Exercise reduces inhibitory neuroactivity and protects myenteric neurons from age-related neurodegeneration

Autonomic Neuroscience: Basic and Clinical 141 (2008) 31 – 37 www.elsevier.com/locate/autneu

Exercise reduces inhibitory neuroactivity and protects myenteric neurons from age-related neurodegeneration Karina Martinez Gagliardo a,⁎, Naianne Kelly Clebis b , Sandra Regina Stabille c , Renata De Britto Mari d , Jacira Maria Andrade De Sousa e , Romeu Rodrigues De Souza f a

Department of Surgery of the Faculty of Veterinary Medicine and Zootechnics, University of São Paulo, Cidade Universitária, São Paulo, Brazil b Department of Morphology of the Centre of Biomedical Sciences, Federal University of Rio Grande do Norte, Natal, RN, Brazil c Department of Biology, Paranaense University, Paranavaí, PR, Brazil d Department of Surgery of the Faculty of Veterinary Medicine and Zootechnics, University of São Paulo, Cidade Universitária, São Paulo, Brazil e Department of Biochemistry of the Centre of Biomedical Sciences, Federal University of Rio Grande do Norte, Natal, RN, Brazil f Department of Surgery of the Faculty of Veterinary Medicine and Zootechnics, University of São Paulo, Cidade Universitária, São Paulo, Brazil Received 26 March 2007; received in revised form 13 April 2008; accepted 22 April 2008

Abstract The practice of regular exercise is indicated to prevent some motility disturbances in the gastrointestinal tract, such as constipation, during aging. The motility alterations are intimately linked with its innervations. The goal of this study is to determine whether a program of exercise (running on the treadmill), during 6 months, has effects in the myenteric neurons (NADH- and NADPH-diaphorase stained neurons) in the colon of rats during aging. Male Wister rats 6 months (adult) and 12 months (middle-aged) old were divided into 3 different groups: AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged submitted to physical activity). The aging did not cause a decline significant (p N 0.05) of the number of NADH-diaphorase stained neurons in sedentary rats (AS vs. MS group). In contrast, a decline of 31% was observed to NADPH-diaphorase stained neurons. Thus, animals that underwent physical activity (AS vs. MT group) rescued neurons from degeneration caused by aging (total number, density and profile of neurons did not change with age – NADH-diaphorase method). On the other hand, physical activity augmented the decline of NADPH-diaphorase positive neurons (total number, density and profile of neurons decreased). Collectively, the results show that exercise inhibits age-related decline of myenteric neurons however, exercise augments the decline of neurons with inhibitory activity (nitric oxide) in the colon of the rats. © 2008 Elsevier B.V. All rights reserved. Keywords: Aging; Physical activity; Myenteric plexus; Neurons; Colon; Rats

1. Introduction Aging is a natural stage of the vital process characterized by general decline in the physiology of several tissues and organs (Wiley, 2002; Rutten et al., 2003; Szweda et al., 2003). The aging process in the gastrointestinal (GI) tract is followed by a series of disorders such as a reduction in the esophageal motility (Hollins and Castell, 1974; Khan et al.,

⁎ Corresponding author. Tel./fax: +55 13 3495 1079. E-mail address: [email protected] (K. Martinez Gagliardo). 1566-0702/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.04.009

1977), delay in the gastric emptying (Moore et al., 1983; Clarkston et al., 1996; Smits and Lefebvre, 1996; Brogna et al., 1999), decrease in the colon motility (Madsen, 1992; Butt et al., 1993) and increase in the intestinal transit (Brocklehurst, 1985; Firth and Prather, 2002). The GI motility alterations observed with senescence are associated to abnormalities of the autonomic innervation and/or the smooth muscles (Camilleri et al., 2000; De Giorgio et al., 2000; Hanani et al., 2004). It is well known that the motility is mainly controlled by the myenteric neurons of the enteric nervous system (intrinsic innervation), mostly the nitrergic inhibitory neurons and the excitatory

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cholinergic (Mitolo-Chieppa et al., 1998; Lomax and Furness, 2000; Phillips et al., 2003). Disturbances in these neurons, such as reduction in its number, may be related to changes in the GI function. Besides the intrinsic innervation component, the GI tract is also modulated by the extrinsic innervation component, whose alterations may also cause a bigger susceptibility to neuronal loss (Phillips and Powley, 2001). Constipation or reduction of the intestinal motility, is a symptom usually reported in senile and sedentary patients (Donald et al., 1985; Dapoigny and Scabies, 1991; Wade and Cowen, 2004). It may result in complications such as intestinal blockage due to fecal impaction, idiopathic megacolon and fecal incontinence (Brocklehurst, 1985). The regular practice of a physical activity, associated with diet rich in fibers, is recommended to stimulate the motor activity of the colon and, consequently, to help with the defecation. However, the results of such therapy are controversial. According to some researchers, the physical activity does not present a consistent effect in the large intestine function (Bingham and Cummings, 1989; Robertson et al., 1993), while others have already reported the beneficial effect of this therapy (Young et al., 1932; Van Liere et al., 1954; Dapoigny and Scabies, 1991). Given the neuron plastic capacity, some possible beneficial effects may be observed with a program of physical activity, since there had already been reports of neurogenesis in some areas of the hippocampus in animals submitted to such activity (Arida et al., 2004, 1999; Brazel and Rao, 2004; Prickaerts et al., 2004). Therefore, the study aimed to clarifying the possible neuronal changes (quantitative and qualitative) observed in the colon of middle-aged rats (12 months) after establishing a physical activity program. Moreover, the research also intended to describe possible changes of the development in myenteric plexus, comparing adult sedentary rats (6 months) with middle-aged sedentary rats (12 months). The choice of rats as animal model is due to the fact that this rodent has similar symptoms to those seen in aging human beings. Therefore, they are considered excellent models to study such changes (Santer and Baker, 1993). The results obtained may help in therapies that will benefit human as well as veterinary medicine. 2. Material and methods Thirty Wistar male rats (Rattus norvergicus) from the Central Biotery of the Federal University of São Paulo (UNIFESP) were used for this study. The animals were kept in polypropylene boxes, at controlled room temperature (22– 24 °C) and photoperiod (12:12 h). All animals received Nuvital® chow and water ad libitum. The rats were divided into three different groups (n = 10/per group): “adult sedentary” (AS; 6 months of age), “middle-aged sedentary” (MS; 12 months of age) and “middle-aged submitted to physical activity” (MT, 12 months of age).

The procedures used in this experiment are in agreement with the ethical principles of animal experimentation of the Bioethical Commission of the Veterinary Medicine College of the University of São Paulo (Protocol no. 463/2004). 2.1. Experimental protocol of physical activity (MT group) The rats in the MT group started their physical activity program on an ergometric treadmill (Inbrasport®) from 6 months (180 days) to 12 months (360 days) of age. They ran on the treadmill 5×/week, for 60 min with a gradual progressive load until they reached 60% of maximum VO2. However, before setting their training protocol, the animals had to get used to performing physical activity on a treadmill for 2 weeks. Only animals classified on the Dishman et al. (1988) scale as three (average runners), four (above-average runners) and five (excellent runners) were used in the experiment. In order to determine the intensity of the physical exercise load, the animals were first submitted to the maximum effort test on an ergometric treadmill (test of exercise with gradual load), following the protocol of Azevedo et al. (2003). The initial treadmill speed was 0.3 km/h (or 5 m/min) and every 3 min the speed was increased in the same ratio (0.3 km/h) until exhaustion. The rats were considered exhausted when they were unable to run at the same speed of the treadmill. The maximum VO2 was determined as the maximum speed reached during the maximum effort test. Effort tests were carried out monthly to adjust the training intensity and to verify its efficiency. 2.2. Material processing The animals were sacrificed with a lethal dose of an anesthetic (Thiopental® — 40 mg/kg, intraperitoneal injection). However, the animals fasted for 12 h prior to the sacrifice, in order to facilitate cleaning of the segment. The colon was removed from the abdomen through a celiotomy along the alba line (cranial limit — ceco-colic junction, caudal-anal sphincter limit) and then measured with the aid of a millimeter ruler. One of the colon faces was copied onto white paper, to determine the colonic surface area. The drawings obtained were scanned along with a millimeter paper and the area was determined through the Pro-Plus 3.0.1® Image software. Fifteen colons (n = five/group) were used for the NADHdiaphorase histochemical technique (Gabella, 1969). Thus, the colons were first washed and filled with Krebs solution (pH 7.3), then submitted to the neuronal staining procedure, i.e., washed twice (10 min/each) in Krebs solution and immersed into Krebs with Triton X-100 (Sigma®, St. Louis, USA) 0.3% (5 min). Later on, they were washed twice (10 min/each) in Krebs, incubated in 25 ml of nitro blue tetrazolium (NBT) (0.5 mg/ml) (Sigma®, St. Louis, USA), 25 ml of sodium phosphate buffer (Sinth®, São Paulo, BR) (0.1 M; pH 7.3), 50 ml of distilled water and 0.05 g of β-

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NADH (Sigma®, St. Louis, USA). The incubation lasted an average of 45 min, and then, the colons were fixed in a 10% buffered formaldehyde solution. The other 15 colons (n = five/group) were used for the NADPH-diaphorase histochemical technique (Scherer-Singler et al., 1983). The colons were initially washed and filled with Krebs solution (pH 7.3). The colons were then washed twice in Krebs solution (10 min each) followed by permeabilization in Krebs with Triton X-100 (Sigma®, St. Louis, USA) 0.3% dissolved in phosphate buffered solution (PBS) (Sigma®, St. Louis, USA) (pH 7.3) during 10 min. Later on, they were washed twice in PBS during 10 min, and incubated in 50 mg of NBT (Sigma®, St. Louis, USA), 100 mg of β-NADPH (Sigma®, St. Louis, USA) and 0.3% of Triton X-100 (Sigma®, St. Louis, USA) in Tris–HCl buffer (Sigma®, St. Louis, USA) (0.1 M pH 7.6) for 2 h. They were then fixed in a 4% paraformaldehyde solution. After the neuronal staining (NADH and NADPHdiaphorase histochemical techniques), four different colon segments, each 15 mm in length, were removed from defined regions throughout the colon length. They were carefully opened at the mesocolic edge and microdissected with the aid of a stereomicroscope (Tecnival®) (4× lens) to remove the mucosa and the submucosa. The wholemounts of the circular and longitudinal smooth muscle, containing the myenteric plexus, were mounted between glass slide and cover slip with PBS buffered glycerin (Sinth®, São Paulo, BR). Processing problems of the NADH-diaphorase technique were responsible for the loss of the colon segments of one animal of the MS group.

regions (mesocolic and antimesocolic). However, an adjustment for each column within the membrane was necessary to keep the same distance between the analysis fields, since the colon (as well as the whole intestine) varies in its circumference (Jarvinen et al., 1999). Thus, 84 fields were analyzed throughout the colon.

2.3. Quantitative study

Important correction factors to carry out the quantitative and morphometric study were analyzed. The first factor has to do with the comparison of animals with different age ranges. It has been reported that older animals may have an intestine with a higher length and width than young ones, thus generating a “neuronal dilution”, i.e., a reduction in the number of neurons per area unit in older animals (Gabella, 1971, 1989; Johnson et al., 1998; Phillips and Powley, 2001; Phillips et al., 2003). This may lead to erroneous conclusions in the results interpretation when comparing the neuronal densities. However, this factor was not used in this work since no area difference was observed in the various age groups studied (6 months and 12 months). The second correction factor aims at preventing the natural differences in the diameter of intestine segments and also those caused by the material processing stages (stretch factor). Thus, the segment area was calculated before processing the material (fresh tissue) and then measured again after the processing, when the segment was already mounted in a glass slide — wholemount (processed tissue) (Gabella, 1971; Souza et al., 1993). Thus, instead of deriving a single factor for the entire organ and applying that one number to all of the counts, we determined correction factor for each sample of tissue. Consequently, it was possible to verify the field increase or

2.3.1. Neuronal density (neurons/mm2) The neuronal density was calculated through four histochemically stained wholemount preparations. The neuronal quantification was carried out with the aid of an Olympus BX60® microscope (40× lens) and of an Axio Cam HRc — Zeiss® digital camera, in which the image was captured and transferred to a computer screen, allowing counting of the neuronal profiles on the screen. The image of the analysis field observed in the computer screen was delimited by a test area, made up of inclusion lines (upper edge and right edge) and exclusion (lower edge and left edge) (Gundersen, 1977). Only the neuronal profiles within the test area and not touching the exclusion lines were counted, like the principles of the dissector method. The sampling fields (21 fields/ wholemounts) were adjusted to fit each wholemount. The counting was done using an unbiased counting frame. With a random start, the projected image was systematically sampled at random, using a predetermined fraction (small frames) of the unbiased sampling frame. Based on the membrane width (approximately 15 mm of length), the 21 fields were distributed in 3 columns with 7 fields each, showing all the

2.3.2. Estimate of the total number of neurons The estimate of the total number of neurons (NADH- and NADPH-diaforase positive) was obtained through the product between the numerical density (neurons/mm2) following correction by the stretch factor and colonic surface area. 2.4. Morphometric study 2.4.1. Neuronal profile area To calculate the neuronal profile area, the same fields used for the neuronal density quantification (21 fields/ wholemount) were analyzed, allowing a systematic and random analysis. The field images were captured and transferred to a computer screen and analyzed with the Image-Pro-Plus 3.0.1® software. The perimeter of each neuronal profile was outlined and, then, the area was automatically calculated by the software. The analysis field was delimited by a test area formed by inclusion land exclusion lines (Gundersen, 1977), as was done for the neuronal density. Only the neurons that did not touch the exclusion lines were outlined and had their area calculated. 2.5. Correction factors

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reductions, being able to perform a factor correction in the quantitative and morphometric results. 2.6. Statistical analysis The GraphPad Prism 4.1® software was used for statistical analysis. The analysis for the significance of the quantification and morphometric results was tested using the ANOVA procedure for analyses variance. If p b 0.05, we used the Tukey test.

Table 2 Results (means ± standard deviation) of the neuronal profile areas of NADHand NADPH-diaphorase stained neurons in AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged rats undergoing physical activity) groups Neuronal profile area (µm2)

Group AS Group MS Group MT

NADH

NADPH

129 ± 26.7 164 ± 24.5 137 ± 18.7

208.7 ± 33.7 161 ± 46 174 ± 78

No statistical significance was observed between the groups in both techniques.

3. Results 3.1. Quantitative study The results, expressed as means ± standard deviation, and its statistical significance concerning the quantitative study are summarized in Table 1. 3.1.1. Neuronal density The age-related changes in the density of NADHdiaphorase stained neurons between the groups (AS, MS and MT) were not statistically significant (p N 0.05). However, in the middle-aged sedentary rats (MS group) the neuronal density was 17% smaller than that from adult sedentary rat (AS group). When compared the AS group with a middle-aged rat submitted to physical activity (MT group), the neuronal density was very similar, being the MT group slight larger (1.4%) than the AS group. There were significant age-related changes (p b 0.05) in the density of NADPH-diaphorase labeled neurons between adult sedentary rats (AS group) and middle-aged sedentary (MS group) and middle-aged submitted to physical activity rats (MT group). The differences were more pronounced when compared AS group with MT group than MS group, being a decrease of 43.7% and 31% respectively. In contrast, no differences were observed between a middle-aged sedentary rat (MS group) and middle-aged rat submitted to physical activity (MT group). 3.1.2. Colonic surface area and total number of neurons No significant differences (p N 0.05) were observed in the colonic surface area among the groups (AS, MS and MT). The total number of NADH-diaphorase labeled neurons reduced with aging (10.4%) (AS vs. MS group), although it

was not significant (p N 0.05). In contrast, when compared the adult sedentary with a middle-age rat submitted to physical activity, a slight increase was observed (0.65%) but this difference was not statistically significant (p N 0.05). The total number of NADPH-diaphorase stained neurons decreased significantly (p b 0.05) when comparing the AS group with the others (MS and MT group). The largest reduction was verified between the AS and MT group, being the number of neurons decreased 45%. When compared the MS with MT group, no significant change (p N 0.05) was observed, although the total number of neurons reduced 14% in the MT group. 3.2. Morphometric study The results, expressed as means ± standard deviation, concerning the morphometric study are summarized in Table 2. The NADH-diaphorase neuronal profile area increased with aging, being higher in the MS group (27% larger than AS group) than MT (6% larger than AS group), although these differences were not significant (p N 0.05). On the other hand, the NADPH-diaphorase neuronal profile area decreased with aging, but not significant (p N 0.05), being more well-defined in the MS group than MT group. The neurons in the AS group were 23% larger than the MS group and 16.6% larger than the MT group. 4. Discussion The major finding in the present study can be summarized as follows: (1) the density and total number of neurons

Table 1 Results (means ± standard deviation) of neuronal density, colonic surface area and total number of neurons for NADH- NADPH-diaphorase techniques in AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged rats undergoing physical activity) groups and its statistical significance Colonic surface area (mm2)

Neuronal density (neurons/mm2)

Group AS Group MS Group MT

NADH

NADPH

276 ± 29.5 228.7 ± 51.5 280 ± 31.6

48 ± 5* 33 ± 4 27 ± 2.5

3861 ± 448 3872 ± 366 3771 ± 487

Asterisks (*) indicate a significant difference (p b 0.05) between the means in the same column.

Total number of neurons NADH

NADPH

993,349 ± 183,872 889,887 ± 236,221 999,808 ± 213,111

200,371 ± 38,986* 128,044 ± 17,791 109,954 ± 19,017

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(NADH labeled neurons) in the colon rats did not change between the AS group (6 months of age) and MS group (12 months of age); (2) in contrast, the density and total number of NADPH labeled neurons decreased; (3) exercise did not change the density and total number of neurons (NADH method) after 6 months as there were no statistical differences between MS and MT group. However, data obtained in this study suggest a trend that exercise may prevent neurodegeneration (density, total number and area profile of NADH neurons, which are metabolically active neurons), although it seems likely that exercise may augment the decline of the total number and density of NADPH neurons. The loss of enteric neurons has been widely reported during the aging (Finch, 2003), being it related to a natural elimination of neurons (Gabella, 1989) or to a neuronal deterioration due to aging (Phillips and Powley, 2001). The literature reports that the colon has a significant neuronal loss due to aging (Santer and Baker, 1988; El-Salhy et al., 1999; Phillips and Powley, 2001; Phillips et al.; 2004) and it can reach up to 64% of the neuronal population (Santer and Baker, 1988). However, the results of the present study did not confirm the existence of age-related decrements in the number of neurons as described in the previous studies (AS vs. MS group). Nevertheless, in accordance with Hall (2002), the effects of aging are more evident in older rats (28-month-old rats) than middle-age rats (12 month-old rats), being it a cause of no significant reduction in the colonic neurons in this study. Despite no evident reduction in the number of the colonic neurons, the present study suggests the beginning of aging process due to the increase in the neuronal body, although not significant. It can reflect the neuronal compensatory hypertrophy in response to the neuronal loss (Phillips et al., 2003). Recent studies affirm that the loss of the enteric neurons with aging is a selective process confined to specific phenotypes (Wade et al., 2002). For instance, the population of cholinergic neurons (excitatory neurons — acetylcholine neurotransmitter) is more vulnerable to aging than nitrergic neurons (inhibitory neurons — nitric oxide neurotransmitter) (Johnson et al., 1998; Phillips et al., 2003; Wade and Cowen, 2004). The loss of excitatory neurons could eventually result in a decline in function of GI tract and not simply a normal part of developmental process suggested by Gabella (1989). However, the data of this study showed also a marked loss of nitrergic neurons (NADPH labeled neurons) with aging (AS vs. MS group). Similar results were observed by Takahashi et al. (2000) in the mid-colon when they compared young rats (4–8 months) to senile rats (22–28 months). Although this research observed a decrease of nitrergic neurons with aging, the data regarding the expression of NO (nitric oxide) in the myenteric plexus of the colon are contradictory. Phillips et al. (2003) verified no reduction in the neuronal density in the subpopulation releasing NO in the large intestine (colon and rectum) when comparing young (3 months) and old rats (24 months). However, Belai et al.

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(1995) observed an increase in the number of neurons expressing NO per ganglion during the aging process. They found that only 76% of the neurons express NO at 6 months of age but this increases to 100% at 26 months of age in the proximal colon of rats. The larger nitrergic neuron sizes for adult rats (AS group) in relation to middle-age rats (MS group) associated to a larger neuronal density in the AS group reflects a greater grouping of neurons in an adult animal than in middle-aged animals. Furthermore, the neurons in the MS group are scarcer and they are probably undergoing the degeneration process (atrophy). According to the literature, the practice of regular physical activity influences neurogenesis in certain regions of the central nervous system (hippocampus) (Arida et al., 2004, 1999; Brazel and Rao, 2004; Prickaerts et al., 2004). However, in the enteric nervous system an aerobic training during 6 months did not show beneficial effects in the number of NADH labeled colonic neurons (AS vs. MT group). In addition, the size of neurons was similar between the groups. Nevertheless, when compared the data of the MS group with the MT group, is seen that the beginning of the age-related signs in the MS group (reduction in the neuronal density and increase of neuronal body size), although they are not significant. Thus, the physical activity might hold up the aging process (neurodegeneration). In contrast, the nitrergic neurons density decreased significantly with the introduction of physical activity to middle-age rats, being more pronounced in the animals submitted to physical activity than sedentary ones. However, when verifying the size of the neuron, a smaller reduction in the animals submitted to physical activity was observed than in sedentary ones. Although the population of metabolically active neurons in rats submitted to physical activity (MT group — NADH) was kept with aging, the population of nitrergic neurons was significantly reduced in these animals, being more pronounced than in sedentary rats (MS group). Therefore, it is impossible to affirm that there is a direct effect of the physical activity in the colonic motility, since other populations of inhibitory neurons could supply the reduction of the inhibitory neurotransmitter (nitric oxide). Nevertheless, the physical activity benefits in the enteric neurons may be closely related to the maintenance of metabolically active neurons of the colon, since the density of the NADHdiaphorase stained neurons (which includes other inhibitory neurons besides the nitrergic) did not decrease significantly when compared to the AS group. The estimate of the total number of stained NADH and NADPH neurons helped to consolidate the data regarding the neuronal density. It also enabled observing that approximately 20% of the metabolically active population of neurons in adult rats is constituted by nitrergic neurons. In the middle-aged sedentary rat group, this number is 14% and 11% in middle-aged rats submitted to physical activity. The data of this study differ from those obtained by Belai et al.

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(1995), who found that 76% and 100% of neurons in the proximal colon of rats aged 6 and 26 months release NO, respectively. 4.1. Methodological approach Firstly, it is important to emphasize that there are more sensitive neuronal markers than NADH-diaphorase histochemistry. However, this technique is a recognized staining method (Gabella, 1987) and the discussion about the “ideal” neuronal marker is beyond the scope of this study. The main objective of the current study is to perform relative quantifications between animal groups, this method being satisfactorily applied here. The second technique (NADPHdiaphorase histochemistry) used is responsible to identify a neuronal subgroup which expresses NO, being identical to the NOS-immunoreactivity (Belai et al., 1992), showing an efficient staining method used in this study. Moreover, the method used allowed the systematic and random collection of the colon segments, permitting that all colon lengths were sampled. Another point was the use of the correct factor to stretch, permitting a real counting of the neuronal density and neuronal profile area. In correcting the results, it was possible to estimate the total number of NADH- and NADPH-diaphorase neurons through the colonic surface area, avoiding inaccuracies caused by distensiveness, as related by Karaosmanoglu et al. (1996). References Arida, R.M., Scorza, F.A., Santos, N.F., Peres, C.A., Cavalheiro, E.A., 1999. Effect of physical exercise on seizure occurrence in a model of temporal lobe epilepsy in rats. Epilepsy Res. 37, 45–52. Arida, R.M., Scorza, F.A., Silva, A.V., Scorza, F.A., Cavalheiro, E.A., 2004. Differential effects of the spontaneous versus forced exercise in rats on the staining of parvalbumin-positive neurons in the hippocampal formation. Neuroscience Lett. 364, 135–138. Azevedo, L.F., Brum, P.C., Mattos, K.C., Junqueira, C.M., Rondon, M.U.P. B., Barreto, A.C.P., Negrão, C.E., 2003. Effects of losartan combined with exercise training in spontaneously hypertensive rats. Braz. J. Med. Biol. Res. 36, 1595–1603. Belai, A., Schmidt, H.H., Hoyle, C.H., Hassal, C.J., Saffrey, M.J., Moss, J., Forstermann, U., Murad, F., Burnstock, G., 1992. Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut. Neurosci. Lett. 31, 60–64. Belai, A., Cooper, S., Burnstock, G., 1995. Effect of age on NADPHdiaforase-containing myenteric neurones of rat ileum and proximal colon. Cell Tissue Res. 279, 379–383. Bingham, S.A., Cummings, J.H., 1989. Effect of exercise and physical fitness on large intestinal function. Gastroenterology 97, 1389–1399. Brazel, C.Y., Rao, M.S., 2004. Aging and neuronal replacement. Ageing Res. Rev. 3, 465–483. Brocklehurst, J.C., 1985. Colonic disease in the elderly. In: James, O.F.W. (Ed.), Clinics in Gastroenterology. W.B. Saunders Company, London, pp. 725–747. Brogna, A., Ferrara, R., Bucceri, A.M., Lanteri, E., Catalano, F., 1999. Influence of aging on gastrointestinal transit time. Invest. Radiol. 34, 357–359. Butt, W.G., Wang, I.S., Kaufman, S.T., Ryan, J.P., Cohen, S., 1993. Agerelated changes in rat colon mechanics. Gastrointest. Mot. 5, 123–128.

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