Aging has been suggested to be associated with disordered gastrointestinal transit that might be implicated in some aging-associated health problems such as anorexia of aging 1. The rate of gastric emptying is a rate-limiting step in the absorption of orally administered drugs 2 and is also a major determinant of the glycemic and cardiovascular responses to oral carbohydrate 3,4. Delayed gastric emptying and disturbed gastric motility are strongly involved in impaired tolerance to gastric feeding in critically ill patients in ICUs 5,6. This leads to an increased risk of pulmonary aspiration with the need for postpyloric feeding or parenteral nutrition 7, which would adversely affect patient morbidity and mortality 8.
Nitric oxide (NO) is an inhibitory neurotransmitter of peripheral nonadrenergic noncholinergic nerves in the gastrointestinal tract that is involved in the reflex accommodation of the gastric fundus to food or fluid 9. NO is also involved in the relaxation of the pylorus and upper duodenum, thereby facilitating gastric emptying 10. Reports on NO changes with aging have been conflicting; Takakhashi et al. 11 reported a decrease in the expression of neuronal nitric oxide synthase (nNOS) in myentric plexus with aging, whereas Domek Łopacinska and Strosznajder 12 reported enhanced expression of nNOS, lowering of endothelial nitric oxide synthase (eNOS) with no alteration in inducible nitric oxide synthase activity (iNOS).
The present study aimed to examine the changes in gastric emptying and motility in aged rats as well as the effect of inhibition or stimulation of NO synthesis on these parameters.
Materials and methods
The present study was carried out on 42 male Sprague–Dawley rats (10 adults, 160–210 g body weight, and 32 aged rats, 300–475 g body weight). The animals were obtained from The Animal House Colony of Faculty of Pharmacy, Ain Shams University. The rats were housed in the animal house of the Physiology Department under standard conditions of boarding and feeding for at least 1 week before starting the experiment. After the acclimation period, rats were housed individually in plastic cages at room temperature (22°C). Rats were fed ad libitum the standard rat chow diet (AIN-93 M diet formulated for adult rodents) prepared according to the National Academy of Science, National Research Council 13 and Reeves et al. 14. Standard rat chow was provided to the rats at 2 p.m. daily. The study was approved by the Ethical Committee of Ain Shams Faculty of Medicine (Cairo, Egypt).
Rats were randomly allocated into the following four groups:
- Adult rats: 10 rats (9–12 months old), used as the adult control group.
- Aged rats: 10 rats (18–30 months). These rats were given normal saline (0.9%) daily by intraperitoneal injections at a dose of 1 ml/kg body weight for 2 weeks, and were used as the aged control group.
- Aged N-ω-nitro-L-arginine methyl ester (L-NAME)-treated rats: 11 rats (18–30 months). These rats were treated with the NOS inhibitor, L-NAME, for 2 weeks. L-NAME was purchased from Sigma (USA). It was freshly dissolved in normal saline (0.9%) before injection (100 mg/1 ml) and injected intraperitoneally at a dose of 10 mg/kg body weight according to Corak and colleagues 15,16.
- Aged L-arginine-treated rats: 11 rats (18–30 months). These rats were treated with the NO precursor, L-arginine, for 2 weeks. L-arginine was purchased from Sigma. It was freshly dissolved in normal saline (0.9%) before injection (300 mg/1 ml), and then injected intraperitoneally at a dose of 300 mg/kg body weight according to Corak and colleagues 15,16.
Throughout the study period, rats were examined for the following:
- body weight, weekly;
- food intake. Daily food intake was calculated as the difference between the amount of food presented to the rat and the amount of food remaining and/or spilt in the cage.
At the end of the study, the rats were assessed for the following parameters.
Study of gastric emptying
Rats were fasted overnight with free access to water and, on the day of experiment, rats were given 25 glass beads 1 mm in diameter from Sigma suspended in 1 ml water by a gavage. After 1 h, the rats were weighed and then anesthetized with sodium thiopental (40 mg/kg body weight). The stomach was exposed by laparotomy and rapidly ligated at both the pylorus and cardia. The stomach was removed and the number of glass beads remaining in the stomach was counted 17. Gastric emptying was calculated using to the following formula:
Recording of gastric motility
The stomach was cut perpendicular to its longer axis parallel to the circular muscle fibers and strips of about 2.0×15.0 mm of gastric antrum were prepared. The muscle strip was placed in a chamber (35 ml) containing a freshly prepared Tyrod’s solution and bubbled with carbogen gas (95% O2+5% CO2) and placed in a water bath (37.0±0.5°C). One end of the strip was fixed to the floor of the chamber through a glass claw; the other end was attached to a D1-isometric force transducer that was connected through a strain gauge coupler FC 117 to a two-channel oscillograph (Washington MD2-Bioscience) to record isometric contractions at attenuation 0.1. The muscle strip was allowed to incubate for at least 40 min before the start of the experiments, with rinsing every 15 min 18,19.
Assessment of gastric motility
Recording of motility was started 1 h after incubation of the isolated gastric strip and continued for 30 min afterwards. The isolated gastric strip showed periods of quiescence and periods of contractile activity during the recording time. The best recording was chosen to calculate the following parameters:
- The average duration of contraction is the average duration of a single contraction in seconds. It was calculated by dividing the sum of contraction durations within the recording time by the number of these contractions 20. The duration of each contraction in seconds was calculated by measuring the distance of contraction in millimeters and then dividing it by 50 as the speed of recording was 50 mm/s.
- Average tension is the average tension of contractions in mg. It was determined by dividing the sum of the tension of all contractions in milligram within the recording time by the number of these contractions 20. The average tension was then divided by the specimen weight in milligram to obtain mg tension/mg tissue. The tension of the recorded contractions was calculated by measuring the height of each contraction in millimeters and the equivalent tension in milligram was obtained from a calibration curve. In this curve, 1 mm height was equivalent to 1 mg tension.
- Frequency of contractions was estimated as the number of contractions per second.
- Motility index/s was calculated by multiplying wave frequency/s by the average tension (mg/mg tissue), modified from Jia et al. 21.
All statistical data and significance tests were performed using the statistical program for social science (SPSS) statistical package (version 15.0; SPSS Inc., Chicago, Illinois, USA). Statistical significance was determined by one-way analysis of variance for differences between the means of different groups; further analysis was carried out using the least significance difference multiple range test to determine intergroup differences. A paired t-test was performed to detect significance from the baseline value. A P value of less than 0.05 was considered statistically significant 22. All results were expressed as mean±SEM.
Changes in body weight
The final body weight of adult rats showed a significant increase compared with their initial body weight (P<0.001). However, aged control, L-NAME-treated, and L-arginine-treated aged rat groups showed a significant decrease in their final body weight values compared with their initial values (P<0.05, P<0.001, and P<0.05), respectively (Table 1).
Compared with the adult control rats, the initial and final body weights of the aged control rat group were significantly higher (P<0.001). However, when the initial and final body weights of both the L-NAME-treated and the L-arginine-treated aged rats were compared with their aged control counterparts, no significant difference was observed (Table 1).
Changes in food intake
Aged control rats showed a significant increase in their average daily food intake throughout the study period when compared with the adult control rats (P<0.001); however, when their average food intake was related to their final body weights, no significant difference was observed (Table 1).
The L-NAME-treated aged rat group showed a significant decrease in their average daily food intake when compared with the aged control rats (P<0.001). However, the L-arginine-treated aged rat group showed an insignificant change in their average daily food intake compared with their aged control counterparts (Table 1).
Changes in gastric emptying (%)
Gastric emptying was not significantly different in the aged control rat group compared with the adult control rat group as well as in both the L-NAME-treated and the L-arginine-treated aged rat groups compared with their aged control counterparts (Table 2).
Changes in the parameters of gastric motility
The aged control group showed a significantly higher frequency of gastric antrum contraction waves compared with the adult control group (P<0.05) (Table 2 and Fig. 1). However, no significant difference was observed in the gastric wave frequency between both the L-NAME-treated and the L-arginine-treated aged rat groups and their aged control counterparts (Table 2 and Fig. 1).
However, wave duration, developed tension (mg/mg tissue) as well as motility index/s were not significantly different in the aged control group from the adult control group as well as in both the L-NAME-treated and the L-arginine-treated aged rat groups from their aged control counterparts (Table 2 and Fig. 1).
Studies on aged animals represent a useful model of healthy aging without the aging-associated diseases frequently encountered in humans such as atherosclerosis, diabetes, etc.
The results of the present study showed that the rate of gastric emptying was not significantly different in the aged control group compared with the adult control group as well as in the L-NAME-treated and the L-arginine-treated aged rat groups compared with the aged control group. These findings were in agreement with those of McDougal et al. 23 on aged rats and the findings of Gainsborough et al. 24 on human volunteers older than 60 years of age, but not in agreement with the findings of Smits et al. 25 on aged rats and those of Di Francesco et al. 26, who reported longer gastric emptying time in elderly human volunteers (∼77 years old) compared with adults (∼32 years old) following the intake of a mixed meal.
The present study showed that the treatment of aged rats with L-NAME or L-arginine had no significant effect on gastric emptying, which is in disagreement with the findings of Orihata and Sarna 10, who reported delayed gastric emptying with L-NAME as well as with an infusion of L-arginine in dogs in vivo and that gastric emptying was more delayed with L-NAME than with the infusion of L-arginine. The author attributed this finding to the fact that the infusion of L-arginine decreased gastric motility and consequently gastric emptying, whereas the infusion of L-NAME increased pyloric and duodenal contractions, leading to more prolonged periods of closure.
The rate of gastric slow wave has been reported to be 3/min in humans 27 and 4–5/min 28 in rats. In the present study, the in-vitro frequency of gastric contractions recorded was much higher (11.4/s in adult control rats and ∼14.5/s in aged control rats), which is in disagreement with the results of Ishiguchi et al. 29, Jia et al. 21, and Fukuta et al. 30, who reported a lower frequency of gastric contraction wave. In the present study, the activity of the gastric antrum strip was observed to be periodic (i.e. few seconds/min) and not continuous throughout 1 min. Also, the muscle strip was excised close to the pyloric sphincter, a region known to have continuous tonic contraction 27. However, the precise mechanism underlying this aging-associated tachygastria should be studied further.
The increased gastric wave frequency observed in the present study was not associated with any significant change in the motility index in the aged control group compared with the adult control group. Reports in the previous literature on gastric motility changes with increased gastric wave frequency have been conflicting. Some authors have reported that tachygastria interfered with gastric peristalsis and emptying 31, whereas others have reported that it enhanced gastric emptying 32.
The observation that the contraction wave frequency was not significantly different in the L-NAME-treated and the L-arginine-treated aged rat groups when compared with the aged control group might suggests that NO was not involved in the mechanisms underlying this aging-associated tachygastria. This finding is in contrast to that of Orihata and Sarna 10, who reported a significant increase in the wave frequency with an infusion of L-NAME and a significant decrease in the wave frequency with an infusion of L-arginine in adult dogs in vivo. The present study showed that the developed tension and contraction wave durations of the gastric antrum muscle strip were not significantly different in the aged control group compared with the adult control group as well as in the L-NAME-treated and the L-arginine-treated aged rat groups compared with the aged control group. These findings suggested that aging as well as variations in the gastric NO content did not adversely affect either Ca++ handling or the myosin ATPase activity of gastric antrum smooth muscle. This was in agreement with the findings of Orihata and Sarna 10, who reported an insignificant effect of an infusion of L-NAME or L-arginine on gastric antrum wave duration, but in contrast with the findings of Janssen et al. 33, who reported that L-NAME significantly increased the intragastric pressure wave amplitude in rats in vivo.
The gastric motility index of the L-NAME-treated and L-arginine-treated aged rat groups was found to be insignificantly different when compared with the aged control group. This finding was in agreement with the findings of Janssen et al. 33 in conscious rats as well as the results of Su et al. 18 and Madsen et al. 34,35 in healthy human volunteers. Nevertheless, it was not in agreement with the findings of Orihata and Sarna 10, who reported increased pyloric and duodenal contractions with L-NAME and decreased gastric contractions with an infusion of L-arginine, and with the findings of Konturek et al. 36, who reported that exogenous NO inhibited antral motor activity, as well as the findings of Glasgow et al. 37, who reported that L-NAME altered spontaneous antrum relaxation in fasted rats. This discrepancy in the results of the parameters of gastric motility between the present study and the previous ones might be because of interspecies differences, age group differences, and variations in the experimental setup, whether in vivo or in vitro, as well as the part of the stomach used for monitoring of gastric motility.
It is known that L-arginine increases NO synthesis only when NOS activity is upregulated 10. Thus, the lack of change in gastric emptying and motility with L-arginine in the present study might indicate unchanged NOS activity in the gastric muscle with aging or the noninvolvement of NO in the regulation of these parameters.
Food intake in aged control rats was significantly higher than that of adult control rats, which was in agreement with the findings of Kaneda et al. 38, who reported increased food intake in rats with advancing age, but was in contrast to the findings of Pu et al. 39, who reported decreased food intake in rats with advancing age. In humans, reports in the literature have reported decreased food intake with aging because of decreased nutritional needs 1,40 and low income 41.
In the present study, although food intake in the aged control group was significantly higher than that of the adult control group, when the average food intake was related to body weight, food intake was not significantly different between the aged control and adult control groups and was found to be significantly decreased only in the L-NAME-treated group. This suggests that the cause of increased food intake in the aged control group was the increased nutritional needs of their higher body masses rather than an actual increase in their appetite.
Monitoring of body weight throughout the study indicated significant weight gain in the adult control group and significant weight loss in all the aged control groups. The short duration of the study (only 2 weeks) might not be conclusive in terms of the changes in body weight with aging and a longer duration of monitoring of body weight is needed. Nevertheless, the aged control group showed significantly higher initial and final body weights compared with their adult controls. Weight loss was highly significant in the L-NAME-treated aged group compared with the aged control group so that their final body weights were significantly decreased compared with their aged control counterparts.
Reports on body weight changes with aging have shown that body mass and body fat increase in middle age (from 40 years onward) and then decrease at the age of 70 years and older 42–44. In rodents, some strains such as Wistar rats show a modest increase in body weight after reaching maturity, whereas other strains such as Sprague–Dawley rats show a constant increase in their body mass throughout their life span 41. This might explain the significantly higher initial and final body weights in the aged control rats in the present study compared with the adult control rats. The observation that food intake in the aged control rat group remained almost unchanged throughout the study period indicates that the cause of weight loss was possibly enhanced energy expenditure because of the stress of social isolation. The reaction of organisms to stress is mediated by the hypothalamic–pituitary–adrenal axis, resulting in elevated circulating levels of adrenocorticotropic hormone and glucocorticoid hormone 45. Most of the stressful stimuli that increase adrenocorticotropic hormone secretion also activate the sympathetic nervous system 46, which is known to increase energy expenditure 27.
The L-NAME-treated aged rat group showed highly significant weight loss at the end of the study compared with their initial body weights as well as a significant decrease in food intake compared with the aged control group. This finding suggests an orexigenic effect of NO on appetite and explains the significant weight loss in the L-NAME-treated aged rat group. L-NAME-associated anorexia has been reported by Li et al. 47, who showed that the intracerebroventricular administration of compounds that altered nitrergic tone decreased food intake and weight gain in normophagic rats by decreasing nNOS. L-NAME has been reported to cross the blood–brain barrier 48, which would explain the anorexia and weight loss in rats in the present study that were administered an intraperitoneal injection of L-NAME.
The present study showed that healthy aging was not associated with significant changes in gastric emptying or motility. The average food intake was comparable with the adult control values when related to body weight. It seems that NO exerted no significant regulatory effect on gastric emptying or gastric antral motility in aged rats. However, inhibition of NO synthesis reduced food intake and enhanced weight loss. The results of the present study show that delayed gastric emptying or disturbed gastric motility should not be interpreted as physiological changes of aging and, if encountered, a pathological cause should be seriously considered.
Conflicts of interest
There are no conflicts of interest.
1. Morley JE. Decreased food intake with aging. J Gerontol A Biol Sci Med Sci. 2001;56(Spec. Iss. 2):81–88
2. Gidal BE. Drug absorption in the elderly: biopharmaceutical considerations for the antiepileptic drugs. Epilepsy Res. 2006;68(Suppl 1):S65–S69
3. Ishii M, Nakamura T, Kasai F, Baba T, Takebe K. Erythromycin derivative improves gastric emptying and insulin requirement in diabetic patients with gastroparesis. Diabetes Care. 1997;20:1134–1137
4. Jones KL, Tonkin A, Horowitz M, Wishart JM, Carney BI, Guha S, et al. Rate of gastric emptying is a determinant of postprandial hypotension in non-insulin-dependent diabetes mellitus. Clin Sci. 1998;94:65–70
5. Heyland DK, Dhaliwal R, Drover JW, Gramlich L, Dodek P. Canadian clinical practice guidelines for nutrition support in mechanically ventilated, critically ill adult patients. J Parenter Enteral Nutr. 2003;27:355–373
6. Mentec H, Dupont H, Bocchetti M, Cani P, Ponche F, Bleichner G. Upper digestive intolerance during enteral nutrition in critically ill patients: frequency, risk factors and complications. Crit Care Med. 2001;29:1955–1961
7. Mutlu GM, Mutlu EA, Factor P. GI complications in patients receiving mechanical ventilation. Chest. 2001;119:1222–1241
8. Ritz MA, Fraser R, Tam W, Dent J. Impacts and patterns of disturbed gastrointestinal function m critically ill patients. Am J Gastroenterol. 2000;95:3044–3052
9. Desai KM, Sessa WC, Vane JR. Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature. 1991;351:477–479
10. Orihata M, Sarna SK. Inhibition of nitric oxide synthase delays gastric emptying of solid meals. J Pharmacol Exp Ther. 1994;271:660–670
11. Takahashi T, Qoubaitary A, Owyang C, Wiley JW. Decreased expression of nitric oxide synthase in the colonic myenteric plexus of aged rats. Brain Res. 2000;883:15–21
12. Domek Łopacińska KU, Strosznajder JB. Cyclic GMP and nitric oxide synthase in aging and Alzheimer’s disease. Mol Neurobiol. 2010;41:129–137
13. Nutrient requirement of domestic animals number 10. 19783rd ed. Washington, DC National academy of science, National Research Council
14. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition and HOC writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939–1951
15. Çorak A, Coşkun T, Alican I, Kurtel H, Yeğen BÇ. The effect of nitric oxide synthase blockade and indometacin on gastric emptying and gastric contractility. Pharmacology. 1997;54:298–304
16. Mizuta Y, Takahashi T, Owyang C. Nitrergic regulation of colonic transit in rats. Am J Physiol Gastrointest Liver Physiol. 1999;277(Pt 1):G275–G279
17. Sugai GCM, De O, Freire A, Tabosa A, Yamamura Y, Tufik S, Mello LEAM. Serotonin involvement in the electroacupuncture- and moxibustion-induced gastric emptying in rats. Physiol Behav. 2004;82:855–861
18. Su YC, Vozzo R, Doran S, Leelakusolvong S, Rayner CK, Chapman IM, et al. Effects of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on antropyloroduodenal motility and appetite in response to intraduodenal lipid infusion in humans. Scand J Gastroenterol. 2001;36:948–954
19. Quintana E, Hernández C, Alvarez Barrientos A, Esplugues JV, Barrachina MD. Synthesis of nitric oxide in postganglionic myenteric neurons during endotoxemia: implications for gastric motor function in rats. FASEB J. 2004;18:531–533
20. Nooh N, Mohammed FA, Ahmed AA, Ahmed MA Selenium induced modulation of intestinal motility stressed rats. 2007 Cairo Faculty of Medicine, Ain Shams University
21. Jia YD, Liu CQ, Tang M, Jiang ZY. Expression of motilin in the hypothalamus and the effect of central erythromycin on gastric motility in diabetic rats. Neurosci Bull. 2007;23:75–82
22. Armitage P, Berry G. Statistical methods in medical reserve in left ventricular hypertrophy. Hypertension. 1987;5:192–197
23. McDougal JN, Miller MS, Burks TF, Kreulen DL. Age-related changes in colonic function in rats. Am J Physiol. 1984;247(Pt 1):G542–G546
24. Gainsborough N, Maskrey VL, Nelson ML, Keating J, Sherwood RA, Jackson SHD, et al. The association of age with gastric emptying. Age Ageing. 1993;22:37–40
25. Smits GJ, Lefebvre RA. Influence of aging on gastric emptying of liquids, small intestine transit and fecal output in rats. Exp Gerontol. 1996;31:589–596
26. Di Francesco V, Zamboni M, Dioli A, Zoico E, Mazzali G, Omizzolo F, et al. Delayed postprandial gastric emptying and impaired gallbladder contraction together with elevated cholecystokinin and peptide YY serum levels sustain satiety and inhibit hunger in healthy elderly persons. J Gerontol A Biol Sci Med Sci. 2005;60:1581–1585
27. Guyton AC, Hall JE Textbook of medical physiology. 200611th ed. Philadelphia ElSevier Saunders
28. Yin J, Chen JDZ. Retrograde gastric electrical stimulation reduces food intake and weight in obese rats. Obes Res. 2005;13:1580–1587
29. Ishiguchi T, Amano T, Matsubayashi H, Tada H, Fujita M, Takahashi T. Centrally administered neuropeptide Y delays gastric emptying via Y2 receptors in rats. Am J Physiol Regulat Integr Comp Physiol. 2001;281:R1522–R1530
30. Fukuta H, Koshita M, Nakamura E, Nakamura H, Yamada A, Kawase Y, et al. Acupuncture modulates mechanical responses of smooth muscle produced by transmural nerve stimulation in gastric antrum of genetically hyperglycemic rats. J Smooth Muscle Res. 2009;45:167–185
31. Won KJ, Sanders KM, Ward SM. Interstitial cells of Cajal mediate mechanosensitive responses in the stomach. Proc Natl Acad Sci USA. 2005;102:14913–14918
32. Le Blanc Louvry I, Guerre F, Songné B, Ducrotté P. Gastric stimulation: influence of electrical parameters on gastric emptying in control and diabetic rats. BMC Surg. 2002;2:5
33. Janssen P, Nielsen MA, Hirsch I, Svensson D, Gillberg PG, Hultin L. A novel method to assess gastric accommodation and peristaltic motility in conscious rats. Scand J Gastroenterol. 2008;43:34–43
34. Madsen JL, Rasmussen SL, Linnet J, Fuglsang S, Rumessen JJ. Effect of sustained-release isosorbide dinitrate on post-prandial gastric emptying and gastroduodenal motility in healthy humans. J Int Med Res. 2004;32:351–358
35. Madsen JL, Søndergaard SB, Fuglsang S, Rumessen JJ, Graff J. Effect of sildenafil on gastric emptying and postprandial frequency of antral contractions in healthy humans. Scand J Gastroenterol. 2004;39:629–633
36. Konturek JW, Thor P, Domschke W. Effects of nitric oxide on antral motility and gastric emptying in humans. Eur J Gastroenterol Hepatol. 1995;7:97–102
37. Glasgow I, Mattar K, Krantis A. Rat gastroduodenal motility in vivo: involvement of NO and ATP in spontaneous motor activity. Am J Physiol Gastrointest Liver Physiol. 1998;275:G889–G896
38. Kaneda T, Makino S, Nishiyama M, Asaba K, Hashimoto K. Differential neuropeptide responses to starvation with ageing. J Neuroendocrinol. 2001;13:1066–1075
39. Pu S, Dube MG, Kalra PS, Kalra SP. Regulation of leptin secretion: effects of aging on daily patterns of serum leptin and food consumption. Regul Pept. 2000;92(1–3):107–111
40. Paquet C, St Arnaud McKenzie D, Kergoat MJ, Ferland G, Dubé L. Direct and indirect effects of everyday emotions on food intake of elderly patients in institutions. J Gerontol A Biol Sci Med Sci. 2003;58:153–158
41. Park YH, De Groot LCPGM, Van Staveren WA. Dietary intake and anthropometry of Korean elderly people: a literature review. Asia Pac J Clin Nutr. 2003;12:234–242
42. Silver AJ, Guillen CP, Kahl MJ, Morley JE. Effect of aging on body fat. J Am Geriatr Soc. 1993;41:211–213
43. Aloia JF, Vaswani A, Ma E, Flaster E. Aging in women – the four-compartment model of body composition. Metabolism. 1996;45:43–48
44. Hauser G, Neumann M. Aging with quality of life – a challenge for society. J Physiol Pharmacol. 2005;56(Suppl 2):35–48
45. Sutanto W, De Kloet ER. The use of various animal models in the study of stress and stress-related phenomena. Lab Anim. 1994;28:293–306
46. Barrett KE, Barman SM, Boitano S, Brooks H Ganong’s review of medical physiology. 200923rd ed. Toronto McGraw-Hill Medical
47. Li M, Vizzard MA, Jaworski DM, Galbraith RA. The weight loss elicited by cobalt protoporphyrin is related to decreased activity of nitric oxide synthase in the hypothalamus. J Appl Physiol. 2006;100:1983–1991
48. Kalayci R, Kaya M, Ahishali B, Arican N, Elmas I, Kucuk M. Long-term L-NAME treatment potentiates the blood-brain barrier disruption during pentylenetetrazole-induced seizures in rats. Life Sci. 2006;79:16–20