- Email: [email protected]
Acute heat stress in growing rats: effect on small intestinal morphometry and in vivo absorption
J. therm. Biol. Vol. 18, No. 3, pp. 145-151, 1993
0306-4565/93 $6.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
ACUTE HEAT STRESS IN GROWING RATS: EFFECT ON SMALL INTESTINAL MORPHOMETRY AND IN VIVO ABSORPTION A. SENGUPTA and R. K. SHARMA Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, U.P., India (Received 10 May 1992; accepted in revised form 18 February 1993)
Abstract--1. Young growing rats (35-45 g) were exposed to high ambient temperature (37 +_ 1°C) for 24 h, while the controls were kept at 24 _+2°C. 2. Quantitative morphometry based on stereological principle and absorptive studies by an in vivo method were performed to evaluate the structural and functional adaptations of the small intestine in rats exposed to heat. 3. The effect of heat stress on various morphometric dimensions of the small intestine shows significant reduction in dry weight, villus height, villus surface area, crypt depth, while the absorptive study shows, significant decrease in absorption per unit dry weight, unit length and unit villus surface area of the small intestine as compared to the control. 4. This adaptive alteration in the structure and function of the small intestine indicates the change in the mucosal dynamics of gut epithelium in heat stress. Key Word Index: High ambient temperature; intestinal stereology; intestinal surface area; intestinal absorption; heat and small intestine
INTRODUCTION Absorption phenomenon in small intestine involves transepithelial nutrient particle movement at the villus epithelial zone and removal of molecules from the basolateral surface and subepithelial interstitial space by intestinal microcirculation. In adaptive regulation of nutrient transport by small intestine, structural and functional alterations occur in this region e.g. number of transporting cells, absorptive surface area, unstirred water layer thickness, circulatory response, specific transport process (Wiiliamson, 1970; Karasov and Diamond, 1983). In unacclimated experimental animals, exposed to high ambient temperature or in the heat acclimated experimental animals, there is evidence of a change in intestinal absorption of sugars (Carpenter and Musacchia, 1974, 1978; Chu et al., 1979; Ciampolini, 1974a, b, 1977; Yousef et al., 1984), and amino acids (Sharma et al., 1982; Sharma, 1985). It is probable that the change in absorptive function of small intestine in heat stressed animals reflects an alteration in structural features of intestinal wall components associated with the absorptive process. Such a possibility arises from the fact that, in the heat stressed animals, alteration in the intestinal absorption was associated with reduction in small intestinal weight (Carpenter and Musacchia, 1974, 1978), although no
studies have been reported so far on the structural and functional adaptation of the small intestine to heat stress. The present study was aimed at elucidating the possible alteration in structural features of the small intestine epithelial layer in heat stressed growing rats, using quantitative morphometry, based on stereological principle, recently applied to the tissue by Mayhew and associates (Mayhew 1984, 1987; Mayhew and Middleton 1985). Along with the morphometric measurements, absorptive function of the small intestine in heat stressed rats was also studied to correlate the possible structural alteration with the associated functional changes. MATERIALS AND METHODS
Animals
The experiment was conducted using 36 rats of Charles-Foster strain (35--45g) divided randomly into two equal groups (A and B) of approximately equal group mean body weight. Following separation from the mother on 21st day of life, the animals were kept in individual cages for 4-5 days for acclimatization to their environment, which included 12h light-dark cycle (light on at 8 a.m.), 24 _+_2°C temperature and 62-74% relative humidity, they were fed lipton India rat diet ad libitum with free access to water. After the 4-5 days acclimatization the group 145
146
A. SENGUPTAand R. K. SHARMA
A animals (experimentals) were weighed and exposed to a temperature at 37 + I°C without altering the other housing conditions mentioned above, while group B animals were weighed and kept at 24 + 2°C and served as controls. Both the groups were kept in the experimental environment for a period of 24 h, with free access to food and water. After 24 h, the animals were weighed again and the amount of food consumed by each of the animal was measured in both the group. The body core temperature (rectal) of all the animals were recorded before, during and after they had been kept in the experimental environment, by a quick registry thermometer approximately 25 mm in the rectum. Group A and B rats were then further divided into subgroups Al and A2 and Bj and B2 respectively; the corresponding subgroup mean body weight being kept approximately equal. The animals of groups Aj and B~ each containing 6 rats, were used for the morphometric studies. Similarly the animals of group A2 and B2 each containing 12 rats were used for the absorptive studies. Measurement of dietary consumption, body weight and the animal experimentation were carried out between 9-11 a.m. each day.
Tissue preparation and sampling Under pentobarbital sodium anaesthesia (40 mg/kg body wt, i.p.) the abdomen was opened. The small intestine lumen washed with warm (37°C) normal saline, stripped of its mesentery and divided into three segments of approximately equal length. Each segment was placed in a separate petri dish containing formalin solution (10%) and sliced further into three smaller pieces of equal length. The proximal piece of first segment, middle piece of second segment and the distal piece of the third segment were selected for the study, so that they represent pieces of duodenum, jejunum and ileum, respectively (designated as proximal, mid and distal for descriptive convenience). The pieces were trimmed to a length of approximately 2 cm and placed in fresh fixative solution at 4°C. After fixation the pieces were dehydrated in graded ethanol and embeded in paraffin. Two to three micron thick transverse sections were cut, stained with Haematoxylin-Eosine and examined under light microscope. One photomicrograph of complete transverse section per segment was recorded and printed to a final magnification of x 50.
Stereological procedure Full details of the principle for estimation of morphometric variables are described by Mayhew and associates (Mayhew 1984, 1987; Meyhew and Middleton 1985). Primary mucosa may be defined as the boundary running between the bases of the villi
and the opening of the crypts, the surface--S(P) roughly corresponds to a smooth bore tube. Secondary mucosal surface area--S(V) is that due to villi. Segmental estimation of the circumference of the primary mucosal tube---B(P), the amplification of primary mucosal surface by villi i.e. villus amplification factor or Ss (V, P) and the surface area of primary mucosa--S(P) and of villi--S(V), were derived by intersection point counting method. For the purpose, quadratic test lines of spacing 5 mm (equivalent to a distance Z of 0.1 mm in the actual specimen, considering x50 final magnification) was independently positioned and oriented on each photomicrograph representing that segment. Intersection between test lines and the surface traces of primary mucosa I(P) and villi I(V) were then counted and summed over horizontal and vertical test lines. The intersection ratio I(V)/I(P) = I(V, P) was used to estimate villus amplification by the relationship Ss (V, P) = 4 / ~ . / ( v , a).
The circumference of the primary mucosal tube was obtained by the relationship B ( P ) = n/4.Z.I(P), where Z = 0.1 mm in this study. The primary mucosal surface area is denoted by S(P)=L.B(P) and the secondary or villus surface area is obtained by the relationship S(V) = S(P)-Ss(V, P).L, being the length of the small intestinal segment (2 cm in this study). The detailed explanation and rationale of all these complex relationship were described by Mayhew (1984, 1987), Mayhew and Middleton (1985) and Ross and Mayhew (1985). The heights of villi and crypt on sections were measured with a micrometer scale. Four profiles per intestinal transverse section were measured in order to estimate the average height in each segment.
Absorption studies An anaesthetized rat was placed o n a heating table to keep the rectal temperature around 37°C. A small intestine loop was prepared either from the jejunum or from the ileal segment for the absorption study. The jejunal loop, 10-12cm in length was prepared from the small intestine lying between 5-20 cm from the ligament of Tr~itz. The ileal loop of identical size was prepared from the small intestine approximately 15 cm proximal to ilcocaecal junction. The method of preparation of jejunal and ileal loop has been described earlier (Sharma and Nagchaudhuri, 1976). 0.5 ml of L-proline solution (200 mM/l at 36-37°C) was injected into the loop through fine needle attached to a tuberculin syringe and the loop w a s replaced to the abdomen. The abdomen was closed and the animal was placed o n a platform inside the perspex chamber placed over a constant temperature
Small intestinal morphometry and absorption in heat stress water bath being kept at 34 _+ l°C. Preliminary experiments showed that this arrangement keeps the body core temperature of the operated and the anaesthetized animal at 37°C within an error of _+0.5°C and prevents dehydration, since the relative humidity inside the perspex chamber was maintained at 100%. After an absorption period of 10 rain, the animal was killed and bled by opening the thorax and incising the heart. The intestinal loop was washed with warm normal saline (37°C) and the loop washing volume was recorded. The washing were analysed for proline content after deproteinization. The absorption of proline from the sac was calculated from the difference between the total amount of proline introduced into the loop and the amount of proline recovered at the end of the experimental period. The length of the loop was measured (Barry et al., 1961) and the dry weight of the loop was recorded after drying in a hot air oven at 90°C till the constant weight was achieved. The villus surface area of each intestinal segment was calculated using data obtained in the morphometric studies. The absorption was expressed in terms of length, dry weight and surface area of the loop.
Biochemical estimation and chemicals Proline was estimated by the method of Wren and Wiggal (1965). L-Proline was obtained from John Baker, U.S.A. All the chemicals used for the estimation of proline were of Anala R grade of BDH laboratory chemical division, England. Student's t-test was used to compare the mean values of the control group with those of the experimental group. A value of P < 0.05 was accepted as indicating significance.
small intestine. In the heat exposed rats a significant fall in the values of villus amplification factor was observed at all regions of the small intestine (Table 2). The values were decreased by 22.4, 28.2 and 24.1%, respectively in the proximal, mid and distal regions. As shown in Fig. 2, the proximodistal gradient was maintained closely to that observed in the controls. Villus surface area was significantly reduced in segments of small intestine in experimental animals when compared with the corresponding control segments (Table 2). The decrease was of the order of 34.2, 32 and 27.6%, respectively in the proximal, mid and distal segments. In the experimental group of animals the villus height decreased significantly in all the segments of the small intestine. The effect was maximum over the mid region, since the region showed 36% reduction as compared to 28.7 and 30% reduction seen in proximal and distal segments, respectively (Table 2 and Fig. 2). The crypt depth follows a similar segmental pattern, comparable to that of the villus height, though the slope of the proximodistal gradient was less (Fig. 2). The mean reduction of all the intestinal segments in the experimental animals was 23% as compared to the controls.
Proline absorption The absorption of proline from the jejunal and ileal segments was normalized to dry weight of the sac, length of the sac and absorptive villus surface area of the sac (Table 2). Irrespective of the parameter chosen to express the results, absorption in the control group was [---] Control Experimental
RESULTS Findings are illustrated in Figs 1 and 2 and summarized in Tables 1-3. At the end of the 24 h experimental period, the control animals had gained 2 + 0.82 g (mean + SD) of body weight, while the heat stressed animals gained only 0.7 + 0.34g. Food consumption was significantly decreased, to 65% in experimental rats, as compared to the controls. Dry weight of the small intestine in the heat exposed rats was less than those of the control rats by 16%, whereas the alteration in the length, cireumference of the primary mucosal tube and primary mucosai surface area of the small intestine were not statistically significant (Figs 1 and 2; Table 2.). The villus amplification factor in the controls showed a declining proximodistal gradient, the gradient being more pronounced between proximal to mid segment than between mid to distal segment of the
147
75
-
611
-
-- 5OO
~ "~. ¢d
/-/ // //
--
41111 .~_ U
/ / 45
-
=
//
--
31111
~
--
2011
--
--
ell 11111 ' ~
Z 311
-
15
-
-5.~
// t'/ // // // // // // // ,'/
0
/./ Length
0 Width
Fig. 1. Changes in the length and the dry weight of the small intestine in two groups of rat. Control animals kept at 24+2°C for 24h and experimental animals kept at 37 _+ I °C for 24 h. Bars denote SD ranges.
148
A. SENGUPTA and R. K. SHARMA
5.0 -
1.0 -
2.5
0.5
-
(b)
(a)
400 --
P
DISCUSSION
1
M
1
I
D
6
P
[ M
I D
200 --
(c) l 5.0--
P
I M
2.5 -- f f ~
P
t M
I
200 - P
___~
~'e'l
(d)
I
D
I M
I D
I00
.... J D
(f) P
t
I M
J D
Fig. 2. Gradients of mucosal architecture revealed in proximal (P), mid (M) and distal (D) segment of the small intestine. Variables illustrated are: (a) primary mucosal tube circumference in mm, (b) primary mueosal surface area in cm2, (c) villus height in/~m, (d) villus amplification factor, (e) villus surface area in em2 and (f) crypt depth in /~m. ( ) Data from control animals kept at 24+2°C for 24 h. (- - -) Data from experimental animals kept at 37 + I°C for 24 h. Bars denote SD.
significantly higher in the jejunal segment than in the ileal segment. However, the proximo-distal gradient w a s least when the results were expressed in terms of unit absorptive surface area. In the experimental group of animals L-proline absorption decreased significantly in the jejunal and the ileal segments, when normalized to unit dry weight, unit length and unit absorptive surface area. Nevertheless, the percentage decrease in absorption was least when the absorption was normalized to unit absorptive surface area.
Exposure of developing rats to 37°C for 24 h lead to significant reductions in food consumption and decrease in the body weight gain as compared to the controls. These observations support findings of the earlier workers (Pennycuik, 1964; Chu et al., 1979; Yousef et al., 1984). This could be explained on the fact that in a hot environment, the body temperature may overshoot the normal limit and the intake of food just sufficient to maintain the body weight may overtax the heat regulating mechanism (Hamilton, 1967). Thus a decrease in food intake may lower heat production in an effort to keep the body temperature within normal limit. In homeotherms, therefore, food consumption bears a negative relation to the environmental heat stress temperatures (Brobeck, 1960). The change in body weight in heat exposed rats was associated with loss of small intestinal dry weight of about 15.5%. This disproportionately greater loss of the small intestinal weight than that of the body weight loss has been also reported by Carpenter and Musacchia (1974, 1978) in heat acclimated rats and hamsters, and by Sharma et al. (1982) and Sharma (1985) in adult rats exposed to acute heat stress. Heat causes loss of tissue mass and leads to an apparent thinning of the gut wall (Carpenter and Musacchia, 1974). The change in length, primary mucosal circumference and primary mucosal surface area of the small intestine in heat stressed rats was not statistically significant (Table 2). Observation in starvation experiments showed that the length and circumference undergo variable alterations when the animals were subjected to food deprivation (Stenling and Helander, 1981; Ross and Mayhew, 1985). Present findings suggest that length and circumference alone are relatively insensitive indicators of the effect of stress. Ross and Mayhew (1985) suggested that the experimental effects be monitored by estimating both variables and then calculating mucosal surface areas.
Table 1. Food intake and body weight gain of control (24 + 2°C) and heat stressed (37+ I°C) rats, measured at the end of 24h period from Day 0 to Day 1 Percentage alteration in heat stress as compared to controls
Variable
Control
Heat stressed
Food consumption (g/animal) Body weight
7 + 4.3
2.4 + 1.4"
65.7
2 + 0.82
0.7 -I-0.34*
65
gain (g/animal)
Values are group means -I-SD (n = 18). *P < 0.01.
Small intestinal morphometry and absorption in heat stress
149
Table 2. Morphometric data for various mucosal dimensions in different small intestinal segments in control (24 + 2°C) and heat stressed (37 + I°C) rats Variable
Group
Primary mucosal circumferenc~ (ram) Primary mucosal surface area (cm2) Villus height (/zm)
Control Heat s t r e s s e d Control Heat stressed Control Heat stressed Control Heat stressed Control Heat stressed Control Heat stressed
Villus amplification factor Villus surface area (cm2) Crypt depth (~m)
Proximal
Mid
3.77 + 0.63 3.22+0.43 ( N S ) 0.75 + 0.13 0.64 + 0.08 (NS) 299 + 19 213 + 26* 5.14 + 0.58 3.98 + 0.73~/ 3.86 ± 0.75 2.57 + 0.62:[: 168 _+ 17 133 ± 12"
3.84 + 0.19 3.49+0.71 (hiS) 0.77 + 0.04 0.69 + 0.14 (NS) 216 + 50 138 + 24t 3.8 + 0.53 2.73 + 0.52t 2.93 + 0.45 1.99 + 0.05t 148 + 17 120 _+ 14~
Distal 4.14 + 0.52 3.91 +0.37 (NS) 0.83 + 0.01 0.78 + 0.07 (NS) 190 + 38 133 + 17t 3.51 + 0.48 2.66 + 0.05:~ 2.88 + 0.25 2.08 + 0.16" 129 ± 15 106 + 8~/
Values are group mean + SD (n = 6). *P < 0.01; ~'P < 0.02; :~P < 0.05; NS = not significant. In the heat stressed rats, a significant reduction in villus amplification factor was observed in all the segments of the small intestine; consequently, a marked reduction in the villus surface area was recorded. In heat exposed rats the small intestine mucosa, responded by a reduction in the absorptive surface area, similar to that seen in states of fasting (Ross and Mayhew, 1985; Mayhew, 1987). All three regions of the small intestine responded to a similar degree and therefore proximodistal gradient in villus surface area, seen in the control animals, persisted in the heat stressed rats (Fig. 2). The decrease in villus surface area in this study, could be due to the villus atrophy and not due to reduction in villus number, which remains constant during the normal life span and various experimental treatments (Clarke, 1972; Forrester, 1972). Villus atrophy in experimental rats was evidenced by approximately 30% reduction in the height of the villus. The observation suggests that measurement of villus shape could be used in heat stress as a variable for mucosal change. It is worthwhile to note that, in spite of the marked degree of villus atrophy, the structural
integrity of the small intestinal mucosa remains well preserved in heat stress. Reduction in the villus height was associated with the reduction in the crypt depth in all the three segments of small intestine. The observation indicates that crypt cell hypoproliferation and alteration in the cellular turnover might result in mucosai hypoplasia. A similar observation has been made in rats responding to semistarvation and fasting (Altmann and Enesco, 1967; Altmann, 1972; Lipscomb and Sharp, 1982). Heat exposure induced morphological change in small intestine mucosa was associated with a decline in its functional performance, as shown by the decrease in L-proline absorption per unit absorptive surface area (Table 3) and consequently total intestinal absorptive capacity also suffered. This is shown by the reduction in proline absorption per unit length of small intestine. All the three intestinal segments, were affected nearly equally. The percent reduction in absorptive capacity per unit length of small intestine far exceeded the percent reduction in proline absorption efficiency per unit absorptive surface area, as compared to the controls. This was expected, since
Table 3. Comparative evaluation of data on intestinal absorption of L-proline in control (24 + 2°C) and heat stressed (37 + 1°C) rats, when normalized to per unit dry weight, per unit length and per unit villus surface area of the small intestinal segments Transport of L-proline normalized to
Segments
Unit weight (~g/mg DW/10 min) Unit length g/ram length/10 min) Unit surface area (~g/cm 2 surface area/10 min)
Jejunum Ileum Jujunum Ileum Jujunum Ileum
Values are group means -1-SD (n = 12). *P < 0.01; t P <0.02; ~P <0.05.
Control
Experimental
Percentage alteration
44.62 + I0.1 23.38 + 7.3 28.51 + 7.8 16.05 + 4. I 147.57+ 42.74 115.04 + 25.2
28.74 + 12.2" 16.07 + 4.9t 15.27 + 6.85* 9.6 + 2.7* 112.74 -I-29.7:1: 93. I + 25.25
35.5 31.2 46.5 40 23.6 19
150
A. SENGUPTAand R. K. SHARMA
absorptive surface area per unit length of intestine was also found to be markedly reduced. Total absorption per unit length of the small intestine (Q) can be expressed as, Q = T.A
where, T = absorption per unit absorptive surface area (in #g/mm 2) and A = absorptive surface area per unit length of the small intestine (in mm2/mm) The fall in total absorption of the small intestine in any given experimental condition was thus, either due to reduction in absorptive surface area or due to decrease in absorptive efficiency per unit surface area or both. The decreased absorption in the small intestine in the heat exposed animals, can be attributed to the reduction of both these variables stated above. In heat acclimated adult rats and hamsters the total amount of glucose transported per everted sac was significantly lower than the control animals (Carpenter and Musacchia, 1974, 1978). Also the glucose uptake in the evened sac of the experimental animals was significantly reduced with associated reduction in apparent Ks (Carpenter and Musacchia, 1978). These observations clearly suggested that reduction in the small intestinal absorption in heat was associated with qualitative functional changes of the intestinal absorptive cell population. In the present study, when proline absorption was normalized to tissue weight of intestinal segment a decreased rate of proline absorption was obtained in experimental rats (Table 3). This confirms previous observation on heat exposed adult rats (Sharma et al., 1982; Sharma, 1985). On the other hand, Chu et al. (1979) and Yousef et al. (1984) reported, increased intestinal absorption for glucose in heat acclimated desert rodents. They speculated that the increased serosal transfer of glucose found in their study may be related to the decreased diffusion barrier due to apparent thinning of the gut wall and decreased metabolism of glucose. This work strongly suggests that in weanling and immature rats (35--45 g), the small intestine undergoes remarkable structural and functional adaptive changes to acute heat exposure at 37 + 1°C for 24 h; the mucosa shows atrophy and decreased capacity for absorption. However, this field requires more intensive work to explain the alteration in the intestinal morphometry and absorption in the animals subjected to heat stress.
Acknowledgements--The work was supported by grants of
the Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
REFERENCES
Altmann G. G. (1972) Influence of starvation and refeeding on mucosal size and epithelial renewal in the rat small intestine. Am. J. Anat. 133, 391--400. Altmann G. G. and Enesco M. (1967) Cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats. Am. J. Anat. 121, 319-336. Barry B. A., Matthews J. and Smyth D. H. (1961) Transfer of glucose and fluid by different parts of the small intestine of the rat. J. Physiol., Lond. 157, 279-288. Brobeck J. R. (1960) Food and temperature. In Recent Progress in Hormone Research, pp. 439-466. Academic Press, New York, N.Y. Carpenter M. and Musacchia X. J. (1974) The effect of chronic heat stress on intestinal function in the rat. Physiologist 17, 371. Carpenter M. and Musacchia X. J. (1978) Chronic heat exposure and intestinal absorption of sugars in hamster. Comp. Biochem. Physiol. 60A, 411-416. Chu D. T., Yousef M. K., Haghani Z. and Hillyard S. D. (1979) Intestinal absorption in heat and cold acclimated desert wood rats. Proc. Soc. exp. Biol. Med. 106, 483-485. Ciampolini M. (1974a) Influence of environmental temperature on xylose absorption in man. In IRCS Medical Science: Alimentary System: Environmental Biology and Medicine; Metabolism and Nutrition; Physiology, Vol. 4,
p. 208. Ciampolini M. (! 974b) Influenceof environmental temperature on intestinal absorption of xylose in rats in vivo. In IRCS Research on Alimentary System: Metabolism and Nutrition; Pediatrics and Perinatal Medicine; Physiology,
Vol. 4, 1545. Ciampolini M. (1977) Variation in intestinal absorption. IV. Environmental temperature, experimental study on rats in relation to rises in temperature. Minerva diet. gastroent. 23, 145. Clarke R. M. (1972) The effectof growth and of fastingon the number of villiand crypts in the small intestineof the albino rat. J. Anat. 112, 27-32. Diamond J. M. and Karasov W. H. 0984) Effect of dietary carbohydrate on monosaccharide uptake by mouse small intestine in vitro. J. Physiol., Lond. 349) 419-440. Forrester J. M. (1972) The number of villiin rats jejunum
and ileum: effect of normal growth, partial enterectomy and tube feeding. J. Anat. 111, 283-291. Hamilton C. L. (1967) Food and temperature. In Handbook o f Physiology. Alimentary Canal. Control of Food and Water Intake, Sect. 6, Vol. 1, pp. 303-317. American
Physiology Society, Washington, D.C. Karasov W. H. and Diamond J. M. (1983) Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am. J. Physiol. 245, G443-G462. Lipscomb H. L. and Sharp J. G. (1982) Effects of reduced food intake on morphometry and cell production in the small intestine of the rat. Virchows. Arch. Cell Path. 41, 285-292. Mayhew T. M. (1984) Geometric model of the rat intestinal mucosa for stereologicalevaluation of villus amplification factors. J. Microsc. 135, 337-346. Mayhew T. M. (1987) Quantitative ultrastructural study on the responses of microvilli along the small bowel to fasting. J. Anat. 154, 237-243. Mayhew T. M. and Middleton C. 0985) Crypts, villi and microvilli in the small intestine of the rat. A stereological
Small intestinal morphometry and absorption in heat stress study of their variability within and between animals. J. Anat. 141, 1-17.
Pennycuik P. R. (1964) The effects on rats of chronic exposure to 34°C. III. Appetite and the efficiency of food conversion. Aust. J. biol. Sci. 17, 236-244. Ross G. A. and Mayhew T. M. (1985) Effects of fasting on mucosal dimensions in the duodenum, jejunum and ileum of the rat. J. Anat. 142, 191-200. Sharma R. K. (1985) Simultaneous measurement of gastric small intestine motility and absorption of proline in rats exposed to heat. Indian J. reed. Res. 81, 330-341. Sharma R. K. and Nagchaudhuri J. (1976) A study of amino acid absorption from small intestine of vinblastin treated rats with altered mucosal morphology. Indian J. med. Res. 64, 1225-34. Sharma R. K., Raj T., Saraswathi M. J. and Nagchaudhuri J. (1982) Effect of high ambient temperature induced heat
151
stress on the functions of small intestine and colon in albino rats. Indian J. med. Res. 75, 593-606. Stenling R. and Helander H. F. (1981) Stereologic studies on the small intestinal epithelium of the rat. 1. The absorptive cells of the normal duodenum and jejunum. Cell. Tissue Res. 217, 11-21. Williamson R. C. N. (1970) Intestinal adaptation, structural, functional and cytokinetic changes. New Engl. J. Med. 298, 1393-1450. Wren J. J. and Wiggall P. H. (1965) An improved colorimettic method for the determination of proline in the presence of other ninhydrin positive compounds. Biochem. J. 94, 216-220. Yousef M. K., Chu D. T. and Haghani Z. (1984) Intestinal transport and thyroid function in heat-acclimated desert rodents. In Thermal Physiology, pp. 491-494, Raven Press, New York, N.Y.
0306-4565/93 $6.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
ACUTE HEAT STRESS IN GROWING RATS: EFFECT ON SMALL INTESTINAL MORPHOMETRY AND IN VIVO ABSORPTION A. SENGUPTA and R. K. SHARMA Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, U.P., India (Received 10 May 1992; accepted in revised form 18 February 1993)
Abstract--1. Young growing rats (35-45 g) were exposed to high ambient temperature (37 +_ 1°C) for 24 h, while the controls were kept at 24 _+2°C. 2. Quantitative morphometry based on stereological principle and absorptive studies by an in vivo method were performed to evaluate the structural and functional adaptations of the small intestine in rats exposed to heat. 3. The effect of heat stress on various morphometric dimensions of the small intestine shows significant reduction in dry weight, villus height, villus surface area, crypt depth, while the absorptive study shows, significant decrease in absorption per unit dry weight, unit length and unit villus surface area of the small intestine as compared to the control. 4. This adaptive alteration in the structure and function of the small intestine indicates the change in the mucosal dynamics of gut epithelium in heat stress. Key Word Index: High ambient temperature; intestinal stereology; intestinal surface area; intestinal absorption; heat and small intestine
INTRODUCTION Absorption phenomenon in small intestine involves transepithelial nutrient particle movement at the villus epithelial zone and removal of molecules from the basolateral surface and subepithelial interstitial space by intestinal microcirculation. In adaptive regulation of nutrient transport by small intestine, structural and functional alterations occur in this region e.g. number of transporting cells, absorptive surface area, unstirred water layer thickness, circulatory response, specific transport process (Wiiliamson, 1970; Karasov and Diamond, 1983). In unacclimated experimental animals, exposed to high ambient temperature or in the heat acclimated experimental animals, there is evidence of a change in intestinal absorption of sugars (Carpenter and Musacchia, 1974, 1978; Chu et al., 1979; Ciampolini, 1974a, b, 1977; Yousef et al., 1984), and amino acids (Sharma et al., 1982; Sharma, 1985). It is probable that the change in absorptive function of small intestine in heat stressed animals reflects an alteration in structural features of intestinal wall components associated with the absorptive process. Such a possibility arises from the fact that, in the heat stressed animals, alteration in the intestinal absorption was associated with reduction in small intestinal weight (Carpenter and Musacchia, 1974, 1978), although no
studies have been reported so far on the structural and functional adaptation of the small intestine to heat stress. The present study was aimed at elucidating the possible alteration in structural features of the small intestine epithelial layer in heat stressed growing rats, using quantitative morphometry, based on stereological principle, recently applied to the tissue by Mayhew and associates (Mayhew 1984, 1987; Mayhew and Middleton 1985). Along with the morphometric measurements, absorptive function of the small intestine in heat stressed rats was also studied to correlate the possible structural alteration with the associated functional changes. MATERIALS AND METHODS
Animals
The experiment was conducted using 36 rats of Charles-Foster strain (35--45g) divided randomly into two equal groups (A and B) of approximately equal group mean body weight. Following separation from the mother on 21st day of life, the animals were kept in individual cages for 4-5 days for acclimatization to their environment, which included 12h light-dark cycle (light on at 8 a.m.), 24 _+_2°C temperature and 62-74% relative humidity, they were fed lipton India rat diet ad libitum with free access to water. After the 4-5 days acclimatization the group 145
146
A. SENGUPTAand R. K. SHARMA
A animals (experimentals) were weighed and exposed to a temperature at 37 + I°C without altering the other housing conditions mentioned above, while group B animals were weighed and kept at 24 + 2°C and served as controls. Both the groups were kept in the experimental environment for a period of 24 h, with free access to food and water. After 24 h, the animals were weighed again and the amount of food consumed by each of the animal was measured in both the group. The body core temperature (rectal) of all the animals were recorded before, during and after they had been kept in the experimental environment, by a quick registry thermometer approximately 25 mm in the rectum. Group A and B rats were then further divided into subgroups Al and A2 and Bj and B2 respectively; the corresponding subgroup mean body weight being kept approximately equal. The animals of groups Aj and B~ each containing 6 rats, were used for the morphometric studies. Similarly the animals of group A2 and B2 each containing 12 rats were used for the absorptive studies. Measurement of dietary consumption, body weight and the animal experimentation were carried out between 9-11 a.m. each day.
Tissue preparation and sampling Under pentobarbital sodium anaesthesia (40 mg/kg body wt, i.p.) the abdomen was opened. The small intestine lumen washed with warm (37°C) normal saline, stripped of its mesentery and divided into three segments of approximately equal length. Each segment was placed in a separate petri dish containing formalin solution (10%) and sliced further into three smaller pieces of equal length. The proximal piece of first segment, middle piece of second segment and the distal piece of the third segment were selected for the study, so that they represent pieces of duodenum, jejunum and ileum, respectively (designated as proximal, mid and distal for descriptive convenience). The pieces were trimmed to a length of approximately 2 cm and placed in fresh fixative solution at 4°C. After fixation the pieces were dehydrated in graded ethanol and embeded in paraffin. Two to three micron thick transverse sections were cut, stained with Haematoxylin-Eosine and examined under light microscope. One photomicrograph of complete transverse section per segment was recorded and printed to a final magnification of x 50.
Stereological procedure Full details of the principle for estimation of morphometric variables are described by Mayhew and associates (Mayhew 1984, 1987; Meyhew and Middleton 1985). Primary mucosa may be defined as the boundary running between the bases of the villi
and the opening of the crypts, the surface--S(P) roughly corresponds to a smooth bore tube. Secondary mucosal surface area--S(V) is that due to villi. Segmental estimation of the circumference of the primary mucosal tube---B(P), the amplification of primary mucosal surface by villi i.e. villus amplification factor or Ss (V, P) and the surface area of primary mucosa--S(P) and of villi--S(V), were derived by intersection point counting method. For the purpose, quadratic test lines of spacing 5 mm (equivalent to a distance Z of 0.1 mm in the actual specimen, considering x50 final magnification) was independently positioned and oriented on each photomicrograph representing that segment. Intersection between test lines and the surface traces of primary mucosa I(P) and villi I(V) were then counted and summed over horizontal and vertical test lines. The intersection ratio I(V)/I(P) = I(V, P) was used to estimate villus amplification by the relationship Ss (V, P) = 4 / ~ . / ( v , a).
The circumference of the primary mucosal tube was obtained by the relationship B ( P ) = n/4.Z.I(P), where Z = 0.1 mm in this study. The primary mucosal surface area is denoted by S(P)=L.B(P) and the secondary or villus surface area is obtained by the relationship S(V) = S(P)-Ss(V, P).L, being the length of the small intestinal segment (2 cm in this study). The detailed explanation and rationale of all these complex relationship were described by Mayhew (1984, 1987), Mayhew and Middleton (1985) and Ross and Mayhew (1985). The heights of villi and crypt on sections were measured with a micrometer scale. Four profiles per intestinal transverse section were measured in order to estimate the average height in each segment.
Absorption studies An anaesthetized rat was placed o n a heating table to keep the rectal temperature around 37°C. A small intestine loop was prepared either from the jejunum or from the ileal segment for the absorption study. The jejunal loop, 10-12cm in length was prepared from the small intestine lying between 5-20 cm from the ligament of Tr~itz. The ileal loop of identical size was prepared from the small intestine approximately 15 cm proximal to ilcocaecal junction. The method of preparation of jejunal and ileal loop has been described earlier (Sharma and Nagchaudhuri, 1976). 0.5 ml of L-proline solution (200 mM/l at 36-37°C) was injected into the loop through fine needle attached to a tuberculin syringe and the loop w a s replaced to the abdomen. The abdomen was closed and the animal was placed o n a platform inside the perspex chamber placed over a constant temperature
Small intestinal morphometry and absorption in heat stress water bath being kept at 34 _+ l°C. Preliminary experiments showed that this arrangement keeps the body core temperature of the operated and the anaesthetized animal at 37°C within an error of _+0.5°C and prevents dehydration, since the relative humidity inside the perspex chamber was maintained at 100%. After an absorption period of 10 rain, the animal was killed and bled by opening the thorax and incising the heart. The intestinal loop was washed with warm normal saline (37°C) and the loop washing volume was recorded. The washing were analysed for proline content after deproteinization. The absorption of proline from the sac was calculated from the difference between the total amount of proline introduced into the loop and the amount of proline recovered at the end of the experimental period. The length of the loop was measured (Barry et al., 1961) and the dry weight of the loop was recorded after drying in a hot air oven at 90°C till the constant weight was achieved. The villus surface area of each intestinal segment was calculated using data obtained in the morphometric studies. The absorption was expressed in terms of length, dry weight and surface area of the loop.
Biochemical estimation and chemicals Proline was estimated by the method of Wren and Wiggal (1965). L-Proline was obtained from John Baker, U.S.A. All the chemicals used for the estimation of proline were of Anala R grade of BDH laboratory chemical division, England. Student's t-test was used to compare the mean values of the control group with those of the experimental group. A value of P < 0.05 was accepted as indicating significance.
small intestine. In the heat exposed rats a significant fall in the values of villus amplification factor was observed at all regions of the small intestine (Table 2). The values were decreased by 22.4, 28.2 and 24.1%, respectively in the proximal, mid and distal regions. As shown in Fig. 2, the proximodistal gradient was maintained closely to that observed in the controls. Villus surface area was significantly reduced in segments of small intestine in experimental animals when compared with the corresponding control segments (Table 2). The decrease was of the order of 34.2, 32 and 27.6%, respectively in the proximal, mid and distal segments. In the experimental group of animals the villus height decreased significantly in all the segments of the small intestine. The effect was maximum over the mid region, since the region showed 36% reduction as compared to 28.7 and 30% reduction seen in proximal and distal segments, respectively (Table 2 and Fig. 2). The crypt depth follows a similar segmental pattern, comparable to that of the villus height, though the slope of the proximodistal gradient was less (Fig. 2). The mean reduction of all the intestinal segments in the experimental animals was 23% as compared to the controls.
Proline absorption The absorption of proline from the jejunal and ileal segments was normalized to dry weight of the sac, length of the sac and absorptive villus surface area of the sac (Table 2). Irrespective of the parameter chosen to express the results, absorption in the control group was [---] Control Experimental
RESULTS Findings are illustrated in Figs 1 and 2 and summarized in Tables 1-3. At the end of the 24 h experimental period, the control animals had gained 2 + 0.82 g (mean + SD) of body weight, while the heat stressed animals gained only 0.7 + 0.34g. Food consumption was significantly decreased, to 65% in experimental rats, as compared to the controls. Dry weight of the small intestine in the heat exposed rats was less than those of the control rats by 16%, whereas the alteration in the length, cireumference of the primary mucosal tube and primary mucosai surface area of the small intestine were not statistically significant (Figs 1 and 2; Table 2.). The villus amplification factor in the controls showed a declining proximodistal gradient, the gradient being more pronounced between proximal to mid segment than between mid to distal segment of the
147
75
-
611
-
-- 5OO
~ "~. ¢d
/-/ // //
--
41111 .~_ U
/ / 45
-
=
//
--
31111
~
--
2011
--
--
ell 11111 ' ~
Z 311
-
15
-
-5.~
// t'/ // // // // // // // ,'/
0
/./ Length
0 Width
Fig. 1. Changes in the length and the dry weight of the small intestine in two groups of rat. Control animals kept at 24+2°C for 24h and experimental animals kept at 37 _+ I °C for 24 h. Bars denote SD ranges.
148
A. SENGUPTA and R. K. SHARMA
5.0 -
1.0 -
2.5
0.5
-
(b)
(a)
400 --
P
DISCUSSION
1
M
1
I
D
6
P
[ M
I D
200 --
(c) l 5.0--
P
I M
2.5 -- f f ~
P
t M
I
200 - P
___~
~'e'l
(d)
I
D
I M
I D
I00
.... J D
(f) P
t
I M
J D
Fig. 2. Gradients of mucosal architecture revealed in proximal (P), mid (M) and distal (D) segment of the small intestine. Variables illustrated are: (a) primary mucosal tube circumference in mm, (b) primary mueosal surface area in cm2, (c) villus height in/~m, (d) villus amplification factor, (e) villus surface area in em2 and (f) crypt depth in /~m. ( ) Data from control animals kept at 24+2°C for 24 h. (- - -) Data from experimental animals kept at 37 + I°C for 24 h. Bars denote SD.
significantly higher in the jejunal segment than in the ileal segment. However, the proximo-distal gradient w a s least when the results were expressed in terms of unit absorptive surface area. In the experimental group of animals L-proline absorption decreased significantly in the jejunal and the ileal segments, when normalized to unit dry weight, unit length and unit absorptive surface area. Nevertheless, the percentage decrease in absorption was least when the absorption was normalized to unit absorptive surface area.
Exposure of developing rats to 37°C for 24 h lead to significant reductions in food consumption and decrease in the body weight gain as compared to the controls. These observations support findings of the earlier workers (Pennycuik, 1964; Chu et al., 1979; Yousef et al., 1984). This could be explained on the fact that in a hot environment, the body temperature may overshoot the normal limit and the intake of food just sufficient to maintain the body weight may overtax the heat regulating mechanism (Hamilton, 1967). Thus a decrease in food intake may lower heat production in an effort to keep the body temperature within normal limit. In homeotherms, therefore, food consumption bears a negative relation to the environmental heat stress temperatures (Brobeck, 1960). The change in body weight in heat exposed rats was associated with loss of small intestinal dry weight of about 15.5%. This disproportionately greater loss of the small intestinal weight than that of the body weight loss has been also reported by Carpenter and Musacchia (1974, 1978) in heat acclimated rats and hamsters, and by Sharma et al. (1982) and Sharma (1985) in adult rats exposed to acute heat stress. Heat causes loss of tissue mass and leads to an apparent thinning of the gut wall (Carpenter and Musacchia, 1974). The change in length, primary mucosal circumference and primary mucosal surface area of the small intestine in heat stressed rats was not statistically significant (Table 2). Observation in starvation experiments showed that the length and circumference undergo variable alterations when the animals were subjected to food deprivation (Stenling and Helander, 1981; Ross and Mayhew, 1985). Present findings suggest that length and circumference alone are relatively insensitive indicators of the effect of stress. Ross and Mayhew (1985) suggested that the experimental effects be monitored by estimating both variables and then calculating mucosal surface areas.
Table 1. Food intake and body weight gain of control (24 + 2°C) and heat stressed (37+ I°C) rats, measured at the end of 24h period from Day 0 to Day 1 Percentage alteration in heat stress as compared to controls
Variable
Control
Heat stressed
Food consumption (g/animal) Body weight
7 + 4.3
2.4 + 1.4"
65.7
2 + 0.82
0.7 -I-0.34*
65
gain (g/animal)
Values are group means -I-SD (n = 18). *P < 0.01.
Small intestinal morphometry and absorption in heat stress
149
Table 2. Morphometric data for various mucosal dimensions in different small intestinal segments in control (24 + 2°C) and heat stressed (37 + I°C) rats Variable
Group
Primary mucosal circumferenc~ (ram) Primary mucosal surface area (cm2) Villus height (/zm)
Control Heat s t r e s s e d Control Heat stressed Control Heat stressed Control Heat stressed Control Heat stressed Control Heat stressed
Villus amplification factor Villus surface area (cm2) Crypt depth (~m)
Proximal
Mid
3.77 + 0.63 3.22+0.43 ( N S ) 0.75 + 0.13 0.64 + 0.08 (NS) 299 + 19 213 + 26* 5.14 + 0.58 3.98 + 0.73~/ 3.86 ± 0.75 2.57 + 0.62:[: 168 _+ 17 133 ± 12"
3.84 + 0.19 3.49+0.71 (hiS) 0.77 + 0.04 0.69 + 0.14 (NS) 216 + 50 138 + 24t 3.8 + 0.53 2.73 + 0.52t 2.93 + 0.45 1.99 + 0.05t 148 + 17 120 _+ 14~
Distal 4.14 + 0.52 3.91 +0.37 (NS) 0.83 + 0.01 0.78 + 0.07 (NS) 190 + 38 133 + 17t 3.51 + 0.48 2.66 + 0.05:~ 2.88 + 0.25 2.08 + 0.16" 129 ± 15 106 + 8~/
Values are group mean + SD (n = 6). *P < 0.01; ~'P < 0.02; :~P < 0.05; NS = not significant. In the heat stressed rats, a significant reduction in villus amplification factor was observed in all the segments of the small intestine; consequently, a marked reduction in the villus surface area was recorded. In heat exposed rats the small intestine mucosa, responded by a reduction in the absorptive surface area, similar to that seen in states of fasting (Ross and Mayhew, 1985; Mayhew, 1987). All three regions of the small intestine responded to a similar degree and therefore proximodistal gradient in villus surface area, seen in the control animals, persisted in the heat stressed rats (Fig. 2). The decrease in villus surface area in this study, could be due to the villus atrophy and not due to reduction in villus number, which remains constant during the normal life span and various experimental treatments (Clarke, 1972; Forrester, 1972). Villus atrophy in experimental rats was evidenced by approximately 30% reduction in the height of the villus. The observation suggests that measurement of villus shape could be used in heat stress as a variable for mucosal change. It is worthwhile to note that, in spite of the marked degree of villus atrophy, the structural
integrity of the small intestinal mucosa remains well preserved in heat stress. Reduction in the villus height was associated with the reduction in the crypt depth in all the three segments of small intestine. The observation indicates that crypt cell hypoproliferation and alteration in the cellular turnover might result in mucosai hypoplasia. A similar observation has been made in rats responding to semistarvation and fasting (Altmann and Enesco, 1967; Altmann, 1972; Lipscomb and Sharp, 1982). Heat exposure induced morphological change in small intestine mucosa was associated with a decline in its functional performance, as shown by the decrease in L-proline absorption per unit absorptive surface area (Table 3) and consequently total intestinal absorptive capacity also suffered. This is shown by the reduction in proline absorption per unit length of small intestine. All the three intestinal segments, were affected nearly equally. The percent reduction in absorptive capacity per unit length of small intestine far exceeded the percent reduction in proline absorption efficiency per unit absorptive surface area, as compared to the controls. This was expected, since
Table 3. Comparative evaluation of data on intestinal absorption of L-proline in control (24 + 2°C) and heat stressed (37 + 1°C) rats, when normalized to per unit dry weight, per unit length and per unit villus surface area of the small intestinal segments Transport of L-proline normalized to
Segments
Unit weight (~g/mg DW/10 min) Unit length g/ram length/10 min) Unit surface area (~g/cm 2 surface area/10 min)
Jejunum Ileum Jujunum Ileum Jujunum Ileum
Values are group means -1-SD (n = 12). *P < 0.01; t P <0.02; ~P <0.05.
Control
Experimental
Percentage alteration
44.62 + I0.1 23.38 + 7.3 28.51 + 7.8 16.05 + 4. I 147.57+ 42.74 115.04 + 25.2
28.74 + 12.2" 16.07 + 4.9t 15.27 + 6.85* 9.6 + 2.7* 112.74 -I-29.7:1: 93. I + 25.25
35.5 31.2 46.5 40 23.6 19
150
A. SENGUPTAand R. K. SHARMA
absorptive surface area per unit length of intestine was also found to be markedly reduced. Total absorption per unit length of the small intestine (Q) can be expressed as, Q = T.A
where, T = absorption per unit absorptive surface area (in #g/mm 2) and A = absorptive surface area per unit length of the small intestine (in mm2/mm) The fall in total absorption of the small intestine in any given experimental condition was thus, either due to reduction in absorptive surface area or due to decrease in absorptive efficiency per unit surface area or both. The decreased absorption in the small intestine in the heat exposed animals, can be attributed to the reduction of both these variables stated above. In heat acclimated adult rats and hamsters the total amount of glucose transported per everted sac was significantly lower than the control animals (Carpenter and Musacchia, 1974, 1978). Also the glucose uptake in the evened sac of the experimental animals was significantly reduced with associated reduction in apparent Ks (Carpenter and Musacchia, 1978). These observations clearly suggested that reduction in the small intestinal absorption in heat was associated with qualitative functional changes of the intestinal absorptive cell population. In the present study, when proline absorption was normalized to tissue weight of intestinal segment a decreased rate of proline absorption was obtained in experimental rats (Table 3). This confirms previous observation on heat exposed adult rats (Sharma et al., 1982; Sharma, 1985). On the other hand, Chu et al. (1979) and Yousef et al. (1984) reported, increased intestinal absorption for glucose in heat acclimated desert rodents. They speculated that the increased serosal transfer of glucose found in their study may be related to the decreased diffusion barrier due to apparent thinning of the gut wall and decreased metabolism of glucose. This work strongly suggests that in weanling and immature rats (35--45 g), the small intestine undergoes remarkable structural and functional adaptive changes to acute heat exposure at 37 + 1°C for 24 h; the mucosa shows atrophy and decreased capacity for absorption. However, this field requires more intensive work to explain the alteration in the intestinal morphometry and absorption in the animals subjected to heat stress.
Acknowledgements--The work was supported by grants of
the Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
REFERENCES
Altmann G. G. (1972) Influence of starvation and refeeding on mucosal size and epithelial renewal in the rat small intestine. Am. J. Anat. 133, 391--400. Altmann G. G. and Enesco M. (1967) Cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats. Am. J. Anat. 121, 319-336. Barry B. A., Matthews J. and Smyth D. H. (1961) Transfer of glucose and fluid by different parts of the small intestine of the rat. J. Physiol., Lond. 157, 279-288. Brobeck J. R. (1960) Food and temperature. In Recent Progress in Hormone Research, pp. 439-466. Academic Press, New York, N.Y. Carpenter M. and Musacchia X. J. (1974) The effect of chronic heat stress on intestinal function in the rat. Physiologist 17, 371. Carpenter M. and Musacchia X. J. (1978) Chronic heat exposure and intestinal absorption of sugars in hamster. Comp. Biochem. Physiol. 60A, 411-416. Chu D. T., Yousef M. K., Haghani Z. and Hillyard S. D. (1979) Intestinal absorption in heat and cold acclimated desert wood rats. Proc. Soc. exp. Biol. Med. 106, 483-485. Ciampolini M. (1974a) Influence of environmental temperature on xylose absorption in man. In IRCS Medical Science: Alimentary System: Environmental Biology and Medicine; Metabolism and Nutrition; Physiology, Vol. 4,
p. 208. Ciampolini M. (! 974b) Influenceof environmental temperature on intestinal absorption of xylose in rats in vivo. In IRCS Research on Alimentary System: Metabolism and Nutrition; Pediatrics and Perinatal Medicine; Physiology,
Vol. 4, 1545. Ciampolini M. (1977) Variation in intestinal absorption. IV. Environmental temperature, experimental study on rats in relation to rises in temperature. Minerva diet. gastroent. 23, 145. Clarke R. M. (1972) The effectof growth and of fastingon the number of villiand crypts in the small intestineof the albino rat. J. Anat. 112, 27-32. Diamond J. M. and Karasov W. H. 0984) Effect of dietary carbohydrate on monosaccharide uptake by mouse small intestine in vitro. J. Physiol., Lond. 349) 419-440. Forrester J. M. (1972) The number of villiin rats jejunum
and ileum: effect of normal growth, partial enterectomy and tube feeding. J. Anat. 111, 283-291. Hamilton C. L. (1967) Food and temperature. In Handbook o f Physiology. Alimentary Canal. Control of Food and Water Intake, Sect. 6, Vol. 1, pp. 303-317. American
Physiology Society, Washington, D.C. Karasov W. H. and Diamond J. M. (1983) Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am. J. Physiol. 245, G443-G462. Lipscomb H. L. and Sharp J. G. (1982) Effects of reduced food intake on morphometry and cell production in the small intestine of the rat. Virchows. Arch. Cell Path. 41, 285-292. Mayhew T. M. (1984) Geometric model of the rat intestinal mucosa for stereologicalevaluation of villus amplification factors. J. Microsc. 135, 337-346. Mayhew T. M. (1987) Quantitative ultrastructural study on the responses of microvilli along the small bowel to fasting. J. Anat. 154, 237-243. Mayhew T. M. and Middleton C. 0985) Crypts, villi and microvilli in the small intestine of the rat. A stereological
Small intestinal morphometry and absorption in heat stress study of their variability within and between animals. J. Anat. 141, 1-17.
Pennycuik P. R. (1964) The effects on rats of chronic exposure to 34°C. III. Appetite and the efficiency of food conversion. Aust. J. biol. Sci. 17, 236-244. Ross G. A. and Mayhew T. M. (1985) Effects of fasting on mucosal dimensions in the duodenum, jejunum and ileum of the rat. J. Anat. 142, 191-200. Sharma R. K. (1985) Simultaneous measurement of gastric small intestine motility and absorption of proline in rats exposed to heat. Indian J. reed. Res. 81, 330-341. Sharma R. K. and Nagchaudhuri J. (1976) A study of amino acid absorption from small intestine of vinblastin treated rats with altered mucosal morphology. Indian J. med. Res. 64, 1225-34. Sharma R. K., Raj T., Saraswathi M. J. and Nagchaudhuri J. (1982) Effect of high ambient temperature induced heat
151
stress on the functions of small intestine and colon in albino rats. Indian J. med. Res. 75, 593-606. Stenling R. and Helander H. F. (1981) Stereologic studies on the small intestinal epithelium of the rat. 1. The absorptive cells of the normal duodenum and jejunum. Cell. Tissue Res. 217, 11-21. Williamson R. C. N. (1970) Intestinal adaptation, structural, functional and cytokinetic changes. New Engl. J. Med. 298, 1393-1450. Wren J. J. and Wiggall P. H. (1965) An improved colorimettic method for the determination of proline in the presence of other ninhydrin positive compounds. Biochem. J. 94, 216-220. Yousef M. K., Chu D. T. and Haghani Z. (1984) Intestinal transport and thyroid function in heat-acclimated desert rodents. In Thermal Physiology, pp. 491-494, Raven Press, New York, N.Y.