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.

Effects of quercetin-supplementation in NADH-diaphorase positive neurons subpopulations in the ileum of rats with experimental diabetes mellitus / Efectos de la suplementación con quercetina en subpoblaciones de neuronas NADH-diaforasa positivas en el Ileon de ratas con diabetes mellitus experimental

The effects of quercetin supplementation in NADH-diaphorase positive (NADH-d) neurons of streptozotocin-induced diabetic rats was carried in this study. Fifteen male rats were divided into three groups: normoglycemic (N), diabetic (D) and diabetic supplemented with quercetin (DQ). Whole mount preparations of the muscular layer of the ileum underwent NADH-d histochemistry for evidencing the NADH-d neuronal subpopulation. Quantitative analyzes were performed on 30 random fields, and morphometric analyzes in 100 neuronal bodies and nuclei per animal. The supplementation promoted a 44 % reduction in the neuronal density in D group when compared to N group (p <0.001); a 24.5 % reduction was observed in the DQ group when compared to N (p <0.01). Animals in D group presented an 18.7 % increase in the cell body areas of myenteric neurons when compared to N (p <0.001); DQ group showed a 14.2 % decrease in neuronal areas when compared to D (p <0.01); the nuclear area were similar among the three groups. We conclude that quercetin supplementation was positive for animals with diabetes mellitus.

Se estudiaron los efectos de la suplementación con quercetina en neuronas NADH-diaforasa positiva (NADH-d) de ratas diabéticas inducidas por estreptozotocina. Quince ratas machos se dividieron en tres grupos: normoglicémico (N), diabéticos (D) y diabéticos suplementados con quercetina (DQ). Las cortes montados de la capa muscular del íleon fueron sometidos a histoquímica de NADH-d para evidenciar la subpoblación neuronal NADH-d. Se realizaron análisis cuantitativos en 30 campos aleatorios y análisis morfométricos en 100 cuerpos y núcleos neuronales, por animal. La suplementación promovió una reducción del 44 % en la densidad neuronal en el grupo D cuando se comparó con el grupo N (p <0,001). Se observó una reducción del 24,5 % en el grupo DQ en comparación con N (p <0,01). Los animales del grupo D presentaron un aumento del 18,7 % en las áreas del cuerpo celular de las neuronas mientéricas cuando se compararon con N (p <0,001). El grupo DQ mostró una disminución de 14,2 % en las áreas neuronales en comparación con D (p <0,01). El área nuclear fue similar entre los tres grupos. Se concluye que la suplementación con quercetina fue positiva para animales con diabetes mellitus.

Antioxidant Effects of the Quercetin in the Jejunal Myenteric Innervation of Diabetic Rats.

PURPOSE: Enteric glial cells (EGCs) exert a critical role in the structural integrity, defense, and metabolic function of enteric neurons. Diabetes mellitus is a chronic disease characterized by metabolic disorders and chronic autonomic neuropathy. Quercetin supplementation, which is a potent antioxidant, has been used in order to reduce the effects of diabetes-induced oxidative stress. The purpose of this research was to investigate the effects of quercetin supplementation in the drinking water at a daily dose of 40 mg on the glial cells and neurons in the jejunum of diabetic rats. MATERIALS AND METHODS: Twenty 90-day-old male adult Wistar rats were split into four groups: normoglycemic control (C), normoglycemic control supplemented with quercetin (Q), diabetic (D), and diabetic supplemented with quercetin (DQ). After 120 days, the jejunums were collected, and immunohistochemical technique was performed to label S-100-immunoreactive glial cells and HuC/D-immunoreactive neurons. RESULTS: An intense neuronal and glial reduction was observed in the jejunum of diabetic rats. Quercetin displayed neuroprotective effects due to reduced cell body areas of neurons and glial cells in Q and DQ groups compared to their controls (C and D groups). Interestingly, quercetin prevented the glial and neuronal loss with a higher density for the HuC/D-immunoreactive neurons (23.06%) and for the S100-immunoreactive glial cells (14.55%) in DQ group compared to D group. CONCLUSION: Quercetin supplementation promoted neuroprotective effects through the reduction of neuronal and glial body areas and a slight prevention of neuronal and glial density reduction.

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.

Vitamins C and E (ascorbate/α-tocopherol) provide synergistic neuroprotection in the jejunum in experimental diabetes.

The present study evaluated the synergistic effects of the association of ascorbic acid and α-tocopherol on myenteric in the jejunum of diabetic rats. The rats were randomly divided into four equal groups: untreated normoglycemic (UC), untreated diabetic (UD), ascorbic acid and α-tocopherol-treated normoglycemic (CAE) and ascorbic acid and α-tocopherol-treated diabetic (DAE). The rats from the CAE and DAE group received supplementation with ascorbic acid (1g/L in water) and α-tocopherol (1% in chow). At 210-days-old, the animals were sacrified and their jejunum was collected and submitted to immunohistochemistry. Quantitative and/or morphometric analysis were performed. Supplementation with ascorbic acid and α-tocopherol prevented the cell loss of myenteric neurons expressing HuC/D and TrkA in an equivalent proportion. We also observed a reduction of the CGRP nerve fiber varicosities and the prevention of the increased cell body size of submucosal VIP neurons (p<0.05). The association of ascorbic acid and α-tocopherol reduced the deleterious effects of diabetes promoting protection on the enteric neurons.

Myosin Va but Not nNOSα is Significantly Reduced in Jejunal Musculomotor Nerve Terminals in Diabetes Mellitus.

Nitric oxide (NO) mediated slow inhibitory junction potential and mechanical relaxation after electrical field stimulation (EFS) is impaired in diabetes mellitus. Externally added NO donor restore nitrergic function, indicating that this reduction result from diminution of NO synthesis within the pre-junctional nerve terminals. The present study aimed to investigate two specific aims that may potentially provide pathophysiological insights into diabetic nitrergic neuropathy. Specifically, alteration in nNOSα contents within jejunal nerve terminals and a local subcortical transporter myosin Va was tested 16 weeks after induction of diabetes by low dose streptozotocin (STZ) in male Wistar rats. The results show that diabetic rats, in contrast to vehicle treated animals, have: (a) nearly absent myosin Va expression in nerve terminals of axons innervating smooth muscles and (b) significant decrease of myosin Va in neuronal soma of myenteric plexus. In contrast, nNOSα staining in diabetic jejunum neuromuscular strips showed near intact expression in neuronal cell bodies. The space occupancy of nitrergic nerve fibers was comparable between groups. Normal concentration of nNOSα was visualized within a majority of nitrergic terminals in diabetes, suggesting intact axonal transport of nNOSα to distant nerve terminals. These results reveal the dissociation between presences of nNOSα in the nerve terminals but deficiency of its transporter myosin Va in the jejunum of diabetic rats. This significant observation of reduced motor protein myosin Va within jejunal nerve terminals may potentially explain impairment of pre-junctional NO synthesis during EFS of diabetic gut neuromuscular strips despite presence of the nitrergic synthetic enzyme nNOSα.

Neuroprotection and neurodegeneration in submucosal VIP-IR neurons in the jejunum of ascorbic acid supplemented aging Wistar rats.

The present work studied the effects of ascorbic acid supplementation (1 mg/ml in water daily) on submucosal vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neurons in the jejunum of aging rats. Twenty-five male rats were divided into the following groups: Y90 (young, 90-day-old rats), A345 (aged, 345-day-old rats), A428 (aged, 428-day-old rats), AA345 (ascorbic acid-supplemented rats, 90-345-day old), and AA428 (ascorbic acid-supplemented rats, 90-428-day old). Whole mounts of the submucosal layer were subjected to immunohistochemistry for determination of VIP-IR. Morphometric analyses were carried out in 100 submucosal VIP-IR neuron cell bodies from each group. At 345 days, neurons from supplemented animals were larger than those of non-supplemented animals of the same age. These results indicate that ascorbic acid neutralized free radicals and played a neuroprotective role. At 428 days, no significant differences between cell body areas were seen with or without ascorbic acid supplementation, indicating that, from a certain age onward, the role of ascorbic acid as a VIP-IR antioxidant was reduced. This supposition is supported by the fact that both supplemented and non-supplemented animals had higher blood concentrations of ascorbic acid on Day 428 compared with Day 345. The possible neuroprotective and neurodegenerative effects of ascorbic acid appear to depend on the age of the animals, dose, and its interaction with other antioxidants.

Immunohistochemical study of vasoactive intestinal peptide (VIP) enteric neurons in diabetic rats supplemented with L-glutamine.

The purpose of this work was to study the area of the varicosities of nerve fibers of myenteric neurons immunoreactive to vasoactive intestinal peptide (VIP-IR) and of the cell bodies of VIP-IR submucosal neurons of the jejunum of diabetic rats supplemented with 2% L-glutamine. Twenty male rats were divided into the following groups: normoglycemic (N), normoglycemic supplemented with L-glutamine (NG), diabetic (D) and diabetic supplemented with L-glutamine (DG). Whole-mounts of the muscle tunica and the submucosal layer were subjected to the immunohistochemical technique for neurotransmitter VIP identification. Morphometric analyses were carried out in 500 VIP-IR cell bodies of submucosal neurons and 2000 VIP-IR varicosities from each group. L-Glutamine supplementation to the normoglycemic animals caused an increase in the areas of the cell bodies (8.49%) and varicosities (21.3%) relative to the controls (P < 0.05). On the other hand, there was a decrease in the areas of the cell bodies (4.55%) and varicosities (28.9%) of group DG compared to those of group D (P < 0.05). It is concluded that L-glutamine supplementation was positive both to normoglycemic and diabetic animals.

Effects of insulin treatment on HuC/HuD, NADH diaphorase, and nNOS-positive myoenteric neurons of the duodenum of adult rats with acute diabetes.

We carried out this investigation with the purpose of verifying whether insulin treatment prevents changes in the density of myoenteric neurons of the duodenum of Wistar rats with streptozotocin short-term diabetes. The animals from the diabetic group (D) lost more weight than the controls (group C), while the insulin treatment (group T) prevented weight loss in three animals and increased visceral fat in all of the animals of this group. Insulin treatment did not prevent the early loss of HuC/HuD myoenteric neurons. The density of nNOS-positive neurons did not change significantly in groups D and T. The density of NADHd-positive neurons in these groups was greater than in group C, indicating that short-term diabetes increases the activity of respiratory chain enzymes.

Vitamin E supplementation in rats with experimental diabetes mellitus: analysis of myosin-V and nNOS immunoreactive myenteric neurons from terminal ileum.

The effect of vitamin E (1 g/kg body weight) supplementation on myosin-V and neuronal nitric oxide synthase (nNOS) immunoreactive myenteric neurons from the ileum of diabetic rats was investigated in the present study. Forty animals were divided into the following groups: normoglycemics (N), normoglycemics treated with vitamin E (NE), diabetics (D), and diabetics treated with vitamin E (DE). Quantitative and morphometric analyses were performed. The area of the tertiary plexus was also determined. Diabetes produced a 24% reduction in the number of myosin-V neurons in group D compared with group N, an effect that was accompanied by an increase in the tertiary plexus area (P < 0.05). Neuronal density was 27% higher in group NE than group N (P < 0.05). Nitrergic neuronal density was not altered as a consequence of either diabetes or vitamin E treatment. Myosin-V and nNOS immunoreactive neuronal cell body area increased significantly in group NE. The area of myosin-V and nNOS myenteric neurons also increased in group D. Vitamin E treatment (group DE) increased only the size of nitrergic neurons. The present results suggest that vitamin E elicited a neuroprotective and neurotrophic effect on the natural aging process, but with regard to diabetes, vitamin E supplementation exerted a neurotrophic effect only on nitrergic neurons.

[NADH-diaphorase positive neurons of the jejunum of disnurtured adult rats (Rattus norvegicus): quantitative aspects]. / Neur nios NADH-diaforase positivos do jejuno de ratos adultos (Rattus norvegicus) desnutridos: aspectos quantitativos.

We aim at contributing with information on the quantitative aspects of the NADH-diaphorase positive myenteric neurons of the jejunum of adult rats subjected to protein desnutrition. Ten rats aging 90 days were divided into two groups: control (n=5, 278 g) and disnurtured (n=5, 280 g). In the following 120 days, the rats from the control group had chow with 22% protein level, and those from the disnurtured group, with 8% protein level. After this period, the control rats weighted 394.4g and the disnurtured 273.5g. The jejunum was subjected to the histochemical technique of the NADH-diaphorase to stain nerve cells in whole-mounts. The neurons found in 80 microscopic fields of both groups were counted. In the control 674.6 neurons were observed, and 1326.8 neurons were counted in the disnurtured group. The low-protein diet did not alter the organization of the neurons, but led to a smaller body growth in the disnurtured animals, preventing neuronal dispersal and leading to a greater density per mm .

Regional differences in the number and type of myenteric neurons in the descending colon of rats.

The purpose of this study was to analyze the neuronal density of the myenteric plexus of the intermediate and antimesocolic regions of the descending colon of rats. Whole-mounts were stained with three different techniques of neuronal evidenciation. Through counts of the number of neurons in an area of 6.64 mm under light microscopy, we found 1,271 +/- 227.54 neurons with Giemsa in the intermediate region and 1,234 +/- 225.92 neurons in the antimesocolic region; with the NADH-diaphorase technique we found 530 +/- 92.97 neurons in the intermediate region and 539 +/- 146.72 neurons in the antimesocolic region; and through the NADPH-diaphorase histochemistry, we found 417 +/- 34.42 neurons in the intermediate region and 547 +/- 84.01 neurons in the antimesocolic region. We conclude that there is a variation in the density of NADPH-diaphorase positive neurons in the intestinal circumference; that the NADH-diaphorase positive neuronal subpopulation represented 42.7% of that stained with Giemsa; and that the NADPH-diaphorase positive neurons represented 37.8% of the whole myenteric population.

Effect of protein and vitamin B deficiency on the morpho-quantitative aspects of the myenteric plexus of the descending colon of adult rats.

We carried out this work with the purpose of studying the effects of protein and vitamin B deficiency on the morphologic and quantitative aspects of the myenteric plexus of the descending colon of adult Rattus norvegicus. Twenty-eight rats were divided in two groups, one of them receiving chow with 22% protein level (control) and the other fed with chow having 8% protein level without vitamin B supplementation, during 120 days. Whole-mounts of the descending colon were prepared and stained with Giemsa, NADH-diaphorase and NADPH-diaphorase. The undernourished rats had a body weight 11.84% less than the control group. Relative to the controls, the experimental group had a colonic area 48% smaller, 51.9% less Giemsa-stained neurons, 28.3% less NADH-diaphorase positive neurons and 24.2% less NADPH-diaphorase positive neurons.

Effect of acetyl-L-carnitine on VIP-ergic neurons in the jejunum submucous plexus of diabetic rats.

The effect of the treatment with acetyl-L-carnitine (ALC) on neurons releasing the vasoactive intestinal polypeptide (VIP) of the submucous plexus in the jejunum of diabetic rats was the purpose of our investigation. Diabetes (DM) was induced by injecting streptozotocin endoveneously (35 mg/kg). After sacrificing the animals, the jejunum was collected and processed for VIP detection. Four groups were used: C (non-diabetic), CC (non-diabetic treated with ALC), D (diabetic), DC (diabetes treated with ALC). We analyzed the immunoreactivity and the cellular profile of 126 cell bodies. The treatment with ALC improved some aspects of DM. However, it promoted a small increase in the area of neurons from group CC, suggesting a possible neurotrophic effect. Neurons from groups D and DC showed a large increase in their cellular profile and immunoreactivity when compared to C and CC, suggesting a larger concentration of this neurotransmitter within the neurons that produce it. This observation constitutes a recurrent finding in diabetic animals, suggesting that ALC does not interfere in the pathophysiological mechanisms that unchain a higher production and/or neurotransmitter accumulation and increase the profile of the VIP-ergic neurons.

Meso and microscopic study of the facial vein and adjacent tissues

El objetivo de esta investigación es estudiar las relaciones de la vena facial con las estructutras adyacentes. Las venas faciales, derecha e izquierda, de 50 cadáveres, fueron disecadas bajo microscopio estereoscopio y descritas sus relaciones con estructuras vecinas. Segmentos de las venas facialesde 10 cadáveres fueron sometidos a cortes histológicos teñidos por el método de weigert modificado por Van Gieson. La vena facial, a pesar de su corto trayecto, sufre importantes variaciones en la constitución de su pared. En sus relaciones de sintopía, se relacionan con músculos, tejido adiposo y glándulas entre otros tejidos. Verificamos la existencia de fibras elásticas y colágenas, conectando la adventicia a los tejidos adyacentes, así como formando una vaina fibrosa alrededor de la vena facial , lo que probablemente protege esta vena de tracciones poroducidas durante los movimientos de las estructuras vecinas. En el trabajo también son discutidas las implicancias de la sintopía de la vena facial y de los requerimientos biodinámicos generados por el movimiento sobre la morfología de esta vena

Study of the myenteric plexus of the ileum of rats subjected to proteic undernutrition

Con el objetivo de verificar los efectos de la desnutrición proteica sobre el plexo mientérico del íleon, fueron utilizados 20 ratas del linaje wistar, cuyas madres fueron desnutridas en los períodos de gestación y/o lactacia sometidas a sacrificio a los 60 días de edad. Se realizaron preparados de membrana del íleon teñidos con GIEMSA, para observación de las neuronas mientéricas y posteriores análisis y cuantificación. Verificamos que la desnutrición proteica no provoca reducción en el número de neuronas mientéricas por cm2 de íleon, y que las neuronas medias con basofilia intermediaria predominan en todos los grupos

Relationship of the facial vein with the submandibular gland

La vena facial constituye una de las principales vías de drenaje sanguíneo del rostro. En su trayecto por el rostro, prácticamente no presenta variaciones, sin embargo, en relación a la región submandibular, informaciones genéricas son encontradas en la literatura. Por lo tanto, el objetivo de este trabajo fue verificar la relación de la vena facial con la glándula submandibular. Las venas faciales derecha e izquierda de 50 cadáveres adultos, de sexo masculino, previamente formolizados, fueron disecadas y analizadas macroscópicamente y bajo estereomicroscopio. En el cuello, en relación a la glándula submandibular fue encontrada: a) sobre su superficie lateral, en ambos antímeros, 20 casos (40 por ciento); unilateralmente a la derecha, 4 casos (8 por ciento) y a la izquierda, 11 casos (22 por ciento); b) posteriormente a la glándula, en ambos antímeros, 9 casos (18 por ciento); unilateralmente a la derecha, 9 casos (18 por ciento) y a la izquierda, 6 casos (12 por ciento); c) medial a la glándula, en ambos antímeros, 2 casos (4 por ciento); y d) no relacionándose directamente con la glándula submandibular, en ambos antímeros, 2 casos (4 por ciento) y apenas en el antímero derecho, 4 casos (8 por ciento). Verificamos, por lo tanto, variaciones con respecto al trayecto recorrido por la vena facial en la region submandibular

Foramen and canal of the mandible of the squirrel monkey (saimiri sciureus - linnaeus, 1758)

Se disecaron 32 hemomandibulas de Saimiri sciureus adultos, de ambos sexos fijadas en solución de formalina al 10 por ciento y desmineralizadas en ácido nítrico. El foramen mandibular se presentó oval, siendo el valor medio de su mayor diámetro 0,91 mm, localizado en el cuadrante antero-inferior de la rama de la mandíbula, a 3,89 mm del 3er molar. El canal de la mandíbula se presentó completo (46,9 por ciento), envuelto por una capa ósea compacta y continua, o incompleto (53,1 por ciento), con capa ósea compacta presente solamente en su origen

Preliminary investigation about the effects of streptozotocin-induced chronic diabetes on the nerve cell number and size of myenteric ganglia in rat colon

Estudiamos el efecto de la diabetes mellitus crónica, inducida por estreptozotocina, en cuanto a los aspectos cuantitativos de las neuronas del plexo mientérico del colon proximal de ratas. Utilizamos cuatro grupos de animales: dos grupos de animales diabéticos (D2 y D8). En dos grupos D2 y D8, sacrificamos animales de dos y ocho meses, respectivamente, después de la inducción de diabetes; y los otros dos grupos C2 y C8, fueron mantenidos como control de los grupos anteriores. Retiramos el colon de las ratas y lo sometimos a cortes histológicos que fueron teñidos con Hematoxilina-Eosina. Se obtuvieron preparados de membrana teñidos con el método Giemsa y NADH-diaphorasa. Constatamos que la mayoría de las neuronas, tanto de animales diabéticos como no diabéticos, poseen núcleo excéntrico y que, por lo tanto, este hallazgo no es indicativo de proceso degenerativo. Verificamos que animales sacrificados dos meses después de la inducción de diabetes, no sufrieron reducción significativa en el número de neuronas por área, mientras que en animales sacrificados ocho meses después de la inducción de diabetes, presentaron reducción significativa en el número de neuronas por área, comparados con el grupo control