Benefits of caloric restriction in the myenteric neuronal plasticity in aging rats

Aging is a biologic process characterized by progressive damage of structures and functions of organic systems. In gastrointestinal tract, it can involve enteric nervous system, which plays an important role in digestion and absorption of nutrients, causing hastening of intestinal transit thus reducing its absorptive function. Caloric restriction has been used in several studies with the intention of delaying deleterious effects of aging. This study aimed to evaluate the effects of caloric restriction on myenteric neurons of ileum by aging in rats. 30 Wistar rats were grouped as follows: GI (animals aged 6 months fed with normal diet), GII (animals aged 18 months fed with normal diet) and GIII (animals aged 18 months subject to 31% of caloric restriction). The rats of the GI group were euthanized at 6 months of age and after experimental period of 12 months animals of the group GII and GIII were euthanized, the ileum of all groups were collected, measured and processed by NADPH-dp and Acetylcholinesterase. Quantitative analysis of neurons revealed that aging promotes the increasing of myenteric neurons NADPH-dp and reduces Acetylcholinesterase neuronal population. However, in the cellular profile area, were not observed significant differences between the groups. The caloric restriction has been efficient and can be used preventively because it minimizes quantitative changes associated with aging on ileum myenteric plexuses.


INTRODUCTION
The increasing of elderly population is considered a global phenomenon (Kapteyn 2010).According to the United Nations the percentage of people aged sixty years or more will be double by the year 2050 (UN 2009).Many factors have contributed to the increasing of life expectancy, some of which are strictly related with medical advances, expansion http://dx.doi.org/10.1590/0001-37652014201300521471-1481 JOICE N.B. PEREIRA et al. of basic sanitation services, practices of physical activity and improvement of feeding habits (Almeida and Fernandes 2001).
Aging is characterized as a biological process with progressive functional and structural damage of many organic systems (Riggs et al. 2004).This process increases the probability of degenerative diseases and is hastened by an interaction between genetic factors (Kenyon 2005).All these factors together especially contribute to increase in the free radical production (Letiembre et al. 2007), thus causing functional and structural alterations in organs (Lukiw 2007).
Some authors reported that in the aging process many changes can occur in the digestive system, such as, decreasing of esophagus motility (Butt et al. 1993) and consequently decreasing of bacterial colonies (Madsen 1992), delayed gastric depletion (Brogna et al. 1999) and increasing of intestinal transit (Anuras and Leoning-baucke 1984).During the entire process of aging can be observed from changes lighter in the gastrointestinal tract, such as constipation, even more serious conditions such as intestinal obstruction (Shamburek and Farrar 1990).
The GI tract is innervated by intrinsic neurons of the enteric nervous system (ENS) (Furness andCosta 1987, Furness 2006) as well as by extrinsic neurons from sympathetic, parasympathetic and sensory nerves.The ENS together with its two main plexuses (the myenteric and submucosal), is responsible for the control of the motility, blood flow and secretion of the digestive tube, as it acts in the digestion and absorption of nutrients (Phillips and Powley 2007).The impairment of enteric neurons, caused by aging, usually implies in the occurrence of diseases and disorders in the GI tract (Hazzard et al. 1999).
The decrease in the total number of enteric neurons, particularly in the myenteric plexus, have been related.These authors also reported that aging influences the changing of the density of nerve fibers, and, in the structure of the enteric ganglia (Souza et al. 1993, Phillips and Powley 2001, Mari et al. 2008).Some studies have been performed with the objective of delaying the deleterious effects of aging in organic systems, thus increasing, among these studies, the caloric restriction (CR) has been investigated (Holloszy and Fontana 2007).
CR is defined as a moderate reduction (20-40%) of calorie intake when compared with an ad libitum diet without damage in the maintenance of essential nutrients (Piper and Bartke 2008).Studies with rodents have shown that many diseases and disorders associated with age decrease visibly by CR usage (Weindruch andWalford 1988, Masoro 2002) and that the CR reduces the degeneration of neurons in animal models suffering from Parkinson disease (Mattson 2000).
The mechanisms of action responsible for the benefic effects of CR include changes in gene expression, reduction of oxidative stress, decreased metabolism and increased capacity of DNA repair (Sohal andWeindruch 1996, Lee et al. 1999).
The studies on ENS have demonstrated that CR promotes reduction of enteric neuronal death during aging (Cowen et al. 2000).In the ENS, authors reported that CR minimized the deleterious effects on total population and nitrergic myenteric neurons in the duodenum of aged rats (Porto et al. 2012).
Owing to the increase in the elderly population and the incessant search for alternatives that reduce the deleterious effects of aging, this study aimed to evaluate the effects of CR in the plasticity of myenteric neurons NADPH-diaforase positive (NADPH-dp) and Acetilcolinesterase positive neurons (AchE+) in the ileum of rats under aging process.

MATERIALS AND METHODS
All the procedures of this study regarding the manipulation and use of animals were in accordance with the ethical principles adopted by the Brazilian School of Animal Experimentation (COBEA) and approved by the Faculty of Veterinary Medicine and Animal Science of the Sao Paulo University Bioethics committee under the Protocol number 1132/2007.

NEURONAL PLASTICITY WITH CALORIC RESTRICTION
Thirty male Wistar rats (Rattus norvegicus) were distributed into three groups: GI: six-month-old animals fed with a normal diet (Diet A) from weaning period (21 days) to six months-old.
GII:18-month-old animals fed with a normal diet (Diet A) from weaning to 18 months-old.
GIII:18-month-old animals fed with a diet with 31% caloric restriction (Diet B) starting as of six months of age.
In this study, two isoproteic diets were performed (diet A and B) (Table I).Diet A (Nuvital®) was considered standard for laboratory animals and was provided to the animals of GI and GII groups.Diet B was provided to the animals of the GIII group; in this group of animals the crude energy was reduced by 31% (2700 kcal/EB/kg) compared to the diet A (3900 kcal/EB/kg).
The animals were individually housed in polypropylene boxes, provided with food and drink under controlled environmental conditions for temperature (24±2°C) and lighting (12 hours light cycle / 12 hours dark cycle); water was given to the animals ad libitum.
To determine the amount of food to be provided daily during the experiment, all the groups of animals were kept in a seven-day adaptation process.During this process, the animals were provided with a normal diet and water ad libitum.The average of the daily consumption during the adaptation phase was used as the daily amount of chow that was administered throughout the duration of the first month of the experiment.Thus, the amount supplied to the animals of GIII was adjusted monthly based on the average of consumption of the animals from GII.This was necessary to ensure that the animals fed a low-calorie diet compensate with increased intake.
After the adaptation period, the animals of the GII were fed with a normal diet (A) with a chemical composition based on the dietary references for laboratory animals, established by the National Research Council (NRC 1995).The GIII animals were fed a diet with 31% less energy than that compared to normal diet.The animals of GII and GIII were subjected to the experiment of six to 18 months of age.
At the end of the experiment (12 months), the animals of the groups GII and GIII fasted for 12 hours and were than anesthetized intraperitoneally with Thiopental ® (40 mg/kg of body weight).The rats of the group GI at six months of age anesthetized were the same way.After, the laparotomy was performed, were collected and measured the retroperitoneal and periepididimal fat together, and the distal ileum of each animal was removed, along the ileocecal fold and ileocecal union.

TABLE I The percentages and chemical compositions
of the experimental diets with respect to their energy levels.
Five ileum from each group were used for acetylcholinesterase histochemistry and the remain ing five samples from each group were subjected to NADPH-diaphorase histochemistry.

DETECTION OF ACETILCOLINESTERASE POSITIVE MYENTERIC NEURONS (ACHE+) (KARNOVSKY AND ROOTS 1964)
The ileum were collected and washed firstly in saline solution and later in 4% paraformaldehyde in phosphate buffer; intestinal samples remained immersion-fixed in this fixative for one hour at 4°C, and later rinsed in Krebs solution containing hyaluronidase (2000 RTU) and tetraisopropil phyrophosphoramide (Isso-OMPA) for twelve hours.The incubation was performed for a period of 24 hours at 4°C in medium containing 0.17 M acetylcholine, 6.5 mL phosphate buffer (0.1 M, pH 7.1), 0.5 mL of sodium citrate (100 mM), 1 mL of copper sulphate (30 mM), 1 mL of distilled water and 1 mL of potassium ferricyanide (5 mM) adding tetraisopropil phyrophosphoramide and hyaluronidase.To detect the nitrergic myenteric neurons, the ileums obtained from five animals of each group were rinsed and filled with PBS (pH 7.4).The sections were sutured to retain the filling solution in situ.Each sample of ileum was soaked in two baths of PBS (10 minutes each) followed by permeabilization in PBS with 0.3% Triton X-100 in sodium phosphate buffer (pH 7.3) for 10 minutes; the samples were then immersed in two baths of PBS for10 minutes each.To observe the NADPHdp myenteric neurons after washing process, each ileum was incubated in reaction medium containing 50 mg NBT, 100 mg β-NADPH and 0.3% Triton X-100 in Tris-HCl (0.1 M, pH 7.6) buffer for two hours.The terminal portions of each ileum were released, and the duodenum was immersed in 4% paraformaldehyde for fixation and storage.
After fixation, each ileum was incised along the longitudinal axis of the mesenteric insertion and subjected to similar micro dissection procedures, for removal of the mucosa and submucosa to obtain wholemounts samples; each sample was then mounted on glass slides using Permount ® synthetic resin.

NEURONAL QUANTIFICATION (MM²)
To quantify the population of the myenteric neurons stained with Acetylcholinesterase (AchE+) and NADPH-diaphorase (NADPH-dp) were use the whole-mount preparation.The neuronal density/mm 2 of the ileum was determined by light microscope (Olympus BX50/Hamburg/Germany) accoupled to a high-resolution digital camera AxioCam HR (Zeiss ® / Jena/ Germany) and photographed in a 40X objective.
The quantitative analysis for each wholemount preparation was adapted from a test system containing 60 equally spaced fields by cut samples of all regions along the circumference of the ileum.A test system using inclusion and exclusion lines was also applied to the images taken.The neurons within the boundaries of the test area and on the top and right edges were counted, and the neurons on the bottom and left edges were excluded.

MORPHOMETRY OF THE NEURONAL CELL BODY AREA (ΜM²)
The measurement of the cell body (CB) area of the ileum myenteric neurons was performed using the computerised image analysis software Image-Pro Plus 3.0.1.(Media Cybernetics, Silver Spring, Maryland, USA).Images of the neurons taken for quantitative analysis were used for 1475 NEURONAL PLASTICITY WITH CALORIC RESTRICTION morphometric analysis.The CB area of 100 neurons was measured for each animal of each group.

STATISTICAL ANALYSIS
The statitical analsyis relative to the body weight and weight of the retroperitoneal and periepididimal fat of the animals was evaluated by T-student test.The morphoquantitative data of the NADPH-dp and AchE+ myenteric neurons, and, intestinal measurements were analyzed by Kruskal-Wallis test.The results were expressed as the average ± standard deviation with a signifi cance level of 5%.

RESULTS
During the experimental period (12 months), the animals were weighed daily and the data referring to the average of the initial and fi nal weight of each group are presented in Table II.From the third month of weighing, a signifi cant reduction (p<0.05) in the weight average (g) of the animals of GIII is clearly visible when compared to the animals of GII.
The weight of the retroperitoneal and periepididimal fat together was higher in animals fed the diet with 3900 Kcal/EB (12.3±1.2 g) compared to animals fed diet 2700 kcal/EB (9.1±1.5 g).However this difference was not signifi cant (p> 0.05).
It was observed in all groups studied (GI, GII e GIII), that the myenteric neurons AchE+ e NADPHdp (Figure 1) was predominantly grouped in ganglia interconnected by bundles of nerve fi bers.No changes caused by aging were observed in the morphological analysis of whole-mount preparations.

GI GII GIII
Initial weight (06 months) 397±1.2398±0.9 a 396±1.(86.4±18.8).Therefore, there was no need to apply correction factors to the results obtained from the quantification and analysis of neuronal morphology.

NEURONAL DENSITY
The data referring to the density of the neurons NADPH-dp and AchE+ per mm ² from the ileum of the three groups studied are presented in Table III.
When GII (18 months) was compared with GI (6 months), a significant increase (p<0.05) was observed in the density of myenteric neurons NADPH-dp during aging process.However, this increase (p<0.05) was minimized in animals subject to caloric restriction (GIII).
Aging promotes a significant reduction (p<0.05) in the density of AchE+ neurons ileum of the rats when compared to GI and GII.It was observed that the animals under caloric restriction diet (GIII) presented a significant increase (p<0.05) in the density of myenteric neurons (49 and 59% higher) when compared to GI and GII, respectively.

MORPHOMETRY OF THE CELLULAR PROFILE
Regarding the cellular profile area of myenteric neurons (µm 2 ), no statistical differences (p>0.05) were observed among the groups (GI, GII and GIII), in two hystochemistry techniques utilized (NADPHdiaforase and Acetylcholinesterase) (Table IV).In the three groups studied, the AchE+ neurons presented cellular profile area predominantly between 100 to 200 µm 2 , whereas the NADPH-dp neurons revealed a cellular profile area lower than100µm 2 .

DISCUSSION
The amount of food (diet A and B) supplied to the GI and GII are controlled to ensure that animals can not compensate for the GIII calorie restriction with an increase in food intake.Therefore, a significant reduction (p<0.05) of 8% in the body weight average (g) was observed in the animals of GIII when compared to the GII group; this results confirm the benefic effects of the CR in the control of body weight during aging.
Studies have demonstratedd reduction of 20% of body weight and fat in mice aged 12 months subject to 50% of food restriction for four weeks (Papa et al. 1997(Papa et al. , 2002)).Additionally, authors demonstrated that the amount of body fat in rats subject to CR from 12 to 28 months of age was clearly different from the rats fed ad libitum (Bailey et al. 1993).
The weight of the retroperitoneal and periepididymal fat, measured together, was higher (p >0.05) in group GII fed diet A than that compared with animals fed diet B. Similar results reported a reduction in body fat in several experiments with animals of other species.Pekas (1993) studying with feed restriction in pigs also observed a reduction in body fat of animals.In birds, Fontana et al. (1993) and Palo et al. (1995) reported a reduction in carcass fat when animals were subjected to food restriction.
The reduction in body fat is the result of CR imposed on animals, demonstrating that there is a mobilization of this energy reserve.Albeit less intense, this effect also occurred in relation to muscle mass of animals, perceived by the lower weight of the animals submitted to the RC, especially at the beginning of the experiment.
No morphological changes were observed in the myenteric plexuses of the three groups of animals studied; this result indicates that no interference occurred during aging and CR in the spatial organization of the myenteric plexus.The inexistence of morphological changes in the plexus of rats during aging process has also been reported in previous studies (Natali et al. 2000, Mari et al. 2008, Porto et al. 2012).
In the present study, the area of the ileum (mm 2 ) was verified to avoid erroneous interpretations of the morphometric and quantitative data.In cases of altered intestinal dimensions, a correction factor corresponding to the ratio between the area of the intestine in a young animal and that of an elderly animal was used to correct quantitative data (Phillips and Powley 2001).However, because the intestinal dimensions in the present study did not differ with age or CR (p>0.05), the use of a correction factor was not necessary.
Similar results on the intestinal area was observed in the duodenum of aged rats, where no change in the area of the duodenum was observed with aging and CR (Porto et al. 2012).
When GII (18 months) and GI (six months) were compared a considerable increase of 19% in the density of nitrergic neurons during aging was observed; this increase of the nitrergic population with aging was also observed in the duodenum (Porto et al. 2012), and colon of rats (Belai et al. 1995, Cowen et al. 2000).
The increase observed in the nitrergic population during aging suggests a high resistance to cellular death of this neuronal population when compared to other neuronal sub populations (Santer 1994).
The NO is responsible for relaxing the smooth intestinal muscles, contributing in a secondary way, towards reducing the intestinal transit and increasing the absorptive capacity of fluids by the intestinal epithelium (Sanders and Ward 1992, Mizuta et al. 1999).
During aging process, the increase of NO production promotes disorders in the intestinal motility, characterized by reduction in the frequency, amplitude and duration of the propulsive contraction of the gastrointestinal tract (Cortesini et al. 1995).
Studies regarding the protective or harmful effect of the NO on neurons have been widely debated (Calabrese et al. 2007).It is known that in many neurodegenerative diseases such as the amyotrophic lateral sclerosis and Parkinson's disease, high concentration of NO may lead to cellular death by apoptosis (Singh and Dikshit 2007).Cirilo et al. (2013), in studies with 50% dietary restriction and aging, analyzed myenteric population HuC/D, subpopulation nNOS and glial cells in rats 7-12 months old, and found that aging did not cause changes in any of the populations studied, however, with dietary restriction, a significant decrease of the population HuC/D and glial cells, without affecting the population nitrergic.In addition, with aging beyond death of neurons, is reported quantitative decrease in the population of enteric glia, which plays an important role in maintaining the enteric nervous system (Phillips et al. 2004).Thus, glial cell death would lead to a reduction of neurotrophic factors during aging (Dolatshad et al. 2001), contributing to neuronal loss myenteric.
However, in this study it was observed that in the NADPH-dp myenteric population the CR revealed minimizing the increase of the density that occurs in aging process.Such effect may be associated to a possible maintenance of the neurotrophic factors or to a high resistance of free radicals promoted by CR (Johnson et al. 1998, Wade andCowen 2004).
Regarding the AchE+ neurons, a reduction of 49% in the neuronal density during aging were observed.Supporting this evidence, some authors reported on a reduction in the neurons that reveal acetylcholine in the small intestine of animals under aging process (Cowen et al. 2000, Phillips et al. 2003, Porto et al. 2012).The cholinergic neurons are responsible for contraction of muscle fibers, so, its reduction leads to a reduction in the intestinal function (Xiong et al. 1995).
In our study we observed that the CR acted on neuronal vulnerability at the expense of the aging process, significantly increasing the density of AchE+ neurons.
Corroborating our results, in the duodenum of rats to CR group observed a significant increase in the population myenteric AchE+ (Porto et al. 2012).According to authors the death of neurons during aging in the nervous central system is attributed to the increasing of free radicals (Merry 2002).
During was reported on the increase of oxidative stress aging in the myenteric neurons population; additionally these authors also determine that the extensive absorptive surface of the intestine contributes to increase the level of exposure of the free radicals in the gastrointestinal tract (Thrasivoulou et al. 2006).
Changes in the cellular profile area of the myenteric neurons are reported under experimental conditions involving diabetes mellitus (Furlan et al. 2002), under nutrition (Castelucci et al. 2002) and dietary restriction (Cirilo et al. 2013).According to authors the increase of neurons size is usually confirmed during animal development (Gabella 1971).Thus, in considering the neuronal changes observed in the aging process, we can check the possible effects of CR on this parameter..
No changes in the cellular profile area with regards to aging and/or CR were observed in both two neuronal populations of this study.In studies of dietary restriction and aging, no differences in cell profile in aged rats in the total population, nitrergic subpopulation and glial cells, so this restriction was responsible for a decrease in the cellular profile in the total population and an increase in area were observed glial cells and no changes in the area of the nitrergic subpopulation.(Cirilo et al. 2013).With the death of myenteric neurons, the remaining nerve cells act in a compensatory manner to supply the same area of innervation with increase of cellular profile (Phillips et al. 2003).Thus, this increase of neuronal metabolic activity could lead to cellular death by apoptosis, characterized by a decrease of cellular profile.
The increase in the cellular profile area during aging process was described by many authors (Phillips et al. 2003, Peck et al. 2009).However, the increase in cellular profile measurements was not evident in the neurons NADPH-dp of the colon of the rats aged twelve months during aging NEURONAL PLASTICITY WITH CALORIC RESTRICTION (Gagliardo et al. 2008).Whereas a progressive increase exists during neuronal development to adulthood and, the plastic capacity of neurons throughout life, we suggest that changes in cell profile are less evident at older ages.
Through the neuronal labeling technique used in this study, we verified that the CR may be utilized as a preventive by minimizing the quantitative changes arising from aging on the ileum myenteric plexus.Thus, the maintenance of the enteric population by CR can prevent disorders of gastrointestinal tract attenuated by age, thus increasing the quality of life of elderly that according to world records is growing continually.
DETECTION OF POSITIVE NADPH-DIAPHORASE MYENTERIC NEURONS (NADPH-DP) (SCHERER-SINGLER ET AL. 1983) Average with different letters in the row differ (p<0.05) by T-Student test.

TABLE III Average of the neurons density NADPH-dp and AchE+ per mm² from the ileum of the rats of the GI (six months
), GII (diet A) and GIII (diet B) groups.a, b , cAverage with different letters in the row differ (p<0.05) by Kruskal-Wallis test.

TABLE IV Measurements average (µm 2 ) of the cellular profile area of the neurons NADPH-dp and AchE+ of animals from GI, GII, GIII groups.
a Average with similar letters in the same column are not different (p<0.05) in the Kruskal-Wallis test.NEURONAL PLASTICITY WITH CALORIC RESTRICTION