Kisspeptin-10 Improves Testicular Redox Status but Does Not Alter the Unfolded Protein Response (UPR) That Is Downregulated by Hypothyroidism in a Rat Model.

Hypothyroidism compromises the testicular redox status and is associated with reduced sperm quality and infertility in men. In this regard, studies have demonstrated the antioxidant potential of kisspeptin in reproductive and metabolic diseases. In this study, we evaluate the effects of kisspeptin-10 (Kp10) on the testicular redox, as well as mediators of the unfolded protein response (UPR) in adult rats with hypothyroidism. Adult male Wistar rats were randomly separated into the Control (n = 15), Hypo (n = 13) and Hypo + Kp10 (n = 14) groups, and hypothyroidism was induced with 6-propyl-2-thiouracil (PTU) for three months. In the last month, half of the hypothyroid animals received Kp10. Testis samples were collected for enzymatic, immunohistochemical and/or gene evaluation of mediators of oxidative stress (TBARs, lipid hydroperoxides (LOOH), ROS, peroxynitrite, SOD, CAT and GPX), endoplasmic reticulum stress (GRP78, ATF6, PERK, CHOP, HO-1 and sXBP1) and antiapoptocytes (BCL-2). Hypothyroidism increased apoptosis index, TBARS and LOOH concentrations, and reduced testicular gene expression of Sod1, Sod2 and Gpx1, as well as the expression of Grp78, Atf6, Ho1 and Chop. Treatment with Kp10, in turn, reduced testicular apoptosis and the production of peroxynitrite, while increased SOD1 and GPX ½ expression, and enzymatic activity of CAT, but did not affect the lower expression of UPR mediators caused by hypothyroidism. This study demonstrated that hypothyroidism causes oxidative stress and dysregulated the UPR pathway in rat testes and that, although Kp10 does not influence the low expression of UPR mediators, it improves the testicular redox status, configuring it as an important antioxidant factor in situations of thyroid dysfunction.

A Rodent Model of Human-Dose-Equivalent 5-Fluorouracil: Toxicity in the Liver, Kidneys, and Lungs.

5-Fluorouracil (5-FU) is a chemotherapy drug widely used to treat a range of cancer types, despite the recurrence of adverse reactions. Therefore, information on its side effects when administered at a clinically recommended dose is relevant. On this basis, we examined the effects of the 5-FU clinical treatment on the integrity of the liver, kidneys, and lungs of rats. For this purpose, 14 male Wistar rats were divided into treated and control groups and 5-FU was administered at 15 mg/kg (4 consecutive days), 6 mg/kg (4 alternate days), and 15 mg/kg on the 14th day. On the 15th day, blood, liver, kidney, and lung samples were collected for histological, oxidative stress, and inflammatory evaluations. We observed a reduction in the antioxidant markers and an increase in lipid hydroperoxides (LOOH) in the liver of treated animals. We also detected elevated levels of inflammatory markers, histological lesions, apoptotic cells, and aspartate aminotransferase. Clinical treatment with 5-FU did not promote inflammatory or oxidative alterations in the kidney samples; however, histological and biochemical changes were observed, including increased serum urea and uric acid. 5-FU reduces endogenous antioxidant defenses and increases LOOH levels in the lungs, suggesting oxidative stress. Inflammation and histopathological alterations were also detected. The clinical protocol of 5-FU promotes toxicity in the liver, kidneys, and lungs of healthy rats, resulting in different levels of histological and biochemical alterations. These results will be useful in the search for new adjuvants to attenuate the adverse effects of 5-FU in such organs.

Global cerebral ischemia followed by long-term reperfusion promotes neurodegeneration, oxidative stress, and inflammation in the small intestine in Wistar rats.

AIMS: Brain ischemia and reperfusion may occur in several clinical conditions that have high rates of mortality and disability, compromising an individual's quality of life. Brain injury can affect organs beyond the brain, such as the gastrointestinal tract. The present study investigated the effects of cerebral ischemia on the ileum and jejunum during a chronic reperfusion period by examining oxidative stress, inflammatory parameters, and the myenteric plexus in Wistar rats. MAIN METHODS: Ischemia was induced by the four-vessel occlusion model for 15 min with 52 days of reperfusion. Oxidative stress and inflammatory markers were evaluated using biochemical techniques. Gastrointestinal transit time was evaluated, and immunofluorescence techniques were used to examine morpho-quantitative aspects of myenteric neurons. KEY FINDINGS: Brain ischemia and reperfusion promoted inflammation, characterized by increases in myeloperoxidase and N-acetylglycosaminidase activity, oxidative stress, and lipid hydroperoxides, decreases in superoxide dismutase and catalase activity, a decrease in levels of reduced glutathione, neurodegeneration in the gut, and slow gastrointestinal transit. SIGNIFICANCE: Chronic ischemia and reperfusion promoted a slow gastrointestinal transit time, oxidative stress, and inflammation and neurodegeneration in the small intestine in rats. These findings indicate that the use of antioxidant and antiinflammatory molecules even after a long period of reperfusion may be useful to alleviate the consequences of this pathology.

Avaliação de neurônios mioentéricos NADPH-diaforase positivos do íleo de ratos diabéticos suplementados com Acetil-L-Carnitina / Evaluation of positive NADPH-diaphorase myenteric neurons in diabetic rats supplemented with Acetyl-L-Carnitine

Objetivou-se avaliar a suplementação com acetil-L-carnitina (ALC) sobre os neurônios mioentéricos do íleo de ratos após a indução de diabetes. Foram usados animais diabéticos suplementados com ALC (DC), diabéticos (D), normoglicêmicos suplementados com ALC (CC) e normoglicêmicos (C). Neurônios NADPH-d foram quantificados e mensurados. Observou-se redução na glicemia e na ingestão de água no grupo DC. A densidade neuronal em 12,72mm² de íleo foi semelhante nos quatro grupos (p>0,05): DC (558,8 ± 220,2), D (513,4 ± 72,01), CC (645,2 ± 144,9) e C (934 ± 248,5). A área média do corpo celular dos neurônios (µm²) nos animais diabéticos, DC (303,9 ± 114,2) e D (285,4 ± 111,8), foram maiores que nos grupos normoglicêmicos, CC (173,6 ± 53,78) e C (158,4 ± 53,73). A área do íleo (mm²) também mostrou-se maior nos animais dos grupos diabéticos, DC (190,96) e D (171,62) quando comparados aos normoglicêmicos: CC (138,04) e C (130,06). Entretanto no grupo DC, ambas as áreas foram maiores que no D (P<0,05). Assim, pode se inferir discreto incremento na população neuronal. Os dados indicaram que a ALC não interferiu nos mecanismos que promovem aumento na produção de óxido nítrico (NO) pelos neurônios mioentéricos do íleo e que a maior dilatação do íleo no grupo DC poderia ser resultante de efeito colateral da dose de carnitina empregada.

The objective was to evaluate supplementation with acetyl-L-carnitine (ALC) on myenteric neurons of the ileum of rats after induction of diabetes. Diabetic animals supplemented with ALC (DC), diabetic (D), normoglycemic animals supplemented with ALC (CC) and normoglycemic (C) were used. NADPH-d neurons were quantified and measured. There was a reduction in blood glucose and water intake in the DC group. The neuronal density in 12.72mm² of ileum was similar in the four groups (p>0.05): DC (558.8 ± 220.2), D (513.4 ± 72.01), CC (645.2 ± 144.9) and C (934 ± 248.5). The mean cell body area of neurons (µm²) in diabetic animals, DC (303.9 ± 114.2) and D (285.4 ± 111.8), were greater than in the normoglycemic groups, CC (173.6 ± 53.78) and C (158.4 ± 53.73). The ileum area (mm²) was larger in animals of the diabetic groups, CD (190.96) and D (171.62) compared to the normoglycemic groups: CC (138.04) and C (130.04). However, in the DC group, both areas were larger than in D (p<0.05). Thus, a slight increase in neuronal population can be inferred. The data indicated that ALC did not interfere with mechanisms that promote an increase in the production of nitric oxide (NO) by myenteric neurons of the ileum and that the greater dilation of the ileum in the DC group could be the result of a side effect of the dose of carnitine used.

Neonatal metformin short exposure inhibits male reproductive dysfunction caused by a high-fat diet in adult rats.

Metformin (Met) is widely used to control blood glucose levels and acts on various organs, including reproductive tissues, to improve reproductive and lifespan. This study evaluated whether neonatal Met exposure prevented male reproductive dysfunction caused by being overweight during adulthood. Randomized Wistar rat pups received an intraperitoneal injection from postnatal days (PNDs) 1 to 12of saline (Sal; 0.9% NaCl/day in 2mL/kg) or Met (100 mg/kg/day in 2 mL/kg). From PNDs 60 to 90, the animals received a regular (R; 4.5% fat; Sal R and Met R groups) or a high-fat (HF; 35% fat; Sal HF and Met HF groups) diet. At PND 90, all animals were euthanized to evaluate their biometric and reproductive parameters. The Sal and Met groups with R showed similar body weights, however, the HF diet increased the body weight in both groups. The Sal HF group showed testicular damage regarding in antioxidant status and inflammatory profile in the epididymal cauda. The HF diet reduced Leydig and Sertoli cells numbers, with lower sperm quality. The Met R animals showed positive reproductive programming, due to improved antioxidant defense, inflammatory biomarkers, and sperm morphology. Met HF prevented HF diet damage to reproductive organs and sperm morphology, but not to sperm motility. Early Met exposure positively affected the male reproductive system of adult rats, preventing reproductive HF disorders. STATEMENT OF NOVELTY AND SIGNIFICANCE: Metformin is used to control type 2 diabetes mellitus and can act to improve metabolism and lifespan. Metformin avoidance is recommended during pregnancy, but there is no information regarding its use when breastfeeding. For the first time, we showed in this current study that metformin positively acts in the male reproductive tissues and helps involved in later life. These data showed a better antioxidant defense and anti-inflammatory profile of Metformin animals than Saline animals and might directly improve reproductive organs morphophysiology and sperm morphology. Also, the neonatal Met application programs the male reproduction to counterbalance damages from an obesogenic environment in later life.

Colonic neuronal loss and delayed motility induced by high-fat diet occur independently of changes in the major groups of microbiota in Swiss mice.

BACKGROUND: Obesity has been linked to gastrointestinal disorders, and the loss of myenteric neurons in the intestine caused by high-fat diets (HFD) has been attributed to changes in microbiota and lipotoxicity. We investigated whether the prebiotic inulin modulates bacterial populations and alleviates neuronal loss in mice fed HFD. METHODS: Swiss mice were fed purified rodent diet or HFD (59% kcal fat), or both diets supplemented with inulin for 17 weeks. Intestinal motility was assessed and a metagenome analysis of the colonic microbiota was performed. The gene expression of inflammatory markers was evaluated, and immunofluorescence was performed for different types of myenteric neurons and glial cells in the distal colon. KEY RESULTS: The HFD caused obesity and delayed colonic motility. The loss of myenteric neurons and glial cells in obese mice affected all of the studied neuronal populations, including neurons positive for myosin-V, neuronal nitric oxide synthase, vasoactive intestinal peptide, and calretinin. Although obese mice supplemented with inulin exhibited improvements in colonic motility, neuronal, and glial cell loss persisted. The HFD did not altered the expression levels of inflammatory cytokines in the intestine or the prevalence of the major groups in microbiota, but inulin increased the proportion of the genus Akkermansia in the obese mice. CONCLUSIONS AND INFERENCES: In Swiss mice, the HFD-induced neuronal loss but did not change the major groups in microbiota. This suggests that, despite the increase in the beneficial bacteria, other factors that are directly linked to excess dietary lipid intake affect the enteric nervous system.

Chronic ingestion of deoxynivalenol-contaminated diet dose-dependently decreases the area of myenteric neurons and gliocytes of rats.

BACKGROUND: Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is commonly found in cereals ingested by humans and animals. Its ingestion is correlated with hepatic, hematologic, renal, splenic, cardiac, gastrointestinal, and neural damages, according to dose, duration of exposure and species. In this work, the effects of the ingestion of DON-contaminated diet at concentrations considered tolerable for human and animal intake were assessed. METHODS: Male Wistar rats aging 21 days were allotted to five groups that were given, for 42 days, diets contaminated with different concentrations of DON (0, 0.2, 0.75, 1.75, and 2 mg kg-1 of chow). Food ingestion, bodyweight, oxidative status and morphometric analyses of gliocytes, and neurons of jejunal myenteric ganglia were recorded. KEY RESULTS: At these concentrations, there was no food rejection, decrease in bodyweight gain, changes in oxidative status, or loss of either neurons or gliocytes. However, DON decreased gliocyte area, general neuronal population, nitrergic, cholinergic and NADH-diaphorase positive subpopulations and, as a result, ganglion area. CONCLUSIONS & INFERENCES: It was concluded that, even in the absence of visible effect, DON exposure reduces cell body area of gliocytes and neurons of the myenteric plexus of the rat jejunum.

Curcumin protects remote organs against injury that is caused by intestinal ischemia and reperfusion

In addition to several local pathophysiological consequences, intestinal injury that is caused by ischemia and reperfusion can result in the development of lesions in remote organs. Curcumin has therapeutic potential because of its antiinflammatory and antioxidant effects. The present study evaluated the effects of curcumin on oxidative and inflammatory parameters in the liver and kidneys in rats that were subjected to intestinal ischemia and reperfusion. The rats were subjected to 45 min. of ischemia followed by 7 days of reperfusion and treated daily with 60 mg kg-1 curcumin. The liver and kidneys were collected, weighed, and biochemically analyzed. Intestinal ischemia and reperfusion increased levels of lipid hydroperoxide (LOOH), decreased levels of reduced glutathione (GSH), and increased the enzymatic activity of superoxide dismutase (SOD), glutathione S-transferase (GST), and myeloperoxidase (MPO) in the liver. Intestinal ischemia and reperfusion decreased kidney weight and increased GST activity in the kidneys. Curcumin prevented these changes in the liver and kidneys. Intestinal ischemia and reperfusion mainly affected the liver, promoting inflammation and oxidative stress. The kidneys underwent repair much earlier than the liver, in which they did not present alterations of MPO or main parameters of oxidative stress after 7 days of reperfusion. Treatment with curcumin had beneficial effects, ameliorating or even preventing injury that was caused by intestinal ischemia and reperfusion in the liver and kidneys in rats

Resveratrol promotes neuroprotection and attenuates oxidative and nitrosative stress in the small intestine in diabetic rats.

Damages to the enteric nervous system caused by diabetes mellitus (DM) are frequently attributed to oxidative and nitrosative stress. We aimed to investigate the effect of Resveratrol (RSV) (10 mg/kg) on oxidative and nitrosative stress in the intestinal wall and morphoquantitative aspects of the myenteric plexus of the duodenum, jejunum and ileum in diabetic rats. Twenty-four rats were distributed into four groups (n = 6/group): control (C group), control treated with RSV (CR group), diabetic (D group), and diabetic treated with RSV (DR group) for 120 days. Immunohistochemical staining techniques for the general neuronal population, nitrergic and calretinin neuronal subpopulations, enteric glial cells and glial fibrillary acid protein were performed in the myenteric plexus. Furthermore, parameters of oxidative and nitrosative stress were analyzed in the intestinal wall. RSV attenuated oxidative and nitrosative stress and prevented neuronal loss and hypertrophy of the HuC/D-IR, nNOS-IR and CALR-IR neuronal subpopulations in the DR group compared with the D group (P < 0.05). In addition, RSV prevented the increase in glial fibrillary acid protein fluorescence in the DR group compared with the D (P < 0.05). These results suggest that RSV has antioxidant and neuroprotective effects in myenteric plexus in rats with experimental DM.

Evaluation of the treatment with resveratrol-loaded nanoparticles in intestinal injury model caused by ischemia and reperfusion.

The gastrointestinal tract is extremely sensitive to ischemia and reperfusion (I/R). Studies have reported that resveratrol (RSV) is able to combat damage caused by intestinal I/R. Because of its effectiveness in increasing the permanence and bioavailability of resveratrol in the intestinal epithelium, we investigated whether the effect of resveratrol-loaded in poly(anhydride) nanoparticles reduce oxidative stress and promote myenteric neuroprotection in the ileum of rats subjected to I/R. Physicochemical evaluations were performed on nanoparticles. The animals were divided into nine groups (n = 6/group) and treated every 48 h. Treatments with resveratrol (7 mg/kg of body weight) were applied 5 days before surgery and continued for 7 days after surgery (reperfusion period). The superior mesenteric artery was occluded to cause I/R injury. Oxidative stress, myeloperoxidase, nitrite, aspartate aminotransferase, alanine aminotransferase, immunolabeling of myenteric neurons and glial cells, and gastrointestinal transit was evaluated. Both nanoparticle formulations presented negative charge with homogeneous distribution, and the payload, showed an encapsulation efficiency of 60%. Resveratrol administered in free form prevented alterations that were caused by I/R. The results of the groups treated with RSV-loaded nanoparticles presented similar results to the group treated with free resveratrol. Treatment with empty nanoparticles showed that poly(anhydride) is not an ideal nanocarrier for application in in vivo models of intestinal I/R injury, because of hepatotoxicity that may be caused by epithelial barrier dysfunction that triggers the translocation of nanoparticles.

Effect of simvastatin on sensorial, motor, and morphological parameters in sciatic nerve crush induced-neuropathic pain in rats.

The present study compares the effects of a low and high doses of simvastatin in a model of peripheral neuropathy by evaluating sensorial, motor, and morphological parameters. First, male Wistar rats were orally treated with vehicle (saline, 1 mL/kg), simvastatin (2 and 80 mg/kg) or morphine (2 mg/kg, s.c.), 1 h before 2.5% formalin injection. Neuropathic pain was induced by crushing the sciatic nerve, and mechanical and cold allodynia, nerve function, histology, MPO and NAG concentrations, as well as mevalonate induced-nociception were evaluated. Animals were orally treated with vehicle, simvastatin, or gabapentin (30 mg/kg) for 18 days. Simvastatin (2 and 80 mg/kg) reduced the inflammatory pain induced by formalin, but failed to decrease the paw edema. Mechanical allodynia was reduced by the simvastatin (2 mg/kg) until the 12th day after injury and until the 18th day by gabapentin. However, both simvastatin and gabapentin treatments failed in attenuated cold allodynia or improved motor function. Interestingly, both doses of simvastatin showed a neuroprotective effect and inhibited MPO activity without altering kidney and hepatic parameters. Additionally, only the higher dose of simvastatin reduced the cholesterol levels and the nociception induced by mevalonate. Our results reinforce the antinociceptive, antiallodynic, and anti-inflammatory effects of oral simvastatin administration, which can strongly contribute to the sciatic nerve morphology preservation. Furthermore, our data suggest that lower and higher doses of simvastatin present beneficial effects that are dependent and independent of the mevalonate pathway, respectively, without causing signs of nerve damage.

Resveratrol promotes myenteric neuroprotection in the ileum of rats after ischemia-reperfusion injury.

AIMS: The present study evaluated the effects of resveratrol in the myenteric plexus after intestinal ischemia-reperfusion (I/R) injury caused by occluding the superior mesenteric artery for 45min, followed by 7days of reperfusion. MAIN METHODS: Forty-two male Wistar rats were divided into seven groups: control (C group), untreated sham surgery control (SC group), sham surgery control treated with resveratrol before surgery (STA group), sham surgery control treated with resveratrol before and after surgery (STAD group), ischemic control (IRC group), ischemic treated before I/R (IRTA group), and ischemic treated before and after I/R (IRTAD group). Resveratrol (10mg/kg) was administered for 4days and 2h prior to surgery and/or 7days later. Morphometric analyses were performed, and the density of the general neuronal population (HuC/D-immunoreactive [IR]), nitrergic subpopulation (neuronal nitric oxide synthase [nNOS]-IR), vasoactive intestinal peptide (VIP)ergic varicosities (VIP-IR), and glial cells (S100-IR) was determined. KEY FINDINGS: Injury that was caused by I/R significantly reduced (p<0.01) the HuC/D-IR general neuronal population. Treatment with resveratrol before and after ischemia had a neuroprotective effect. Morphometric changes caused by I/R in nitrergic neurons and varicosities were also attenuated by resveratrol. Ischemia/reperfusion promoted the proliferation of enteric glial cells, and resveratrol treatment before and after I/R reversed this effect. SIGNIFICANCE: Resveratrol had neuroprotective effects, showing promise for application in intestinal surgery and transplants.

Resveratrol Reduces Morphologic Changes in the Myenteric Plexus and Oxidative Stress in the Ileum in Rats with Ischemia/Reperfusion Injury.

BACKGROUND: Intestinal ischemia/reperfusion injury can be caused by surgical procedures and inflammatory bowel disease. It is normally associated with the increased production of free radicals and changes in the enteric nervous system. AIMS: Given the antioxidant and neuroprotective properties of resveratrol, the present study assessed its influence on oxidative stress in the intestinal wall and the morphology of myenteric neurons in the ileum of rats subjected to ischemia/reperfusion. METHODS: Resveratrol was orally administered daily at a dose of 10 mg/kg for 5 days. Changes in the ileum response to ischemia after 45 min were investigated followed by 3 h reperfusion. Lipoperoxide and carbonylated protein levels, and the activity of the antioxidant enzymes glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured following ischemia/reperfusion injury. RESULTS: The density and morphometry of the general neuronal population, nitrergic neurons and glial cells, and morphometry of VIP varicosities in the ileum were also studied. Lipoperoxide and carbonylated protein levels were 171 and 40% higher during the ischemia/reperfusion, respectively, compared to control cohorts, and resveratrol attenuated these values. The glutathione ratio was 64% lower during ischemia/reperfusion, compared to control cohorts. Resveratrol increased the reduced/oxidized glutathione ratio, attenuated the changes in the activity of the antioxidant enzymes and the detrimental morphologic changes caused by ischemia/reperfusion in the general neuronal population and nitrergic neurons. CONCLUSIONS: Oral treatment with resveratrol reduced the oxidative stress in the ileum and attenuated the morphologic changes that occurred in the myenteric plexus of the ileum in rats subjected to ischemia/reperfusion.

High-fat diet promotes neuronal loss in the myenteric plexus of the large intestine in mice.

BACKGROUND: Obesity is considered a risk factor for other chronic diseases, and diets rich in lipids can cause alterations in the intestinal functions. AIM: The aim of this study was to investigate the effects of a high-fat diet (HFD) on the myenteric plexus of the large intestine in mice. METHODS: Swiss mice were distributed into four groups: Control animals fed standard chow for 8 and 17 weeks (C8 and C17 groups) and hyperlipidic animals fed HFD for 8 and 17 weeks (Ob8 and Ob17 groups). Immunofluorescence was performed in the large intestine for the morphologic and quantitative analysis of neuronal populations. RESULTS: Animals in the Ob17 group exhibited increased body weight and visceral fat gain compared with the C17 group. The intestinal area was also reduced in the two Ob groups. In the proximal colon, the Ob17 group exhibited 16.1 % reduction of the general neuronal density and 33 % reduction of the VIP-immunoreactive (IR) subpopulation. The general neuronal density in the distal colon was reduced by 45 % in the Ob17 group, and the nNOS-IR density was reduced by 35 %. The morphometry of neuronal cell bodies in the Ob17 group exhibited a reduction of the neuronal area of all of the neuronal populations studied in the proximal colon, with a reduction of the subpopulations of nNOS-IR and VIP-IR neurons in the distal colon. CONCLUSIONS: The HFD caused neuronal loss in the myenteric plexus, and nitrergic neurons were more resilient. The changes were more pronounced in the distal colon after 17 weeks.