Evaluation of immunohistochemical expression of ghrelin-producing rectal cells in Wistar rats underwent to the cafeteria diet 1

PURPOSE: To investigate the impact of cafeteria diet on ghrelin expression in rectal tissue and identify the morphologic cell type. METHODS: Twenty-four male Wistar rats were divided into four subgroups of six animals each: RC1 (rat chow 1) and CAF1 (cafeteria diet 1) for a period of 30 days; RC2 (rat chow 2) and CAF2 (cafeteria diet 2) for a period of 60 days. The animal and rectal weight, the number and the type of immunoreactive ghrelin cells were recorded and compared between the subgroups. The statistical study was established by ANOVA and Student’s t test. RESULTS: There was no difference in the total of immunoreactive cells (p=0.685) between the subgroups nor between weight and presence or absence of ghrelin expression (p=0.993). All the immunoreactive cells identified were closed-type. CONCLUSION: The cafeteria diet did not have influence on the amount of immunoreactive rectal cells of ghrelin and only one type (closed-type) of immunoreactive cells was expressed in the rectum.


Introduction
Nowadays obesity and overweight are an epidemic and a public health problem and its prevalence is increasing in developed and developing countries [1][2] .
In 2008, 1.5 billion adults were overweight, and one third of them were obese.Among children up to the 5th year of life, 43 million were overweight in 2010.The World Health Organization (WHO) estimates that, in 2015, there will be over 700 million people with body mass index (BMI) over 30 3 .
The main causes of obesity are defined by WHO as: increase intake of high energy density foods, salt and sugar, and few vitamins and minerals; associated with sedentary habits manly due to urbanization and technological advances 4 .
Obesity is identified as an important colorectal cancer risk factor in several studies published in the literature, that demonstrates a risk increase of up to twice in obese people in comparison with non-obese [5][6] .
Ghrelin, a hormone discovered and isolated by Kojima et al. 7 in the X/A like cells of the fundus of rat stomach 7 .It exhibits a potent orexigenic action, stimulating appetite and food intake by direct action on hypothalamic neurones 8 .In humans, it is peptide composed of 28 amino acids and contains a fatty acyl group on the third amino acid from the N-terminal which is responsible for its physiological functions, this structure is homologous to the rats except for exchange in two amino acids (arg 11 -val 12 ) 7 .The major producing source of serum ghrelin is the gastrointestinal tract (GIT), and the stomach is the mainly producer, followed by small intestine 9 .Through immunohistochemical methods and in situ hybridization, ghrelin producing cells were identified in the mucosal layer of the gastric body, duodenum, ileum, cecum and colon at different levels of expression 10 .Despite this Teive et al. 11 showed no compensatory increase in the number of duodenal ghrelin cells in the post-operative period of the sleeve gastrectomy in rats.
There are two distinct morphological cells types, "opened type" and "closed-type", with identical immunoreactive characteristics, but with different distribution along the gastrointestinal segments, decreasing the frequency of closed-type as it progresses toward the distal segments of the GIT 12 .
Ghrelin is considered to be a gastrointestinal peptide and presumably the opened-type cells are regulated by intraluminal information as nutrients and pH, and the closed-type, by neuronal and hormonal stimulation or mechanical distention.Also there is no literature consensus whether they are the same cells at distinct stages of development or two different cells types 13 .
Waseem et al. 14  Cefeteria diet is a diet model used in experimental studies with rats.It is that best reproduces and induces the metabolic syndrome, the weight gain, the multiple organ dysfunctions and the diseases caused by obesity.This is a type of diet in which the animals have ad libitum intake of human foods with high energy density and tasty as chocolates, bacon, stuffed biscuits, soft drinks, along with water and rat chow 15 .
Recently, the same diet was used for a period of 35 days (short-term) by Colombo et al. 16 in research on effect on insulin resistance and serum levels of adiponectin and ghrelin and showed a significant weight gain on the cafeteria group when compared with controls.The purpose of this work was to evaluate the impact of cafeteria diet on the amount of rectal ghrelin producing cells and analyzed the morphological cell type found in the rectum by imunohistochemical method.

Methods
The Federal University of Santa Catarina Committee of Ethics in the Use of Animals (CEUA) approved all the animal experimental procedures in May 11, 2011 (protocol # PP00591 -23080.009228/2011-89).

Dietary management
Following a one week period of adaptation the rats in the CAF subgroups were fed on a cafeteria-style diet consisting of 45% carbohydrates, 45% lipids, 10% proteins, while the rats in the RC subgroup were fed on standard rat chow consisting of 55% carbohydrates, 22% proteins and 5% lipids (Nuvilab Cr-1, Nuvital, PR, Brazil).All rats were given water ad libitum.
The CAF1 and RC1 subgroups were fed over a period of 30 days and the CAF2 and RC2 subgroups were fed over a period of 60 days.In order to determine the development of the animals, the weights values were recorded weekly and on the day of surgery (Figure 1).

Surgical procedures
After the end of feeding period the rats were anesthetized with an intramuscular injection of 100mg/kg of ketamine and 10mg/kg xylazine.
The animals were submitted to a midline laparotomy for resection of the rectum.The specimens were preserved in a 10% buffered formaldehyde solution.All the resected rectal segments were numbered and coded by Arabic numbers (1 to 24).The rats were euthanized by intramuscular injection of ketamina 300mg/kg and 30mg/kg xylasina.

Histophatological procedures
A midline longitudinal strip of rectal specimens, 2mm wide on anterior rectal wall was cut to be fi xed on a microscopic slide.The specimens were processed, and paraffi n blocks were prepared to make slides for both hematoxylin-eosin and immunohistochemical staining.

Immunohistochemistry
Tissues were stained immunohistochemically using the labeled streptavidin biotin method (LSAB).Briefl y, the specimens were deparaffi nized, rehydrated and incubated with 0.3% hydrogen peroxide in methanol for 20 minutes to block endogenous peroxidase activity.Antigen retrieval was performed using citrate buffer (pH 6.0) for 40 minutes at 95-98ºC, and the specimens were left to cool for 30 minutes at room temperature.
The specimens were incubated with primary rabbit anti-ghrelin (rat-mouse) antibody (Phoenix Pharmaceuticals, CA, USA) diluted 1:10.000for 12 hours at 8ºC in a humidifi ed The rat stomach, which is known to express ghrelin, was used as a positive control for the experiment, and the negative control was a specimen that received phosphate buffer solution (PBS) instead of primary antibody.The positive result was indicated by the appearance of brown color in the antibodybinding site.

Morphmetric analysis, cell counting, and statistical analysis
The microscopic slides contained the strips of the rectum were entirely examined by two pathologists blinded to the experimental conditions.They used a light microscope (Optiphot-2, Nikon, Japan) and counted all the immunopositive ghrelin cells.Only the nucleated cells localized in the mucosal layer with brown chromogen in the cytoplasm or in any part of the cell membrane were considered for counting.Three full counting were performed and considered only the cells which had concordance of both observes.The opened and closed-types cells were counted.
ANOVA test was used to assess statistical signifi cance, which was set at p<0.05.All the data were expressed as the means and SD and SPSS 17 statistics software (SPSS, Inc., IL, USA) was used for data processing.

Results
Ghrelin cells were observed in the rectal mucosal layer and not found in other layers.All the immunopositive cells foud were small and rounded shaped and classifi ed as closed-type cells

Evaluation of immunohistochemical expression of ghrelin-producing rectal cells in Wistar rats underwent to the cafeteria diet
Acta Cirúrgica Brasileira -Vol.28 (8) 2013 -617 (Figure 2).Weight gain was recorded weekly and the averages of final weight (operation day) in the subgroups were: CAF1 349.50g;RC1 339.83g;CAF2 411.33g and RC2 327.33g.There was statistical difference in comparison between CAF2 and the other groups and there was no difference in other comparisons (Table 1).There were similar values of immunopositive cell count for all subgroups and statistical analysis showed no significant difference between them (p=0.685)as shown in the Table 2.
Final mean weight compared to the presence or absence of ghrelin expression also had no statistically significant difference (p=0.993)(Table 3).Ghrelin has many physiologic functions including the carcinogenesis acting in some steps of cell growing and apoptosis [17][18] .We investigated the hypothesis that weight gain could alter the number of producing ghrelin cells in the segments of the gastrointestinal tract.
Wassen et al. 14 demonstrated an increase of the ghrelin expression, mainly of the growth hormone secretagogue receptors 1b (GHS-R 1b), in malignant colorectal cells and this condition promoted colorectal malignancy by advancing stage of tumor.
That increase was significant when compared CRC obese patients with non-obese.
Based on these this data and on the characteristics of human and rat ghrelin, we designed this study because we believed that obesity could modify the number of ghrelin cells specifically in the rectum leading to local changes that promote premalignant disorders.
Sahin et al. 19 studied several segments of the gastrointestinal rat tract and showed decrease on ghrelin tissue expression and ghrelin serum levels in rats submitted to diet-induced obesity, however these difference was not significant on rectal segment.These results do not explain the hypothesis that there are a local involvement of ghrelin expression and rectal carcinogenesis.
We analyzed the entire length of the rectum (proximal, middle and distal) and also found no differences between the subgroups that this fact leads us to conclude that obesity do not have influence on ghrelin cells in the rectum.
Although another study demonstrates presence of two distinct types of ghrelin cells in distal segments of GIT with predominance of opened-type 12 we have found only closed-type cells in the rectum.Further and more detailed studies are needed to identify and determine the ghrelin-producing cells in colorectal segments and its relation to obesity.

Conclusion
Cafeteria diet did not have impact on the amount of ghrelin-producing cells and only one type (closed-type) was found in the rectum.

FIGURE 1 -
FIGURE 1 -Schematic drawing showing the feeding period.
chamber and with the biotinylated secondary antibody (Dako, CA, USA) for 30 minutes at room temperature.The specimens were incubated with streptavidin conjugated to peroxidase (Dako, CA, USA) for 40 minutes at room temperature and were treated for one minute with 3-3diaminobenzidine (DAB), which acted as a chromogen.The specimens were counterstained with Harris hematoxylin for nine minutes, dehydrated in a graded ethanol series, clarifi ed with xylene and mounted under coverslips ERV-MOUN (EasyPath, SP, Brazil).

TABLE 1 -
Mean and standard deviation (SD) of body

TABLE 3 -
Mean and standard deviation (SD) of final weight (g) compared with expression or non-expression of ghrelin.
a Mean ± Standard deviation b Student's t test Discussion Obesity and overweight induced by diet and lifestyle are considered a global epidemic by the World Health Organization (WHO), especially in western countries.They are important risk factors for several conditions, including colorectal cancer.