Effects of cancer-induced cachexia and administration of L-glutathione on the intestinal mucosa in rat.

Walker-256 tumor is an experimental model known to promote cachexia syndrome, oxidative stress, and systemic inflammation. This study evaluated the duodenal mucosa of rats with Walker-256 tumor administered with 1% L-glutathione, intending to evaluate the damage caused by cancer-associated cachexia in the gastrointestinal tract and the effects of antioxidant administration on mucosal protection. Twenty-four 55-day-old male Wistar rats were distributed into four groups: control (C); control administered with 1% L-glutathione (C-GSH); Walker-256 tumor (W) and Walker-256 tumor administered with 1% L-glutathione (W-GSH). After 14 days of treatment, the duodenum was harvested for morphometric analysis of the mucosa, proliferation, apoptosis, immunostaining of varicosities immunoreactive (IR) to vasoactive intestinal peptide (VIP) and 5-HT-IR cells, and quantification of mast cells and goblet cells. Walker-256 tumor-bearing rats showed cachexia syndrome, mucosal atrophy, reduced cell proliferation, reduced 5-HT-IR cells, and increased goblet cells and VIPergic varicosities, which were not reversed by L-glutathione. On the other hand, L-glutathione caused a reduction of cells in apoptosis and mast cell recruitment, demonstrating a partial recovery of the damage detected in the intestinal mucosa.

Cancer-induced morphological changes in enteric glial cells in the jejunum of Walker-256 tumor-bearing rats.

Cancer-induced cachexia is associated with systemic inflammation and gastrointestinal dysfunction. How changes to cells of the enteric nervous system contribute to gut dysfunction in tumor development and cancer cachexia is unknown. Here, we tested the hypothesis that changes to enteric glia, a type of peripheral glia that surround enteric neurons and regulate gut homeostasis, are associated with tumor development and that supplementing with the antioxidant L-glutathione is protective against the changes induced. Immunohistochemistry for neurons, enteric glial cells and immune cells was performed in whole-mount preparations and frozen histological sections of the jejunum from 20 Wistar rats, distributed in 4 groups: control, tumor of Walker-256, control administered with 1 % L-glutathione, and tumor of Walker-256 administered with 1 % L-glutathione. Morphoquantitative analyses were made using Image-Pro® Plus 4.5 and ImageJ® 1.43° software. Tumor development significantly reduced neuronal and glial cell populations in the myenteric and submucosal plexuses and enlarged glial cell body area in the submucosal plexus. In contrast, tumors increased glia in the jejunal mucosa and this effect was accompanied by B-lymphocyte recruitment. GSH-supplemented diet was not sufficient to protect against changes to neurons and glia in the submucosal plexus but was partially protective in the myenteric plexus. L-glutathione had no effect on physiological parameters of cachexia but was sufficient to preserve enteric glial cell density in the myenteric plexus. These results suggest that changes to both enteric neurons and glia likely contribute to the gastrointestinal effects of tumor development and that oxidative stress contributes to these effects in the enteric nervous system.

L-glutathione 1% promotes neuroprotection of nitrergic neurons and reduces the oxidative stress in the jejunum of rats with Walker-256-bearing tumor.

AIMS: Our main goals were to investigate the effects of L-glutathione (1%) treatment in Walker-256 tumor-bearing rats by analyzing immunoreactive neurons (IR), responsive to the nNOS enzyme and 3-Nitrotyrosine, in their jejunum myenteric plexus. Moreover, the oxidative state and inflammatory process in these animals were investigated. METHODS: Four experimental groups were utilized: control (C), control treated with L-glutathione (CGT), Walker-256 tumor-bearing rats (TW), and Walker-256 tumor-bearing rats treated with L-glutathione (TWGT). After 14 days of tumor inoculation, the jejunum was collected for immunohistochemical techniques and assessment of oxidative status. Plasma was collected to evaluate oxidative status and measure cytokines. RESULTS: The TW group exhibited a decrease of reduced glutathione in their jejunum, which was prevented in the L-glutathione treated TWGT group. TW animals presented pronounced oxidative stress by increasing levels of lipoperoxidation in their jejunum and malondialdehyde in their plasma; however, the L-glutathione treatment in TWGT group was not able to avoid it. The total antioxidant capacity was altered in groups TW and TWGT, yet the last one had a better index in their plasma. The IL-10, and TNF-α levels increased in TWGT animals. The nNOS-IR neuron density decreased in the jejunum myenteric plexus of the TW group, which was avoided in the TWGT group. The nNOS +3-Nitrotyrosine neurons quantification did not show significative alterations. CONCLUSION: The treatment with L-glutathione (1%) imposed an important defense to some parameters of oxidative stress induced by TW-256, leading to neuroprotection to the loss in the nNOS-IR neuron density.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor.

PURPOSE: To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. METHODS: Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). RESULTS: WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). CONCLUSIONS: L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor

ABSTRACT Purpose: To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. Methods: Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). Results: WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). Conclusions: L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

Combined use of systemic quercetin, glutamine and alpha-tocopherol attenuates myocardial fibrosis in diabetic rats.

This study aimed to analyze the effects of the quercetin (100 mg/kg), 1% glutamine and 1% α-tocopherol antioxidants in the myocardium of rats with streptozotocin-induced diabetes mellitus. Twenty male rats were subdivided into four groups (n = 5): N (normoglycemic); D (diabetic); NT (normoglycemic treated with antioxidants); and DT (diabetic treated with antioxidants) treated for 60 days. Clinical parameters, oxidative stress markers, inflammatory cytokines, myocardial collagen fibers and immunoexpression of superoxide dismutase 1 (SOD-1), glutathione peroxidase-1 (GPx-1), interleukin-1ß (IL-1-ß), transforming growth factor-beta (TGF-ß), and fibroblast growth factor-2 (FGF-2) were evaluated. Results showed reduced body weight, hyperphagia, polydipsia and hyperglycemic state in groups D and DT. The levels of glutathione (GSH) were higher in NT and DT compared to N (p < 0.01) and D (p < 0.001) groups, respectively. Greater GSH levels were found in DT when compared to N animals (p < 0.001). In DT, there was an increase in IL-10 in relation to N, D and NT (p < 0.05), while GPx-1 expression was similar to N and lower compared to D (p < 0.001). TGF-ß expression in DT was greater than N (p < 0.001) group, whereas FGF-2 in DT was higher than in the other groups (p < 0.001). A significant reduction in collagen fibers (type I) was found in DT compared to D (p < 0.05). The associated administration of quercetin, glutamine and α-tocopherol increased the levels of circulating interleukin-10 (IL-10) and GSH, and reduced the number of type I collagen fibers. Combined use of systemic quercetin, glutamine and alpha-tocopherol attenuates myocardial fibrosis in diabetic rats.

Testicular morphometry of rats with Walker 256 tumor supplemented with L-glutamine.

Glutamine is often used to treat metabolic changes associated with anorexia-cachexia syndrome in patients with malignant neoplasms. Walker 256 tumor is an excellent model for studying these changes associated with cancer in different organs, including injuries in testicular functions. However, the effects of supplementing glutamine on testicular morphometry in this model have not yet been investigated. Thus, the objective of this study was to evaluate the effect of L-glutamine supplementation on testicular morphometry in rats transplanted with Walker 256 tumor cells. Forty puberty Wistar rats were divided into four groups: control without L-glutamine (C); control supplemented with L-glutamine (CG); inoculated with Walker 256 tumor cells (WT) and inoculated with Walker 256 tumor cells and supplemented with L-glutamine (WTG). The testicles were removed, weighed, fixed in Bouin, and included in paraffin for histomorphometric analysis. Walker 256 tumor caused quantitative changes in the tubular and intertubular compartments and tunica albuginea, with reductions in the percentages of lumen and tunica albuginea, number of Sertoli cells per gram of testis; number of Leydig cells; percentage of blood vessels and connective tissue in intertubule. However, glutamine supplementation prevented part of these changes caused by the tumor, presenting mainly a protective effect on the tunica albuginea and percentage of blood and lymph vessels in the intertubule. These results indicate the potential of L-glutamine was able to recover for testicular dysfunction associated with cancer.

Intestinal changes associated with fluoride exposure in rats: Integrative morphological, proteomic and microbiome analyses.

Gastrointestinal signs and symptoms are the first signs of toxicity due to exposure to fluoride (F). This suggests the possibility that lower levels of subchronic F exposure may affect the gut. The aim of this study was to evaluate changes in the morphology, proteome and microbiome of the ileum of rats, after subchronic exposure to F. Male rats ingested water with 0, 10, or 50 mgF/L for thirty days. Treatment with F, regardless of the dose, significantly decreased the density of HuC/D-IR neurons, whereas CGRP-IR and SP-IR varicosities were significantly increased compared to the control group. Increased VIP-IR varicosities were significantly increased only in the group treated with 50 mgF/L. A significant increase in thickness of the tunica muscularis, as well as in the total thickness of the ileum wall was observed at both F doses when compared to controls. In proteomics analysis, myosin isoforms were increased, and Gastrotopin was decreased in F-exposed mice. In the microbiome metagenomics analysis, Class Clostridia was significantly reduced upon exposure to 10 mgF/L. At the higher F dose of 50 mg/L, genus Ureaplasma was significantly reduced in comparison with controls. Morphological and proteomics alterations induced by F were marked by changes associated with inflammation, and alterations in the gut microbiome. Further studies are needed to determine whether F exposure increases inflammation with secondary effects of the gut microbiome, and/or whether primary effects of F on the gut microbiome enhance changes associated with inflammation.

Protective effects of quercetin-loaded microcapsules on the enteric nervous system of diabetic rats.

Quercetin-loaded microcapsules (QLM) promote controlled release and higher bioavailability of quercetin, an antioxidant and neuroprotective agent. We evaluated the antioxidant effect of QLM on enteric innervation and in the oxidative status of the ileum of diabetic rats. Wistar adult rats (Rattus norvegicus) were used in six groups containing normoglycemic (N), diabetic (D) and either normoglycemic or diabetic groups treated with QLM at a dose of 10 mg/kg (NQ10 and DQ10, respectively) or 100 mg/kg (NQ100 and DQ100, respectively). DQ10 e DQ100 did not prevent overall neuronal loss in the total and cholinergic populations. Nitrergic population showed differences regarding the treatments: DQ10 preserved neurons in the nitrergic population whilst DQ100 increased nitrergic loss. Evaluation of the redox status showed pro-oxidant effects in NQ100 by t-butyl-induced chemiluminescence analysis. We observed a reduction in the carbonylic content and an increase of low molecular weight antioxidants for DQ10 e DQ100. Therefore, QLM treatment at a dose of 10 mg/kg acted positively on nitrergic neurons reducing oxidative damage induced by diabetes.

Effects of acute fluoride exposure on the jejunum and ileum of rats: Insights from proteomic and enteric innervation analysis.

Fluoride (F) is largely employed in dentistry, in therapeutic doses, to control caries. However, excessive intake may lead to adverse effects in the body. Since F is absorbed mostly from the gastrointestinal tract (GIT), gastrointestinal symptoms are the first signs following acute F exposure. Nevertheless, little is known about the mechanistic events that lead to these symptoms. Therefore, the present study evaluated changes in the proteomic profile as well as morphological changes in the jejunum and ileum of rats upon acute exposure to F. Male rats received, by gastric gavage, a single dose of F containing 0 (control) or 25 mg/Kg for 30 days. Upon exposure to F, there was a decrease in the thickness of the tunic muscularis for both segments and a decrease in the thickness of the wall only for the ileum. In addition, a decrease in the density of HuC/D-IR neurons and nNOS-IR neurons was found for the jejunum, but for the ileum only nNOS-IR neurons were decreased upon F exposure. Moreover, SP-IR varicosities were increased in both segments, while VIP-IR varicosities were increased in the jejunum and decreased in the ileum. As for the proteomic analysis, the proteins with altered expression were mostly negatively regulated and associated mainly with protein synthesis and energy metabolism. Proteomics also revealed alterations in proteins involved in oxidative/antioxidant defense, apoptosis and as well as in cytoskeletal proteins. Our results, when analyzed together, suggest that the gastrointestinal symptoms found in cases of acute F exposure might be related to the morphological alterations in the gut (decrease in the thickness of the tunica muscularis) that, at the molecular level, can be explained by alterations in the gut vipergic innervation and in proteins that regulate the cytoskeleton.

Quercetin increases bioavailability of nitric oxide in the jejunum of euglycemic and diabetic rats and induces neuronal plasticity in the myenteric plexus.

Considering the antioxidant, neuroprotective, inflammatory and nitric oxide modulatory actions of quercetin, the aim of this study was to test the effect of quercetin administration in drinking water (40 mg/day/rat) on neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), overall population of myenteric neurons (HuC/D) and nitric oxide (NO) levels in the jejunal samples from diabetic rats. Male Wistar rats were distributed into four groups (8 rats per group): euglycemic (E), euglycemic administered with quercetin (E+Q), diabetic (D) and diabetic administered with quercetin (D+Q). Rats were induced to diabetes with streptozotocin (35mg/kg/iv) and, after 120 days, the proximal jejunum were collected and processed for immunohistochemical (VIP, nNOS and HuC/D) and chemiluminescence (quantification of tissue NO levels) techniques. Diabetes mellitus reduced the number of nNOS-IR (immunoreactive) (p <0.05) and HuC/D-IR (p <0.001) neurons, however, promoted an increased morphometric area of nNOS-IR neurons (p <0.001) and VIP-IR varicosities (p <0.05). In D+Q group, neuroplasticity effects were observed on HuC/D-IR neurons, accompanied by a reduction of cell body area of neurons nNOS- and VIP-IR varicosities (p <0.05). The NO levels were increased in the E+Q (p <0.05) and D+Q group (p <0.001) compared to the control group. In conclusion, the results showed that quercetin supplementation increased the bioavailability of NO in the jejunum in euglycemic and mitigate the effects of diabetes on nNOS-IR neurons and VIP-IR varicosities in the myenteric plexus of diabetic rats.

Anti- and pro-oxidant effects of quercetin stabilized by microencapsulation on interstitial cells of Cajal, nitrergic neurons and M2-like macrophages in the jejunum of diabetic rats.

Given the well-known antioxidant and neuroprotective properties of quercetin, the aim of this work was to evaluate the effects of quercetin stabilized by microencapsulation at two doses (10 mg kg-1 and 100 mg kg-1) on the oxidative/antioxidant status, number and morphological features of ICC, nitrergic neurons and M2-like macrophages in jejunum of diabetic rats. The rats were randomly distributed into six groups: normoglycemic control (N), diabetic control (D) and either normoglycemic or diabetic groups treated with quercetin-loaded microcapsules at a dose of 10 mg kg-1 (NQ10 and DQ10, respectively) or 100 mg kg-1 (NQ100 and DQ100, respectively). After 60 days, the jejunum was collected. Whole mounts were immunostained for Ano1, nNOS and CD206, and oxidative stress levels and total antioxidant capacity of the jejunum were measured. Diabetes led to a loss of ICC and nitrergic neurons, but increased numbers of M2-like macrophages and elevated levels of oxidative stress were seen in diabetic animals. High-dose administration of quercetin (100 mg kg-1) further aggravated the diabetic condition (DQ100) but this treatment resulted in harmful effects on healthy rats (NQ100), pointing to a pro-oxidant activity. However, low-dose administration of quercetin (10 mg kg-1) gave rise to antioxidant and protective effects on ICC, nNOS, macrophages and oxidative/antioxidant status in DQ100, but NQ100 displayed infrequent negative outcomes in normoglycemic animals. Microencapsulation of the quercetin may become promising alternatives to reduce diabetes-induced oxidative stress but antioxidant therapies should be careful used under healthy status to avoid toxic effects.

QUERCETIN SUPPLEMENTATION PREVENTS CHANGES IN THE SEROTONIN AND CASPASE-3 IMMUNOREACTIVE CELLS OF THE JEJUNUM OF DIABETIC RATS.

BACKGROUND: Serotonin (5-HT) is present in the epithelial enterochromaffin cells (EC), mast cells of the lamina propria and enteric neurons. The 5-HT is involved in regulating motility, secretion, gut sensation, immune system and inflammation. OBJECTIVE: Evaluate the effects of diabetes and quercetin supplementation on serotoninergic cells and its cell loss by apoptosis in jejunal mucosa of streptozotocin-induced diabetic rats (STZ-rats). METHODS: Twenty-four male Wistar rats were divided into four groups: normoglycemic (C), normoglycemic supplemented with 40 mg/day quercetin (Q), diabetic (D) and diabetic supplemented with 40 mg/day quercetin (DQ). After 120 days, the jejunum was collected and fixated in Zamboni's solution for 18 h. After obtaining cryosections, immunohistochemistry was performed to label 5-HT and caspase-3. Quantification of 5-HT and caspase-3 immunoreactive (IR) cells in the lamina propria, villi and crypts were performed. RESULTS: The diabetic condition displayed an increase of the number of 5-HT-IR cells in villi and crypts, while decreased number of these cells was observed in lamina propria in the jejunum of STZ-rats. In the diabetic animals, an increased density of apoptotic cells in epithelial villi and crypts of the jejunum was observed, whereas a decreased number of caspase-3-IR cells was observed in lamina propria. Possibly, quercetin supplementation slightly suppressed the apoptosis phenomena in the epithelial villi and crypts of the STZ-rats, however the opposite effect was observed on the 5-HT-IR cells of the lamina propria. Quercetin supplementation on healthy animals promoted few changes of serotoninergic function and apoptotic stimuli. CONCLUSION: These results suggest that quercetin supplementation mostly improved the serotonergic function affected by diabetes maybe due to antioxidant and anti-inflammatory properties of quercetin.

Rheumatoid arthritis induces enteric neurodegeneration and jejunal inflammation, and quercetin promotes neuroprotective and anti-inflammatory actions.

AIMS: The aim of our study was to study the pathological mechanisms induced by the rheumatoid arthritis (RA) on the Enteric Nervous System (ENS). MAIN METHODS: We evaluated the effect of the chronic arthritis and its treatment with 50 mg/kg quercetin alone (AQ) and combined with 17.5 mg/kg ibuprofen (AIQ) for 60 days on neurons, glial cells and intestinal wall. Other groups were used: control (C), arthritic (A) and arthritic treated with 17.5 mg/kg ibuprofen (AI). After 60 days, the jejunum was removed and processed for immunohistochemical techniques. Immunostainings were performed for HuC/D and S100 (myenteric and submucosal plexuses), and GFAP (only myenteric plexus), while immunolabeling for CD45 and CD20 lymphocytes was performed using cryosections. Western blot was performed for GDNF, S100 and GFAP. KEY FINDINGS: A group yielded a remarkable density decrease of the neurons and glial cells with morphometric changes in the myenteric and submucosal plexuses, reduction of the GDNF expression and GFAP-related parameters (GFAP expression, occupancy area and GFAP-expressing glial cells) and intestinal inflammation and atrophy of the mucosa and intestinal wall. AQ group substantially reversed most of these effects, except for intestinal atrophy of the jejunum. The AI and AIQ groups displayed lower beneficial results than AQ for parameters related to the neurons and glial cells, although AIQ did not prevent the inflammation of the mucosa. SIGNIFICANCE: The severe chronic rheumatoid arthritis induced severe effects on ENS and mucosa, and quercetin treatment continues to be an important antioxidant supplement preventing the progression of the RA severity.

A suplementação com quercetina previne mudanças em células imunorreativas à serotonina e caspase-3 do jejuno de ratos diabéticos / Quercetin supplementation prevents changes in the serotonin and caspase-3 immunoreactive cells of the jejunum of diabetic rats

ABSTRACT BACKGROUND: Serotonin (5-HT) is present in the epithelial enterochromaffin cells (EC), mast cells of the lamina propria and enteric neurons. The 5-HT is involved in regulating motility, secretion, gut sensation, immune system and inflammation. OBJECTIVE: Evaluate the effects of diabetes and quercetin supplementation on serotoninergic cells and its cell loss by apoptosis in jejunal mucosa of streptozotocin-induced diabetic rats (STZ-rats). METHODS: Twenty-four male Wistar rats were divided into four groups: normoglycemic (C), normoglycemic supplemented with 40 mg/day quercetin (Q), diabetic (D) and diabetic supplemented with 40 mg/day quercetin (DQ). After 120 days, the jejunum was collected and fixated in Zamboni's solution for 18 h. After obtaining cryosections, immunohistochemistry was performed to label 5-HT and caspase-3. Quantification of 5-HT and caspase-3 immunoreactive (IR) cells in the lamina propria, villi and crypts were performed. RESULTS: The diabetic condition displayed an increase of the number of 5-HT-IR cells in villi and crypts, while decreased number of these cells was observed in lamina propria in the jejunum of STZ-rats. In the diabetic animals, an increased density of apoptotic cells in epithelial villi and crypts of the jejunum was observed, whereas a decreased number of caspase-3-IR cells was observed in lamina propria. Possibly, quercetin supplementation slightly suppressed the apoptosis phenomena in the epithelial villi and crypts of the STZ-rats, however the opposite effect was observed on the 5-HT-IR cells of the lamina propria. Quercetin supplementation on healthy animals promoted few changes of serotoninergic function and apoptotic stimuli. CONCLUSION: These results suggest that quercetin supplementation mostly improved the serotonergic function affected by diabetes maybe due to antioxidant and anti-inflammatory properties of quercetin.

RESUMO CONTEXTO: A serotonina (5-HT) está presente nas células epiteliais enterocromafins (CE), nos mastócitos da lâmina própria e nos neurônios entéricos. A 5-HT está envolvida na regulação da motilidade, secreção, nocepção intestinal, sistema imunológico e inflamação. Objetivo: Avaliar os efeitos do diabetes e da suplementação de quercetina sobre a função serotoninérgica e a perda celular por apoptose na mucosa jejunal de ratos diabéticos induzidos por estreptozotocina (ratos STZ). MÉTODOS: Vinte e quatro ratos Wistar machos foram divididos em quatro grupos: normoglicêmico (C), normoglicêmico suplementado com quercetina 40 mg/dia (Q), diabético (D) e diabético suplementado com quercetina 40 mg/dia (DQ). Após 120 dias, o jejuno foi coletado e fixado na solução de Zamboni por 18 horas. Após a obtenção de cortes em criostato, a imuno-histoquímica foi realizada para marcar 5-HT e caspase-3. A quantificação de células imunorreativas (IR) à 5-HT e caspase-3 foram realizadas na lâmina própria, vilosidades e criptas. RESULTADOS: A condição diabética ocasionou um aumento do número de células 5-HT-IR nas vilosidades e criptas, enquanto que na lâmina própria houve uma redução dessas células, no jejuno de ratos STZ. Nos animais diabéticos, foi observada uma densidade aumentada de células apoptóticas no epitélio do jejuno, tanto nas vilosidades quanto nas criptas, por outro lado um número reduzido de células caspase-3-IR foi observado na lâmina própria. Possivelmente, a suplementação de quercetina suprimiu ligeiramente os fenômenos de apoptose no epitélio de vilosidades e criptas do jejuno de ratos STZ, no entanto, o efeito oposto foi observado nas células 5-HT-IR da lâmina própria. A suplementação com quercetina em animais saudáveis promoveu poucas alterações na função serotoninérgica e nos estímulos apoptóticos. CONCLUSÃO: Estes resultados sugerem que a suplementação de quercetina melhorou principalmente a função serotoninérgica afetada pelo diabetes, talvez devido às propriedades antioxidantes e anti-inflamatórias da quercetina.

DOES THE RHEUMATOID ARTHRITIS AFFECT THE ENTERIC NERVOUS SYSTEM?

BACKGROUND: Few studies regarding arthritic diseases have been performed to verify the presence of the neurodegeneration. Given the increased oxidative stress and extra-articular effects of the rheumatoid arthritis, the gastrointestinal studies should be further investigated aiming a better understanding of the systemic effects the disease on enteric nervous system. OBJECTIVE: To determine whether the rheumatoid arthritis affects the nitrergic density and somatic area of the nNOS- immunoreactive (IR) myenteric neurons, as well as the morphometric areas of CGRP and VIP-IR varicosities of the ileum of arthritic rats. METHODS: Twenty 58-day-old male Holtzmann rats were distributed in two groups: control and arthritic. The arthritic group received a single injection of the Freund's Complete Adjuvant in order to induce arthritis model. The whole-mount preparations of ileum were processed for immunohistochemistry to VIP, CGRP and nNOS. Quantification was used for the nitrergic neurons and morphometric analyses were performed for the three markers. RESULTS: The arthritic disease induced a reduction 6% in ileal area compared to control group. No significant differences were observed in nitrergic density comparing both groups. However, arthritic group yielded a reduction of the nitrergic neuronal somatic area and VIP-IR varicosity areas. However, an increase of varicosity CGRP-IR areas was also observed. CONCLUSION: Despite arthritis resulted in no alterations in the number of nitrergic neurons, the retraction of ileal area and reduction of nitrergic somatic and VIP-IR varicosity areas may suggest a negative impact the disease on the ENS.

Ethyl Acetate Fraction from Trichilia catigua Confers Partial Neuroprotection in Components of the Enteric Innervation of the Jejunum in Diabetic Rats.

BACKGROUND/AIMS: Diabetes causes damage to the enteric nervous system. The enteric nervous system consists of neurons and enteric glial cells (EGCs). The present study evaluated the effects of an ethyl-acetate fraction (EAF) from Trichilia catigua (T. catigua; 200 mg/kg) on the total population of enteric neurons (HuC/D-immunoreactive [IR]) and EGCs (S100-IR and glial fibrillary acidic protein [GFAP]-IR) in the total preparation and jejunal mucosa in diabetic rats. METHODS: The animals were distributed into four groups: normoglycemic rats (N), diabetic rats (D), normoglycemic rats that received the EAF (NC), and diabetic rats that received the EAF (DC). The jejunum was processed for immunohistochemistry to evaluate HuC/D, S100, and GFAP immunoreactivity. The expression of S100 and GFAP proteins was also quantified by Western blot. RESULTS: The D group exhibited a decrease in the number of neurons and EGCs, an increase in the area of cell bodies, an increase in S100 protein expression, a decrease in GFAP protein expression, and a decrease in S100-IR and GFAP-IR EGCs in the jejunal mucosa. The DC group exhibited a decrease in the number of neurons and EGCs, a decrease in the area of cell bodies, a decrease in S100 and GFAP protein expression, and a decrease in S100-IR and GFAP-IR EGCs in the jejunal mucosa. The NC group exhibited maintenance of the number of neurons and EGCs, an increase in the area of cell bodies, and a decrease in S100 and GFAP protein expression. CONCLUSION: The EAF from T. catigua partially conferred protection against diabetic neuropathy in the enteric nervous system.

Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine.

BACKGROUND: The intestinal wall has a complex topographical architecture. The multi-layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section-based or whole-mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in-depth analysis has been performed yet on the potential of different clearing techniques for the small intestine. AIM: The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue. METHODS: Five aqueous-based clearing protocols (SeeDB2, CUBIC, ScaleS, Ce3D, and UbasM) and four organic reagent-based clearing protocols (3DISCO, iDISCO+, uDISCO, and Visikol® ) were assessed in segments of small intestine from CX3CR1GFP/GFP and wild-type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno-)labeling were analyzed. KEY RESULTS: All organic reagent-based clearing protocols-except for Visikol-rendered tissue highly transparent but led to substantial tissue shrinkage and deformation. Of the aqueous-based protocols, only Ce3D yielded full-thickness tissue transparency. In addition, Ce3D displayed excellent GFP retention and preservation of tissue morphology. CONCLUSIONS: Ce3D emerged as a most efficient protocol for enabling rapid full-thickness 3D mapping of the mouse intestinal wall.

Evaluation of the effects of autohemotransfusion in arthritic rats induced by two doses of complete Freund's adjuvant

The aim the study was to evaluate the effects of autohemotransfusion in adjuvant-induced arthritis model by injections of high and low doses of Complete Freund ́s Adjuvant (CFA). Male Holtzman rats (200-230g) were distributed in six groups: control (C); control treated by autohemotransfusion (CT); CFA induced arthritis 0.5% w/v (AIA); CFA induced arthritis 0.5% w/v treated with autohemotransfusion (AIAT); CFA induced arthritis 0.1% w/v (AS) and CFA induced arthritis 0.1% w/v treated with autohemotransfusion (AST). The number of leukocytes, the weight of different organs and the paw volume were analyzed. The autohemotransfusion without erythrocytes promoted a reduction in the number of leukocytes in AIAT and AST when compared to AIA (p 0.05). The autohemotransfusion used in this work presented positive effects on AIA as they promoted a reduction in the number of leukocytes and an increase in thymus weight and body growth. However, other types of autohemotransfusion must be tested to determine the true efficacy of this alternative method of treatment.

In vitro antioxidant potential and in vivo effects of Schinus terebinthifolia Raddi leaf extract in diabetic rats and determination of chemical composition by HPLC-ESI-MS/MS.

The present study investigated the in vitro and in vivo antioxidant potential and phytochemical composition of Schinus terebinthifolia, which is widely used in folk medicine for various therapeutic purposes. The in vitro analyses indicated that the hydroethanolic extract (HE) had 312.50 ± 0.50 mg GAE/g of total phenols. It also presented anti-DPPH• and anti-ABTS•+ activity, reduced phosphomolybden and metal ions and blocked the bleaching of ß-carotene. The HE at concentrations of 3.0 and 2.0 µg/mL had TRAP values of 2.223 ± 0.018 and 1.894 ± 0.026 µM Trolox, respectively. The HE increased the availability of antioxidants in plasma in treated animals in vivo. HPLC-ESI-MS/MS indicated the presence of 11 phenols: cumaric acid, (+)-catechin, myricetin-3-O-glicuronide, kaempferol-3-O-glucoside, myricetin, myricitrin, quercetin, gallic acid, methyl galate, pentagalloyl glucose and ethyl galate. Thus, S. terebinthifolia has potential for the prevention or treatment of diseases that are related to oxidative stress, such as diabetes mellitus.