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Surface hydrophobicity of the gastric mucosa in the developing rat
GASTROENTEROLOGY
1988;94:57-81
Surface Hydrophobicity of the Gastric Mucosa in the Developing Rat Effects of Corticosteroids, Prostaglandin E2 ELIZABETH
J. DIAL and LENARD
Department of Physiology Houston, Texas
and Cell Biology,
n an earlier report we obtained evidence that the gastric mucosa of the suckling rat is intrinsically more sensitive to damage by luminal HCl than is the adult (l), and that the neonate also lacks a sensitivity to 16,16-dimethyl prostaglandin Ez (dmPGEJ for a gastric protective response (2,3). Treatment of the suckling rat with hydrocortisone results in the precocious induction of an intrinsic gastric protective property, but has no influence on the ontogenic appearance of a “cytoprotective” response to dmPGEz (l-3). As a result of these findings we have
and
M. LICHTENBERGER
University
Surface hydrophobicity of the gastric mucosa and its variation in response to treaiments with corticosteroids, thyroxine, and 16,16-dimethyl prostaglandin Ez were measured in developing rats. A d&elopmenttil increase in the hydrophobicity of the lumitial surface of the gastric mucosa ,was r6corded between the first and third weeks of life. The hydrophobicity bf the stomach was ncit consistently influenced by an acute adininistration of 16,16-dimethyl prostaglandin Ez (5 &kg, 30 min before examination) until the end of the third week of life, at which time a significant 40% increase was recorded. Similarly, the decrease in surface hydrophobicity that resulted from luminal administration of an ulcerogenie dose of HCl (0.6 N, 6 ml/kgj Gas blocked by i6,l&dimethyl prostaglandin Ez only in 3-wk-old rats and not ifi rats 1 and 2 wk of age. Neither the normal developmental increase nor the 16,16dimethyl piostaglandin Ez-induced enhancement in gastric surface hydiophobicity was induced precociousiy by corticosterone or thyrdxine. The posiible importance of these findings on the development of gastric surface hydrophobicity to the ontogeny of both gastric barrier function and prostaglandininduced gastric protection is discussed.
I
Thyroxine,
of Texas
Medical
School
at Houston,
proposed that the “gastric barrier” to acid is immature in suckling rats, and consequently these stomachs do not possess the ability to either intrinsically protect themselves from luminal damaging agents or to mount a cytoprotective response after prosttiglandins are administered exogenously. In the adult dog, the gastric mucosa posse&es a relatively hydrophobic, or nonwettable, surface (4). Using an in vitro system our laboratory has demonstrated that gastric damaging agents reduce this hydrophobic property, whereas it is increased by dmPGEz treatment (5). It was postulated that dmPGE, may exert its gastric protective action, in part, by maintaining or elevating luminal hydrophobicity in the presence of damaging agents. In the present study we have investigated the surface hidrophobicity of the developing rat gastric mucosa to determine whether increased sensitivity of the neonatal stomach to damage by HCl may be related to a more wettable (lower hydrophobicity) gastric muizosal surface. In addition, we have examined the postnatal changes in gastric mucosal hydrophobicity (intrinsic and dmPGE,-stimulated) of suckling rats treated with corticosteroids or thyroxine, hormones that are known to influence the maturation of the gastrointestinal tract. Methods Animals Pregnant Sprtigue-Dawley rats (Harlan-SpragueDawley, Houston, Tex.) were obtained several days before Abbreviations used in this paper: CO, corticosterone; dmPGE,, 18,18-dimethyl prostaglandin E,; HC, hydracortisone; T4, thyroxine. 0 1988 by the American Gastroenterological Association 0018~5085/88/$3.50
58 DIAL AND LICHTENBERGER
giving birth. The age of newborn rats could thus be accurately determined. Litter size was limited to 10 rats per female. For most experiments the animals were fasted for 18 h before use by either removing the pups from the mother (before weaning] or removing the Rat Chow (Purina Formula 5008, Ralston Purina, St. Louis, MO.) from the cages (after weaning).
Gastric Mucosal Hydrophobicity Measurements The surface hydrophobicity of the gastric mucosa was determined on a goniometer (Rame-Hart, Inc., Mountain Lakes, N.J.) by measurement of the contact angle that formed between a droplet of water (0.5 ~1) and the stomach surface. As previously described, the contact angle measurement serves as an estimate of the degree of hydrophobicity of the mucosal surface (4,6). The stomachs were taken from animals anesthetized and then killed with ether, and the antrum and forestomach were removed. The remaining corpus was opened along the lesser curvature and spread flat onto a piece of filter paper with the mucosal side upward. The tissue was gently blotted twice with a thin paper wipe to remove adherent mucus and excess fluid, and the stomach was air-dried for 10 min. This partial drying procedure was necessitated as contact angles cannot be measured on a wet surface. Contact angle readings were made in at least four different locations along the oxyntic mucosa and the average was used as one value for that stomach.
Statistical
Methods
All values are reported as the mean * standard error. Differences between treatment groups were analyzed by Student’s t-test using p < 0.05 as the level of significance.
Experimental
Protocols
The four studies outlined below were designed to investigate the ontogenic alterations in the surface hydrophobicity of the gastric mucosa of both a control population of rat pups (study 1) and suckling rats treated with hormones to induce the precocious maturation of gastric barrier properties (study 2). In addition, the effect of a cytoprotective dose of dmPGE2 was investigated in control (study 3) and hormone-treated rats (study 4) during the first 3 wk of life. Study 1. Developmental changes in gastric mucoOne rat from each of three to sol surface hydrophobicity. four litters was removed daily to measure gastric hydrophobicity in fully fed rats. In addition, fasted rats from other litters were examined every other day. Weaning was performed at an age of 22 days. Study 2. Effect of corticosteroids and thyroxine on gastric mucosal surface hydrophobicity. Four litters of rats were used for each of the following three experiments. At the age of 7 days, 1 rat from each litter was used for control hydrophobicity measurements. Each litter was then divided; half of the pups (4-5 rats) were injected with saline (controls] and the remainder (4-5 rats] with the following test hormones: hydrocortisone (HC, 10 mg/kg),
GASTROENTEROLOGYVol. 94, No. 1
study 2-l; thyroxine (T4, 0.1 mg!kg, daily), study 2-2; and corticosterone (CO, 10 m&g, daily) plus thyroxine (0.1 mg/kg, daily), study 2-3. On days E&9,10, and 11 one litter from each study (4-5 rats for control and test) was examined for changes in gastric mucosal, hydrophobicity. Study 3. Efiect of 16,16-dimethyl prostaglandin Ez on gastric mucosal surface hydrophobicity in control and acid-challenged developing rats. Fasted littermate rats at 7, 14, and 21 days were used. The animals were injected subcutaneously with either dmPGEz at 5 pg!kg or an equivalent volume of saline (controls). After 30 min, half of the rats in each group were anesthetized and their stomachs were prepared for contact angle measurements, while the other half received an oral dose of 0.6 N HCl (6 ml/kg) and had their gastric contact angles read 60 min later. Study 4. E#ect of 16,16-dimethyl prostaglandin E2 on gastric mucosal surface hydrophobicity in corticosterone- and thyroxine-treated rats. Four litters of rats were treated on days 7-10 of life with either saline (control), T4, CO, or T4 + CO at doses described above in study 2. On day 11 of life, each litter was divided, half were injected with saline (4-5 rats) and half with dmPGEz (5@kg s.c., 4-5 rats), and gastric contact angles were measured 30 min later.
Results Study 1. Developinental Changes in Gastric Mucosal Surface Hydrophobicity Figure 1 contains the combined results of gastric mucosal contact angle measurements made in fed and fasted rats from 1 day of age to 44 days. The contact angle values were not statistically different between fed and fasted rats at any one developmental period. The contact angle readings increased between days 7 and 21 and were greater by approximately twofold at the end of the third week of life. Regression analysis of the data between 7 and 22 days revealed a positive relationship between age 60
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Figure
I. Gastric mucosal hydrophobicity in developing rats. The stomachs of fed or fasted rats were measured for contact angles as described in Methods. Each point is the mean of 3-1 animals from separate litters with at least four readings averaged per animal.
HYDROPHOBICITY AND DEVELOPMENT
January 1988
59
and surface hydrophobicity with r = 0.526 and p < 0.05. This elevation in the hydrophobicity of the surface of the gastric mucosa by day 21 was coincident with the end of the period of weaning. Study 2. Effect of Corticosteroids and Thyroxine on Gastric Mucosal Surface Hydrophobicity Hydrocortisone treatment of 7-day-old rats (study 2-1) did not appear to influence the ontogenic increase in gastric mucosal surface hydrophobicity (Figure 2). Similarly, T4 treatment (study 2-2) had no real influence on this developmental parameter. The combination of CO and T4 (study 2-3), likewise, did not affect the ontogenic development of a nonwettable gastric mucosal surface. Study 3. Effect of 16,16-Dimethyl Prostaglandin Ez on Gastric Mucosal Surface Hydrophobicity of Control and AcidChallenged Developing Rats Control rats at 7, 14, and 21 days (Figure 3) showed a developmental increase in contact angle readings in confirmation with the results in Figure 1. . Sable 0 Hydrocartlsone
4orA
6 ”
n Thyroxw? o-
10
0
7
c
8
7 days
0
Saline
dm PGE,
control
(5 rg/kg)
dmPGE,
plus
acld
challenge
Figure 3. Effect of dmPGE, on gastric mucosal surface hydrophobicity in control and acid-challenged developing rats. Gastric contact angles were measured in rats 30 min after treatment with saline or dmPGE2 (5 &kg s.c.) and 60 min after an oral dose of 0.6 N HCl (6 ml/kg). Values are expressed as the mean degrees t SE for (3-8) rats as in Figure 1.
Treatment with dmPGEz alone had no significant effect until day 21, when an acute increase in contact angle readings occurred 30 min after drug administration. However, it should be noted that at day 14 an initial, but nonsignificant increase (p < 0.2) in surface hydrophobicity was observed in response to dmPGE, treatment. The ulcerogenic challenge with acid produced gross hemorrhagic lesions in suckling rats and, in confirmation with our earlier report, this was not clearly prevented by treatment with dmPGEz until the third week of life (3). In addition, acid treatment significantly reduced the gastric contact angles in both control and dmPGEz-treated rats on days 7 and 14 of life. However, on day 21 the dmPGEa-treated rats maintained their gastric mucosal surface hydrophobicity in the presence of acid, whereas control rats showed a significant reduction.
0 Cortlcosterone +Thyraxw > # 1 9
10
11
Age (days)
Figure 2. Effect of corticosteroids and T4 on gastric mucosal surface hydrophobicity. A. Study 2-l. Half of each rat litter was injected with saline and half with HC (10 m&g) on day 7 of age. B. Study Z-Z. Half of each litter was injected with saline and half with T4 (0.1 mg/kg) on days 7, 8,9, and 10 of age. C. Study 2-3. Half of each litter was injected with saline and half with T4 (daily) plus CO (10 mg/kg, daily) for days 7, 8, 9, and 10 of life. Gastric contact angles were measured daily. Values are expressed as the mean degrees f SE for 4-5 rats as in Figure 1.
Study 4. Effect of 16,16-Dimethyl Prostaglandin E2 on Gastric Surface Hydrophobicity in Corticosteroneand Thyroxine-Treated Rats Table 1 shows dmPGE, treatment on developing rats. On response to dmPGE,, treatment.
the results the surface day 11 of either alone
of hormonal plus hydrophobicity of life there was no or after CO or T4
60
DIAL AND LICHTENBERGER
GASTROENTEROLOGY Vol. 94, No. 1
Table 1. Effect of 16,16-Dimethyl Gastric Surface Corticosterone-
Prostaglandin Hydrophobicity of and Thyroxine-Treated Saline
Saline control co T4 CO + T4
100 100 100 100
* 2 f +
13 17 8 10
Ez on Rats dmPGEz 80 83 107 92
k t f k
9 4 22 8
dmPGEp, 16,16-dimethyl prostaglandin Ez; CO, corticosterone; T4, thyroxine. Separate litters were treated on days 7,8,9, and 10 of life with either saline, CO (10 mg/kg), T4 (0.1 mg/kg), or CO plus T4. On day 11 of age half of each litter was injected with saline and half with dmPGEz (5 &kg s.c.). Gastric contact angles were measured 30 min later. Values are expressed as the percentage of saline (control) t SE for 4-6 animals per group (see Methods].
Discussion In previous reports (l-31, we have obtained evidence that the ability of the rat stomach to intrinsically protect itself against damaging agents is immature in the suckling rat. Similarly, the neonatal rat stomach is unresponsive to the cytoprotective action of prostaglandins until the end of the second week of life. It is our contention that the development of these protective properties in the rat stomach is, in part, related to age-dependent increases in surface hydrophobicity, which we report here. The use of contact angle measurements as assessments of surface hydrophobicity was previously reported by our laboratory for dog gastric tissue, both freshly excised (4) and after in vitro incubation (5). In each case the tissue was lightly blotted to remove excess moisture and was allowed to air-dry for a short time before contact angle readings. More recent work (6) has indicated that control blotted and dried dog tissue remains intact, as viewed at the lightmicroscopic level. Thus, the partially dried tissue may be a reasonable reflection of the state of the tissue surface at death. As discussed by Goddard et al. (S), contact angle measurements may be the only technique available to study surface properties of sheets of cells. Therefore, the changes seen here in contact angle measurements of the rat gastric mucosa represent a new and unique procedure for the study of developmental phenomena. The gradual twofold increase in gastric surface hydrophobicity seen by the third week of life may be triggered or associated with a number of factors. We have investigated three of these: (a) the maturational increase in circulating corticosteroids, (b) the increase in circulating thyroxine, and (c) the ontogenic appearance of the ability of dmPGEz to induce a gastric protective response. A fourth factor, the presence of food in the stomach, was studied only for an acute effect by comparing contact angles of gastric
mucosa excised from fasted and fed animals. Longterm effects of early or late weaning were not investigated in this study. It was determined that the ontogenic increase in gastric mucosal hydrophobicity appeared to be independent of the acute presence of food, as fasted rats showed no clear differences from fully fed animals. We cannot eliminate the possibility that factors in the milk diet could be affecting surface hydrophobicity on a more chronic basis (7). Circulating levels of CO rise in the young rat after day 12 of age (8) and thus initiate many maturational processes in the gastrointestinal tract (9). Because we have previously noted a profound effect of HC on the resistance of the suckling rat gastric mucosa to luminal acid (3), we examined the possibility that HC may be acting through an increase in the hydrophobicity of the mucosal surface. However, that possibility does not appear to be the case, as administration of HC to Y-day-old rats failed to precociously induce the developmental increase in gastric hydrophobicity. Thyroxine also shows developmental increases in circulating levels during the suckling period (10). However, T4 alone or in combination with CO (the endogenous rat corticosteroid) did not influence the developmental appearance of gastric mucosal surface hydrophobicity. Thus, we feel that neither T4 nor CO has a direct role in regulating the ontogenic appearance of this effect. These results do not eliminate a possible indirect effect, however, through permissive actions of either of these hormones. In earlier reports (2,3) we indicated that dmPGEz failed to induce an adult cytoprotective response in suckling rats until the third week of life. Earlier ages (14 days) showed proportionally smaller responses. In this study we have presented evidence that, similar to its antiulcer efficacy, dmPGEz initiated an acute increase in surface hydrophobicity of the rodent gastric mucosa between the second and third week of life. The dmPGE,-induced increase in hydrophobicity at 14 days of age (25%) corresponded with the attenuated gastric protective effect at this age (30% of adult values), although it did not reach statistical significance (p < 0.2). The similar ontogenie time-courses of these two prostaglandindependent responses (surface hydrophobicity and gastric protection) thus suggest that they may be linked in some manner. Indeed, we would speculate that one possible mechanism by which dmPGEl can induce epithelial protection is by increasing the hydrophobicity of the gastric surface, thus preventing the movement of luminal acid into the underlying tissue. Although prostaglandins clearly have a variety of actions that contribute to protection of the gastric mucosa (ll), we believe that the effect on
January 1988
hydrophobicity may be of considerable importance, as it is the first line of defense of the tissue against a luminal damaging agent. However, whether the developmental increase in surface hydrophobicity of the gastric mucosa is causally or only temporally linkedto the development of gastric mucosal protection remains to be determined. Consistent with our earlier report on protection of the gastric mucosa by HC and dmPGE2, which appear to possess additive and somewhat independent effects, we note here that dmPGEz affects surface hydrophobicity whereas HC does not. In the previous study we reported that HC failed to precociously induce the appearance of a cytoprotective response to prostaglandins. In the present study it was demonstrated that the normal ontogenic appearance of a hydrophobic response to prostaglandins similarly was not influenced if 1-wk-old rats were treated with exogenous HC. In addition, we can now state that T4 also does not affect the appearance of this hydrophobic response to dmPGE,. These findings, therefore, emphasize the point that surface hydrophobicity represents one of many gastric properties that contribute to the intrinsic acid resistance of the stomach. The property that is influenced by corticosteroids resulting in a precocious development of acid resistance has yet to be resolved, but appears to be unrelated to the ontogenic changes in surface wettability. A possible explanation for this steroidinduced effect may lie with the mucous cell, as Yeomans et al. (12) have reported that mucous cell differentiation in the rat gastric mucosa in organ culture is influenced by the adrenal corticosteroids. In summary, we have found that there is a developmental increase in gastric mucosal surface hydrophobicity in the rat that occurs by the end of the third week of life and is coincident with the end of the period of weaning. Concurrently, there is an increased responsiveness to dmPGE2, reflected in both an acute increase in surface hydrophobicity and in protection against luminal damaging agents. The developmental signal(s) that influence these gastric properties to ensure that the stomach of the young
HYDROPHOBICITY
rat is protected against luminal to be fully characterized.
AND DEVELOPMENT
ulcerogens
61
have yet
References 1. Dial EJ, Lichtenberger LM. Development of the gastric mucosal barrier to acid in the rat (abstr). Dig Dis Sci 1985;30:372. 2. Dial EJ, Lichtenberger LM. Development of the cytoprotective response to 16,16-dimethyl prostaglandin EZ in the rat (abstr). Gastroenterology 1985;88:1365. LM. Development of gastric mucosal 3. Dial EJ, Lichtenberger protection against acid in the rat: role of corticosteroids and prostaglandins. Gastroenterology 1986;91:318-25. 4. Hills BA, Butler BD, Lichtenberger LM. Gastric mucosal barrier: hydrophobic lining to the lumen of the stomach. Am 1Physiol 1983;244:G561-8. 5. Lichtenberger LM, Richards JE, Hills BA. Effect of 16,16dimethyl prostaglandin Ez on the surface hydrophobicity of aspirin-treated canine gastric mucosa. Gastroenterology 1985;88:308-14. 6. Goddard PJ, Hills BA, Lichtenberger LM. Does aspirin damage canine gastric mucosa by reducing its surface hydrophobicity? Am J Physiol 1987;252:G421-30. 7. Dial EJ, Lichtenberger LM. A role for milk phospholipids in protection against gastric acid. Gastroenterology 1984;87: 379-85. of total and free corticos8. Henning SJ. Plasma concentrations terone during development in the rat. Am J Physiol 1978; 235:E451-6. 9 Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 1981;241:G199-214. 10. Walker P, Dubois JD, Dussault JH. Free thyroid hormone concentrations during postnatal development in the rat. Pediatr Res 1980;14:247-9. 11 Miller TA. Protective effects of prostaglandin against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol 1983;245:G601-3. 12. Yeomans ND, Trier JS, Moxey PC, Markezin ET. Maturation and differentiation of cultured fetal stomach. Effects of corticosteroids, pentagastrin, and cytochalasin B. Gastroenterology 1976;71:770-7.
Received August 4, 1986. Accepted July 27, 1987. Address requests for reprints to: Elizabeth J. Dial, Ph.D., Department of Physiology and Cell Biology, University of Texas Medical School, P.O. Box 20708, Houston, Texas 77225. This work was supported by grants HLI 13021 and AM 33239 from the National Institutes of Health.
1988;94:57-81
Surface Hydrophobicity of the Gastric Mucosa in the Developing Rat Effects of Corticosteroids, Prostaglandin E2 ELIZABETH
J. DIAL and LENARD
Department of Physiology Houston, Texas
and Cell Biology,
n an earlier report we obtained evidence that the gastric mucosa of the suckling rat is intrinsically more sensitive to damage by luminal HCl than is the adult (l), and that the neonate also lacks a sensitivity to 16,16-dimethyl prostaglandin Ez (dmPGEJ for a gastric protective response (2,3). Treatment of the suckling rat with hydrocortisone results in the precocious induction of an intrinsic gastric protective property, but has no influence on the ontogenic appearance of a “cytoprotective” response to dmPGEz (l-3). As a result of these findings we have
and
M. LICHTENBERGER
University
Surface hydrophobicity of the gastric mucosa and its variation in response to treaiments with corticosteroids, thyroxine, and 16,16-dimethyl prostaglandin Ez were measured in developing rats. A d&elopmenttil increase in the hydrophobicity of the lumitial surface of the gastric mucosa ,was r6corded between the first and third weeks of life. The hydrophobicity bf the stomach was ncit consistently influenced by an acute adininistration of 16,16-dimethyl prostaglandin Ez (5 &kg, 30 min before examination) until the end of the third week of life, at which time a significant 40% increase was recorded. Similarly, the decrease in surface hydrophobicity that resulted from luminal administration of an ulcerogenie dose of HCl (0.6 N, 6 ml/kgj Gas blocked by i6,l&dimethyl prostaglandin Ez only in 3-wk-old rats and not ifi rats 1 and 2 wk of age. Neither the normal developmental increase nor the 16,16dimethyl piostaglandin Ez-induced enhancement in gastric surface hydiophobicity was induced precociousiy by corticosterone or thyrdxine. The posiible importance of these findings on the development of gastric surface hydrophobicity to the ontogeny of both gastric barrier function and prostaglandininduced gastric protection is discussed.
I
Thyroxine,
of Texas
Medical
School
at Houston,
proposed that the “gastric barrier” to acid is immature in suckling rats, and consequently these stomachs do not possess the ability to either intrinsically protect themselves from luminal damaging agents or to mount a cytoprotective response after prosttiglandins are administered exogenously. In the adult dog, the gastric mucosa posse&es a relatively hydrophobic, or nonwettable, surface (4). Using an in vitro system our laboratory has demonstrated that gastric damaging agents reduce this hydrophobic property, whereas it is increased by dmPGEz treatment (5). It was postulated that dmPGE, may exert its gastric protective action, in part, by maintaining or elevating luminal hydrophobicity in the presence of damaging agents. In the present study we have investigated the surface hidrophobicity of the developing rat gastric mucosa to determine whether increased sensitivity of the neonatal stomach to damage by HCl may be related to a more wettable (lower hydrophobicity) gastric muizosal surface. In addition, we have examined the postnatal changes in gastric mucosal hydrophobicity (intrinsic and dmPGE,-stimulated) of suckling rats treated with corticosteroids or thyroxine, hormones that are known to influence the maturation of the gastrointestinal tract. Methods Animals Pregnant Sprtigue-Dawley rats (Harlan-SpragueDawley, Houston, Tex.) were obtained several days before Abbreviations used in this paper: CO, corticosterone; dmPGE,, 18,18-dimethyl prostaglandin E,; HC, hydracortisone; T4, thyroxine. 0 1988 by the American Gastroenterological Association 0018~5085/88/$3.50
58 DIAL AND LICHTENBERGER
giving birth. The age of newborn rats could thus be accurately determined. Litter size was limited to 10 rats per female. For most experiments the animals were fasted for 18 h before use by either removing the pups from the mother (before weaning] or removing the Rat Chow (Purina Formula 5008, Ralston Purina, St. Louis, MO.) from the cages (after weaning).
Gastric Mucosal Hydrophobicity Measurements The surface hydrophobicity of the gastric mucosa was determined on a goniometer (Rame-Hart, Inc., Mountain Lakes, N.J.) by measurement of the contact angle that formed between a droplet of water (0.5 ~1) and the stomach surface. As previously described, the contact angle measurement serves as an estimate of the degree of hydrophobicity of the mucosal surface (4,6). The stomachs were taken from animals anesthetized and then killed with ether, and the antrum and forestomach were removed. The remaining corpus was opened along the lesser curvature and spread flat onto a piece of filter paper with the mucosal side upward. The tissue was gently blotted twice with a thin paper wipe to remove adherent mucus and excess fluid, and the stomach was air-dried for 10 min. This partial drying procedure was necessitated as contact angles cannot be measured on a wet surface. Contact angle readings were made in at least four different locations along the oxyntic mucosa and the average was used as one value for that stomach.
Statistical
Methods
All values are reported as the mean * standard error. Differences between treatment groups were analyzed by Student’s t-test using p < 0.05 as the level of significance.
Experimental
Protocols
The four studies outlined below were designed to investigate the ontogenic alterations in the surface hydrophobicity of the gastric mucosa of both a control population of rat pups (study 1) and suckling rats treated with hormones to induce the precocious maturation of gastric barrier properties (study 2). In addition, the effect of a cytoprotective dose of dmPGE2 was investigated in control (study 3) and hormone-treated rats (study 4) during the first 3 wk of life. Study 1. Developmental changes in gastric mucoOne rat from each of three to sol surface hydrophobicity. four litters was removed daily to measure gastric hydrophobicity in fully fed rats. In addition, fasted rats from other litters were examined every other day. Weaning was performed at an age of 22 days. Study 2. Effect of corticosteroids and thyroxine on gastric mucosal surface hydrophobicity. Four litters of rats were used for each of the following three experiments. At the age of 7 days, 1 rat from each litter was used for control hydrophobicity measurements. Each litter was then divided; half of the pups (4-5 rats) were injected with saline (controls] and the remainder (4-5 rats] with the following test hormones: hydrocortisone (HC, 10 mg/kg),
GASTROENTEROLOGYVol. 94, No. 1
study 2-l; thyroxine (T4, 0.1 mg!kg, daily), study 2-2; and corticosterone (CO, 10 m&g, daily) plus thyroxine (0.1 mg/kg, daily), study 2-3. On days E&9,10, and 11 one litter from each study (4-5 rats for control and test) was examined for changes in gastric mucosal, hydrophobicity. Study 3. Efiect of 16,16-dimethyl prostaglandin Ez on gastric mucosal surface hydrophobicity in control and acid-challenged developing rats. Fasted littermate rats at 7, 14, and 21 days were used. The animals were injected subcutaneously with either dmPGEz at 5 pg!kg or an equivalent volume of saline (controls). After 30 min, half of the rats in each group were anesthetized and their stomachs were prepared for contact angle measurements, while the other half received an oral dose of 0.6 N HCl (6 ml/kg) and had their gastric contact angles read 60 min later. Study 4. E#ect of 16,16-dimethyl prostaglandin E2 on gastric mucosal surface hydrophobicity in corticosterone- and thyroxine-treated rats. Four litters of rats were treated on days 7-10 of life with either saline (control), T4, CO, or T4 + CO at doses described above in study 2. On day 11 of life, each litter was divided, half were injected with saline (4-5 rats) and half with dmPGEz (5@kg s.c., 4-5 rats), and gastric contact angles were measured 30 min later.
Results Study 1. Developinental Changes in Gastric Mucosal Surface Hydrophobicity Figure 1 contains the combined results of gastric mucosal contact angle measurements made in fed and fasted rats from 1 day of age to 44 days. The contact angle values were not statistically different between fed and fasted rats at any one developmental period. The contact angle readings increased between days 7 and 21 and were greater by approximately twofold at the end of the third week of life. Regression analysis of the data between 7 and 22 days revealed a positive relationship between age 60
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40
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Age (days)
Figure
I. Gastric mucosal hydrophobicity in developing rats. The stomachs of fed or fasted rats were measured for contact angles as described in Methods. Each point is the mean of 3-1 animals from separate litters with at least four readings averaged per animal.
HYDROPHOBICITY AND DEVELOPMENT
January 1988
59
and surface hydrophobicity with r = 0.526 and p < 0.05. This elevation in the hydrophobicity of the surface of the gastric mucosa by day 21 was coincident with the end of the period of weaning. Study 2. Effect of Corticosteroids and Thyroxine on Gastric Mucosal Surface Hydrophobicity Hydrocortisone treatment of 7-day-old rats (study 2-1) did not appear to influence the ontogenic increase in gastric mucosal surface hydrophobicity (Figure 2). Similarly, T4 treatment (study 2-2) had no real influence on this developmental parameter. The combination of CO and T4 (study 2-3), likewise, did not affect the ontogenic development of a nonwettable gastric mucosal surface. Study 3. Effect of 16,16-Dimethyl Prostaglandin Ez on Gastric Mucosal Surface Hydrophobicity of Control and AcidChallenged Developing Rats Control rats at 7, 14, and 21 days (Figure 3) showed a developmental increase in contact angle readings in confirmation with the results in Figure 1. . Sable 0 Hydrocartlsone
4orA
6 ”
n Thyroxw? o-
10
0
7
c
8
7 days
0
Saline
dm PGE,
control
(5 rg/kg)
dmPGE,
plus
acld
challenge
Figure 3. Effect of dmPGE, on gastric mucosal surface hydrophobicity in control and acid-challenged developing rats. Gastric contact angles were measured in rats 30 min after treatment with saline or dmPGE2 (5 &kg s.c.) and 60 min after an oral dose of 0.6 N HCl (6 ml/kg). Values are expressed as the mean degrees t SE for (3-8) rats as in Figure 1.
Treatment with dmPGEz alone had no significant effect until day 21, when an acute increase in contact angle readings occurred 30 min after drug administration. However, it should be noted that at day 14 an initial, but nonsignificant increase (p < 0.2) in surface hydrophobicity was observed in response to dmPGE, treatment. The ulcerogenic challenge with acid produced gross hemorrhagic lesions in suckling rats and, in confirmation with our earlier report, this was not clearly prevented by treatment with dmPGEz until the third week of life (3). In addition, acid treatment significantly reduced the gastric contact angles in both control and dmPGEz-treated rats on days 7 and 14 of life. However, on day 21 the dmPGEa-treated rats maintained their gastric mucosal surface hydrophobicity in the presence of acid, whereas control rats showed a significant reduction.
0 Cortlcosterone +Thyraxw > # 1 9
10
11
Age (days)
Figure 2. Effect of corticosteroids and T4 on gastric mucosal surface hydrophobicity. A. Study 2-l. Half of each rat litter was injected with saline and half with HC (10 m&g) on day 7 of age. B. Study Z-Z. Half of each litter was injected with saline and half with T4 (0.1 mg/kg) on days 7, 8,9, and 10 of age. C. Study 2-3. Half of each litter was injected with saline and half with T4 (daily) plus CO (10 mg/kg, daily) for days 7, 8, 9, and 10 of life. Gastric contact angles were measured daily. Values are expressed as the mean degrees f SE for 4-5 rats as in Figure 1.
Study 4. Effect of 16,16-Dimethyl Prostaglandin E2 on Gastric Surface Hydrophobicity in Corticosteroneand Thyroxine-Treated Rats Table 1 shows dmPGE, treatment on developing rats. On response to dmPGE,, treatment.
the results the surface day 11 of either alone
of hormonal plus hydrophobicity of life there was no or after CO or T4
60
DIAL AND LICHTENBERGER
GASTROENTEROLOGY Vol. 94, No. 1
Table 1. Effect of 16,16-Dimethyl Gastric Surface Corticosterone-
Prostaglandin Hydrophobicity of and Thyroxine-Treated Saline
Saline control co T4 CO + T4
100 100 100 100
* 2 f +
13 17 8 10
Ez on Rats dmPGEz 80 83 107 92
k t f k
9 4 22 8
dmPGEp, 16,16-dimethyl prostaglandin Ez; CO, corticosterone; T4, thyroxine. Separate litters were treated on days 7,8,9, and 10 of life with either saline, CO (10 mg/kg), T4 (0.1 mg/kg), or CO plus T4. On day 11 of age half of each litter was injected with saline and half with dmPGEz (5 &kg s.c.). Gastric contact angles were measured 30 min later. Values are expressed as the percentage of saline (control) t SE for 4-6 animals per group (see Methods].
Discussion In previous reports (l-31, we have obtained evidence that the ability of the rat stomach to intrinsically protect itself against damaging agents is immature in the suckling rat. Similarly, the neonatal rat stomach is unresponsive to the cytoprotective action of prostaglandins until the end of the second week of life. It is our contention that the development of these protective properties in the rat stomach is, in part, related to age-dependent increases in surface hydrophobicity, which we report here. The use of contact angle measurements as assessments of surface hydrophobicity was previously reported by our laboratory for dog gastric tissue, both freshly excised (4) and after in vitro incubation (5). In each case the tissue was lightly blotted to remove excess moisture and was allowed to air-dry for a short time before contact angle readings. More recent work (6) has indicated that control blotted and dried dog tissue remains intact, as viewed at the lightmicroscopic level. Thus, the partially dried tissue may be a reasonable reflection of the state of the tissue surface at death. As discussed by Goddard et al. (S), contact angle measurements may be the only technique available to study surface properties of sheets of cells. Therefore, the changes seen here in contact angle measurements of the rat gastric mucosa represent a new and unique procedure for the study of developmental phenomena. The gradual twofold increase in gastric surface hydrophobicity seen by the third week of life may be triggered or associated with a number of factors. We have investigated three of these: (a) the maturational increase in circulating corticosteroids, (b) the increase in circulating thyroxine, and (c) the ontogenic appearance of the ability of dmPGEz to induce a gastric protective response. A fourth factor, the presence of food in the stomach, was studied only for an acute effect by comparing contact angles of gastric
mucosa excised from fasted and fed animals. Longterm effects of early or late weaning were not investigated in this study. It was determined that the ontogenic increase in gastric mucosal hydrophobicity appeared to be independent of the acute presence of food, as fasted rats showed no clear differences from fully fed animals. We cannot eliminate the possibility that factors in the milk diet could be affecting surface hydrophobicity on a more chronic basis (7). Circulating levels of CO rise in the young rat after day 12 of age (8) and thus initiate many maturational processes in the gastrointestinal tract (9). Because we have previously noted a profound effect of HC on the resistance of the suckling rat gastric mucosa to luminal acid (3), we examined the possibility that HC may be acting through an increase in the hydrophobicity of the mucosal surface. However, that possibility does not appear to be the case, as administration of HC to Y-day-old rats failed to precociously induce the developmental increase in gastric hydrophobicity. Thyroxine also shows developmental increases in circulating levels during the suckling period (10). However, T4 alone or in combination with CO (the endogenous rat corticosteroid) did not influence the developmental appearance of gastric mucosal surface hydrophobicity. Thus, we feel that neither T4 nor CO has a direct role in regulating the ontogenic appearance of this effect. These results do not eliminate a possible indirect effect, however, through permissive actions of either of these hormones. In earlier reports (2,3) we indicated that dmPGEz failed to induce an adult cytoprotective response in suckling rats until the third week of life. Earlier ages (14 days) showed proportionally smaller responses. In this study we have presented evidence that, similar to its antiulcer efficacy, dmPGEz initiated an acute increase in surface hydrophobicity of the rodent gastric mucosa between the second and third week of life. The dmPGE,-induced increase in hydrophobicity at 14 days of age (25%) corresponded with the attenuated gastric protective effect at this age (30% of adult values), although it did not reach statistical significance (p < 0.2). The similar ontogenie time-courses of these two prostaglandindependent responses (surface hydrophobicity and gastric protection) thus suggest that they may be linked in some manner. Indeed, we would speculate that one possible mechanism by which dmPGEl can induce epithelial protection is by increasing the hydrophobicity of the gastric surface, thus preventing the movement of luminal acid into the underlying tissue. Although prostaglandins clearly have a variety of actions that contribute to protection of the gastric mucosa (ll), we believe that the effect on
January 1988
hydrophobicity may be of considerable importance, as it is the first line of defense of the tissue against a luminal damaging agent. However, whether the developmental increase in surface hydrophobicity of the gastric mucosa is causally or only temporally linkedto the development of gastric mucosal protection remains to be determined. Consistent with our earlier report on protection of the gastric mucosa by HC and dmPGE2, which appear to possess additive and somewhat independent effects, we note here that dmPGEz affects surface hydrophobicity whereas HC does not. In the previous study we reported that HC failed to precociously induce the appearance of a cytoprotective response to prostaglandins. In the present study it was demonstrated that the normal ontogenic appearance of a hydrophobic response to prostaglandins similarly was not influenced if 1-wk-old rats were treated with exogenous HC. In addition, we can now state that T4 also does not affect the appearance of this hydrophobic response to dmPGE,. These findings, therefore, emphasize the point that surface hydrophobicity represents one of many gastric properties that contribute to the intrinsic acid resistance of the stomach. The property that is influenced by corticosteroids resulting in a precocious development of acid resistance has yet to be resolved, but appears to be unrelated to the ontogenic changes in surface wettability. A possible explanation for this steroidinduced effect may lie with the mucous cell, as Yeomans et al. (12) have reported that mucous cell differentiation in the rat gastric mucosa in organ culture is influenced by the adrenal corticosteroids. In summary, we have found that there is a developmental increase in gastric mucosal surface hydrophobicity in the rat that occurs by the end of the third week of life and is coincident with the end of the period of weaning. Concurrently, there is an increased responsiveness to dmPGE2, reflected in both an acute increase in surface hydrophobicity and in protection against luminal damaging agents. The developmental signal(s) that influence these gastric properties to ensure that the stomach of the young
HYDROPHOBICITY
rat is protected against luminal to be fully characterized.
AND DEVELOPMENT
ulcerogens
61
have yet
References 1. Dial EJ, Lichtenberger LM. Development of the gastric mucosal barrier to acid in the rat (abstr). Dig Dis Sci 1985;30:372. 2. Dial EJ, Lichtenberger LM. Development of the cytoprotective response to 16,16-dimethyl prostaglandin EZ in the rat (abstr). Gastroenterology 1985;88:1365. LM. Development of gastric mucosal 3. Dial EJ, Lichtenberger protection against acid in the rat: role of corticosteroids and prostaglandins. Gastroenterology 1986;91:318-25. 4. Hills BA, Butler BD, Lichtenberger LM. Gastric mucosal barrier: hydrophobic lining to the lumen of the stomach. Am 1Physiol 1983;244:G561-8. 5. Lichtenberger LM, Richards JE, Hills BA. Effect of 16,16dimethyl prostaglandin Ez on the surface hydrophobicity of aspirin-treated canine gastric mucosa. Gastroenterology 1985;88:308-14. 6. Goddard PJ, Hills BA, Lichtenberger LM. Does aspirin damage canine gastric mucosa by reducing its surface hydrophobicity? Am J Physiol 1987;252:G421-30. 7. Dial EJ, Lichtenberger LM. A role for milk phospholipids in protection against gastric acid. Gastroenterology 1984;87: 379-85. of total and free corticos8. Henning SJ. Plasma concentrations terone during development in the rat. Am J Physiol 1978; 235:E451-6. 9 Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 1981;241:G199-214. 10. Walker P, Dubois JD, Dussault JH. Free thyroid hormone concentrations during postnatal development in the rat. Pediatr Res 1980;14:247-9. 11 Miller TA. Protective effects of prostaglandin against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol 1983;245:G601-3. 12. Yeomans ND, Trier JS, Moxey PC, Markezin ET. Maturation and differentiation of cultured fetal stomach. Effects of corticosteroids, pentagastrin, and cytochalasin B. Gastroenterology 1976;71:770-7.
Received August 4, 1986. Accepted July 27, 1987. Address requests for reprints to: Elizabeth J. Dial, Ph.D., Department of Physiology and Cell Biology, University of Texas Medical School, P.O. Box 20708, Houston, Texas 77225. This work was supported by grants HLI 13021 and AM 33239 from the National Institutes of Health.