- Email: [email protected]
Cecectomized rat
Cecectomized A Model Animals
Rat
of Experimental
JOSEPH D.
FONDACARO,*
Secretory
DAVID C.
Diarrhea
in Conscious
AND
KOLPAK, DANIEL B. BURNHAM,
GERALD P. MCCAFFERTY
of antisecretory antidiarrheal agents in animal models is limited primarily to extrapolations of efficacy from enteropooling studies in vivo, isolated intestinal loops in situ, and Ussing flux chamber preparations in vitro. While these standard techniques are useful, they do not mimic secretory diarrhea. Our studies indicate that in rats, the cecum may serve a “reservoir” function in response to secretagogue administration. Thus, diarrhea is not observed consistently and reEvaluation
liably
in this species
Therefore, lizing
conscious
compromising gogues. ologic
ileocecal
nificantly
3.5
periods
of small
as a useful, posed
p.o.) Oral
and
by the
antidiarrheal
of secretory
normal
administration
E2, cholera induces
diarrhea
administration within
30-60
antidiarrheal
hypersecretion
and reliable
of
(1 mg/kg),
that in the rat, the cecum intestinal
within
chow,
known
min following
physi-
secretory secreta-
Dimethyl
1 hr that continues antidiarrheal
or morphine
prosfor apagents
(IO mg/kg)
all sig-
administration.
may serve as a fluid
These
reservoir
and that the cecectomized
tool for evaluating
secreta-
and other intestinal
or carbachol).
uti-
without
intestinal
growth
of standard
toxin,
agents.
diarrhea
of the cecum,
use of potent
and maintain
clonidine
fecal output
accurate,
Key Words: tomized
hr.
reduce indicate
resection
by oral
(IO mg/kg),
studies
rats by surgical
prostaglandin
E2 (300 pg/kg,
chlorpromazine
model
recover
can be induced
proximately
of potential
for as long as 60 days. After 48 hr on standard
(dimethyl
taglandin
evaluation
a reproducible
patency,
quickly
parameters
gogues
valid
cecectomized
Animals
diarrhea
to allow
we have developed
during
rat serves
new compounds
with
pro-
activity.
Secretory
diarrhea;
Drug
evaluation;
Antidiarrheal
agents;
Cecec-
rat
INTRODUCTION An integral
part of the drug
discovery
agents in laboratory models of the targeted of potential antidiarrheal drugs has relied
process
is the careful
screening
disease. The pharmacologic primarily on extrapolations
of new
evaluation of efficacy
From the Department of Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania. * Current address: Merreli Dow, Inc., Kansas City, MO, USA. Address reprintrequests to: Dr. Joseph D. Fondacaro, Merrell Dow, Inc., Park B-P.O. Box9627, Kansas City, MO 64134. Received December 14, 1989; revised and accepted March 29, 1990.
59 Journal of Pharmacological
Methods
0 1990 Elsevier Science Publishing
24, 59-71 (1990) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
0160~5402/90/$3.50
60
j. D. Fondacaro from studies using the enteropooling technique (Di~oseph et al., 1984)‘ isolated intestinal loops (Nakati et al., 1982), and Ussing flux chamber preparations (Dharmsathaphorn et al., 1984; Fondacaro et al., 1988b). The Thiry-Vella loop preparation in dogs also has been used to evaluate antidiarrheal activity of agents with various mechanisms of action (Guilikson et al., 1981). These techniques are valuable as initial screens for antisecretory, proabsorptive, or motility effects that may contribute in varying degrees to antidiarrheal activity. However, they do not mimic adequately secretory diarrhea in humans, i.e., the pathophysioiogic condition where intestinal secretion exceeds the absorptive capacity of the colon. Our attempts to develop such a model in the rat have been unsuccessful largely because of failure to achieve the criteria of reliability and reproducibility, which are important in evaluating efficacy of new drugs. The rat has a pronounced cecum as part of its gastrointestinal tract. The rat cecum has been characterized as a reservoir for intestinal contents where high concentrations of various microbial flora assist in the digestion of carbohydrates, cellulose, and peptides through microbial fermentation processes (Ambuhl et al., 1979; Williams and Senior, 1982; Yang et al., 1969, 1970). We have observed, in preliminary studies, an increase in cecal fluid content following oral secretagogue administration (Fondacaro et al., 1988a), suggesting that the cecum also appears to play a role in handling of excess ~ntest;na~fluid. Therefore, the present studies were conducted in the rat to determine the role of the cecum under conditions of intestinal hypersecretion. This has led to the development of the cecectomized rat as a model of diarrhea and its demonstration as a reliable screen for evaluation of antidiarrheal agents. METHODS All of the animal studies described in this report adhere to the standards established by the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National institutes of Health Publication #85-21). The animals used in these studies were male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing between 200 and 250 g. Cholera Toxin Studies Studies were conducted in rats to determine the fate (i.e., the location within the intestine) of fluid secreted in response to cholera toxin. Rats fasted 24 hr were administered 0.5 mglkg cholera toxin orally in 1.0 mL of 2% NaHC03 plus 2% casamino acid solution. This dose of toxin is five times that required to elicit an enhanced enteropooling within 4 hr in the rat (unpublished observation). Animals were sacrificed with an overdose of pentobarbita~ and the small jntestine, cecum and colon were immediately isolated with appropriately placed ligatures and removed. The total fluid volumes in the small intestine, cecum, and colon were determined gravimetrically and compared to vehicle-treated controls. Cecectomy Cecectomies were performed in unfasted rats similar to the procedure described by Ambuhl et al. (1979). Under anesthesia with methohexital (60 mg/kg, i.p.jl the
Secretory Diarrhea in Cecectomited
Rats
cecectomy was initiated with a 2-cm rnidve~~ra~ incision. The cecum was lifted from the abdominal cavity and exteriorized onto a gauze drape (Figure IA), In shamoperated rats, the intact cecum was replaced in the abdominal cavity immediately. In rats to be cecectomized, the cecal apex was freed by severing the avascular area of the mesocecum. Next, a ligature of no. 1 silk suture was positioned so as to occlude the cecum and its vasculature without compromising ileocolonic patency (Figure IB).After th e Iigature was secured and ileocolonic patency confirmed, the cecum was resected, and the remaining exposed cecai mucosa was washed with saline and cauterized (Figure ICI. The intestinal segment was then returned to the abdominal cavity, and the abdominal muscle fascia closed with interrupted 410 chromic-gut sutures. The dermal incision was closed with g-mm stainless steel wound clips that were removed approximately 1 wk postsurgery. Immediately foilowing the surgical procedure, animals were returned to their cages and allowed free access to food and water. Animals were permitted at least a 48-hr recovery period before being used in experiments. Serum Na was determined by flame photometry. Hematocrits were determined from packed cell volume of centrifuged capillary tube whole blood sampfes. Diarrhea
Assay in Cecectomized
Rats
Cecectomized rats were put into individua! wire-bottomed cages placed over sheets of clean paper, and deprived of food and water for the duration of the assay. Rats were given a 2-hr acclimatization period prior to the start of the assay in order to eliminate sporadic episodes of anxiety-induced defecation. During this period, they were observed also for consistent occurrences of peileted feces; an animal producing other than a pelleted stool was disqualified from the study. In cecectomized rats, diarrhea was induced with oral administration of secretagogue: either 16,16 djmethyl prostagJandin EZ fdmPGE2f in 3.5% EtOH, carbachol b.
INTACT CECUM
&
BEFORE CECAL RESECTION
c.
AFTER
CECECTOMY
HGURE 1. An illustration of the surgical procedures for cecectomy in the rat. (A) The intact cecum showing small intestine and colon. iB) Isolation of the cecum by a ligation at the cecafcolonic junction and cutting of the avastufar portion ,of the mesocecum. (C) Continuous ileocolonic junction with cecum removed showing cut end of cecal tissue that has been ligated and cauterized.
62
J. D. Fondacaro
in water, or cholera toxin in an aqueous vehicle of 2% NaHC03 plus 2% casamino acids. Antidiarrheal agents were administered by gavage after the onset of diarrhea1 episodes. Selected doses of all secretagogues and antidiarrheal agents used were based on enteropooling studies in the rat performed previously (unpublished observations). The cage papers were removed and examined at ‘l5-min intervals for carbachol-induced diarrhea, 30”min intervals for dmPGE2-induced diarrhea, and hourly when cholera toxin was used as the secretagogue. Sampling times were selected based on enteropooling data. Fecal output was recorded at each interval and scored in a manner similar to that used by Doherty and Hancock (19831, where fecal consistency is assigned a numerical score in each experimental group as follows: 1 = normal pelleted stool, 2 = soft-formed stools, 3 = water stool and/or diarrhea. The fecal output index (FOI) is defined as the summation of the number of defecation episodes and their ranked consistency score within an observation period and is expressed as a mean _t SEM for each group. Materials The compounds used in this study and their source are carbachol, Aldrich Chemical Co., Milwaukee, WI; casamino acids, Difco Laboratories, Detroit, MI; cholera Boehringer Ingelheim Ltd., toxin, Behring Diagnostics, Lajolla, CA; clonidine, Ridgefield, CT; ~6,16-dimethyl prostaglandin Ez, Upjohn Diagnostics, Kalamazoo, Ml; methohexital, Eli Lilly Co., Indianapolis, IN; morphine, Mallinckrodt, Inc., Paris, KY. Chlorpromazine was synthesized at Smith Kline and French Laboratories. Statistics Student’s t test and analysis of variance were used for statistical comparisons of data points. Significance is accepted at p < 0.05 or less. All results are expressed as the mean I SEM. RESULTS lnitiaf~y, studies were conducted in intact rats to determine the fate of intestinal fluid secreted in response to the administration of cholera toxin (0.5 mglkg, p.o.1. As seen in Figure 2, small intestinal fluid content significantly increases from 0.8 + 0.1 g at the onset of the study to 2.9 f 0.2 g at 3 hr and remains elevated to 5 hr. At 6 and 7 hr after cholera toxin administration, this value falls to 2.3 +- 0.3 and 1.7 I+ 0.3, respectively, yet are still significantly elevated above prestimulation values (p < 0.05). Fluid content in the large intestine is minimally affected by cholera toxin (Figure 2), with the exception of the 6-hr point (0.2 + 0.04 g), which is significantly elevated above the 0 time control value (0.09 3- 0.01 g). Total cecal content increased three-fold at 3 hr after cholera toxin administration and remains significantly elevated throughout the duration of the observation period (Figure 2). Diarrhea was not observed in any of the animals receiving cholera toxin. From these studies, it appears that the cecum plays an important physiologic role in fluid conservation by storing secreted fluid and preventing diarrhea. To substantiate this, the cecum of rats was surgically removed as described in Methods,
Secretory Diarrhea in Cecectomized
4-
n 0 A
Rats
=cecum q small intestine = colon
;;lc +--Y
0
2
4
8
6
Hours (post cholera toxin, 0.5mg/kg,p.o.) FIGURE 2. Cholera toxin-stimulated fluid accumulation in the small intestine, cecum, and colon of intact rats. Values at the mean + SEM of six animals at each time point. All values for the small intestine and the cecum are significantly elevated compared to controls at time 0 (p < 0.05). There is no significant change in the colon except at 6 hr.
and studies were conducted with intestinal secretagogues. A comparison of some physiologic parameters between sham-operated intact and cecectomized rats (Table 1) indicates that over a 30-day period, body weight gain, hematocrit, and serum Na concentrations are not altered in cecectomized animals. In fact, the average percent increase in body weight over 30 days in the two groups was nearly identical (64.2% in controls versus 64.8% in the cecectomized group; Table 1). Observation of these
TABLE 1 Rats
Comparison of Physiologic Parameters of Cecectomized
DAY 10
DAY 0 Body weight
Versus Sham-operated
DAY 30
(g)
Sham-op
291 k 4
357 2 7
453
Cecectomized
287 t
4
339 2 5
442 f
Sham-op
43 2 1
45 2 1
43 k 1
Cecectomized
42 -r- 1
43 2 1
41 k 1
Hematocrit
Serum
2 5 5
(pcv)
Na (mEq/L)
Sham-op
135 2 1
140 k 4
134 f
Cecectomized
140 k 2
137 t
138 k 2
Values
are the mean
f
SEM of six animals
in each group.
2
1
63
64
J. 0. Fondacaro
rats over this time period indicates normal activity, pelleted stool output, and normal food intake (data not shown). The intestinal response to oral administration of secretagogues in cecectomized rats is presented in panels A, B, and C of Figure 3, and diarrhea is quantitated and expressed as FOI as described in Methods. As seen, dmPCE2 (0.3 mgikg, p.0.) produces a significant increase in FOI beginning 30 min after administration and lasting up to 2.5 hr (Figure 3, panel A). In sham-operated intact controls, there is only a slight increase in FOI between 1.5 and 2.5 hr after dmPGEz administration. The response to carbachol (15 mg/kg, p.o.1 is more rapid, with a significant rise in FOI occurring 15 min after administration (Figure 3, panel B), reaching a peak at 30-45 min and steadily declining to 2 hr. In sham-operated controls, carbachol also produces a smaller and shorter lasting increase in FOI within the first hour following administration. Cholera toxin (0.5 mgikg, p.0.) produces a significant increase in FOI at 5 and 6 hr after administration to cecectomized rats (Figure 3, panel C). This response is not sustained (as with dmPGE,; panel A) and immediately returns to control values by 7 hr. There is no effect of cholera toxin on FOI in intact, shamoperated rats.
c(m
14-
cecectomized sham-operated
l2z ‘0 E S o_ 3 0
Tii : L
loB-
642O0
3
1
4
Hours*
(post dmPGE,; 0.3 mg/kg, p.o.) FIGURE 3. Panel A-PGE,-induced increase in FOI in cecectomized versus sham-operated control rats. Values for cecectomized rats from 0.5 to 2.5 hr are significantly elevated above increase in FOI in cecontrols t* = p < 0.05 versus control). Panel B-Carbachof-induced cectomized versus sham-operated control rats. Values for cecectomized rats from 0.25 to 1.50 hr are significantly elevated above controls (* = p < 0.05 versus control). Panel CCholera toxin-induced increase in FOI in cecectomized versus sham-operated control rats. Values for cecectomized rats from 5 to 7 hr are significantly elevated above controls (* = p < 0.05 versus control). With all three secretagogues, the data points represent the mean + SEM of six to nine animals at each point.
Secretory Diarrhea in Cecectomized
Rats
20 18g
16-
z
14-
3
12-
4
lo-
o
8-
z : IL
6-
J
42O-
2
1 Hours
0
(post carbachol;
15 mg/kg, p-0.)
1412IO8642O-
? 4
2
0
a
6
Hours (post cholera toxin; 0.5 mg/kg, p.o.) FIGURE 3.
In evaluating these three
the onset,
secretagogues
the best profile
duration,
(Continued)
and magnitude
in cecectomized
for the testing
and evaluation
of the diarrheal
rats, the response of antidiarrheal
response
to
to dmPGEz provides agents.
Therefore,
to
establish a nominal dose of dmPGE, to be used in further studies, a dose-response relationship was established for the FOI response to dmPGEz in cecectomized rats.
65
66
J. D. Fondacaro
As seen in Figure 4, cumulative (4-hr) FOI increases with increasing oral doses of dmPGE, between 0.1 and 1.0 mg/kg. With the use of an intermediate dose of dmPGE, (0.3 mgikg, p.o.1, the effects of three known antidiarrheal agents, chlorprom~ine, clonidine, and morphine, were studied against dmPGE,-induced diarrhea in cecectomized rats in order to test the reliability and validity of this model as a potential screen for antidiarrheal drugs. Chlorpromazine (IO mg/kg, p.0.) administered to cecectomized rats after the onset of diarrhea (i.e., ? hr after dmPGEz administration) causes a significant reduction in FOI and an abrupt cessation of diarrhea (Figure 5). Those cecectomized animals given dmPGEZ alone continue to experience diarrhea up to 3 hr after secretagogue administration, while vehicle-treated, cecectomized rats maintained normal stool output for the duration of the study. The alpha-2 adrenoceptor agonist clonidine (1 .O mg/kg, p.o.) administered to cecectomized rats 1 hr after the onset of dmPGE,induced increase in fecal output likewise produces a rapid fall in FOI to control level and a cessation of diarrhea (Figure 6). As in the chiorprom~ine study, cecectomized rats given dmPGEz alone continue to express diarrhea up to 3 hr, while vehicle-treated, cecectomized rats maintain normal FOI. Likewise, morphine produces qualitatively and quantitatively similar results as clonidine. Morphine (10 mg/ kg, p.o.) reduces FOI to control levels within 30 min when administered 1 hr after dmPGE, (Figure 7), while elevated FOI and diarrhea are seen in untreated cecectomized rats given dmPGE* at time 0. Thus, in this model the action of these wellknown antidiarrheal drugs has been substantiated. Finally, we have observed that these animals recover very rapidly from being used in the diarrhea1 model assay. We have reused animals in the model 7, 10, 30, and
C
0.1 dm PGE,
0.3
0.5
1.0
(mg/kg)
FIGURE 4. The dose-response relationship of 4 hr-cumulative FOI to orally administered dmPGE, in cecectomized rats. The controls were cecectomized rats given vehicle (3.5% EtOH). The FOI at all doses of dmPCEz is significantly elevated above the control value @ < 0.05).
12 t
10 Z ‘0 s
8-
3 Q
6-
2
4
i6 : L
2-
+--+ H
dm PGE2 dm PGE2 plus chlorpromazine
A-A
vehicle control
I
O0
1
2
3
4
Hours FIGURE 5. The effect of chlorpromazine (10 mg/kg, p.0.) given 1 hr (arrow) after the onset of dmPCE,-induced diarrhea in cecectomized rats versus untreated dmPCEz-administered and vehicle control cecectomized rats. All points represent the mean + SEM of at least six rats in each group. Values for dmPCEz plus chlorpromazine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
12-
M H
dm PGE;! dm PGE2 plus clonidine
A-A
vehicle control
lo-
8-
6-
1
2
3
4
Hours FIGURE 6. The effect of clonidine (1.0 mg/kg, p.o.) given 1 hr (arrow) after the onset of dmPCEz-induced diarrhea in cecectomized rats versus untreated dmPCE,-administered and vehicle control cecectomized rats. All points represent the mean & SEM of at least six rats in each group. Values for dmPCEz plus clonidine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
67
68
J. D. Fondacaro
0
1
2
3
4
Hours FIGURE 7. The effect of morphine (10 mg/kg, p.o.1 given 1 hr (arrow) after onset of dmPGEzinduced diarrhea in cecectomized rats versus untreated drnPG~~-administered and vehicle control cecectomized rats. All points represent the mean Z? SEM of at least six rats in each group. Values for dmPGE, plus morphine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
70 days after initial use and observe no difference
cretagogue or in response to antidiarrheal
in fecal output response to se-
drugs (data not shown).
DISCUSSION The purpose of the current study was to establish an experimental model of secretory diarrhea in rats that can be used to accurately evaluate the efficacy of antisecretory antidiarrheal agents. In the course of these studies, we have determined that the cecum of the rat may serve a reservoir function during periods of hypersecretion of the small intestine. Cholera toxin, a potent intestinal secretagogue, was used to create the hypersecretory state and the location of secreted and accumulated intestinal fluid volume was determined over a 7-hr period. As expected, small intestinal fluid volume increased markedly at 3 hr and remained elevated for 2-3 hr (Robert et al., 1976). In contrast, the large bowel &as generally unaffected over this time period in that fluid volume in the colon was unaltered. However, total fluid volume in the cecum of these rats was markedly elevated for the entire duration of the study. Thus, it appears that the cecum serves a reservoir function in the rat during periods of small intestinal hypersecretion. This would make it extremely difficult to elicit secretory diarrhea in these animals and appears to be the reason for our inability in previous attempts to develop a satisfactory model diarrhea in the intact rat (unpublished observations).
Secretory Diarrhea in Cecectomized
Rats
It is well known that the human colon has a sizeable absorptive reserve (Binder and Sadler, 1987), which also has been termed colonic “salvage” (Read, 1982). The colon has the capacity to absorb quantities of fluid far above what is normally presented to it in a normal 24-hr period, Thus, it is possible for the human small intestine to experience
mild transient
hypersecretory
states without
producing
accompanying
diarrhea. preciable
Our studies suggest that the rat colon apparently does not have an apabsorptive reserve. Instead, it appears as though the cecum, functioning
as a fluid
reservoir,
gradually
titrates
fluid
into the colon
so as not to exceed
the
absorptive capacity of that organ. It is conceivable also that the rat cecum absorbs large volumes of fluid, but data on the fluid absorption function of this portion of the
rat intestinal
Furthermore,
tract
stools. This is contrary (Scarpello
is not available,
cecectomized et al., 1978),
and this was not the focus
rats unchallenged
with secretagogue
to what has been reported in which
of this study.
produce
with other cecectomy
rats had persistent
unprovoked
pelleted
procedures
diarrhea
2-3
wk
postsurgery. With our procedure, cecectomized rats in this study express diarrhea only after secretagogue administration. This difference may be explained by the fact that the other of the terminal
cecectomy
ileum
procedures
and proximal
involved
colon,
removal
whereas
of significant
our procedure
resection of the cecum and leaves the ileum and colon intact. Removal of the cecum is a relatively simple surgical procedure. rapidly, feed secretagogue
portions
involves Animals
only
recover
normally, and have normal growth patterns. However, when given a challenge, these cecectomized rats respond very differently from their
sham-operated,
intact littermates.
Cecectomized
rats elicit diarrhea
that, depending
on the secretory stimulus, has a predictable onset, intensity, and duration. Although intact rats generally do not express exteriorized diarrhea, carbachol may elicit some diarrhea1
response
in intact
cholinergic stimulation addition to stimulation variable,
however,
required
for confident
It is of interest small intestine
rats (Figure
and does
not provide
evaluation
As an experimental tages over other rats, at the time
the
necessary
and testing of potentially
model
compounds
may be the
result
of
experimental
“window”
new antidiarrheal
drugs.
of diarrhea,
following
stimulation
the cecectomized
of secretion rat has several
(Tapper advan-
models. First, even though surgery has been performed on these of study, animals are healthy. We have shown that body weight
gain, hematocrit (as an indirect index indirect index of water and electrolyte of these
B), which
to note that in Ussing chamber studies, the in vitro response of the to carbachol likewise is very abrupt and does not readily permit
evaluation of antisecretory et al., 1978).
Some
3, panel
of propulsive motor activity in the small and large bowel in of small bowel secretion. This response is unacceptably
cecectomized
rats were
of plasma volume), absorption) remain followed
and serum Na (as an normal over 30 days.
for 60 days with
no observable
abnormalities noted. Second, drug evaluation can be conducted in conscious, noninstrumented animals, thus eliminating a possible variable introduced by anesthesia. Third, the diarrhea1 response is consistently reproducible by anesthesia. Third, the diarrhea1 response is consistently reproducible in these animals. Also, drug treatment can be instituted after the onset of diarrhea and evaluated at several time
69
70
J. D. Fondacaro points, an advantage which many in vivo models do not offer. Likewise, by avoiding pretreatment with a test drug, one reduces the likelihood of potential drug-induced compromises in other systems that may modify the desired response. Furthermore, working with conscious rats allows gross observation of potential behavioral, postural, and other changes that are important parameters in drug evaluation. Finally, animals can be reused in future experiments (up to 10 wk in our studies). Thus, the cecectomized rat provides an excellent model of secretory diarrhea for the evaluation of antidiarrheal drugs. Agents from the phenothiazine, alpha-2 adrenoceptor agonist and opiate classes of compounds are known to have antidiarrheal activity (Fondacaro, 1986). Therefore, we used known standard agents from these categories of compounds to test the validity and usefulness of the PGE,-stimulated cecectomized rat as a model for evaluating antisecretory antidiarrheal therapies. All three compounds, chlorpromazine, clonidine, and morphine, produced an abrupt fall in fecal output to control values (i.e., suppressed the ongoing diarrhea). These data demonstrate the usefulness of this model for evaluating the efficacy of various antidiarrheal agents and suggest the utility of this technique for testing proposed new antisecretory antidiarrheal compounds. In summary, the cecum of the rat gastrointestinal tract may serve as a reservoir for excess fluid that is secreted by the small intestinal epithelium and emptied into the lower bowel. Furthermore, the colon of the rat is unlike that of the human in that it does not appear to possess an appreciable fluid absorptive reserve. Instead, it may be that excess fluid is titrated into the colon from the cecum at a rate that does not exceed the absorptive capacity of the colon. Thus, removal of the rat cecum has provided an experimental model in which diarrhea can be secretagogue-induced. This model has been validated as a useful means to reliably evaluate novel compounds having potential utility for treating secretory diarrhea. The authors express their sincere thanks to Kiki Schiller and Sally Weber for their secretarial assistance in the preparation of this manuscript. This study was presented as a poster presentation at the annual meetings of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1988, and The American Gastroenterological Association, New Orleans, May 1988.
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Secretory Diarrhea in Cecectomized
Rats
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Gastrointest
Physiol4):E402-E409.
Williams Vi, Senior W (1982) Effects of cecectomy on the digestibility of food and rate of passage of digesta in the rat. Aust / Biol Sci 35:373-379. Yang MC, Manoharan K, Young AK (1969) Influence and degradation of dietary cellulose in cecum of rats. j Nutr 97:260-264. Yang MC, Manoharan K, Michelsen 0 (1970) Nutritional contribution of volatile fatty acids from the cecum of rats. 1 Nutr 100:545-550.
71
Rat
of Experimental
JOSEPH D.
FONDACARO,*
Secretory
DAVID C.
Diarrhea
in Conscious
AND
KOLPAK, DANIEL B. BURNHAM,
GERALD P. MCCAFFERTY
of antisecretory antidiarrheal agents in animal models is limited primarily to extrapolations of efficacy from enteropooling studies in vivo, isolated intestinal loops in situ, and Ussing flux chamber preparations in vitro. While these standard techniques are useful, they do not mimic secretory diarrhea. Our studies indicate that in rats, the cecum may serve a “reservoir” function in response to secretagogue administration. Thus, diarrhea is not observed consistently and reEvaluation
liably
in this species
Therefore, lizing
conscious
compromising gogues. ologic
ileocecal
nificantly
3.5
periods
of small
as a useful, posed
p.o.) Oral
and
by the
antidiarrheal
of secretory
normal
administration
E2, cholera induces
diarrhea
administration within
30-60
antidiarrheal
hypersecretion
and reliable
of
(1 mg/kg),
that in the rat, the cecum intestinal
within
chow,
known
min following
physi-
secretory secreta-
Dimethyl
1 hr that continues antidiarrheal
or morphine
prosfor apagents
(IO mg/kg)
all sig-
administration.
may serve as a fluid
These
reservoir
and that the cecectomized
tool for evaluating
secreta-
and other intestinal
or carbachol).
uti-
without
intestinal
growth
of standard
toxin,
agents.
diarrhea
of the cecum,
use of potent
and maintain
clonidine
fecal output
accurate,
Key Words: tomized
hr.
reduce indicate
resection
by oral
(IO mg/kg),
studies
rats by surgical
prostaglandin
E2 (300 pg/kg,
chlorpromazine
model
recover
can be induced
proximately
of potential
for as long as 60 days. After 48 hr on standard
(dimethyl
taglandin
evaluation
a reproducible
patency,
quickly
parameters
gogues
valid
cecectomized
Animals
diarrhea
to allow
we have developed
during
rat serves
new compounds
with
pro-
activity.
Secretory
diarrhea;
Drug
evaluation;
Antidiarrheal
agents;
Cecec-
rat
INTRODUCTION An integral
part of the drug
discovery
agents in laboratory models of the targeted of potential antidiarrheal drugs has relied
process
is the careful
screening
disease. The pharmacologic primarily on extrapolations
of new
evaluation of efficacy
From the Department of Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania. * Current address: Merreli Dow, Inc., Kansas City, MO, USA. Address reprintrequests to: Dr. Joseph D. Fondacaro, Merrell Dow, Inc., Park B-P.O. Box9627, Kansas City, MO 64134. Received December 14, 1989; revised and accepted March 29, 1990.
59 Journal of Pharmacological
Methods
0 1990 Elsevier Science Publishing
24, 59-71 (1990) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
0160~5402/90/$3.50
60
j. D. Fondacaro from studies using the enteropooling technique (Di~oseph et al., 1984)‘ isolated intestinal loops (Nakati et al., 1982), and Ussing flux chamber preparations (Dharmsathaphorn et al., 1984; Fondacaro et al., 1988b). The Thiry-Vella loop preparation in dogs also has been used to evaluate antidiarrheal activity of agents with various mechanisms of action (Guilikson et al., 1981). These techniques are valuable as initial screens for antisecretory, proabsorptive, or motility effects that may contribute in varying degrees to antidiarrheal activity. However, they do not mimic adequately secretory diarrhea in humans, i.e., the pathophysioiogic condition where intestinal secretion exceeds the absorptive capacity of the colon. Our attempts to develop such a model in the rat have been unsuccessful largely because of failure to achieve the criteria of reliability and reproducibility, which are important in evaluating efficacy of new drugs. The rat has a pronounced cecum as part of its gastrointestinal tract. The rat cecum has been characterized as a reservoir for intestinal contents where high concentrations of various microbial flora assist in the digestion of carbohydrates, cellulose, and peptides through microbial fermentation processes (Ambuhl et al., 1979; Williams and Senior, 1982; Yang et al., 1969, 1970). We have observed, in preliminary studies, an increase in cecal fluid content following oral secretagogue administration (Fondacaro et al., 1988a), suggesting that the cecum also appears to play a role in handling of excess ~ntest;na~fluid. Therefore, the present studies were conducted in the rat to determine the role of the cecum under conditions of intestinal hypersecretion. This has led to the development of the cecectomized rat as a model of diarrhea and its demonstration as a reliable screen for evaluation of antidiarrheal agents. METHODS All of the animal studies described in this report adhere to the standards established by the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National institutes of Health Publication #85-21). The animals used in these studies were male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing between 200 and 250 g. Cholera Toxin Studies Studies were conducted in rats to determine the fate (i.e., the location within the intestine) of fluid secreted in response to cholera toxin. Rats fasted 24 hr were administered 0.5 mglkg cholera toxin orally in 1.0 mL of 2% NaHC03 plus 2% casamino acid solution. This dose of toxin is five times that required to elicit an enhanced enteropooling within 4 hr in the rat (unpublished observation). Animals were sacrificed with an overdose of pentobarbita~ and the small jntestine, cecum and colon were immediately isolated with appropriately placed ligatures and removed. The total fluid volumes in the small intestine, cecum, and colon were determined gravimetrically and compared to vehicle-treated controls. Cecectomy Cecectomies were performed in unfasted rats similar to the procedure described by Ambuhl et al. (1979). Under anesthesia with methohexital (60 mg/kg, i.p.jl the
Secretory Diarrhea in Cecectomited
Rats
cecectomy was initiated with a 2-cm rnidve~~ra~ incision. The cecum was lifted from the abdominal cavity and exteriorized onto a gauze drape (Figure IA), In shamoperated rats, the intact cecum was replaced in the abdominal cavity immediately. In rats to be cecectomized, the cecal apex was freed by severing the avascular area of the mesocecum. Next, a ligature of no. 1 silk suture was positioned so as to occlude the cecum and its vasculature without compromising ileocolonic patency (Figure IB).After th e Iigature was secured and ileocolonic patency confirmed, the cecum was resected, and the remaining exposed cecai mucosa was washed with saline and cauterized (Figure ICI. The intestinal segment was then returned to the abdominal cavity, and the abdominal muscle fascia closed with interrupted 410 chromic-gut sutures. The dermal incision was closed with g-mm stainless steel wound clips that were removed approximately 1 wk postsurgery. Immediately foilowing the surgical procedure, animals were returned to their cages and allowed free access to food and water. Animals were permitted at least a 48-hr recovery period before being used in experiments. Serum Na was determined by flame photometry. Hematocrits were determined from packed cell volume of centrifuged capillary tube whole blood sampfes. Diarrhea
Assay in Cecectomized
Rats
Cecectomized rats were put into individua! wire-bottomed cages placed over sheets of clean paper, and deprived of food and water for the duration of the assay. Rats were given a 2-hr acclimatization period prior to the start of the assay in order to eliminate sporadic episodes of anxiety-induced defecation. During this period, they were observed also for consistent occurrences of peileted feces; an animal producing other than a pelleted stool was disqualified from the study. In cecectomized rats, diarrhea was induced with oral administration of secretagogue: either 16,16 djmethyl prostagJandin EZ fdmPGE2f in 3.5% EtOH, carbachol b.
INTACT CECUM
&
BEFORE CECAL RESECTION
c.
AFTER
CECECTOMY
HGURE 1. An illustration of the surgical procedures for cecectomy in the rat. (A) The intact cecum showing small intestine and colon. iB) Isolation of the cecum by a ligation at the cecafcolonic junction and cutting of the avastufar portion ,of the mesocecum. (C) Continuous ileocolonic junction with cecum removed showing cut end of cecal tissue that has been ligated and cauterized.
62
J. D. Fondacaro
in water, or cholera toxin in an aqueous vehicle of 2% NaHC03 plus 2% casamino acids. Antidiarrheal agents were administered by gavage after the onset of diarrhea1 episodes. Selected doses of all secretagogues and antidiarrheal agents used were based on enteropooling studies in the rat performed previously (unpublished observations). The cage papers were removed and examined at ‘l5-min intervals for carbachol-induced diarrhea, 30”min intervals for dmPGE2-induced diarrhea, and hourly when cholera toxin was used as the secretagogue. Sampling times were selected based on enteropooling data. Fecal output was recorded at each interval and scored in a manner similar to that used by Doherty and Hancock (19831, where fecal consistency is assigned a numerical score in each experimental group as follows: 1 = normal pelleted stool, 2 = soft-formed stools, 3 = water stool and/or diarrhea. The fecal output index (FOI) is defined as the summation of the number of defecation episodes and their ranked consistency score within an observation period and is expressed as a mean _t SEM for each group. Materials The compounds used in this study and their source are carbachol, Aldrich Chemical Co., Milwaukee, WI; casamino acids, Difco Laboratories, Detroit, MI; cholera Boehringer Ingelheim Ltd., toxin, Behring Diagnostics, Lajolla, CA; clonidine, Ridgefield, CT; ~6,16-dimethyl prostaglandin Ez, Upjohn Diagnostics, Kalamazoo, Ml; methohexital, Eli Lilly Co., Indianapolis, IN; morphine, Mallinckrodt, Inc., Paris, KY. Chlorpromazine was synthesized at Smith Kline and French Laboratories. Statistics Student’s t test and analysis of variance were used for statistical comparisons of data points. Significance is accepted at p < 0.05 or less. All results are expressed as the mean I SEM. RESULTS lnitiaf~y, studies were conducted in intact rats to determine the fate of intestinal fluid secreted in response to the administration of cholera toxin (0.5 mglkg, p.o.1. As seen in Figure 2, small intestinal fluid content significantly increases from 0.8 + 0.1 g at the onset of the study to 2.9 f 0.2 g at 3 hr and remains elevated to 5 hr. At 6 and 7 hr after cholera toxin administration, this value falls to 2.3 +- 0.3 and 1.7 I+ 0.3, respectively, yet are still significantly elevated above prestimulation values (p < 0.05). Fluid content in the large intestine is minimally affected by cholera toxin (Figure 2), with the exception of the 6-hr point (0.2 + 0.04 g), which is significantly elevated above the 0 time control value (0.09 3- 0.01 g). Total cecal content increased three-fold at 3 hr after cholera toxin administration and remains significantly elevated throughout the duration of the observation period (Figure 2). Diarrhea was not observed in any of the animals receiving cholera toxin. From these studies, it appears that the cecum plays an important physiologic role in fluid conservation by storing secreted fluid and preventing diarrhea. To substantiate this, the cecum of rats was surgically removed as described in Methods,
Secretory Diarrhea in Cecectomized
4-
n 0 A
Rats
=cecum q small intestine = colon
;;lc +--Y
0
2
4
8
6
Hours (post cholera toxin, 0.5mg/kg,p.o.) FIGURE 2. Cholera toxin-stimulated fluid accumulation in the small intestine, cecum, and colon of intact rats. Values at the mean + SEM of six animals at each time point. All values for the small intestine and the cecum are significantly elevated compared to controls at time 0 (p < 0.05). There is no significant change in the colon except at 6 hr.
and studies were conducted with intestinal secretagogues. A comparison of some physiologic parameters between sham-operated intact and cecectomized rats (Table 1) indicates that over a 30-day period, body weight gain, hematocrit, and serum Na concentrations are not altered in cecectomized animals. In fact, the average percent increase in body weight over 30 days in the two groups was nearly identical (64.2% in controls versus 64.8% in the cecectomized group; Table 1). Observation of these
TABLE 1 Rats
Comparison of Physiologic Parameters of Cecectomized
DAY 10
DAY 0 Body weight
Versus Sham-operated
DAY 30
(g)
Sham-op
291 k 4
357 2 7
453
Cecectomized
287 t
4
339 2 5
442 f
Sham-op
43 2 1
45 2 1
43 k 1
Cecectomized
42 -r- 1
43 2 1
41 k 1
Hematocrit
Serum
2 5 5
(pcv)
Na (mEq/L)
Sham-op
135 2 1
140 k 4
134 f
Cecectomized
140 k 2
137 t
138 k 2
Values
are the mean
f
SEM of six animals
in each group.
2
1
63
64
J. 0. Fondacaro
rats over this time period indicates normal activity, pelleted stool output, and normal food intake (data not shown). The intestinal response to oral administration of secretagogues in cecectomized rats is presented in panels A, B, and C of Figure 3, and diarrhea is quantitated and expressed as FOI as described in Methods. As seen, dmPCE2 (0.3 mgikg, p.0.) produces a significant increase in FOI beginning 30 min after administration and lasting up to 2.5 hr (Figure 3, panel A). In sham-operated intact controls, there is only a slight increase in FOI between 1.5 and 2.5 hr after dmPGEz administration. The response to carbachol (15 mg/kg, p.o.1 is more rapid, with a significant rise in FOI occurring 15 min after administration (Figure 3, panel B), reaching a peak at 30-45 min and steadily declining to 2 hr. In sham-operated controls, carbachol also produces a smaller and shorter lasting increase in FOI within the first hour following administration. Cholera toxin (0.5 mgikg, p.0.) produces a significant increase in FOI at 5 and 6 hr after administration to cecectomized rats (Figure 3, panel C). This response is not sustained (as with dmPGE,; panel A) and immediately returns to control values by 7 hr. There is no effect of cholera toxin on FOI in intact, shamoperated rats.
c(m
14-
cecectomized sham-operated
l2z ‘0 E S o_ 3 0
Tii : L
loB-
642O0
3
1
4
Hours*
(post dmPGE,; 0.3 mg/kg, p.o.) FIGURE 3. Panel A-PGE,-induced increase in FOI in cecectomized versus sham-operated control rats. Values for cecectomized rats from 0.5 to 2.5 hr are significantly elevated above increase in FOI in cecontrols t* = p < 0.05 versus control). Panel B-Carbachof-induced cectomized versus sham-operated control rats. Values for cecectomized rats from 0.25 to 1.50 hr are significantly elevated above controls (* = p < 0.05 versus control). Panel CCholera toxin-induced increase in FOI in cecectomized versus sham-operated control rats. Values for cecectomized rats from 5 to 7 hr are significantly elevated above controls (* = p < 0.05 versus control). With all three secretagogues, the data points represent the mean + SEM of six to nine animals at each point.
Secretory Diarrhea in Cecectomized
Rats
20 18g
16-
z
14-
3
12-
4
lo-
o
8-
z : IL
6-
J
42O-
2
1 Hours
0
(post carbachol;
15 mg/kg, p-0.)
1412IO8642O-
? 4
2
0
a
6
Hours (post cholera toxin; 0.5 mg/kg, p.o.) FIGURE 3.
In evaluating these three
the onset,
secretagogues
the best profile
duration,
(Continued)
and magnitude
in cecectomized
for the testing
and evaluation
of the diarrheal
rats, the response of antidiarrheal
response
to
to dmPGEz provides agents.
Therefore,
to
establish a nominal dose of dmPGE, to be used in further studies, a dose-response relationship was established for the FOI response to dmPGEz in cecectomized rats.
65
66
J. D. Fondacaro
As seen in Figure 4, cumulative (4-hr) FOI increases with increasing oral doses of dmPGE, between 0.1 and 1.0 mg/kg. With the use of an intermediate dose of dmPGE, (0.3 mgikg, p.o.1, the effects of three known antidiarrheal agents, chlorprom~ine, clonidine, and morphine, were studied against dmPGE,-induced diarrhea in cecectomized rats in order to test the reliability and validity of this model as a potential screen for antidiarrheal drugs. Chlorpromazine (IO mg/kg, p.0.) administered to cecectomized rats after the onset of diarrhea (i.e., ? hr after dmPGEz administration) causes a significant reduction in FOI and an abrupt cessation of diarrhea (Figure 5). Those cecectomized animals given dmPGEZ alone continue to experience diarrhea up to 3 hr after secretagogue administration, while vehicle-treated, cecectomized rats maintained normal stool output for the duration of the study. The alpha-2 adrenoceptor agonist clonidine (1 .O mg/kg, p.o.) administered to cecectomized rats 1 hr after the onset of dmPGE,induced increase in fecal output likewise produces a rapid fall in FOI to control level and a cessation of diarrhea (Figure 6). As in the chiorprom~ine study, cecectomized rats given dmPGEz alone continue to express diarrhea up to 3 hr, while vehicle-treated, cecectomized rats maintain normal FOI. Likewise, morphine produces qualitatively and quantitatively similar results as clonidine. Morphine (10 mg/ kg, p.o.) reduces FOI to control levels within 30 min when administered 1 hr after dmPGE, (Figure 7), while elevated FOI and diarrhea are seen in untreated cecectomized rats given dmPGE* at time 0. Thus, in this model the action of these wellknown antidiarrheal drugs has been substantiated. Finally, we have observed that these animals recover very rapidly from being used in the diarrhea1 model assay. We have reused animals in the model 7, 10, 30, and
C
0.1 dm PGE,
0.3
0.5
1.0
(mg/kg)
FIGURE 4. The dose-response relationship of 4 hr-cumulative FOI to orally administered dmPGE, in cecectomized rats. The controls were cecectomized rats given vehicle (3.5% EtOH). The FOI at all doses of dmPCEz is significantly elevated above the control value @ < 0.05).
12 t
10 Z ‘0 s
8-
3 Q
6-
2
4
i6 : L
2-
+--+ H
dm PGE2 dm PGE2 plus chlorpromazine
A-A
vehicle control
I
O0
1
2
3
4
Hours FIGURE 5. The effect of chlorpromazine (10 mg/kg, p.0.) given 1 hr (arrow) after the onset of dmPCE,-induced diarrhea in cecectomized rats versus untreated dmPCEz-administered and vehicle control cecectomized rats. All points represent the mean + SEM of at least six rats in each group. Values for dmPCEz plus chlorpromazine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
12-
M H
dm PGE;! dm PGE2 plus clonidine
A-A
vehicle control
lo-
8-
6-
1
2
3
4
Hours FIGURE 6. The effect of clonidine (1.0 mg/kg, p.o.) given 1 hr (arrow) after the onset of dmPCEz-induced diarrhea in cecectomized rats versus untreated dmPCE,-administered and vehicle control cecectomized rats. All points represent the mean & SEM of at least six rats in each group. Values for dmPCEz plus clonidine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
67
68
J. D. Fondacaro
0
1
2
3
4
Hours FIGURE 7. The effect of morphine (10 mg/kg, p.o.1 given 1 hr (arrow) after onset of dmPGEzinduced diarrhea in cecectomized rats versus untreated drnPG~~-administered and vehicle control cecectomized rats. All points represent the mean Z? SEM of at least six rats in each group. Values for dmPGE, plus morphine from 1.5 to 3.0 hr are significantly reduced from those of dmPGEz alone (* = p < 0.05).
70 days after initial use and observe no difference
cretagogue or in response to antidiarrheal
in fecal output response to se-
drugs (data not shown).
DISCUSSION The purpose of the current study was to establish an experimental model of secretory diarrhea in rats that can be used to accurately evaluate the efficacy of antisecretory antidiarrheal agents. In the course of these studies, we have determined that the cecum of the rat may serve a reservoir function during periods of hypersecretion of the small intestine. Cholera toxin, a potent intestinal secretagogue, was used to create the hypersecretory state and the location of secreted and accumulated intestinal fluid volume was determined over a 7-hr period. As expected, small intestinal fluid volume increased markedly at 3 hr and remained elevated for 2-3 hr (Robert et al., 1976). In contrast, the large bowel &as generally unaffected over this time period in that fluid volume in the colon was unaltered. However, total fluid volume in the cecum of these rats was markedly elevated for the entire duration of the study. Thus, it appears that the cecum serves a reservoir function in the rat during periods of small intestinal hypersecretion. This would make it extremely difficult to elicit secretory diarrhea in these animals and appears to be the reason for our inability in previous attempts to develop a satisfactory model diarrhea in the intact rat (unpublished observations).
Secretory Diarrhea in Cecectomized
Rats
It is well known that the human colon has a sizeable absorptive reserve (Binder and Sadler, 1987), which also has been termed colonic “salvage” (Read, 1982). The colon has the capacity to absorb quantities of fluid far above what is normally presented to it in a normal 24-hr period, Thus, it is possible for the human small intestine to experience
mild transient
hypersecretory
states without
producing
accompanying
diarrhea. preciable
Our studies suggest that the rat colon apparently does not have an apabsorptive reserve. Instead, it appears as though the cecum, functioning
as a fluid
reservoir,
gradually
titrates
fluid
into the colon
so as not to exceed
the
absorptive capacity of that organ. It is conceivable also that the rat cecum absorbs large volumes of fluid, but data on the fluid absorption function of this portion of the
rat intestinal
Furthermore,
tract
stools. This is contrary (Scarpello
is not available,
cecectomized et al., 1978),
and this was not the focus
rats unchallenged
with secretagogue
to what has been reported in which
of this study.
produce
with other cecectomy
rats had persistent
unprovoked
pelleted
procedures
diarrhea
2-3
wk
postsurgery. With our procedure, cecectomized rats in this study express diarrhea only after secretagogue administration. This difference may be explained by the fact that the other of the terminal
cecectomy
ileum
procedures
and proximal
involved
colon,
removal
whereas
of significant
our procedure
resection of the cecum and leaves the ileum and colon intact. Removal of the cecum is a relatively simple surgical procedure. rapidly, feed secretagogue
portions
involves Animals
only
recover
normally, and have normal growth patterns. However, when given a challenge, these cecectomized rats respond very differently from their
sham-operated,
intact littermates.
Cecectomized
rats elicit diarrhea
that, depending
on the secretory stimulus, has a predictable onset, intensity, and duration. Although intact rats generally do not express exteriorized diarrhea, carbachol may elicit some diarrhea1
response
in intact
cholinergic stimulation addition to stimulation variable,
however,
required
for confident
It is of interest small intestine
rats (Figure
and does
not provide
evaluation
As an experimental tages over other rats, at the time
the
necessary
and testing of potentially
model
compounds
may be the
result
of
experimental
“window”
new antidiarrheal
drugs.
of diarrhea,
following
stimulation
the cecectomized
of secretion rat has several
(Tapper advan-
models. First, even though surgery has been performed on these of study, animals are healthy. We have shown that body weight
gain, hematocrit (as an indirect index indirect index of water and electrolyte of these
B), which
to note that in Ussing chamber studies, the in vitro response of the to carbachol likewise is very abrupt and does not readily permit
evaluation of antisecretory et al., 1978).
Some
3, panel
of propulsive motor activity in the small and large bowel in of small bowel secretion. This response is unacceptably
cecectomized
rats were
of plasma volume), absorption) remain followed
and serum Na (as an normal over 30 days.
for 60 days with
no observable
abnormalities noted. Second, drug evaluation can be conducted in conscious, noninstrumented animals, thus eliminating a possible variable introduced by anesthesia. Third, the diarrhea1 response is consistently reproducible by anesthesia. Third, the diarrhea1 response is consistently reproducible in these animals. Also, drug treatment can be instituted after the onset of diarrhea and evaluated at several time
69
70
J. D. Fondacaro points, an advantage which many in vivo models do not offer. Likewise, by avoiding pretreatment with a test drug, one reduces the likelihood of potential drug-induced compromises in other systems that may modify the desired response. Furthermore, working with conscious rats allows gross observation of potential behavioral, postural, and other changes that are important parameters in drug evaluation. Finally, animals can be reused in future experiments (up to 10 wk in our studies). Thus, the cecectomized rat provides an excellent model of secretory diarrhea for the evaluation of antidiarrheal drugs. Agents from the phenothiazine, alpha-2 adrenoceptor agonist and opiate classes of compounds are known to have antidiarrheal activity (Fondacaro, 1986). Therefore, we used known standard agents from these categories of compounds to test the validity and usefulness of the PGE,-stimulated cecectomized rat as a model for evaluating antisecretory antidiarrheal therapies. All three compounds, chlorpromazine, clonidine, and morphine, produced an abrupt fall in fecal output to control values (i.e., suppressed the ongoing diarrhea). These data demonstrate the usefulness of this model for evaluating the efficacy of various antidiarrheal agents and suggest the utility of this technique for testing proposed new antisecretory antidiarrheal compounds. In summary, the cecum of the rat gastrointestinal tract may serve as a reservoir for excess fluid that is secreted by the small intestinal epithelium and emptied into the lower bowel. Furthermore, the colon of the rat is unlike that of the human in that it does not appear to possess an appreciable fluid absorptive reserve. Instead, it may be that excess fluid is titrated into the colon from the cecum at a rate that does not exceed the absorptive capacity of the colon. Thus, removal of the rat cecum has provided an experimental model in which diarrhea can be secretagogue-induced. This model has been validated as a useful means to reliably evaluate novel compounds having potential utility for treating secretory diarrhea. The authors express their sincere thanks to Kiki Schiller and Sally Weber for their secretarial assistance in the preparation of this manuscript. This study was presented as a poster presentation at the annual meetings of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1988, and The American Gastroenterological Association, New Orleans, May 1988.
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Secretory Diarrhea in Cecectomized
Rats
Fondacaro JD, Kolpak DC, Burnham DB, McCafferty CP (1988a) Cecectomized rat: A model of experimental diarrhea in conscious animals. Gastroenterology 95:A132.
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Gullikson GW, Dajani EZ, Bianchi RG (1981) Inhibition of intestinal secretion in the dog: A new approach for the management of diarrhea1 states. J Pharmacol Exp Ther 219:591-597. Nakaki T, Nakadati T, Yamamoto S, Kato R (1982) Alpha-2 adrenergic inhibition of intestinal secretion induced by prostaglandin Et, vasoactive intestinal peptide and dibutyryl cyclic AMP in rat jejunum. / Pharmacol Exp Ther 220:637-641. Read NW (1982) Diarrhea: The failure of colonic salvage. Lancet 2:481-483.
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