Neurally mediated gastric mucosal damage in hypophysectomized rats LYNNE. HIERLIPIY Department of Physiology, Queen 3 University, Kingston, Qnt. , Canada K7L 3N6

JOHN L. WALLACE Department of Medical Physiology, University of Calgary, Calgary, Alta. , Canada 7 2 N 4Nl AND

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ALASTAIR V. FERGUSBN Deparpment of Physiology, Queert 's University, Kingston, Ont. , Canada K7L 3NB Received February 20, 1992 NIERLIHY, k. E., WALLACE, J. L., and FERGUSON, A. V. 1992. Neurally mediated gastric mucosal damage in hypophysectomized rats. Can. 9. Bhysiol. Phamacsl. 70: 1189- 1116. The role of the pituitary hormones in the development of neurally mediated gastric mucosal damage was examined in both normal and hypophysectomized urethane-anaesthetized male Sprague -Dawley rats. Gastric mucosal damage was elicited either by electrical stimulation of intact vagal nerves or by electrical stimulation in the paraventricular nucleus. Macroscopic damage was scored following the stimulation period and samples of the stomach were fixed for histological assessment. Damage scores were assigned based on a 8 (normal) to 3 (severe) scale. Control experiments in which the vagi were not stimulated did not result in any significant gastric damage in either normal (0.56) or sham surgery (0.14) animals, whereas hypophysectomized animals were observed to have significant damage (1.44, p < 0.05). Stimulation of the vagi in hypophysectomized animals resulted in damage that was not significantly different compared with the hypophysectomized control animals (1.25, p > 0.05). In normal animals, stimulation of vagal nerves resulted in mean damage scores of 2.00, values that were not significantly different from those observed in hypophysectomized animals (1.25, p > 8.05). Similarly, stimulation irn the paraventricular nucleus of hypophysectomized animals resulted in gastric lesions (2.80) that were not significantly different from those observed in nomal animals (1.91, p > 0.05). These data suggest that such neurally mediated gastric damage does not depend upon neurosecretory projections to the pituitary gland, but that the maintenance of an intact gastric mucosa under normal conditions requires the presence of pituitary hormones. Key words: vagus, paraventricular nucleus, hypophysectomy, gastric. HHERLIHY, L. E., WALLACE, J. L., et FERGUSON, A. V. 1992. Neurally mediated gastric mucosal damage in hypophysectomi z e d rats. Can. J. Pkysiol. Bharmacol. 78 : 1189-1116. On a examin6 le r61e des hormones hypophysaires dans le dtveloppement d'une alteration de la muqueuse gastrique par mkdiation neurale ckez des rats Sprague -Dawley mlles, anesthCsiCs B l'urCthane, hypopkysectomisCs et normaux. L'aBtCration de la rnuqueuse gastrique a CtC provoquCe par une stimulation Clectrique des nerfs vagues intacts ou par une stimulation Clectrique dans le noyau paraventriculaire. L'altCration macroscopique a CtC kvaluCe aprks la pCride de stimulation et des Cchantillons de l'estornac ont Ctt fixCs pour une Cvaluation histologique. Les altbrations ont CtC cotkes selon une Cchelle comprise entre 8 (normal) et 3 (sCvke). Les expkriences tCmoins, dans lesquelles les nerfs vagues n'ont pas CtC stimulCs, n'ont provoquC aucune altCration gastrique significative chez les animaux normaux (0,56) et les animaux opCrCs de manihe factice (0,14), alors que ces alterations ont CtC significatives (1,44, p < 0,05) chez les animaux hypophysectomis6s. Chez les animaux hypophysectomisds, la stimulation des nerfs vagues a prsvoquk une altkration qui n9apas differ6 significativement de celle des animaux tdmoins hypsghysectomisCs (1,25, p > 0.05). Chez les animaux normaux, la stimulation des nerfs vagues a provoquC des altkrations msyennes (2,00), qui n'ont pas diff6rC significativement de celles observkes chez les anirnaux hypophysectomises (1,25, p > 0'05). Similairement, la stimulation dans le noyau paraventriculaire des animaux hypophysectomisCs a p r o v q u t des lCsions gastriques (2,00) qui n90ntpas differ6 significativement de celles observCes chez les animaux nomaux (1,91, p > 0,05). Ces rksultats suggkrent qu'une altkration gastrique par mCdiation neurale ne dCpend pas des projections neurosCcrttrices B la glande hypophysaire, mais que, dans des conditions normales, le maintien d'un muqueuse gastrique intacte requiert la prksence d'hormones hypophysaires. Mots c1ix : vague, noyau paraventriculaire, hypophysectomie, gastrique. [Traduit par la r6dactionI

Introduction The vagus nerve is perhaps the most commonly h s w n peripheral nervous structure involved in gastric function. Its established parasympathetic effects on acid secretion resulted in its past popularity as a target for ulcer therapy (Hirschowitz 1982). It is well known that prior to the development of antisecretory agents, the elimination of vagal effects on acid secretion through the surgical procedure of vagotorny was widely used as the treatment for ulcer disease (Hirschowitz 1982). Vagal influences on gastric function include stimulation of acid secretion as well as significant effects on gastric motility and blood flow (Martinson 1965). Yano et al. (1983) have demonstrated both cholinergic excitation and noncholinergic Printed in Canadn ! Imprim6 au Canada

inhibition of gastric motility following vagal stimulation and have also reported vagal stimulation to cause an immediate increase in gastric blood flow. This increase in gastric mucosal blood flow following vagal nerve stimulation has been demonstrated to be an indirect result of increased acid secretion as well as a direct consequence of vagal stimulation, since prior treatment with orneprazole will abolish the secretory response while the blood flow response is maintained (Thiefin et al. 1990). The use of stereotaxic techniques enabling specific manipulations of components of the central nervous system (CNS) has established that higher CNS structures &so exert control over the autonomic nervous system including the gastrointestinal

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BAY FIG.1. The graph in (A) demonstrates the daily weight gain pattern of a group of animals following hypophysectomy. Animals were shipped within 1 week of surgery, thus Day I represents the date of arrival from Charles River Inc. Properly hypophysectomized animals did not gain any significant amount of weight over an $-day period. An improperly hypophysectomized animal was identified by the significant degree of daily weight gain ( >4 -6 g per day). The mean body weights (fstandard error of the mean) of both properly hypophysectomized and normal animals are presented in (B). H Y P O X ~ hypophysectomized.

(GI) tract, Although most commonly known for the production of the posterior pituitary hormones, electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus has been shown to increase gastric acid secretion (Rogers and Hermann 1986) and influence gastric motility (Rogers and Hermann 1987). The secretory response was attenuated following microinjection of an oxytocin antagonist into the dorsal motor nucleus of the vagus in the medulla, demonstrating the involvement of a descending pathway from the PVN to vagal motor neurons that utilized oxytocin as a neurotransmitter. These PVN-induced responses on motility are presumably mediated by the vagus nerve via anatomically verified descending projections to the dorsal vagal complex of the medulla (Swanson and Sawchenko 1983, 1980) since they are eliminated following bilateral vagotomy. Electrical stimulation of both hypothalamic and extrakypthalmic CNS structures has been demonstrated to induce GI

ulceration, while specific lesions can affect the degree s f GI damage that is observed in several experimental stress models in rats. Studies in our laboratory have shown that a $0-min period of BVN stimulation will induce GI erosions and that such PVN-induced damage is prevented by vagotomy (Ferguson et a%.1988). A similar period s f vagal nerve stimulation also results in the formation of gastric mucosal lesions (Hierlihy et al. 1991). These vagal-induced lesions are only observed following intact vagal nerve stimulation as opposed to isolated stimulation of either the peripheral or central cut ends of the vagi, experimental manipulations that do not induce any gastric mucosal damage. Furthermore, such vagal-induced gastric damage is not observed in animals in which the PVN has been ablated. Together these data indicate a major role for both afferent and efferent vagal projections, with the afferent component specifically involving the PVN, in the development of such mucosal damage. Double retrograde tracing studies have established that the BVN contains distinctly arranged populations of cells that project either to the posterior pituitary, the median eminence for the control of anterior pituitary function, or to autonomic centres in the brain stem and spinal cord (Swanson and Sawchenko 1983). Evidence also exists to suggest that these three separate populations of PVN neurons contain similar populations s f peptidergic and adrenergic neurons (Swanson and Sawchenko 1983). For instance, corticotropin-releasing hormone (CRH) , commonly known for its effect on the anterior pituitary, is also found to be present within a small number of PVN neurons that project to the dorsomedial medulla and (or) the spinal cord as well as within neurons projecting to the posterior pituitary (Sawchenko 1987). It is clear that PVN stimulation induced gastric damage depends at least in part upon projections to autonomic centres in the brain stem, and that vagal stimulation induced gastric damage requires a neural connection with the PVN. The present studies were designed to examine whether such vagal- and PVN-induced gastric damage depends on the neurosecretory projections of the PVN controlling the pituitary gland.

Materials and methods Hypophysectomy Normal male Sprague-Dawley rats, hypophysectomized and appropriate sham surgery rats were purchased from Charles River Cs. (Boston, Mass.). Animals were housed under conditions of controlled temperature (24 f 1°C) and lighting (06:00--20:OO h) and were allowed 7 days to recover from shipping. The completeness of hypophysectomy was subjectively evaluated by monitoring the loss of guard hairs (Williams et a!. 1988) and the degree of weight gain, i.e., properly hypophysectomized animals lack guard hairs and gain no more than 1-2 g per day whereas improperly hypophysectomized animals exhibit guard hairs and gain at least 4. -6 g per day (Fig. 1). Animals that retained guard hairs and gained a significant amount of weight were not used in this study. All animals were fed Purina laboratory rodent chow; hypophysectomized and sham surgery animals also received a banana cereal and drinking solution of 10% dextrose - 1% saline to improve survival. The night before experimentation, food was withdrawn and in the case of hypophysectomized animals and sham surgery animals, the drinking solution was replaced with water. Experirnen~aIprofocoI Animals (I00 - 120 g hypophysectomized animals, 850 -200 g normal and sham surgery animals) were anaesthetized with urethane (1.4 g/kg i.p.) then fitted with both an indwelling femoral arterial catheter (PE-SO, Intramedic, Barsippany, NJ) to monitor blood pres-

HIERLIHY ET AL.

sure and heart rate and an indwelling tracheal tube to facilitate the animals9 breathing. Body temperature was maintained at 37.0 1.OaC throughout experiments using a feedback-controlled heating blanket. Animals were divided into two experimental groups: the first to examine the effect of hypophysectomy on vagal stimulation induced gastric damage and the second to examine the effect of hypophysectomy on PVN stimulation induced gastric damage.

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Vagab sdrnuhtion Cervical vagus nerves were surgically isolated and laid over hypophysectombipolar hook electrodes in groups of normal (NORM), ized (HYPOX), and sham surgery animals (SHAMHYPOX). A Grass SB9 stimulator was used to deliver supramaximal stimulation at a frequency of 5 Hz (5 V, 1 ms pulse duration) for 1 h. Supramaximal stimulation was confirmed by observed blood pressure changes (- 30 to -70 mmHg; 1 mmHg = 133.3 Pa), where any further increase in stimulation intensity did not elicit any further change in blood pressure. Such stimulation parameters applied to intact vagus nerves have been previously shown to induce significant gastric mucosal damage in the rat (Hierlihy et ral. 1991). Within all groups of animals (NORM, SHAM HY mx,and H Y ~ Xone ) , further experimental group of animals was utilized in which animals were prepared for vagal nerve stimulation but stimulation was not applied to these groups (NO STHM).

P KV stimulation Anaesthetized animals were placed in a stereotaxic frame. A small burr hole was made in the skull and a parylene-coated tungsten monopolar electrode (MPI-LFQlG, Microprobe Inc., Clarksburg, MB) was advanced toward the region of the PVN using a micromanipulator according to coordinates of Paxinos and Watson (1982). Starting at a point 7.0 mrn ventral to the brain surface, the stimulating electrode was advanced in 0.2-mm increments. At each increment the area was electrically stimulated at 200 pA, $dB Hz, and 100 ,us pulse duration for 5 - 14) s and the resultant effects on blood pressure were assessed. Stimulating currents were determined by measuring the voltage drop across a known resistor. Upon blood pressure changes indicative of stimulation in the PVN (Donevan and Ferguson 1988; Ferguson and Renaud 1984), a stimulation (200 PA, 60 Hz, I00 ps pulse duration) period of 1 h was initiated. Such electrical stimulation in the PVN has been previously shown to induce significant gastric lesions in the rat (Ferguson et al. 1988). After this stimulation period a marking lesion was made at the site of stimulation by passing direct current (0.1 d, 10 s) through the electrode.

Macroscopic damage Hollowing both vagal nerve and PVN stimulation periods, the abdominal cavity was opened to remove the stomach and proximal duodenum. The stomach was cut along the greater curvature and examined macroscopically. An observer unaware of the experimental treatment assigned damage scores based on an arbitrary scale from 0 to 3, where 0 indicates normal mucosal appearance and a score of 3 indicates extensive gastric damage including regions of overt hemorrhage. Stomachs were then immersed in 10% Formalin in preparation for later histological assessment sf gastric damage. Gastric histology Samples of dorsal and ventral corpus regions of the stomach (2 X 10 mm) were excised. These tissues were then immersed in 10% Fomalin and processed by routine procedures. Hematoxylin- and eosin-stained sections were coded and histological analysis was performed in random order so that the observer was unaware as to which experinlend group each tissue belonged. Central nervous system histology Following PVN stimulation animals were perfused with 0.9% sdine followed by 10% Formalin administered through the left ventricle of the heart. The brain was removed and placed in 10% Formalin for at least 12 h. Coronal 100-prn sections were cut through the forebrain using a Vibratome and such sections were strained with

PIG. 2. This histogram demonstrates individual control gastric damage scores from normal (NORMAL), sham surgery (SHAM),and animals following a period of sham hypophysectomized (MYPOX) vagal stimulation. *p < 0.05 compared with either normal or sham surgery group (Mann -Whitney U-test) .

cresyl violet for later determination of the anatomical location of stimulation sites.

Sfafisbih'alanalysis All data are expressed in the text as the average gastric damage score, and graphically utilizing damage scores of individual animals from each group. Comparisons of damage scores were made using the Mann -Whitney U-test for anonparametric data. An associated probability ( p value) of 5% or less was considered significant.

Results Eflecf of hgpsphysectcamy can the gastric rnucssa Sham stimulation experiments in which no electrical stimulation was applied to the vagus nerves did not result in any significant gastric damage to sham surgery animals (average damage score, 0.14, n = 7) as compared with normal animals also undergoing no nerve stimulati~n(0.56, n = 9, p > 8.05). In contrast, significant mucosal damage was observed in hypoghysestomized animals that underwent sham vagal stimulation (1.44, n = 8, p < 0.05, Fig. 2) as compared with both normal and sham surgery animals. Observed damage was apparent by a generalized hyperemia and the focal appearance of hemorrhagic erosions throughout the corpus region, although some damaged areas were occasionally observed in the antmrn. The gastric mucosd surface s f hygophysectomized animals lacked rugd folds compared with both normal and sham surgery animals. A significant thinning s f the stomach wall was noted with some areas being somewhat translucent. No consistent damage was observed in the duodenum apart from local hyperemia. Such macroscopic gastric damage was confirmed histslogicdly as m c o s a l erosions where the lesions did not penetrate the muscularis rnucosae (Fig. 3).

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FIG.3. Photomicrograph of gastric mucosa sf a hypophysectomized rat in which vagal stimulation was applied for 60 min. Slides were stained with hematoxylin and eosin. Note destruction of the epithelium with cellular debris, mucus, and blood visible in the lumen. Damage involved the upper one third of the mucosa and did not penetrate the muscularis mucosae.

FIG.4. These histograms represent individual damage scores from normal (NORMAL), sham surgery (SHAM H Y ~ X and ) , hypophysectomized (HYFOX) animals following a period of either no stimulation (NO STIM)or electrical vagal stimulation (STIM).In (A) normal animals undergoing vagal stimulation were observed to have significantly higher gastric damage scores compared with normal animals not undergoing stimulation. Similarly, in (B), sham surgery animals also displayed significantly higher damage scores compared with sham surgery control aniinals. In contrast, there is no significant difference in the range of damage scores between the nonstimulated and stimulated groups of hypophysectomized animals. *p < 0.05 compared with nonstimulated group (Mann-Whifney U-test).

Eflect ofhypophysectomy on vagal stimulation induced gastric damage Electrical stimulation of intact vagal nerves resulted in gastric damage scores (2.8, n = 9) that were significantly different compared with normal animals undergoing no nerve stimulation (p < 0.85, Fig. 4/11. Similarly, vagal stimulation elicited gastric damage scores in sham surgery animals (1.5,

n = 10) that were significantly different compared with COPresponding sham stimdation control animals ( p < 0.05, Fig. 4B). These vagal stimulation induced damage scores obtained from sham surgery animals were not significantly different ( p > 0.05) from gastric damage scores obtained from normal animals following vagd stimulation. Gastric mucosd damage was again observed following

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FIG.5. The photomicrographs presented in (A) and (B) show typical examples s f stimulation sites both within and outside the PVN, respectively. Hn (C) a schematic representation of two different coronal sections through the hypothalamus in the region of PVN is presented. The anatomical Bwations of both PVN (m, normal and a, hypophysectomized) and non-PVN ( A and 0)stimulation sites are presented. Scale bar, 0.5 mm. V, 3rd ventricle; SON, supraoptic nucleus; OT, optic tract.

vagal nerve stimlalation in hypophysectomized animals (1-25, n = 8). These damage scores were not significantly different compared with vagal induced damage observed in both normal and sham surgery animals ( p > 0.05). Both normal and sham surgery animals exhibited vagd induced damage that was significantly different compared with their corresponding control groups. In contrast, vagal induced damage scores from hypophysectomized animals were not significantly different from those of the corresponding hypophysectomized control group that did not undergo vagal stimulation (g > 0.05, Fig. 4C). Eflect ofhjpophg?sectomy ore P W stimulation-induced gastric damage Hypophysectomized animals in which the PVN was stimulated were assigned to one of two experimental groups according to the anatomically verified location of the stimulating electrode tips, either in the PVN, or in adjacent regions (Fig. 5). Such divisions were carried out prior to knowledge of gastric damage scores. Animals in which stimulation sites were within 0.3 mrn of the nucleus were assigned to the PVN stimulation group (PVN). All other sites were designated as non-PVN sites (non-BVN). Removal of the pituitary gland did not appear to cause any detrimental effects to the PVN, as determined from its morphologicd appearance during histological assessment of stimulation sites.

Hypophysectomized animals exhibited gastric damage following electrical stimulation in non-PVN sites (1. 17, n = 18). These gastric damage scores were significantly different compared with normal animals following stimulation in non-PVN regions ( p < 8.05, Fig. 6B). Gastric mucosal lesions were again observed in hypophysectomized animals following activation of neurons within the PVN. These damage scores (2.00, pa = 6) were not significantly different from gastric damage scores from normal animals following similar PVN stimulation (1.91, n = 1I , p > 0.05, Fig. 6A). PVN induced gastric damage in normal animals was significantly different compared with normal animals in which stimulation was applied in non-PVN regions (0.40, n = 10, g < 8.05). This is in accordance with our previous report that nonspecific stimulation of this hypothalamic region does not induce significant changes to the gastric mucosa of normal animals. On the other hand, PVN induced gastric damage in hypophysectomized animals was not significantly different from damage observed following nonspecific stimulation in non-PVN sites (g > 0.05). Discussion

These studies have demonstrated that gastric damage observed in hypophysectomized animals following electrical stimulation

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FIG. 6. These histograms represent individual gastric damage scores obtained from bath normal (NORMAL) and hypophysectomized Qwu~ox) animals following electrical stimulation in either BVN QPVN) or won-PVN (naon-PVN) regions. Stimulation within non-PVN sites resulted in significantly reduced damage scores in normal animals compared with hypophysecto~nizedanimals (B). In contrast, electrical stimulation within the PVN resulted in significantly higher damage scores in both normal and hypophysectomized animals (A). *g < 0.05 compared with all other groups QMann- Whitney U-test).

of either intact vagal nerves or the hypothalamic PVN is similar in nature and severity to that observed in normal animals following identical neural manipulations. Our previous studies demonstrating that stimulation in PVN induces gastric damage, and that lesion of BVN abolishes the ability of vagal stimulation to cause damage, suggest a comnon role for the PVN in the induction of such gastric damage. The present studies were undertaken to determine whether the BVN may exert such effects through its well-established role in controlling pituitary hormone secretion. However, the results of the present studies suggest that the mechanisms underlying gastric damage induced by such neural manipulations are not dependent upon neural projections to the pituitary gland nor the presence of pituitary hormones. Whereas endocrine states such as hypothyroidism have been shown to significantly increase susceptibility to stress ulcer formation induced by cold-restraint stress (Hernandez el al. 1988), the lack sf pituitary hormones does not appear to enhance the susceptibility of the gastric mucosa to either vagal or PVN stimulation with respect to gastric injury. Considering that neither vagal nor PVN stimulation induced any significant gastric damage compared with the appropriate control groups, such data could be interpreted as suggesting

that hypophysectomy itself removes hormonal factors that are potentially injurious to the gastric mucosa. It has recently been shown that endogenous vasopressin plays a major role in the development of ethanol-induced gastric erosions in rats (Laszlo el a&.1991). Thus perhaps the removal of vasopressin partidy accounts for the lack of further damage to the gastric mucosa compared with control animals. On the other hand hypophysectomy does appear to have an adverse effect on the gastric mucosa under control conditions, suggesting the pituitary hormones play a major role in the maintenance of a normal gastric mucosa. Histological analysis of the gastric mucosa revealed similar damage in both stimulated and nonstimulated groups of hypophyse~tomizedanimals that involved the destruction of the epithelium with cellular debris, blood, and mucus visible in the lumen. Such mucssal damage was confined to the upper one third of the mucosa and was therefore classified as mucosal erosions. Previous studies by other investigators have examined both functional and morphological effects of hypophysectomy on the GI tract. Jacobson and Magnani (1964) have reported a decrease in all indices of gastric secretion with no histological observation of cellular atrophy 6 weeks following hypophysectomy in dogs. Robert et al. (1966) also demonstrated an inhibition of gastric juice secretion with reductions in secretory volume, acidity, and pepsin concentration 3 -4 weeks following hypophysectomy in the rat, with growth hormone treatment restoring these changes nearly completely except for those affecting pepsin, which were unaffected. Morphological changes have been reported by Crean et al. (1968) who observed reductions in the weight of the stomach. surface area, and volume of the hndic mucosa, as well as a reduction in the total parietal cell population, 3 weeks following hypophysectomy in rats. These observations were believed to be a specific effect of pituitary hormone deprivation on the stomach and not a consequence of reduced somatic growth, since underfed sham-surgery animals did not exhibit similar changes. The experiments in the present study were performed within 14 days of hypophysectomy. Thus, it is difficult to assess the specific effects of hypsphysectomy on mucosal morphology and function, considering the relative lack of studies examining the short-term effects of hypophysectomy. Nevertheless, in the present study both vagal and PVN stimulatisn resulted in the formation of gastric mucosal damage in hypophysectomized animals that was similar in nature to that induced in normal animals. Given that gastric acid is required for the formation of gastric lesions and that both neural manipulations are known to cause an immediate increase in acid secretion (Martinson 1965; Rogers and Hermann 1986), the present results suggest an adequate secretory response in the presence of compromised endogenous defence mechanisms. The failure of underlying protective mechanisms to normal stresses ofthe gastric anapcosa was demonstrated by the finding sf significant mucosal damage in control hypophysectomized animals in contrast to normal control animals that exhibited no significant gastric damage. As sham-surgery animals underwent similar surgery for hypophysectomy and did not demonstrate any significant gastric damage, it is clear that gastric injury observed in hypophysectomized control animals was a consequence of the removal of the pituitary gland and its hormones and not a result of the surgery itself. However, it should be recognized that sham-surgery animals were not restricted with respect to weight gain, thus the effect of reduced somatic growth was not specifically addressed. One

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of the first lines of defence of the GI tract mucosa is undoubtedly the ability of the mucosa to replace desquamated or injured surface epithelial cells (Kauffman 1984). The gastric epithelium undergoes constant and rapid renewal, with a turnover rate of approximately 3 days in the rat (Grant et al. 1953). The rapid turnover rate of the GI tract mucosa requires that such proliferation and growth are balanced by cell loss so that under normal conditions cell populations are maintained in a steady state. In his review concerning the regulation of GI mucosal growth, Johnson (1988) reminds us that GI mucosal growth involves not only various GI peptides and luminal factors but is also affected by the same hormones that alter metabolism in other tissues, which include insulin, cortisol, thyroxine, and growth hormone. The integrity of the GI tract mucosa is therefore undoubtedly at risk following the removal of the pituitary gland due to the removal of these hormones that affect the replication process. As a consequence of the rapid turnover rate, tissue morphology as well as mucosal function are quickly affected following the loss of such factors. Atrophy, hyposecretion, and decreases in absorption and perhaps ulceration can therefore occur due to insufficient cell production as a result of hypophysectomy (Johnson 1988). In a study examining the healing of ulcers produced by thermal injury, a delay in epithelial proliferation and thus a delay in regeneration of the mucosa was shown in adrenalectomized but to a greater extent in hypophysectomized animals compared with normal animals thought to be due to a general slowing down of growth as a result of the lack of pituitary hormones (Skoryna et al. 1958). Therefore it appears hypophysectomy renders the mucosa more susceptible to the normal stresses placed upon it, owing to its limited proliferative activity. Indeed gastrin, a potent tropic hormone in the bndic mucosa, has been shown to prevent stress-induced ulcer formation in the rat (Takeuchi and Johnson 1979), while restraint stress-induced gastric mucosal ulceration can be partially prevented by growth hormone (Vanamee et al. 1991). More importantly to the development of mucosal erosion during short-term electrical stimulation of vagal nerves or PVN is the mucosal response to injury termed epithelial restitution. Restitution refers to the process of rapid repair of the mucosa, which is different from the healing process (Ito et a&. 1984). The healing process involves an inflammatory component, and occurs over a relatively long period of time during which the necrotic cells are replaced by healthy cells via mitosis as discussed earlier. On the other hand, restitution involves migrating epithelial cells and occurs from minutes to several hours but clearly in a short enough time to exclude mitosis. Such restitution of the epithelium occurs in areas of superficial injury as opposed to the repair of deep injury (Morris and Wallace 1981). The process of restitution following superficial gastric mucosal epithelium most likely takes place in the stomach on a regular basis. Any impairment in the migration of cells, as is possible following hypophysectomy where the lack of pituitary hormones adversely affects individual cell metabolism and thus function, impairs the ability of the mucosa to protect itself from injury. A relationship between the pituitary and GI lesions has been suggested in previous reports, but the data have been too conflicting and incomplete to warrant a definitive statement regarding the role of the pituitary in the pathogenesis of gastric ulcers. Acute ulcerations and hemorrhagic erosions have been reported in 8 of 18 dogs following hypophysectomy however hypothalamic damage was present (Keller and D' Amour 1934),

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whereas posterior pituitary extracts injected into rats have been found to cause gastric lesions (Dodds et al. 1935). As well, gastric biopsies from patients with hypopituitarism have demonstrated gastric erosions with atrophy or a normal mucosa (Schapiro eb al. 1970). The effect of the pituitary may also affect experimentally induced ulcers and appears to vary according to the method used to produce the ulcers. Although hypophysectomy does not influence restraint ulcers in the rat (Menguy 1960), hypophysectomy does cause a delay in the healing of ulcers produced by punch-out excision of gastric mucosa in the rat (Skoryna et al. 1958). Robert et al. (1966) demonstrated that the incidence of steroid-induced ulcers was reduced in animals 3 weeks following hypophysectomy, and further, that the administration of growth hormone increased the healing of such ulcers. In this study, hypophysectomized animals tliat were administered vehicle alone were not found to have any mucosal injury 3 weeks following surgery as opposed to the results of the present study. More recently, a study examining intracisternal neurotensininduced gastric protection following cold-restraint stress demonstrated that hypophysectomized rats expressed a reduced rate of protection in this response to neurotensin (Hernandez st a&. 1987). This study demonstrated a time-dependent effect of hypophysectomy in that neurotensin was partially effective in its protective effects 5 days following hypophysectomy but completely ineffective in hypophysectomized animals 14 days following removal of the pituitary gland. Stomach weight was significantly reduced 14 days, but not 5 days, aker hypophysectomy in agreement with previous studies suggesting hypophysectomy decreases stomach weight as well as other indices of gastric function. These results again suggest that long-term modulation of the gastric mucosa by the pituitary is required for tissue resistance against stress. In conclusion, these studies suggest that the development of vagal stimulation- and PVN stimulation-induced damage does not directly involve the neurosecretory projections of the PVN to either the posterior pituitary or median eminence and develops independently from this gland's hormonal effects on the gastric mucosa. It is clear from these studies, however, that the maintenance of an intact healthy mucosa requires the presence of the pituitary gland and its hormones. Considering these effects of pituitary hormones on the GI rnucosal integrity and growth, the use of hypophysectomized animals in studies examining the effect of pituitary hormones should be interpreted with caution and in certain circumstances m y in fact be inappropriate.

Acknowledgments This research was supported by the Medical Research Council sf Canada (MRC). k.E. Hierlihy is supported by an MRC Studentship. Thanks also are extended to Pauline Smith for technical assistance. Crean, G. P. 1968. Effect of hypophysectomy on the gastric mucosa of the rat. Gut, 9: 332-342. Dodds, E. C., Hills, G. M., Noble, R. L., and Williams, P. C. 1935. The posterior lobe of the pituitary gland: Hts relationship to the stomach and to the blood picture. Lancet, 228: 2099 - 1180. Donevan, S. D., and Ferguson: A. V. 1988. Subfornical organ and cardiovascular influences on identified septa1 neurons. Am. J. Physiol. 254: R544 -R55 I . Ferguson, A. V., and Renaud, %. P. 1984. Hypothalamic paraventricular nucleus lesions decrease pressor responses to subfornical organ stimulation. Brain Res. 305: 361 -364.

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