Reflexion

Int. J. Morphol.,

27(2):387-392, 2009.

Analysis of Myenteric Neurons of the Cecum of Diabetic

Rats After Supplementation with Ascorbic Acid

Análisis de Neuronas Mientéricas del Ciego de Ratas Diabéticas

Después de Suplementación con Ácido Ascórbico

Jacqueline Nelisis Zanoni; Priscila Gonçalves Cardoso Pereira & Marli Aparecida dos Santos Pereira

ZANONI, J. N.; PEREIRA, P. G. C. & DOS SANTOS, M. A. Analysis of myenteric neurons of the cecum of diabetic rats after supplementation with ascorbic acid. Int. J. Morphol., 27(2):387-392, 2009.

SUMMARY: The objective of this work was to investigate the neuroprotective action of the ascorbic acid over the myenteric neurons in the cecum of Wistar rats, four months after induction of the diabetes mellitus experimental with streptozotocin. Three groups with five rats each were used: C- controls, D- diabetic, DA- diabetic treated with ascorbic acid. For evidentiation of the myenteric neurons was carried out to Giemsa´s technique. Were evaluated the areas of cell bodies of 500 neurons in each group studied. The quantitative analysis was carried out in an area of 16.6 mm2 in each cecum studied. In the animals diabetic observed elevation of the glycemia and glycated hemoglobin. The supplementation with ascorbic acid was effective under the myenteric neurons of the cecum of diabetics rays, since was presented the effect neuroprotective and neurotrofic.

KEY WORDS: Ascorbic acid; Diabetes mellitus; Oxidative stress; Cecum; Streptozotocin.

INTRODUCTION

The diabetes mellitus is one of the chronic illnesses more diffused in the world (Booya et al., 2005). Several are the alterations provoked by the diabetes mellitus, among them shine through the neuropathy, however the mechanisms that generate the wounds in the nerves still do not be completely understood and explored.

Due to the physiology of the diabetes mellitus the gastrointestinal tract intensely is attacked, are observed big enlargement of the stomach, small and large intestines (Diane et al., 1979; Zanoni et al., 1997; 2003). The manifestations observed in gastrointestinal tract are generated mainly due to the neurological alterations that are located in particular in the Autonomic Nervous System (Hosking et al., 1978).

The neuronal damage due to diabetes mellitus has been attributed mainly to sorbitol, a polyol synthesized from the glucose reduction by the aldose reductase (Vinson et al.,

1989). The sorbitol is transformed into fructose and they both, in high concentrations, cause an increase of intracellular osmolality with edema formation, neuronal lesion and a consequent reduction of the nervous conduction speed (Sil- va & Teixeira, 1999). The free radicals are also responsible

for provoking neuronal damage due to the increase of the oxidative stress (Kuyvenhoven & Meinders, 1999) because of an increase of the non-enzymatic glycosylation, increase of the auto oxidation, increase of the metabolic stress, reduction of the levels of antioxidant as, by example, the acid ascorbic (Young et al., 1992). Therefore, they are involved in reactions that can cause irreversible damages to the cells such as loss of cellular function and death by necrosis or apoptosis (Hirotaka & Yasuhito, 2002).

The diabetes mellitus provokes death of the myenteric neurons, as shown by the reduction in their number and alteration in their size, after long periods of diabetes, in several intestinal segments (Romano et al., 1996; Zanoni et al., 1997, 2003; Hernandes et al., 2000; Fregonesi et al.,

2005).

Drugs that reduce the concentration of sorbitol and the oxidative stress are prominent in the treatment of the chronic complications of the diabetes mellitus, being that the acid ascorbic is some of these substances, therefore acts in the inhibition of the enzyme aldose reductase, responsible by the reduction of the glucose in sorbitol, and also in the

Morphophysiological Sciences Department (DCM/UEM), State University of Maringá, 87020900 Maringá, PR, Brazil.

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ZANONI, J. N.; PEREIRA, P. G. C. & DOS SANTOS, M. A. Analysis of myenteric neurons of the cecum of diabetic rats after supplementation with ascorbic acid.

Int. J. Morphol., 27(2):387-392, 2009.

reduction of the species reactivate to the oxygen, suggesting a prominent paper in the neuroprotection (Yue et al., 1989; Cunningham et al., 1994; Cunningham, 1998; Will & Byers,

1996).

Thus, the purpose of this work was to determine the neuroprotective effect of ascorbic acid about the density and size of myenteric neurons in the cecum of diabetic rats.

MATERIAL AND METHOD

Animal procedures. All experiments described in this work are according to the ethical beginnings adopted by the Brazilian College of Animal Experimentation (COBEA), and were supervised and approved by the Committee of Ethics on Animal Experimentation of the State University of Maringá.

Fifty male Wistar rats (Rattus norvegicus), 90 days of age, were used. The rats were divided into three groups: untreated controls (C), untreated diabetes (D) and diabetes treated with ascorbic acid (DA). Diabetes mellitus was induced through intravenous administration of streptozotocin (35 mg/kg body weight; Sigma, St. Louis, MO, USA), dissolved in a 10 mM citrate buffer solution (pH 4.5), after a

14-hr fast. The animals were kept in individual cages, receiving water and food (Nuvital lab) ad libitum, with a controlled photoperiod (06:00–18:00 hr) and room temperature (24ºC ± 2ºC).

Ascorbic acid (Sigma, St. Louis, MO, USA) was added daily to the animal's water from DA group for four months (1 g/L prepared fresh each day) (133.6 mg/ml) (Young et al.). The amounts of food intake, water consumption, and eliminated urine were measured during the treatment of rats.

At 210 days of age the animals were anesthetized with thiopental (40 mg/kg body weight). Blood was collected by cardiac puncture to measure the blood levels of glycemia, glycated hemoglobin and ascorbic acid.

Subsequently, the cecum were washed with saline solution and filled with fixing solution of Giemsa respecting the physiology of the organ, therefore without occur distensions. Right away the body was dissected under stereomicroscopy and stained according to the technical of Giemsa for evidentiation of the myenteric neurons (Barbosa,

1978).

Quantitative analysis of myenteric neurons. The cell

bodies of the myenteric neurons of cecum were counted in microscope of light, with a 40x objective lens. The quantification was carried out by sample in the two thirds near to the antimesocolic region utilizing Olympus BX50 microscope. In each segment of each animal were counted

102 fields. The area of the field of the microscope was measured with a micrometered ruler Zeiss. The results were expressed as neurons by mm2. The total area quantificated was of 16.6 mm2.

Size of cecum and calculation for enlargement proportion obtaining of cecum of diabetic rats (group D and DA).

The results obtained for the neuronal quantification should be corrected, since alterations in the size of the intestine, provoked by the intestinal growth with the natural ageing process and/or by pathological process, are able to "dilute" the number of present neurons in the area analyzed (Cowen et al., 2000).

The cecum was collected and positioned about sulfite white paper and the contours of the longitudinal average section was carried out through tracing, for obtaining of the circumference of the area of the cecum (mm2). The area was obtained through images analysis software Image Pro Plus

4 (Average Cybernetics, USA) (Table I). To the correction of the neuronal density was calculated the proportion of the dilatation of the cecum of groups D and DA in relation the group C, where quantitative results were multiplied.

Morphometric analysis. The images were obtained with a high-resolution, transmitted to a microcomputer and recorded into a compact disc. The cell body area (µm2) of 100 cell bodies in each animal, in a total of 500 neurons for each group studied was measured through images analysis soft- ware Image Pro Plus 4 (Average Cybernetics, USA).

Statistical analysis. Results were subjected to statistical analyses through the programs Statistica and GraphPad Prism, being expressed as media ± standard error. Morphometric data were set in delineation blocks followed by Tukey’s test. For the other results, we used the variance analysis One-way ANOVA, followed by Tukey’s test. Significance level was set at 5%.

RESULTS

The streptozotocin promoted to diabetic syndrome in the animal of the group D and DA, therefore all of the rats were severely hyperglycemia shown up by the blood glucose

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ZANONI, J. N.; PEREIRA, P. G. C. & DOS SANTOS, M. A. Analysis of myenteric neurons of the cecum of diabetic rats after supplementation with ascorbic acid.

Int. J. Morphol., 27(2):387-392, 2009.

levels and rate of glycated haemoglobin (p < 0.05) (Fig. 1). Also they were observed hyperfagia, polydypsia, polyuria (Figure 1).

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