key: cord-0728570-toy8lt0i authors: Marc Rhoads, J; John Macleod, R; Richard Hamilton, J title: Effect of Glucocorticoid on Piglet Jejunal Mucosa during Acute Viral Enteritis date: 1988 journal: Pediatr Res DOI: 10.1203/00006450-198803000-00010 sha: 310015d35bccde94297fb3046ffb70437c03b4be doc_id: 728570 cord_uid: toy8lt0i ABSTRACT: We measured the effect of pharmacological doses of glucocorticoid on piglet jejunal structure and function during acute viral diarrhea. Weaned piglets, infected experimentally with transmissible gastroenteritis virus, a coronavirus that induces a diarrheal illness similar to human rotavirus infection, received methylprednisolone (30 mg/kg) or saline intramuscularly at 48 and 72 h after infection; noninfected littermate controls were similarly injected with methylprednisolone. Animals were killed at 96 h, at the height of diarrhea, and jejunal epithelium was studied in vitro. Transmissible gastroenteritis, as expected, induced structural, enzyme, and Na transport abnormalities. Methylprednisolone did not affect small intestinal structure or function of noninfected control piglets. In transmissible gastroenteritis-infected piglets, jejunal villi were longer and glucose-facilitated Na absorption was greater after methylprednisolone than after saline treatment. Increased glucose stimulation of Na flux in vitro in the methylprednisolone-treated infected group was not attributable to enhanced Na(+)-K(+)-ATPase activity and occurred despite persistence of the virus within mucosal cells, shown by immunofluorescense microscopy. In this piglet model of viral diarrhea, early regeneration of absorptive surface that precedes recovery of disaccharidase function is accelerated by glucocorticoid therapy. ABSTRACT. We measured the effect of pharmacological doses of glucocorticoid on piglet jejunal structure and function during acute viral diarrhea. Weaned piglets, infected experimentally with transmissible gastroenteritis virus, a coronavirus that induces a diarrheal illness similar to human rotavirus infection, received methylprednisolone (30 mg/kg) or saline intramuscularly at 48 and 72 h after infection; noninfected littermate controls were similarly injected with methylprednisolone. Animals were killed at 96 h, at the height of diarrhea, and jejunal epithelium was studied in vitro. Transmissible gastroenteritis, as expected, induced structural, enzyme, and Na transport abnormalities. Methylprednisolone did not affect small intestinal structure or function of noninfected control piglets. In transmissible gastroenteritis-infected piglets, jejunal villi were longer and glucose-facilitated Na absorption was greater after methylprednisolone than after saline treatment. Increased glucose stimulation of Na flux in vitro in the methylprednisolone-treated infected group was not attributable to enhanced Na+-K+-ATPase activity and occurred d e s~i t e ~ersistence of the virus within mucosal cells. . . shown by immunofluorescense microscopy. In this piglet model of viral diarrhea, early regeneration of absorptive surface that precedes recovery of disaccharidase function is accelerated by glucocorticoid therapy. (Pediatr Res 23: [279] [280] [281] [282] 1988) Abbreviations Isc, short-circuit current Na, sodium MP, methylprednisolone Jz, Na flux from mucosa to serosa JS;' ,", Na flux from serosa to mucosa Jyi, Net Na flux AJyS, increment in net Na flux Na+-K+-ATPase, sodium-potassium-stimulated adenosine triphosphatase TGE, transmissible gastroenteritis ANOVA, analysis of variance G, conductance resembles that of human rotavirus (1, 2). Watery diarrhea is attributable to a loss of functioning differentiated enterocytes and dominance of the epithelium by relatively undifferentiated cells ( 1-4). Glucocorticoids in pharmacological doses may influence small intestinal epithelial differentiation. In suckling rabbits, glucocorticoid accelerates the appearance in the intestinal brush border of active disaccharidases and stimulates precocious development of glucose-facilitated Na transport (5) . Furthermore, under some experimental conditions, pharmacological doses of glucocorticoid augment the activity of rat intestinal mucosal Na+-K+-ATPase (5-7); in both rats and humans they enhance in vivo absorption of Na, water, and glucose (6; 8). Therefore, we studied the effect of pharmacological doses of MP on the response of piglet small intestine to experimentally induced infection with TGE virus. These experiments focused on the jejunal epithelium, its structure, enzyme activities, and transport function. Thirteen York-Landrance piglets, 17-21 days old, were weaned and fed evaporated cow's milk for 3 days. Eight piglets received a standard intragastic inoculum of Purdue strain of TGE virus (I) and five uninfected littermates were used as controls. Three uninfected piglets received MP (30 mg/kg intramuscularly) on the 17th and 18th day of life (control MP). Of the eight TGE-infected pigs, four received MP (30 mg/kg intramuscularly) 48 and 72 h after TGE inoculation (TGE-MP) and the other four were injected with an identical volume of saline (TGE-saline). For intestinal studies, piglets were killed by a parenteral injection of 325 mg pentobarbital sodium. Beginning 10 cm distal to the ligament of Treitz, the first 10-12 cm of jejunum were removed quickly, flushed with normal saline, stripped of muscle, and mounted in conventional Ussing chambers (exposing an area of 1.29 cm2) for studies of ion transport (4). The next 25 cm was removed, flushed with saline, and processed as follows: the first l-cm segment was quick-frozen for viral immunofluorescence; the next cm was fixed in Bouin's solution, blocked in paraffin, and stained with hematoxylin and eosin; and from the remaining 23 cm, mucosa was scraped with a glass slide, homogenized with a Dounce homogenizer, and frozen at -70" C. This frozen homogenate was assayed for sucrase, lactase (9), Na+-K+-TGE is an invasive viral enteritis o c c u i n g in Young pigs (1). ATPase (2) , and thymidine kinase (4); results are expressed as The impact of this coronavirus infection on the small intestine specific activities. For ion flux measurements, mucosal and serosal sides of the 280 RHOADS ET AL. ences and conductances were stable indicating tissue viability during each of these 2-h studies. We added 22Na to mucosal or serosal chambers initially and, after a 15-min equilibration period, withdrew 1-ml samples from each chamber at 10-min intervals. We calculated undirectional Na fluxes from the rate of appearance of label in the opposite chamber. After 55 min, we added 5 ml of Krebs buffer containing D-glucose (30 mM final concentration) to the mucosal and serosal chambers, and again measured unidirectional Na fluxes, sampling from the chambers at 10-min intervals for 1 h. We calculated J E and J,N," using jejunal segments paired so that G did not differ by more than 25%. Net Na flux was calculated by the formula Jr$= JE-Jg. Preliminary studies of TGE viral antigen in the mucosa used direct immunofluorescence with fluorescein-conjugated porcine anti-TGE globulin, as previously described (3). Jejunal sections were interpreted, without prior identification, by an observer who routinely performs TGE immunofluorescence. Results were reported as positive (diffuse fluorescence of enterocytes), weakly positive (weak fluorescence of occasional enterocytes), or negative (3) . For light microscopic studies, all specimens were examined by the same observer without prior identification, using a calibrated micrometer eye piece to measure 10-1 5 representative crypt villus units. 22Na was obtained from Amersham (Montreal, Canada), Dglucose from BDH Chemicals (Toronto, Ontario, Canada). The remaining reagents were obtained from Sigma Chemical (St. Louis, MO). Mucosal structure and enzymes were compared in the four groups of piglets using one-way ANOVA. For data involving comparisons of means between several groups, we used Scheffe's multiple comparisons method (1 1). For transport studies, Na fluxes under basal conditions were compared with those after adding D-glucose using the t test for paired variates. Based on the hypothesis that MP would increase transport (5-8), we compared jejunal Na transport in MP-treated pigs (uninfected, TGE- infected) with Na transport in their corresponding controls using Student's one-tailed t test. were noted in uninfected piglets treated with MP. Infected piglets all developed anorexia and appeared ill. Each of the four infected pigs given saline developed diarrhea and demonstrated a fluidfilled small bowel at 96 h. Of the four infected animals injected with MP, three developed diarrhea but only one showed a fluidfilled intestine at 96 h. The four MP-treated infected piglets weighed approximately the same (2390 f 150 g) as their salineinjected counterparts (2530 f 160 g). Also, the weights of the proximal and distal small bowel after flushing with saline did not differ in the two groups. Microscopic studies (Table I) . MP treatment did not alter jejunal structure of uninfected piglets compared with controls. Ninety-six h after TGE infection jejunal villi were blunted ( p < 0.05) regardless of whether animals had received saline or MP. However, in comparing the two TGE-infected groups, we observed taller villi ( p < 0.05) and, in most preparations, less inflammation of the lamina propria in MP-treated animals than in saline-treated controls. In 96-h TGE jejunum, TGE immunofluorescence was only weakly positive in two and negative in the other two preparations from saline-treated piglets. In MP-treated TGE-jejunum at 96 h, three preparations were available for immunofluorescence staining, and, in all of these, immunofluorescence was strongly positive. These preliminary observations suggested MP had delayed viral clearance. Jejunal mucosal enzymes (Table 2 ). In uninfected pigs, activities of disaccharidases, Na+-K+-ATPase, and thymidine kinase were not altered by MP iniection. Reductions of sucrase and Na'-K'-ATPase activities were observed in the jejuna of both groups of 96-h TGE-injected piglets (saline-injected and MPinjected) ( p < 0.05, compared with uninjected controls). While mean thymidine kinase activity tended to be higher and lactase tended to be lower in TGE jejunum than in controls, the results were not statistically significant. MP had no significant effect on sucrase, lactase, Na+-Kt-ATPase, or thymidine kinase activities in TGE-infected pigs, regardless of whether activities were related to mucosal protein, intestinal wet weight, or length. Na transport data. Measured in vitro in Ussing chambers, transmucosal unidirectional Na fluxes (Jg, JJ,N and G in uninfected MP-treated pigs did not differ from those of controls (Table 3) . After D-glucose (30 mM), Jf;: and Isc increased in both groups of uninfected piglets ( p < 0.05), and there was no difference in the magnitude of these responses between MP-treated (AJ;; = 4.2 k 1.0 pEq. cm-'. h-') and untreated control tissue (AJ?; = 3.9 k 0.7 pEq.~rn-~.h-') (Fig. 1) . Ussing chamber transport data from 96-h TGE-infected piglets are shown in Table 4 and Figure 1 , which compare jejunal Na transport response to D-glucose of MP-injected animals with that of saline-injected litter mates. Basal mucosal to serosal Na flux was modestly enhanced in MP-treated animals, but net Na flux was secretory and did not differ from controls. After 30 mM Dglucose, J; ; increased in both saline-injected and MP-treated TGE jejunum ( p < 0.01); however, only in the glucocorticoid- h-') (Fig. I) . There were small increases in JP"," and G after Dglucose in both groups, which were significant only in salinetreated piglets; however, the magnitude of these changes (AJP: , AG) did not differ between the two groups. Short-term, high-dose, glucocorticoid treatment of normal piglets did not alter jejunal structure, enzymes, or glucose-facilitated Na transport in the current experiments. Previous studies using similar doses in rats found increased ileal villus height (12) , increased disaccharidase activities (12) , and enhanced electroneutral NaCl transport (1 3) in response to corticosteroid. Furthermore, MP (20-30 mg/kg) has been shown to stimulate basal ileal Na and C1 transport (13, 14) and ileal response to combined absorptive stimuli (glucose + alanine + epinephrine) (14) after only 24-48 h of treatment. Because we found MP treatment had no impact on basal jejunal Isc, we conclude that glucocorticoid treatment did not stimulate electrogenic chloride secretion, as reported in rat (15) and rabbit (16) ileum by others. These discrepancies between our data and those reported previously could be attributed to many possible factors, including species specificity, differences in the responses of jejunum and ileum, and the relatively brief period of MP treatment we used. We gave MP at 48 and 72 h after TGE inoculation because previous studies showed maximal jejunal injury and viral shedding before 40 h after infection (3), when the epithelium is most undifferentiated and presumably most responsive to glucocorticoid (1 7, 18). As recovery from TGE-induced injury in untreated pigs is complete by 144 h (4) we studied jejunal tissue at 96 h, expecting animals at this stage to show partial villus recovery but little or no virus remaining in the epithelium. Glucocorticoid treatment clearly did not alter disaccharidase activity in either normal pigs or in those with viral diarrhea. In the normal group, the lack of effect of MP probably reflects a functionally mature mucosal disaccharidase pattern in the weaned animal. However, in the TGE-infected epithelium, low disaccharidase activities persisted after MP treatment, despite a lengthening of villi in MP-treated animals. Studies in suckling rats showed the effects of glucocorticoid require emergence of cells from the crypts, with maximal effects on mucosal sucrase after 120 h (1 9). Consequently, we expected an earlier response in TGE epithelia, because enterocyte transit time decreases from approximately 55 to 18 h in this disease (3) . TGE jejunal cells, therefore, differ from those of the developing gut not only in their rapid proliferation but also in their failure to respond by 48 h to adrenocorticoid by synthesis or activation of brush border digestive enzymes. In the virus-damaged epithelium, glucocorticoid treatment did enhance functional maturity as reflected by jejunal villus structure and the Na-absorptive response to glucose. Accelerated enterocyte migration, observed after long-term corticosteroid treatment (12) , could have contributed to this observation. In previous studies of suckling rabbits treated with cortisone (5) we attributed precocious development of jejunal glucose-facilitated Na transport to increased lumen to cell driving forces resulting from enhanced Na+-K'-ATPase activity. However, our current data demonstrate increased glucose-stimulated Na absorption in the weaned piglet in the absence of altered Naf-K+-ATPase act!Vlty, suggesting that the glucocorticoid effect observed was independent of the activity of the basolateral membrane Na pump. Rapid activation of the Na+-K+ pump can occur as a result of a sudden increase in intracellular Na (20) . In rabbit descending colon glucocorticoid treatment was recently shown to enhance apical Na permeability (21) , which in turn would rapidly activate Na+-K+-ATPase. Our data do not preclude MP effects on brush border permeability and/or basolateral pump function but they do indicate that MP does not alter Na+-K+-ATPase specific activity in the jejunum and that it has no major effect on basal Na transport in control or TGE jejunum. The augmented jejunal response to o-glucose we observed in the MP-treated infected piglet is compatible with the hypothesis that glucocorticoid alters brush border glucose-Na cotransport, as observed in intestinal brush border membrane vesicles from rats injected with triamcinolone (22) . Alternatively, MP may alter jejunal glucose-Na cotransport simply by increasing absorptive surface area. Increased glucose-stimulated Na absorption in the treated pigs was not due to enhanced elimination of virus from the epithelium. Our data showing villus blunting and a 1 O-f old reduction in mucosal disaccharidases in MP-treated infected piglets suggest the drug did not interfere with exfoliation of infected villus cells. However, the finding of homogeneous immunofluorescence of enterocytes at 96 h, in the MP-treated group only, suggests that glucocorticoid treatment may interfere with viral clearance from the mucosa. Determination of the effects of MP on viral clearance was not the aim of the present study and would require sequential quantitation of virus in the mucosa and intestinal fluid. Our finding, however, is compatible with the theory that MP treatment could facilitate persistence of virus beyond the 40h stage, when it is normally cleared (3) . Although the TGE model in the piglet represents a major human disease, human rotavirus enteritis, our data should not be interpreted to suggest that corticosteroid treatment of babies with severe viral enteritis would be beneficial. In fact, the observation that virus may be retained in the epithelium after MPtreatment suggests such treatment is contraindicated. Our findings do indicate that the relatively undifferentiated epithelium occurring in this acute viral enteritis is responsive to hormonal influences. Transmissible gastroenteri tis. Mechanisms responsible for diarrhea in an acute viral enteritis in piglets Transmissible gastroenteritis of piglets: a model of infantile viral diarrhea The mucosal lesion in viral enteritis: extent and dynamics of the epithelial response to virus invasion in transmissible gastroenteritis of piglets Tran smissible gastroenteritis: sodium transport and the intestinal epithelium during the course of viral enteritis Effect of cortisone on postnatal development of ion transport in rabbit small intestine Gots RE 1975 Na+-K+-activated adenosine triphosphatase and intestinal electrolyte transport. Effect of adrenal steroids Effect of dexamethasone on electrolyte transport in the large intestine of the rat Effect of intraluminal hydrocortisone on solute and water absorption in the human jejunum Method for assay of intestinal disaccharidases Ion transport in isolated rabbit ileum. I. Shortcircuit current and Na fluxes Comparing the mean of several groups Effects of prednisolone on the small intestinal mucosa of the rat Stimulation of neutral NaCI absorption by glucocorticoids and Na depletion in rat ileum in vivo Methylprednisolone increases absorptive capacity of rabbit ileum in vitro Effects of methylprednisolone on electrolyte transport by in vitro rat ileum Physiologic and pharmacologic effects of glucocorticoids on ion transport across rabbit ileal mucosa in vitro Postnatal development: coordination of feeding, digestion, and metabolism Coordinate loss of glucocorticoid responsiveness by intestinal enzymes during postnatal development Koldovsky 0 1972 Cell migration and cortisone induction of sucrase activity i'n jejunum and ileum Serum stimulates the Na+, K+ pump in quiescent fibroblasts by increasing Na+ entry Relationships among sodium current, permeabi lity, and Na activities in control and glucocorticoid-stimulated rabbit descending colon Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and I ,25-dihydroxy vitamin D-3 Acknowledgments. The authors are grateful to Krys Streich, Mahmood Khan, Helen Conway, and William Wilson for expert technical assistance, to Shelley Goodman and Martha Long for secretarial assistance; to Kathy Holland for assistance with the statistical analysis; and to Drs. Brian Darbyshire and Douglas Key, Department of Microbiology, Ontario Veterinary College, Guelph, Ontario for assistance in fluorescent antibody studies.