Experimental Gerontology 51 (2014) 54–64

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Food restriction enhances oxidative status in aging rats with neuroprotective effects on myenteric neuron populations in the proximal colon João Paulo Ferreira Schoffen a,⁎, Ana Paula Santi Rampazzo b, Carla Possani Cirilo b, Mariana Cristina Umada Zapater b, Fernando Augusto Vicentini b, Jurandir Fernando Comar c, Adelar Bracht c, Maria Raquel Marçal Natali b a b c

Center of Biological Sciences, State University of the North of Paraná (UENP), Bandeirantes, Paraná, Brazil Department of Morphological Sciences, State University of Maringá (UEM), Maringá, Paraná, Brazil Department of Biochemistry, State University of Maringá (UEM), Maringá, Paraná, Brazil

a r t i c l e

i n f o

Article history: Received 10 August 2013 Received in revised form 3 January 2014 Accepted 7 January 2014 Available online 14 January 2014 Section Editor: Christian Humpel Keywords: Food restriction Aging Oxidative stress Myenteric plexus Muscle layer

a b s t r a c t Food restriction may slow the aging process by increasing the levels of antioxidant defenses and reducing cell death. We evaluated the effects of food restriction on oxidative and nutritional status, myenteric cell populations, and the colonic muscle layer in aging rats. Wistar rats were distributed into control groups (7, 12, and 23 months of age) and subjected to food restriction (50% of normal diet) beginning at 7 months of age. The animals were sacrificed, and blood was collected to evaluate its components and markers of oxidative status, including thiobarbituric acid-reactive substances, reduced glutathione, catalase, glutathione peroxidase, and total antioxidant capacity. The proximal colon was collected to evaluate HuC/D and neuronal nitric oxide synthase (nNOS)-positive and -negative myenteric neurons, S-100 glial cells, and the muscle layer. Age negatively affected oxidative status in the animals, which also increased the levels of total cholesterol, protein, and globulins and increased the thickness of the muscle layer. Aging also reduced the number and hypertrophied glial cell bodies, HuC/D neurons, and nNOSnegative and -positive neurons. An improvement was observed in oxidative status and the levels of total cholesterol and triglycerides with food restriction, which also provided neuroprotection of the intrinsic innervation. However, food restriction accentuated the loss of enteric glia and caused hypertrophy in the muscle layer at 23 months. Food restriction improved oxidative and nutritional status in rats and protected HuC/D neurons and nNOS-negative and -positive neurons against neuronal loss. Nevertheless, food restriction caused morphoquantitative changes in glial cell populations, with possible interference with colonic neuromuscular control. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Aging is a complex process that involves progressive and deleterious changes in cellular function, partially resulting from the increased production of reactive oxygen species (ROS), senescence, cellular dysfunction, or cell death (Finkel and Holbrook, 2000; Oliveira and Schoffen, 2010). Oxidative and metabolic changes that occur during aging increase the vulnerability of cells and organs to several diseases (Friedlander, 2003; Mattson and Magnus, 2006). Most studies in animals and humans have reported a quantitative decrease in the number of neurons in the enteric nervous system (ENS) with aging (Bernard et al., 2009; El-Salhy et al., 1999; Phillips et al., 2003; Saffrey, 2013), but not in all studies (Gamage et al., 2013; Peck ⁎ Corresponding author at: Center of Biological Sciences, State University of the North of Paraná (UENP), Rodovia BR-369 Km 54, Vila Maria-Bandeirantes CEP: 86360-000, Paraná, Brazil. Tel.: +55 43 3542 8042; fax: +55 43 3542 8008. E-mail address: [email protected] (J.P.F. Schoffen). 0531-5565/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2014.01.001

et al., 2009). Changes in the structure or quantity of enteric neurons may explain disorders of the digestive system (Bernard et al., 2009; Wade, 2002). Common changes include intrinsic innervation and alterations in smooth muscle (i.e., target tissue responsible for neuronal development, maintenance, and plasticity) that subsequently alter gastrointestinal motility and colonic transit time, which is often slowed in elderly people (Madsen and Graff, 2004; Wiskur and Meerveld, 2010). Thus, chronic constipation affects up to 27% of the elderly population (Bouras and Tangalos, 2009). Variations in the density of different myenteric neuronal populations in the gastrointestinal tract (GIT) during aging have been reported (Belai et al., 1995; Gagliardo et al., 2008; Phillips et al., 2003; Porto et al., 2012), indicating differences in the susceptibility of certain types of neurons according to the subpopulation and segment analyzed. Notably, the colon is one region of the GIT that is more vulnerable to aging (Phillips and Powley, 2001, 2007). With regard to the enteric glial cell population, changes in the number and properties of these cells as age advances have been poorly investigated (Cirilo et al., 2013; Phillips et al., 2004).

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Neuronal loss may be attributable to several causes, including the increased production and accumulation of free radicals (Thrasivoulou et al., 2006), the reduction of neurotrophic factors derived from glial cells (Wade, 2002), the activation of apoptosis, changes in gene expression or mutations in certain genes (Wiley, 2002), changes in Ca2+ signaling (Mattson, 2007), and mitochondrial dysfunction (Wade, 2002). Studies have indicated that a reduction of caloric intake may slow aging by reducing the incidence of age-related chronic diseases (e.g., diabetes, cancer, cardiovascular disease, and neurodegenerative disease), extending the lifespan of various species, including mammals (Bordone and Guarente, 2006; Sohal and Weindruch, 1996). Food restriction, also referred to as caloric restriction, may also reduce the rate of tumor formation and increase the efficiency of the immune system (Wachsman, 1996), slow metabolism, reduce apoptosis (Zhang and Herman, 2002), enhance neurotrophic signaling (Thrasivoulou et al., 2006), increase the plasticity, longevity, and survival of neurons (Cowen et al., 2000), and minimize the effects of oxidative stress generated during cellular respiration by reducing the levels of free radicals (Barja, 2002). The literature indicates that myenteric neuron neurodegeneration begins in adulthood and continues through middle age and old age, with different times of degeneration among intestinal segments (Phillips and Powley, 2001, 2007). However, few studies have investigated the possible beneficial effects of food restriction on the behavior of myenteric intrinsic innervation in aging (Cirilo et al., 2013; Cowen et al., 2000; Porto et al., 2012; Thrasivoulou et al., 2006), and fewer studies have correlated this behavior with oxidative status (Cirilo et al., 2013; Thrasivoulou et al., 2006). No studies of which we are aware of have morphoquantitatively investigated glial and myenteric neurons in the large intestine in animals subjected to prolonged food restriction. Given the possible occurrence of alterations in the myenteric plexus during the aging process and beneficial effects of reduced food intake, we evaluated the effects of food restriction on morphoquantitative aspects of the overall neuronal population (HuC/D-positive), nitrergic neuron subpopulation (nNOS-positive), cholinergic neuron subpopulation (nNOS-negative), and glial cell population (S-100) in the myenteric ganglia and muscle layer of the proximal colon and examined nutritional and oxidative status in rats during the aging process. We sought to provide data that may minimize the deleterious effects of aging. 2. Material and methods 2.1. Animals and treatment

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(Thionembutal; 40 mg/kg of body weight). The naso-anal distance was measured to determine the Lee index (body weight1/3 [g]/naso-anal distance [cm] × 1000) (Bernardis and Patterson, 1968). Blood was collected by cardiac puncture to measure biochemical parameters, including total protein, albumin, globulin, glucose, triglycerides, total cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), and analyze oxidative status in the animals. Laparotomy was then performed to remove and weigh retroperitoneal and periepididymal adipose tissues. The large intestine was collected, and the length was measured. Samples of the proximal colon (characterized by the end of the ileum–cecum–colic ampoule until the disappearance of mucosal oblique folds) underwent immunohistochemical double staining with HuC/D (i.e., a pan-neuronal marker) and nNOS, double staining with HuC/D and S-100 (i.e., a marker of glial cells), and histological processing to morphometrically analyze the intestinal muscle layer. All of the procedures were approved by the Ethics Committee for Animal Experimentation of the State University of Maringá, Brazil. 2.3. Biochemical analysis of blood components To analyze total protein, albumin, globulin, triglycerides, and total cholesterol, blood was collected and placed in test tubes to obtain serum. For the glucose assay and determination of the activity of AST and ALT, blood sample was added to test tubes that contained 3 mmol/L fluoride ethylenediaminetetraacetic acid (EDTA). Both samples were centrifuged at 3000 rotations per minute for 15 min to obtain plasma using an Analisa kit (Gold Analisa Diagnóstica, Minas Gerais, Brazil). 2.4. Evaluation of oxidative status 2.4.1. Blood preparation The collected blood was placed in tubes that contained 3 mmol/L EDTA as an anticoagulant. The blood was then centrifuged at 1000 ×g for 10 min, and the supernatant (plasma) was separated and frozen in liquid nitrogen for the analysis of lipid peroxidation and total antioxidant capacity. The residual sediment that contained erythrocytes was subjected to two cycles of washes by resuspension and centrifugation at 1000 ×g with 0.9% NaCl. The erythrocyte suspension was hemolyzed with 10 volumes of chilled deionized water and centrifuged at 4000 ×g for 10 min. The supernatant was used to determine reduced glutathione and the activity of catalase and glutathione peroxidase. All of the procedures were conducted at temperatures below 4 °C.

Thirty male Wistar rats (Rattus novergicus; 7, 12, and 23 months of age) were used. They were kept in polyethylene boxes on a 12 h/12 h light/dark cycle under controlled temperature (22 ± 2 °C). The animals were assigned to five groups: control groups (7 months of age [C7 group], 12 months of age [C12 group], and 23 months of age [C23 group]) and experimental groups (food restriction from 7 to 12 months of age [RA12 group] and food restriction from 7 to 23 months of age [RA23 group]). The control groups were fed ad libitum with standard NUVILABNUVITAL rodent chow. The amount of food consumed was controlled by supplying 100g daily for each animal and weighing the remainder, thus obtaining daily intake. In the experimental groups beginning at 7 months of age, the animals received half of the average daily intake of the rats fed ad libitum for 5 and 16 months (RA12 and RA23 groups, respectively). The body weights of the animals were recorded every 2 weeks, and food intake was monitored every month.

2.4.2. Analytical procedures for assessing oxidative stress The levels of lipid peroxidation were determined by the thiobarbituric acid-reactive substance (TBARS) method as described by Buege and Aust (1978) to evaluate oxidative injury. The total antioxidant capacity (TAC) of the plasma was determined by the colorimetric method using 2,2′azinobis (3-etylbenzthiazoline-6-sulphonic acid) (ABTS) as described by Erel (2004). The levels of reduced glutathione (GSH) in erythrocytes were determined by spectrofluorimetry as described by Hissin and Hilf (1976). Catalase activity in erythrocytes was assessed by the enzymatic decomposition of H2O2 measured by spectrophotometry at 240 nm as described by Aebi (1974). The activity of glutathione peroxidase in erythrocytes was determined by the decrease in absorbance caused by the decomposition of NADPH-dependent H2O2 at 340 nm at 25 °C (Paglia and Valentine, 1967; Tappel, 1978).

2.2. Tissue collection

Samples of the proximal colon were washed with 0.1 M phosphatebuffered saline (PBS; pH 7.4) to remove residues, and its ends were tied off, filled and distended with Zamboni's fixative, and immersed in the same fixative for 18 h at 4 °C. The samples were then opened and

At 7, 12, and 23 months of age, after fasting for 10 h, the rats were weighed and anesthetized with intraperitoneal sodium thiopental

2.5. Immunohistochemistry of myenteric neurons and glia

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washed with 80% alcohol to remove the fixative, dehydrated in an ascending series of alcohol (95% to 100%), cleared in xylene, rehydrated in a descending series of alcohol (100%, 90%, 80%, and 50%), and stored at 4 °C in 0.1 M PBS (pH 7.4) with 0.08% sodium azide for dissection. Whole-mount preparations were obtained by removing the mucosal, submucosal, and circular muscle layers of the samples of the proximal colon using a stereomicroscope with transillumination. The samples were subjected to immunohistochemical double staining with HuC/D and nNOS to evaluate the HuC/D neuronal and nitrergic subpopulations and double staining with HuC/D and S-100 to evaluate the HuC/D neuronal and enteric glia subpopulations, respectively. 2.5.1. Double staining with HuC/D-nNOS and HuC/D-S-100 Whole-mount preparations of the muscle layer of the proximal colon were washed twice in 0.1 M PBS (pH 7.4) with 0.5% Triton X-100 for 10 min and blocked for 1 h in a solution that contained 0.1 M PBS (pH 7.4) with 0.5% Triton X-100, 2% bovine serum albumin (BSA), and 10% goat serum to avoid nonspecific binding. The tissues were then incubated for 48 h in a solution that contained 0.1 M PBS (pH 7.4) with 0.5% Triton X-100, 2% BSA, and 2% goat serum and primary antibodies. After incubation, the membranes were washed three times in 0.1 M PBS (pH 7.4) with 0.5% Triton X-100 for 5 min and incubated with secondary antibodies for 2 h at room temperature. After new washes with PBS to remove excess antibody, the membranes were mounted between slides and coverslip with buffered glycerol. Double staining with HuC/D-nNOS and HuC/D-S-100 occurred with the incubation of tissues with the primary and secondary antibodies described in Table 1.

analyses were performed using the ImagePro Plus 4.5 image analysis software (Media Cybernetics, Silver Springs, MD, USA). 2.7. Morphometric analysis of the muscle layer To evaluate the thickness of the muscle layer, samples of the proximal colon were opened at the mesenteric edge, washed with saline, fixed in Bouin's solution for 6 h, dehydrated in an ascending series of alcohol (80%, 90%, and 100%), cleared in xylene, and embedded in paraffin to obtain 6-μm-thick semi-serial sections using a Leica RM 2145 microtome, which were stained with hematoxylin–eosin. The measurement of 100 points of the muscle layer (10 sections/ animal) was performed from images captured with a 10× objective on an Olympus BX41 light microscope coupled to a Q Color 3 camera (Olympus America) and analyzed with Image-Pro Plus 4.5. The results are expressed in micrometers. 2.8. Statistical analysis The experiment was a completely randomized design. The numerical data were subjected to the Kolmogorov–Smirnov test to verify normality. When this assumption was met, we used one-way analysis of variance (ANOVA) to test the differences between the means and the Tukey's post hoc test to identify the differences. Data were analyzed using Statistica 7.1 and GraphPad Prism 5 software. The level of significance was set at 5% (p b 0.05), and the results are expressed as mean ± standard error.

2.6. Morphoquantitative analysis of the myenteric plexus 3. Results To quantify the myenteric neuronal population, HuC/D neurons, nNOS-positive nitrergic neurons, and glial cells (S-100), immunofluorescent was detected using an Olympus BX40 light microscope equipped with filters for immunofluorescence and coupled to a Moticam 2500 camera. We quantified neuronal and glial cell bodies by sampling 48 ganglia per animal in microscopic images captured in the mesenteric, intermediate, and anti-mesenteric regions of the intestinal circumference (16 images/region) using a 20 × objective. The number of cholinergic neurons (nNOS-negative) was calculated as the difference between the number of nitrergic neurons (nNOS-positive) and HuC/D-immunoreactive cells (Phillips et al., 2003). From the images captured, we measured the area of 48 ganglia (μm2) per animal that contained cell bodies of HuC/D-positive neurons. A myenteric ganglion was defined by considering the peripheral neurons visualized as the limit of the ganglia examined. This criterion was adopted to determine the number of neurons per ganglion, the number of glial cells per ganglion, and the ganglion area. Individual neurons or small groups of 1–3 neurons were excluded from the counts. We measured the areas (μm2) of cell bodies of 150HuC/D and nNOS neurons and S-100 glial cells per animal. To estimate the size of cholinergic neurons, we performed morphometry of the cell bodies of 150nNOSnegative, HuC/D-positive neurons by overlapping the images of the same double immunostaining (HuC/D-nNOS) ganglia. Morphoquantitative Table 1 Antibodies and dilutions used in double immunostaining techniques with HuC/D-nNOS and HuC/D-S-100. Primary antibody

Supplier

Dilution

Secondary antibodya

Anti-HuC/D (mice) Anti-nNOS (rabbit) Anti-S-100 (rabbit)

Invitrogen, USA

1:500

Santa Cruz Biotechnology, USA

1:500

Sigma, USA

1:200

Alexa fluor 488 (anti-mice) Alexa fluor 546 (anti-rabbit) Alexa fluor 546 (anti-rabbit)

a

Secondary antibodies were used with 1:500 dilution and supplied by Invitrogen, USA.

3.1. Animal treatment The results for food intake, body weight, adipose tissue weight, the Lee index, and the length of the large intestine are presented in Table 2. The aging process caused a significant reduction (p b 0.05) of food consumption in the C12 and C23 groups compared with the C7 group, without significant changes in final body weight. Food restriction significantly decreased the weight of the rats in the RA groups compared with normally fed rats. The Lee index is considered similar to body mass index. In the present study, these values remained constant between ages, but the RA12 and RA23 groups exhibited a reduction (p b 0.05) compared with the respective control groups. A significant reduction of adipose tissue weight (periepididymal and retroperitoneal) was also observed in the food restriction groups compared with the other groups. In the normally fed groups, retroperitoneal adipose tissue weight in the C23 group was high compared with the C12 and C7 groups (p b 0.05). The summation of the weights of the adipose tissues confirmed a gradual increase in visceral fat with advancing age, which was reduced (p b 0.05) by food restriction. With regard to large intestine length, an increase (p b 0.05) was detected in 23-month-old animals (C23 and RA23 groups) compared with 7-month-old animals, but no changes (p N 0.05) were observed in the RA12 group compared with the C12 and C7 groups. 3.2. Biochemical analysis of blood components The biochemical analysis of blood components is presented in Fig. 1. A significant increase in total cholesterol was verified in the 23-month-old group compared with 7- and 12-month-old animals, and this parameter was reduced by food restriction (RA23 group). We also observed a decrease in triglycerides (p b 0.05) by food restriction at 23 months compared with controls. Age and food restriction increased (p b 0.05) the levels of total protein and globulins compared with the C7 group, with maintenance of albumin levels. The analysis of glucose levels showed

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Table 2 Final body weight, food intake, Lee index (body weight1/3/naso-anal distance × 1000), weight of periepididymal (PER) and retroperitoneal (RET) adipose tissues, summation of tissues (PER + RET), and the length of the large intestine in rats in the normally fed control groups at 7 months of age (C7 group), 12 months of age (C12 group), and 23 months of age (C23 group) and food-restricted groups at 12 months of age (RA12 group) and 23 months of age (RA23 group). The data are expressed as mean ± standard error (n = 6). Parameters

Groups C7

Body weight (g) Food intake (g) Lee index PER (g) RET (g) PER + RET (g) Large intestine (cm) (a)

489.5 28.08 295.6 7.80 7.87 15.68 14.63

C12 ± ± ± ± ± ± ±

8.72a(a) 0.43a 2.72a 1.10a 0.93a 1.98a 0.42a

492.9 21.34 301.7 9.32 10.11 19.43 15.58

C23 ± ± ± ± ± ± ±

4.32a 0.38b 3.83a 0.33a 1.02a 1.09ab 0.55ac

510.3 22.66 307.3 8.81 13.47 22.29 17.17

RA12 ± ± ± ± ± ± ±

7.67a 0.47b 1.55a 0.75a 0.72b 1.45b 0.65bc

358.5 14 272.5 3.16 1.55 4.71 14.33

RA23 ± ± ± ± ± ± ±

5.05b 0.00c 3.86b 0.21b 0.12c 0.32c 0.84a

299.8 13.5 269.4 1.07 0.21 1.29 18.17

± ± ± ± ± ± ±

10.40c 0.00c 4.02b 0.06b 0.05c 0.11c 0.30b

Means followed by different letters in the same row differ significantly (p b 0.05).

no alterations with age or food restriction compared with the groups of the same age. Enzymes that indicate liver damage were analyzed in the C7, C23, and RA23 groups. The levels of AST were similar between groups (C7 group: 142.3 ± 11.31 U/L; C23 group: 127.5 ± 1.42 U/L; RA23 group: 121.1 ± 3.00 U/L), but food restriction significantly increased ALT levels (C7 group: 99.52 ± 3.69 U/L; C23 group: 75.95 ± 6.52 U/L; RA23 group: 115.2 ± 4.05 U/L).

3.3. Oxidative status The oxidative status results are shown in Fig. 2. As shown in Fig. 2A, plasma TBARS levels gradually increased with age (95.37% and 163.58% at 12 and 23 months, respectively) compared with the 7-month-old group. The RA rats had a significantly smaller increase (54.94% and 64.20% at 12 and 23 months, respectively) compared with the 7-month-old group. The compound reduced glutathione (GSH) is a component of the intracellular antioxidant system; therefore, its levels were determined in erythrocytes (Fig. 2B). Contrary to TBARS, GSH levels progressively decreased with age (16.92% and 51.74% lower at 12 and 23 months, respectively) compared with 7-month-old animals. With food restriction, GSH levels decreased by only 27.86% at 23 months but were not reduced at 12 months. The levels of GSH increased at 12 months with food restriction (25.87% higher than controls). Catalase activity decreased with age (12.21% and 38.26% smaller at 12 and 23 months, respectively; Fig. 2C). With food restriction, catalase activity remained lower at 23 months but increased by 20.07% at 12 months compared with controls. The activity of glutathione peroxidase was not changed with age and mildly increased (p b 0.05) with food restriction (Fig. 2D). Total antioxidant capacity was additionally determined in plasma but only at 7 and 23 months. Aging reduced plasma TAC (0.50 ± 0.01 and 0.31 ± 0.02 μmol · mL−1 at 7 and 23 months, respectively). Food

restriction increased TAC in aged rats, which reached the values of controls (0.53 ± 0.02 μmol/mL).

3.4. Morphoquantitative analysis of the myenteric plexus The morphology of myenteric ganglia was preserved, regardless of treatment. Double staining with HuC/D-nNOS revealed all immunostained neurons within the ganglia, with the nitrergic subpopulation located predominantly in the peripheral region of the ganglion, whereas the other neurons were often located in the central region of the same ganglion. In the ganglion, the mean percentage of nNOS-positive neurons (nitrergic) was 24.44%, and the mean percentage of nNOS-negative neurons (cholinergic) was 75.56% (Fig. 3A,C,E). Aging quantitatively reduced all neuronal populations. From 12 months of age onward, a significant reduction of the number of HuC/D-positive (9.92%) and nNOS-negative (12.23%) neurons was observed in the ganglia. At 23 months of age, neuronal loss (p b 0.05) compared with the C12 group was 11.69% in the HuC/D population and 11.99% in the cholinergic subpopulation. Comparisons of the C23 and C7 groups indicated that neurodegeneration (p b 0.05) amounted to 20.45% in HuC/D-positive cells and 22.75% in nNOS-negative cells (Fig. 3B,F). For nNOS-positive neurons, the numerical reduction (p b 0.05) was observed at 23 months but with a lower proportion (12.57%), indicating greater resistance to the effects of aging in this neuronal subpopulation (Fig. 3D). With 50% food restriction, the neuronal density of the HuC/D population and cholinergic subpopulation (nNOS-negative) was high (p b 0.05; 10.76% and 16.25% higher in the RA12 group compared with the C12 group), with maintenance of neuronal density in the RA23 group compared with the C23 group (Fig. 3B,F). Numerical preservation (p b 0.05) of 18.75% was also found in the nitrergic subpopulation (nNOS-positive) but only in the RA23 group (Fig. 3D).

Fig. 1. Effects of aging and food restriction on blood components in normally fed control rats at 7, 12, and 23 months of age (C7, C12, and C23 groups) and food-restricted rats at 12 and 23 months of age (RA12 and RA23 groups). (A) Levels of total cholesterol, triglycerides, and glucose. (B) Levels of total protein, globulin, and albumin. *p b 0.05, significant difference compared with C7 group; #p b 0.05, significant difference compared with C7 and C12 groups; ≠p b 0.05, significant difference compared with C23 group.

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Fig. 2. Effects of aging and food restriction on oxidative stress in blood in normally fed control rats at 7, 12, and 23 months of age (C7, C12, and C23 groups) and food-restricted rats at 12 and 23 months of age (RA12 and RA23 groups). (A) Levels of plasma lipid peroxidation (TBARS). (B) Levels of erythrocyte reduced glutathione (GSH). (C) Catalase activity. (D) Glutathione peroxidase activity. *p b 0.05, significant difference compared with C7 group; #p b 0.05, significant difference compared with C7 and C12 groups; ≠p b 0.05, significant difference compared with C23 group.

Double staining with HuC/D and S-100 indicated that glial cell bodies were smaller than neuronal bodies, presenting numeric variation according to age in a proportion of approximately 1 neuron to 1.25, 1.08, and 0.97 glial cells in the C7, C23, and RA23 groups, respectively (Fig. 4A–C). Enteric glia underwent significant numerical reductions of 31.33% in ganglia in the C23 group compared with the ganglia in the C7 group, 10.34% in the RA23 group compared with the C23 group, and 38.43% in the RA23 group compared with the C7 group (Fig. 4D). Age and food restriction led to an increase (p b 0.05) in the cell profile (CP) area of glial cells (C7 group: 45.97 ± 0.47 μm2; C23 group: 67.02 ± 0.72 μm2; RA23 group: 69.33 ± 0.86 μm2). Glial cells showed high variability in the cell body area, from 17 to 194.88 μm2. The neuron and ganglion morphology results are presented in Table 3. The neuronal morphometric analysis revealed an increase (p b 0.05) in the area of CP of the HuC/D-positive population, nNOSnegative subpopulation, and nNOS-positive subpopulation in the C23 group compared with the C12 and C7 groups, indicating an effect of age. However, in animals subjected to food restriction (RA12 and RA23 groups), a significant reduction was detected in the CP area for all neuronal populations, but the reduction was not significant for cholinergic neurons (nNOS-negative) in the RA12 group compared with the C12 group. Neurons of the HuC/D-positive population showed variability in the CP area, from 26.87 to 1641.86 μm2. In the nNOSnegative subpopulation, the area ranged from 60.76 to 1216.19 μm2, and the nNOS-positive subpopulation exhibited variations between 47.20 and 1406.18 μm2. No significant alterations in the area of ganglia were observed according to age or food restriction. 3.5. Analysis of the muscle layer An increase (p b 0.05) in the muscle layer thickness of the proximal colon was observed in the normally fed animals with advancing age (C7 group: 164.78 ± 1.81 μm; C12 group: 188.76 ± 2.06 μm; C23

group: 202.77 ± 2.80 μm). In the groups subjected to food restriction, the muscle layer thickness was maintained (p N 0.05) in the RA12 group (183.60 ± 2.55 μm) compared with the C12 group (188.76 ± 2.06 μm). However, a significant increase in this parameter was found in the RA23 group (230.36 ± 2.74 μm) compared with the C23 group (202.77 ± 2.80 μm). 4. Discussion 4.1. Experimental model and nutritional status of animals The present study used adult rats (7 months of age), aging rats (12 months of age), and old rats (23 months of age). Despite some disagreement in the literature about the age of rodents, the average life expectancy of a rat is approximately 24 months. At 6 months, they are considered adults. At 12 months, they are middle-age. From 18 months onward, they are considered elderly. This classification has been adopted by several authors (Johnson et al., 1998; Phillips and Powley, 2001). The food restriction model used in the present study was a 50% reduction of normal consumption. This has been one of the most widely discussed forms of nutritional intervention to extend the lifespan of different animal species (Bordone and Guarente, 2006; Sohal and Weindruch, 1996; Speakman and Mitchell, 2011). Research using caloric restriction by lowering the amount of food without manipulating calorie quantity or the quality of diets is common (Cowen et al., 2000; Johnson et al., 1998; Thrasivoulou et al., 2006). We chose the term “food restriction” because we considered it to be more appropriate because we imposed a reduction of the supply of calories and other components, such as proteins, amino acids, and minerals (Cirilo et al., 2013). According to Phillips and Powley (2001), body weight varies with age, and rats gain weight until approximately 21 months of age, which rapidly declines after this age. In contrast, the comparison of body weight

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Fig. 3. Neurons of the myenteric plexus of the proximal colon in rats. (A) HuC/D neuronal population. (C) nNOS-positive subpopulation. (E) Overlap of images from A and C. Mean number of (B) HuC/D-positive, (D) nNOS-positive, and (F) nNOS-negative neurons per ganglion between the C7, C12, C23, RA12, and RA23 groups. Scale bar = 100 μm. *p b 0.05, significant difference compared with C7 group; #p b 0.05, significant difference compared with C7 and C12 groups; p b 0.05, significant difference compared with C12 group; ≠p b 0.05, significant difference compared with C23 group.

between the groups of animals fed ad libitum (C7, C12, and C23 groups) demonstrated maintenance of this parameter, reflected by the absence of growth in adulthood. This determination was based on measuring the naso-anal distance and the lower food intake in control animals at 12 and 23 months of age compared with 7-month-old animals. A greater accumulation of fat in the body occurs with aging (Barzilai et al., 1998), which was confirmed by the increase in the total weight of periepididymal and retroperitoneal fat (Gommers et al., 1983; Valle et al., 2010). A reduction of food intake was observed in the C12 and C23 groups. Increased body fat may have occurred because of several factors, such as reduced spontaneous physical activity, the reduced basal energy requirement of cells (Coggan, 1999), and decreased muscle mass, which occur during the aging process (Bross et al., 2000). Food restriction caused a reduction of body weight, together with a decrease in the weights of retroperitoneal and periepididymal adipose

tissues. This is attributable to the increased mobilization of body fat, which directly reflects body composition (Duffy et al., 1997). The reductions of body weight and adipose tissue in food-restricted animals are frequent findings in the literature (Barzilai et al., 1998; Valle et al., 2010). Considering the food intake in the RA12 and RA23 groups, this should not be taken as a reference because it was an imposed condition. The final body weight associated with the measurement of the nasoanal distance allowed us to calculate the Lee index (Bernardis and Patterson, 1968), which is analogous to body mass index. The Lee index remained constant with aging, but it was reduced in foodrestricted animals, which proved the effectiveness of our model, especially when considering the weight of fatty tissues. This parameter is often used in models that interfere with metabolism, such as obesity (Soares et al., 2006) and dietary restriction (Oliveira et al., 2011).

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Fig. 4. Neurons and glial cells in the myenteric plexus of the proximal colon in rats. (A) HuC/D-positive neuronal population. (B) S-100-positive glial cells. (C) Overlap of images from A and B. (D) Mean number of HuC/D-positive neurons and glial cells per ganglion between C7, C23, and RA23 groups. Scale bar = 100 μm. *p b 0.05, significant difference compared with C7 group; ≠p b 0.05, significant difference compared with C23 group.

The present study found an increase in the large intestine length in the C23 and RA23 groups compared with the C7 group, with no influence of the restrictive diet. Considering the ages of the animals, similar results were reported by Phillips and Powley (2001) in Fisher rats, in which a significant increase was observed in the large intestine length after 21 months. Other studies also reported a gradual increase in intestinal length in advanced age in rats (Johnson et al., 1998; Phillips et al., 2003, 2006), mice (Gamage et al., 2013), and guinea pigs (Peck et al., 2009), confirming our results. With regard to blood components, an increase was observed in the levels of total cholesterol (p b 0.05) and triglycerides (p N 0.05) at the age of 23 months. Lower levels of these parameters were found in animals subjected to food restriction, suggesting a positive effect of reduced food intake that may decrease the likelihood of developing chronic degenerative diseases. The elevation of blood glucose levels with aging and reduction with dietary restriction has been described in the literature (Sharma and Kaur, 2008). In the present study, age did not significantly affect glucose levels. Similar results were obtained by Barzilai et al. (1998), who compared glucose levels in rats at 4 and 18 months of age. Despite the

maintenance of glucose levels observed between food-restricted animals and controls, we found that the RA23 group had higher glucose levels compared with the other groups. We can exclude the possible occurrence of a diabetic state because the values were b 300 mg/dL, a value above which indicates diabetes in Wistar rats (Liu et al., 2010). Increases in the levels of total protein and globulins were observed with age and food restriction compared with the C7 group. Serum albumin concentrations were maintained in all of the groups. We consider that food restriction did not cause malnutrition, which can occur with diets with protein levels reduced by 8% (Natali et al., 2000) or 4% (Araújo et al., 2005) in rats. The values obtained for total protein, albumin, and globulin were within reference values for Wistar rats (Hillyer and Quesenberry, 1997). With regard to the evaluation of transaminases and the data obtained by Palomero et al. (2001) in Wistar rats, the levels of AST and ALT were maintained with age, but increased levels of ALT were observed with food restriction (RA23 group compared with the C23 group). We consider that liver function was preserved with age and the diet imposed because high ALT levels in the RA23 group remained statistically similar to adult animals (7 months of age), although they differed from the control

Table 3 Profile of cell bodies of HuC/D-positive, nNOS-negative, and nNOS-positive (μm2) neurons and area of myenteric ganglia (μm2) of the proximal colon in rats in the normally fed control groups at 7 months of age (C7 group), 12 months of age (C12 group), and 23 months of age (C23 group) and food-restricted groups at 12 months of age (RA12 group) and 23 months of age (RA23 group). The data are expressed as mean ± standard error (n = 6). Parameters

Groups C7

HuC/D neurons nNOS-neurons nNOS + neurons Ganglia (a)

374.47 370.90 362.22 21,272

C12 ± ± ± ±

(a)

5.88a 6.01a 4.7a 424.74a

375.16 356.11 344.03 20,139

C23 ± ± ± ±

6.06a 5.90ac 4.56ad 461.10ac

Means followed by different letters in the same row differ significantly (p b 0.05).

402.66 424.21 398.29 20,480

RA12 ± ± ± ±

7.07b 6.92b 5.21b 377.27ac

333.54 338.22 315.56 20,488

RA23 ± ± ± ±

5.48c 5.94c 4.63c 386.64ac

365.68 330.85 375.13 19,015

± ± ± ±

6.27a 5.09c 5.48ae 367.16bc

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group at 23 months of age. Notably, the levels of the enzyme AST were not altered by food restriction, which can rule out possible liver damage. 4.2. Oxidative status In the present study, oxidative status parameters were determined in blood to simultaneously compare the changes that occurred in the intrinsic intestinal innervation with the evolution of oxidative stress in blood (see Section 4.3). The aging process is directly related to increased ROS formation and consequent tissue injury, a well-established process (Finkel and Holbrook, 2000), and mainly attributed to decreased antioxidant capacity, such as the activity of superoxide dismutase, catalase, and glutathione peroxidase (Kuyvenhoven and Meinders, 1999). Food restriction has been previously shown to improve antioxidant capacity and decrease oxidative tissue injury during aging (Sohal and Weindruch, 1996). Accordingly, the present results showed an increase in tissue oxidative stress and a reduction of the antioxidant system during aging, based on parameters determined in blood: increased plasma TBARS, decreased TAC in plasma, decreased erythrocyte GSH content, and decreased erythrocyte catalase activity. Food restriction also reduced oxidative stress during aging, in which it decreased plasma TBARS levels and increased TAC, GSH content, and the activity of catalase and glutathione peroxidase. These results are supported by previous studies of both aging (Cho et al., 2003; Mehdi et al., 2012; Siqueira et al., 2005) and food restriction (Cho et al., 2003; Hamden et al., 2009; Starnes et al., 1989). 4.3. Morphoquantitative analysis of the myenteric plexus The present study found a significant reduction of the number of HuC/D-positive, nNOS-positive, and nNOS-negative neurons in the proximal colon in rats with aging. A progressive reduction of enteric neurons is often reported with age in myenteric neurons in the small intestine (Cowen et al., 2000; Marese et al., 2007; Phillips and Powley, 2001; Porto et al., 2012; Thrasivoulou et al., 2006) and large intestine (Bernard et al., 2009; El-Salhy et al., 1999; Gagliardo et al., 2008; Gomes et al., 1997; Phillips and Powley, 2001; Phillips et al., 2003, 2004) in different species. This reduction begins in adulthood and continues throughout middle-age and old-age, and neurons in the large intestine are affected earlier than neurons in the small intestine (Phillips and Powley, 2001, 2007). Phillips and Powley (2001) analyzed the number of neurons in Fischer rats aged 3 to 27 months and estimated that neuronal loss begins from the age of 12 months and the neuronal stability begins at 3 months. Contrary to these results, Marese et al. (2007) reported that neuronal loss in Wistar rats begins at 7 months of age. These results demonstrate that the strain and lifetime of the animal are important in neuronal morphoquantitative evaluations (Johnson et al., 1998; Wu et al., 2003). In the myenteric ganglia of the proximal colon, we found a reduction of the HuC/D population and cholinergic subpopulation at 12 months of age, whereas we observed a reduction of the nitrergic subpopulation only at 23 months. The reduction of the density of HuC/D-positive neurons was previously obtained in segments of the large intestine in Fischer rats (Phillips et al., 2004), and humans (Bernard et al., 2009) and also with the use of other pan-neuronal markers (El-Salhy et al., 1999; Gomes et al., 1997; Phillips and Powley, 2001; Phillips et al., 2003). The quantification of the cholinergic subpopulation (nNOS-negative) was estimated by calculating the difference between the number of nitrergic neurons (nNOS-positive) and HuC/D-immunoreactive cells, according to Phillips et al. (2003). Nitrergic neurons are responsible for the synthesis of nitric oxide through NOS, and cholinergic neurons that secrete acetylcholine synthesized by choline acetyltransferase represent two distinct subpopulations that together can be used to quantify almost the entire myenteric neuronal population in rats (Nakajima et al., 2000).

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Previous studies revealed that the loss of enteric neurons during aging is a selective process that is limited to specific phenotypes (Cowen et al., 2000; Phillips and Powley, 2007; Phillips et al., 2003; Wade, 2002). In fact, a subpopulation of cholinergic neurons (excitatory neurons) is more vulnerable to the effects of aging than nitrergic neurons (inhibitory neurons) in rats and humans (Bernard et al., 2009; Cowen et al., 2000; Phillips et al., 2003; Porto et al., 2012), but not in mice (Gamage et al., 2013). A decrease in the number of cholinergic neurons in the myenteric plexus in the large intestine in rats was also found by Phillips et al. (2003) and in humans by Bernard et al. (2009). With regard to the nitrergic subpopulation, previous studies (Gagliardo et al., 2008; Takahashi et al., 2000) also observed a reduction of the amount of these neurons in the large intestine with age in Wistar and Fischer rats, respectively. With aging, an increase in the number of nitrergic neuronal phenotype was found in the duodenum in Wistar rats (Porto et al., 2012) and colon in Sprague–Dawley rats (Belai et al., 1995). Most studies have reported maintenance of the density of the nitrergic subpopulation with age in Wistar rats [jejunum (Santer, 1994), ileum (Cirilo et al., 2013)], Sprague–Dawley rats [ileum (Belai et al., 1995; Cowen et al., 2000)], and Fisher rats [small intestine and colon (Phillips et al., 2003, 2006)]. The discrepancies in these results may be explained by regional and species–specific differences (Wu et al., 2003) and methodological differences in the preparation and analysis of the samples, among other reasons (see Saffrey, 2013). We consider that neurodegeneration in the nitrergic subpopulation of the proximal colon in Wistar rats is justified by regional differences in neuronal responses against aging. Thus, a possible mechanism of protection against neuronal loss in advanced age selectively in nitrergic myenteric neurons, which has been suggested in previous studies in Sprague–Dawley (Cowen et al., 2000) and Fisher (Phillips et al., 2003, 2006) rats, cannot be presented as a general phenomenon throughout the GIT for all animal species. In food-restricted animals, we detected a neuroprotective effect on the HuC/D- and nNOS-negative subpopulations at 12 months of age and nNOS-positive subpopulation at 23 months of age. The enteric neuroprotection achieved by reducing food intake or caloric restriction has been described in Sprague–Dawley rats (Cowen et al., 2000; Johnson et al., 1998; Thrasivoulou et al., 2006) and Wistar rats (Porto et al., 2012) during the aging process. Cowen et al. (2000) suggested that cholinergic motor neurons, which express the enzyme choline acetyltransferase, are the main beneficiaries of caloric restriction because neurons that express the enzyme NOS are protected from cell death with advancing age. These authors posited that this resistance indicates that neurons that utilize nitric oxide may have enhanced mechanisms of defense against the damage caused by free radicals. Our results partially agree with Cowen et al. (2000). The neuroprotection provided by food restriction occurred for cholinergic neurons (nNOS-negative) at 12 months of age and declined at 23 months, whereas the protection for nitrergic neurons (nNOS-positive) was only apparent at 23 months. Considering the time of neuroprotection exerted by food or caloric restriction, Johnson et al. (1998) and Cowen et al. (2000) found that a restricted diet imposed at 4–6 months prevented the expected loss of cholinergic neurons at 24 and 30 months of age, respectively, in the ileum in Sprague–Dawley rats. Porto et al. (2012) reported that caloric restriction reduced the loss of NADPH-negative neurons (cholinergic) in the duodenum at 18 months of age in Wistar rats. Positive effects of food restriction observed at 12 months of age suggest neuroprotection for the cholinergic subpopulation in middle-aged animals, which did not last until 23 months, in contrast to the response of the nitrergic subpopulation at 23 months in the proximal colon, which demonstrated significant neuroprotection. These results show that nutrition and perhaps other environmental factors (e.g., physical activity) influence the pattern of gastrointestinal

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innervation in aging and may increase longevity and the survival of myenteric neurons (Cowen et al., 2000; Gagliardo et al., 2008). However, such an influence may present variations over time, and the type of response may be different, depending on the intestinal segment and neuronal population and subpopulation studied, the severity of food restriction, and the species or strains of animals investigated. The observed neuroprotection in HuC/D-positive and nNOS-negative subpopulations at 12 months and the nNOS-positive subpopulation at 23 months indicates that neuroprotection that was promoted by the imposed diet decreased neuronal vulnerability to the effects of aging, but this protection was not complete. Thrasivoulou et al. (2006) found significant preservation promoted by food restriction in the PGP-positive neuronal population in the ileum in Sprague–Dawley rats between 12 and 15 months of age. At 24 months of age, however, the neuroprotection was lower, indicating a drop in the efficiency of this type of diet with aging. Thus, the 50% restriction of food intake appears to promote different peaks of protection between myenteric neuronal populations with advancing age. In addition to the death of myenteric neurons with age, we found a significant numerical reduction of the enteric glial population (S-100-immunoreactive), which plays a key role in maintaining the ENS (Bassotti et al., 2007; Neunlist et al., 2008; Ruhl, 2005). Similar results were obtained by Phillips et al. (2004) using the same glial marker in the small intestine and colon in Fischer rats at 26 months of age compared with 5- to 6-month-old animals. These authors considered the proportionality between glial loss and the death of neurons in the ganglia of the myenteric plexus, suggesting interdependence between these two cell types. The reduction of glial cells led to a reduction of neurotrophic factors produced during aging (Wade, 2002), thus contributing to myenteric neuron loss. Several functions have been attributed to glia, including the regulation of homeostasis and the maintenance of the structural and functional integrity of enteric neurons through the secretion of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GNDF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) (Cabarrocas et al., 2003; Ruhl, 2005; Von Boyen and Steinkamp, 2006). Glial cells are also involved in supporting the ENS, stabilizing structural and metabolic deficiencies of the intestinal wall, enteric neurotransmission, intestinal motility, and inflammatory bowel disease (Neunlist et al., 2008; Ruhl, 2005), and protecting enteric neurons against oxidative stress (Abdo et al., 2010, 2012). Despite observing positive effects of food restriction on the neuronal population as a result of aging, the same was not observed in the enteric glial population, with possible impairment of the production of neurotrophic factors (Cowen et al., 2000) and impairment of the mechanism of neuroprotection against oxidative stress promoted by enteric glia (Abdo et al., 2010, 2012). This may be responsible for the neuronal loss observed in the C23 and RA23 groups in the present study because the loss of glial cells precedes neuronal loss (Phillips and Powley, 2007). A hypothesis that could explain the increase in glial cell loss with food restriction is that the imposed nutritional status could have impaired the homeostasis of glial cells, thus affecting their survival and consequently the neurons with which they are related. Another hypothesis is that glia may be more vulnerable to free radicals or show a decrease in antioxidant defense, thus increasing the possibility of cell death. A final consideration is that diet may not exert any protective effect on enteric glia in the proximal colon in Wistar rats at 23 months of age. Studies that have investigated the behavior of colonic enteric glia before food restriction are not currently found in the scientific literature. The loss of glial cells is a factor that contributes to the death of myenteric neurons. Oxidative status is also involved in the survival and longevity of these two cell types in aging and with food restriction. The increase in oxidative stress in the control animals fed ad libitum indicates that this factor contributed to the loss of glia cells and the myenteric neuronal population with age at different times, reflected by greater or lesser vulnerability of these cells. The improvement in

oxidative status observed in food-restricted animals demonstrates that the nutritional design influenced neuronal death induced by oxidative stress because it increased the survival of cholinergic and nitrergic neurons with advancing age. Unlike enteric neurons, however, the diet negatively influenced the population of glial cells in old rats. Thrasivoulou et al. (2006) confirmed the selective loss of myenteric neurons in rats during aging and considered that the mechanism involved in neuronal death with advancing age is induced by high intracellular levels of free radicals, which are accentuated with ad libitum feeding. With regard to the morphometric analysis, we verified hypertrophy in the CP area of HuC/D-positive, nNOS-negative, and nNOS-positive neurons and glial cells at 23 months of age. These results support the hypothesis that neuronal hypertrophy caused by the stress of aging is associated with the numerical loss of these neurons that seek to maintain peristalsis, secretion, and intestinal absorption (Phillips et al., 2003). We infer that the increase in the CP of glial cells is also a compensatory mechanism that maintains their functions and is related to the loss of glial cells with age. Hypertrophy in myenteric cell bodies associated with age has been observed in the small and large intestines in rats (Cowen et al., 2000; Marese et al., 2007; Phillips et al., 2003), demonstrating the extraordinary neuronal plasticity in fully differentiated nervous tissue. With regard to the hypertrophy of enteric glia in the aging process, the present results are corroborated by a previous study (Cirilo et al., 2013) in the small intestine. Variations in the cell area of enteric neurons indicate the high plasticity of these cells prior to different experimental models and in response to changes in the luminal environment of the GIT (Giancamillo et al., 2010). Increased cell body areas are often observed in diabetic rats (Zanoni et al., 2003). Reductions or maintenance is observed in malnourished rats (Natali et al., 2003; Schoffen et al., 2005) and obese rats (Soares et al., 2006). Based on the increase in glial cell area and the smaller area of the cell body of myenteric neurons, we can infer that there was increased activity of the remaining glial cells and consequent hypertrophy of their cell body, whereas neurons showed atrophy of their CP, possibly attributable to a reduction of ability to synthesize structural proteins, a decrease in cellular activity, or an adaptive response to the lower nutrient input received by the nutritional condition imposed. Brain glial cells (astrocytes) that hypertrophy with greater proliferation of their extensions is associated with the increased synthesis of glial fibrillary acidic protein (GFAP) and vimentin after brain injury (Vasiljković et al., 2009). These central nervous system results support our hypothesis that the hypertrophy of enteric glia could also occur because of an increase in the intracellular synthesis of cytoskeletal proteins, raising its functional capacity (Cirilo et al., 2013). No significant changes were detected in the area of the ganglia according to age or food restriction, and all ganglia were preserved and compressed, which disagrees with the literature. Phillips et al. (2004) found that the size of the ganglia in the small and large intestines was smaller in Fischer rats at 26 months of age compared with 5–6 months of age. Similar results were also reported in the colon of mice (El-Salhy et al., 1999) and old guinea pigs (Peck et al., 2009). Hanani et al. (2004) described an increase in the proportion of ganglia that contain empty spaces, possibly attributable to neuronal loss. Maintenance of the size of ganglia, independent of age or nutritional condition, is mainly attributable to neuronal and glial hypertrophy associated with the numerical loss of these cell types in the ganglion in the case of aging and neuroprotection and glial hypertrophy in the case of diet restriction, indicating internal adjustments in ganglionic organization. This adjustment was highlighted by Gomes et al. (1997), who reported an increase in collagen and elastic fibers in the capsule of colonic myenteric ganglia with age. The present study found a gradual and significant increase in the thickness of the external muscle layer with advancing age, with no

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influence of food restriction at 12 months. This is frequently observed in the aging process when associated with colonic myenteric neuronal loss (Peck et al., 2009) and complete denervation (Buttow et al., 2003; Zucoloto et al., 1997), which would lead to a decrease in intestinal motility typical of aging (Madsen and Graff, 2004; Wiskur and Meerveld, 2010). Muscle hypertrophy under these conditions could be considered a compensatory response (Souza et al., 1993) caused by a decrease in muscle innervation in the GIT. The RA23 group exhibited augmentation of the muscle layer compared with the C23 group and other groups, indicating an effect of food restriction at this age. Two hypotheses may be raised. Initially, these results highlight the involvement of enteric glial cells. Our results indicated marked glial loss in the RA23 group compared with the control groups. Evidence suggests the involvement of these cells in neuromuscular control (Bassotti et al., 2007; Cabarrocas et al., 2003), in which the development of intestinal pathologies is associated with abnormalities in glial density and function. Another possibility is the numerical reduction of the cholinergic subpopulation and survival of the nitrergic neuronal phenotype observed only in 23-month-old animals under food restriction likely affects the colonic enteric neurotransmitter system. This would reduce cholinergic excitatory activity and potentiate nitrergic inhibitory activity in the intestine (Wiskur and Meerveld, 2010), which may have caused the muscle hypertrophy observed in the RA23 group, leading to possible delayed colonic transit. However, a recent study by Hoyle and Saffrey (2012) compared cholinergic neuromuscular transmission in the longitudinal muscle of the ileum in 24-month-old rats fed ad libitum and subjected to 50% caloric restriction and found that the cholinergic system might not be affected by age or diet. However, these authors emphasized that these results should not be extrapolated to the entire gastrointestinal tract where other regulatory components of the cholinergic and noncholinergic innervations are present. Further electrophysiological and pharmacological studies are needed to evaluate the functional capacity of neuromuscular transmission associated with morphoquantitative changes in the neuronal and enteric glial populations caused by aging and food restriction. 5. Conclusions The food restriction (i.e., a 50% reduction of food intake) initiated in adulthood minimized changes in nutritional and oxidative status in rats that result from the aging process and protected HuC/D-positive, nNOSnegative (cholinergic), and nNOS-positive (nitrergic) subpopulations of the proximal colon against neuronal loss. Nevertheless, the imposed diet increased the loss of glial cells and hypertrophied the muscle layer, possibly interfering with neuromuscular control in the colon. Conflict of interest The authors have no conflicts of interests. Acknowledgments The authors were financially supported by the Fundação AraucariaSecretariat of Science, Technology and Higher Education of Paraná State and the National Council for Scientific and Technological Development (CNPq). The authors appreciate the technical support from the laboratories of Animal Histotechnique, Hepatic Metabolism, and Clinical Biochemistry of the State University of Maringá, Brazil. References Abdo, H., Derkinderen, P., Gomes, P., Chevalier, J., Aubert, P., Masson, D., et al., 2010. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione. FASEB J. 24, 1082–1094.

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