Vitamin E prevents cold wrap restraint stress , induced intestinal fluid transport alterations in rats

S BURDICK, N Cul, LR EMPEY, RN FEDORAK. Vitamin E prevents cold wrap restraint stress-induced intestinal fluid transport alterations in rats. Can J Gastroenterol 1994;8(7):417-421. Psychological stress may alter gastrointestinal absorptive function by increasing the quantity of intestinal free radicals or by lowering endogenous intestinal free radical scavenging capacity. Vitamin E has been shown to be a potent en<logenous antioxidant and free radical scavenger under both physiological and pathological conditions. The purpose of this study was to determine whether cold wrap restraint stress altered in vivo intestinal fluid absorption in rats, and whether vitamin E administration prior to the induction of cold wrap restraint stress could prevent such changes in intestinal secretion. Jejuna[, ileal and colonic fluid and electrolyte transport rates were measured in vivo using an isolated loop technique. Cold wrap restraint stress reduced in vivo fluid absorption in the ileum and colon, but not in the jejunum. Administration of vitamin E prior to the cold wrap restraint stress procedure completely prevente<l this alteration of ilea! and colonic fluid absorption.

T HE EFFECTS OF PSYCHOLOGICAL stress on gastrointestinal functions have been known for more than a century and a half ( 1 ).Stress can alter gastric acid secretion, intestinal motility, and fluid and electrolyte absorption rates in nondiseased intestine as well as in the intestine displaying inflammatory changes, irritable bowel syndrome or celiac sprue (2)(3)(4).While an endogenous mediator regulating and coordinating gastrointestinal secretory and motor responses following stre s has not been identified, recent evidence has shown that stress is capable of upregulating the generation of reactive oxygen metabolites and that these reactive oxygen metabolites have physiological consequence• (5,6).
Reactive oxygen metabolites exert a multi rude of biological effect in the gastrointestinal tract, ranging from cell death to nontoxic alterations in the intestinal fluid and electrolyte transport, motility and mutagenic activity (7)(8)(9)(10)(11).Application of physiological, noncytotoxic concentration of hydrogen peroxide (HzOz), hypochlorous acid (HOC!) or monochloramine (NHzCl) to the serosal surface of rat colon mounted in Ussing C hambers elicits marked increases in net water and chloride sercetion (11,12).Furthermore, the HzOz-and NH2Clmediated increases in chloride secretion are inhibited by atropine, tetradotoxin and piroxicam, suggesting that the cholinergic nervous system and prostaglandins may be important in this response (11,13).Taken to-  Reactive oxygen pecies are continuously produced as metabolic byproducts by virtually all tissues in relatively small amounts.Under basal conditions, oxidant-generating systems in the intestine are compensated for by complex sets of protective mechanisms that prevent or limit oxidative damage.The e mechanisms include antioxidant enzymes ( uperoxide dismutase, catala e and glutathione peroxidase) and nonenzymatic free radical scavengers (14,15).A free radical scavenger represents one type of antioxidant chat is defined as any substance that will donate an electron to a free radical, thus inactivating the radical species.Alpha-tocopherol (vitamin E) has recently been identified as one of a number of biologically significant, endogenous, nonenzymatic free radical scavengers (15).
This study investigated whether alterations in functional intestinal fluid and electrolyte absorption occurred as a result of the induction of cold wrap restraint stress in rats and whether the admini Cration of vitamin E, perhaps through it free radical scavenging effect, prevented the occurrence of the e fluid transport abnormalities.

MATERIALS AND METHODS
L-alpha-tocopherol (vitamin E), Lot 86F-0370, purity 95%, wa purchased from Sigma Chemical Company (Missouri).Alpha-tocopherol tandard was purchased from Ea tman Organic Chemicals (New York).Xylazine, 20 mg/mL, wa purcha ed from the Bayvert Division of Chemagro Limited.Ketamine hydrochloride, 100 mg/mL, was purchased from MTC Pharmaceutical.The remainder of the chemicals used were reagent grade and were purcha ed from Sigma Chemical Co.The L-alpha-tocopherol was diluted with 25 mL of methyl cellulose to a final concentration of 5 µg/µL.Animals: Male Sprague-Dawley rats, 250 to 300 g, were randomly divided into four groups: group l, nonvitamin E (sham)-treated nonstres ed controls; group 2, nonvitamin E (sham)-treated cold wrap re traint stressed animals; group 3, vitamin E-treated (1.25 mg/kg/day) cold wrap re traint seres ed animal and; group 4, vitamin E-treated (2.5 mg/kg/day) cold wrap restraint stressed animals.All animals were given acces to standard rat chow (Wayne Rodent Blox, Continental Grain Co, Illinois) anJ water ad libitum.A 12-h light-dark cycled animal care facility was u ed to hold the animals.Animal were allowed seven days to acclimatize to the environment before the studies began.Vitamin E administration: Vitamin E (1.25 or 2.5 mg/kg/day in 1 % methyl cellulo e) was given once daily by intraperitoneal injection for seven consecutive days prior to seres administration.The control group received intraperitoneal injection of 1 % methyl ccllulo e for the same length of time.Cold wrap restraint stress: Cold wrap re traint stress was carried out as previously de cribed (5).Briefly, nonfasting rats were lightly anesthetized with ether, and the forelimbs and shoulders were attached to the thoracic trunk with a harness of paper ma king tape.The hind limbs were then taped to the body in imilar fashion.The animals were put in the cage in a prone position and kept at 4°C for 2 h.While restrained, the animal were completely immobilized and remained in one position without struggling.Care was taken during the restraint sere s pr ce s to prevent injury and to allow the limbs to rest in a neutral po ition in order to avoid providing a painful stimulus.These studies were carried out in accordance with the guideline established by the Univer icy of Alberta Animal Health Science Committee.In vivo intestinal fluid transport studies: Immediately following restraint tres the animals were anesthetized with a mixture ofketamine and xylazine (2:1, volume:volume) and kept warm with a thermostatic heat lamp.In vivo intestinal fluid absorption studies were then performed as previously described (16).The inte tinal tract wa expo ed through a midline incision.An occluding ligature was placed at the cecal ascending junction, and the colonic luminal contents were flu hed out the rectum with a warm 154 mM sodium chloride olution instilled via a cannula inserted though an incision just distal to the occluding ligature.Re idual saline was emptied by gentle manual expres i n.A colonic loop approximately 12 cm long, beginning 2 cm distal to the  cecal-colonic junction and extending to the peritoneal reflection, was created with ligatures.In a similar fashion, the small inte tine was occluded with ligatures placed at the ligament of Treitz and the ileocecal valve.A cannu la was in erted distal to the ligature, and the contents were flushed with warm 154 mM odium chloride.Loops approximately 12 to 15 cm long were created with ligatures in the jejunum (beginning 2 cm distal to the ligament of Treitz) and in the ileum (beginning 2 cm proximal to the ileal-cecal valve).Care was taken not to compromise mesenteric, vascular or neural continuity.A 26-gauge needle was inserted obliquely through the outer muscle layer along the antimesenteric border, and 2 ml of 154 mM sodium chloride prewarmed to 37°C was instilled into each empty loop.The loops were checked for leakage.Previous studies using dye markers had confirmed that leakage does not occur from the intestinal loops over the 1 h duration of the experiment (16) .The viscera were returned to the abdominal cavity and the incision was closed.The rats then were placed in a recovery chamber for 1 h.Animals were given an intraperitoneal overdose of pentobarbitol (250 mg/kg), and the loops were removed.The length of each loop was measured, and the weight of the loop (both full and empty) was recorded to determine the residual intraluminal volume.Results were expre sed as the difference b tween initial and residual luminal volume per centimetre of bowel per hour.
Serum vitamin E determination: Blood samples were taken at the time of pentobarbitol overdose and serum vitamin E levels were determined using method established by Catignani and Bieri (17) .Briefly, serum was deproteinized with ethanol that contained the internal standards (retinyl acetate and alpha-tocophery l acetate), and the lipid was extracted with hexane.After an aliquot of the so lvent phase was evaporated, the residue was dissolved in diethyl ether and diluted with methanol.A portion of this solution was injected onto a Cl8 reversed-phase chromatographic column, and absorbance of the vitamin and internal standards measured at 280 nm.Peak-height ratios were used to quantify vitamin concentration.Statistics: Statistica l analysis of the data was comp leted using the Student-Newman-Keuls multiple range test.
Values are presented as mean± SEM.

RESULTS
Animal groups: Cold wrap restrained rats were complete ly immobilized and remained in the prone position at the bottom of the cage without moving throughout the restraint period.Body temperature remained con tant at 3 7°C.Following the period of restraint, stress animals were immediately anethetized and in vivo fluid transport measurement carried out.Cold wrap restraint stre s has previously been shown CAN ] GASTROENTEROL V OL 8 No 7 DECEMBER 1994 to cause gastric ulceration (5); therefore, the authors carefu lly searched for gro s ulceration along the small and large intestine.There was no evidence of macroscopic ulceration in any animal used in these experiments.Administration of vitamin E to shamoperated control animals: Administration of vitamin E to sham-operated control anima ls did not alter in vivo fluid and electrolyte absorption in the jeJunum (148±15 µL/cm/h), ileum (118±12 µL/cm/h) or colon (163±14 µL/cm/h).Colonic in vivo fluid absorption: As shown in Figure 1, in vivo net colonic fluid absorption was significantly decreased in the nonvitamin E (sham)treated cold wrap restraint seres ed group of rats relative to the level found in nonstressed controls.In contrast, cold wrap restraint stressed animals that received vitamin E supplementation for seven days prior to the period of stress did not show a decrease in colonic fluid absorption.This protective effect of vitam in E was seen at both doses (1.25 and 2.5 mg/kg/day) examined.Ilea! in vivo fluid absorption: Figure 2 shows that in vivo net ilea[ fluid absorption was sign ificantly decreased in the nonvitamin E (sham)-treated cold wrap restraint stressed group.Similar to the results seen in the colon, supplementation with vitamin E for seven days prior to the period of stress prevented the ileal fluid absorption from decreasing.Jejunal in vivo fluid absorption: As shown in Figure 3, in vivo net jejuna!

DISCUSSION
UnJcr physiological conditions free radicals are continuou ly being pro-Juced.In uncontrolled states, the free radicals are able to induce memhrane lipid peroxidatton.As a consequence of lipid peroxidation, mtestinal membrane functions (including transport proces es) are markedly impaired.The lipid hydroperoxides, which me formeJ during lipid peroxidation, can form clusters which create pores in the memhrane through which ions cnn diffuse into the cells; enzymes are inactivated, membrane fluidity decreases, signal transduction is decreased anJ cytotoxic aldehydes are formed ( 18).
In orJer to prevent or minimize free radical-induced tissue Jamage, antioxi-Jant enzymes such ns superoxide Jismutase, catalase and glutathione peroxidase mop up excess free raJicals.In aJdition, antioxidants can prevent the formatton of free radicals or interfere with the propagation steps of the lipid peroxiJation process.
Vitamin Eis the major lipiJ-soluble, peroxyl radical-trapping, chain-breaking antioxidant in human blooJ (19,20).Vitamin E consists of four ro-copherols and four tocotricnob which arc able to inte1fcrc with the propagation sters on the lipid peroxidation process.L-alpha-tocopherol is, in vivo, the most abundant and the most bioactive tocopherol (21 ).The tocotrienols have an unsaturated side-chain which likely functions to retain the molecule in the memhranc, while the tocopbcrol, with its chmman head-group, is responsible for the majority of the antiox iJan t activity ( 6,22 ,23).
Cold stress has been shown to alter both the production of free radicals and the function of the free radical-trapping defence system (24).In rat exposed to cnld stress for short intervals of time, physiological free radical-trapping is reduced as a consequence of dimini hed superoxide dismuta e activity (6).In rats exposed to cold stress for longer intervals of time, the activity of glutathione-S-transferase, a major antioxidant, is decreased in liver and adipose tissue, while glutathione levels are markedly reduced in plasma (24 ).
Physical and psychological stresses have been shown to alter gastrointestinal physiological function in humans and animals (25)(26)(27)(28)(29). Psychological stress experienceJ Juring dichotomous listening significantly reduced human jejuna!fluid absorption through a function of the cholinergic parasympathetic nervous mechanism (25).In animal studies, restraint-based stress-induced changes in mtestinal transit and transport were partially meJiated by endogenous central nervous system corticotrophin-releasing factor (28,29).Recently, psychological stress has been shown to reduce intestinal bloodflow in animals (30).Indeed, oxygen free radicals represent a critical factor 111 the development of gastrointestimd injury after intestinal ischemia and repe1fusion have occurred (31).
ln the present study, cold wrap restraint stress reduced ba •al in vivo intestinal fluid absorption in the ileum and colon, but not in the jejunum.This rcgionalization of beneficial effects by mucosa!protective agents in the intestine are similar to those previously described in other experimental modPls of intestinal injury (16).In addition, divergent effects of seres on the small intestine have heen previously described an<l emphasize the independent regulation that exist in different regions of the gastrointestinal tract (29).The decrease in ileal and colonic absorption in the stre ed animals occurred in the absence of gross macroscopic ulceration, an<l thus likely represent changes at the membrane level; perhaps related to alterations in either the transport carrier or in membrane permeability.
Table I shows that cold restraint stress does not alter the vitamin Econtent in serum, and previous investigations have confirmed that cold stress does not alter vitamin E levels in the rat small intestine (24 ).This result suggests that, under the conditions of cold restraint stress, the vitamin E concentration is maintained at sufficient levels to ensure the integrity of the cellular membrane against the action of basal antioxidant enzymes.'J(/hether a functional disturbance in vitamin E antioxidant activity exists or whether an elevated level of oxidant activity is present under conditions of restraint sere remains to be determined.Vitamin E adrnini tration for seven consecutive days prior to the administration of cold wrap restraint stress resulted in a significant elevation in serum vitamin E levels.le is thus possible that this elevated level of vitamin E might have overridden any functional deficit or elevated oxidant activity induced in the stressed rats.
In the present study, intraperitoneally administered vitamin E reversed the net intestinal secretory response in the ileum and colon of cold wrap restraint stressed rats to control level .This effect was seen at doses of both 1.25 and 2.5 mg/kg/day over seven days.The doses are equivalent to those that might be administered to humans under physiological conditions.le is un-1 likely that the vitamin E directly stimulated intestinal sodium and chloriJe absorption because vitamin E alone ad,! ministered to control rats did not alter intestinal fluid absorption rates.It i1 possible that, Juring the cold wrap re-_ straint stress, the antioxidant defencemechanisms provided by endogenoui levels of normally functioning vitamin E are inadequate to scavenge increased levels of oxygen free radicals or, alternative ly, that vitamin E antiox idant activ ity is functionally impaired and cannot scavenge baseline levels of oxygen free radicals.Oxygen free radicals in the intestine under these conditions could thus be responsible for the observed alterations in fluid and electrolyte tran port.Nevertheles , the exact

Figure 2 )
Figure 2) Effect of vitamin Eon in vivo ileal fluid absorption in rats with cola wrap restraint-induced stress.Values are mean± SEM for n=6 rats.+P<0.05 compared with nonvitamin E (sham) -treated nonstressed group.*p <0.05 compared with non vitamin £-treated cold wrap restraint stressed group Values are mean± SEM for n=6 rats.Effect of vitamin Eon in vivo jejuna!fluid abs01•ption in rats with cold wrap restraim-induced stress.Values are mean± SEM for n=6 rats .+ P<0.05 compared with nonvitamin E ( sham)-treated nonscressed group.*P<0.05c0111pared with nom,itamin £-treated coldwra/J restraint stressed group

TABLE 1
Serum vitamin E levels after seven days of vitamin E administration