findings broaden our understanding of the immune mechanisms involved in chronic cutaneous candidiasis.

Oral dependent-dose toxoplasmic infection model induced by oocysts in rats: Myenteric plexus and jejunal wall changes.

Toxoplasmosis is a widely distributed disease caused by the protozoan Toxoplasma gondii that is mainly transmitted orally. Once ingested, the parasite crosses the intestinal barrier to reach the blood and lymph systems to migrate to other regions of the host. The objective of this study was to evaluate the changes in the myenteric plexus and the jejunal wall of Wistar rats caused by oral infection with T. gondii oocysts (ME-49 strain). Inocula of 10, 100, 500 and 5000 oocysts were used. The total population of myenteric neurons and the most metabolically active subpopulation (NADH-diaphorase positive - NADH-dp) exhibited a decrease proportional to the dose of T. gondii. There was also a quantitative increase in the subpopulation of NADPH-diaphorase-positive (NADPH-dp) myenteric neurons, indicating greater expression of the NOS enzyme. Neuronal atrophy was observed, and morphological and morphometric alterations such as jejunal atrophy were found in the infected groups. Hypertrophy of the external muscle with the presence of inflammatory foci was observed in the group infected with 5000 oocysts. The changes observed in the infected groups were proportional to the number of oocysts inoculated.

Intestinal morphology adjustments caused by dietary restriction improves the nutritional status during the aging process of rats.

During the aging process, the body's systems change structurally and loss of function can occur. Ingesting a smaller amount of food has been considered a plausible proposal for increased longevity with the quality of life. However, the effects of dietary restriction (DR) during aging are still poorly understood, especially for organs of the digestive system. This study aimed to describe the body weight, oxidative status and possible morphological changes of the intestinal wall of rats submitted to DR during the aging process (7 to 18months old). Twelve 7-month-old male Wistar rats fed ad libitum since birth were assigned to two groups: control group (CG, n=6) fed ad libitum from 7 to 18months old; and dietary restriction group (DRG, n=6) fed 50% of the amount of chow consumed by the CG from 7 to 18months old. The body weight, feed and water intake were monitored throughout the experiment. Blood, periepididymal adipose tissue (PAT) and retroperitoneal adipose tissue (RAT), and the small intestine were collected at 18months old. The blood was collected to evaluate its components and oxidative status. Sections from the duodenum and ileum were stained with HE, PAS and AB pH2.5 for morphometric analyses of the intestinal wall components, and to count intraepithelial lymphocytes (IELs), goblet cells and cells in mitosis in the epithelium. DR rats showed a reduction in weight, naso-anal length, PAT, RAT and intestinal length; however, they consumed more water. Blood parameters indicate that the DR rats remained well nourished. In addition, they showed lower lipid peroxidation. Hypertrophy of the duodenal mucosa and atrophy of the ileal mucosa were observed. The number of goblet cells and IELs was reduced, but the mitotic index remained unaltered in both duodenum and ileum. In conclusion, 50% dietary restriction for rats from 7 to 18months old contributed to improving their nutritional parameters but, to achieve this, adjustments were required in the structure of the body weight and morphology of the small intestine.

Physical exercise protects myenteric neurons and reduces parasitemia in Trypanosoma cruzi infection.

To evaluate the parasitemia, nitrergic neurons, and cytokines in Trypanosoma cruzi-infected mice subjected to moderate physical exercise, forty male Swiss mice, 30days of age, were divided: Trained Control (TC), Trained Infected (TI), Sedentary Control (SC), and Sedentary Infected (SI). The moderate physical exercise program on a treadmill lasted 8weeks. Three days after completing the moderate physical exercise program, the TI and SI groups were inoculated with 1300 blood trypomastigotes of the Y strain of T. cruzi, and parasitemia was evaluated from day 4 to day 22 after inoculation. After 75days of infection, cytokines were measured and colonic neurons were quantified using immunofluorescence to identify neuronal nitric oxide synthase (nNOS). The results were analyzed using analysis of variance - Tukey and Kruskal-Wallis tests, to 5% significance. Moderate physical exercise reduced the parasite peak on day 8 of infection and total parasitemia (p<0.05), contributed to survival of number of nNOS-immunoreactive neurons (p<0.01) and promoted neuronal hypertrophy of the neurons (p<0.05), increased the synthesis of tumor necrosis factor-α (p<0.01) and transforming growth factor-ß (p>0.05), providing beneficial effects to the host by acting on the immune system to preserve nitrergic neurons.