- Email: [email protected]
Effect of supplemental vitamin A on the healing of colon anastomosis
JOURNAL
OF SURGICAL
RESEARCH
Effect of Supplemental
36,470-474
Vitamin
(1984)
A on the Healing
of Colon Anastomosis’
STAFFAN BARK, M.D.,* GIUSEPPE RETTURA,PH.D.,**~ DANIELGOLDMAN,M.D.,* ELI SEW-I-ER,PH.D.,*,~ STANLEY M.LEvENsoN,M.D.,* AND ACHILLES A. DEMETRIOU,M.D., PH.D.* Combined *Department of Surgery, Albert Einstein College of Medicine, and Montefrore Hospital and Medical Center, and TDepartment of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 Presented at the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 The effect of dietary supplementation with vitamin A on the healing of colon anastomoses was studied. Fifty adult male Sprague-Dawley rats were divided into two groups: (1) rats fed a standard chow which contains the equivalent of about 15 IU vitamin A/g diet: (2) rats fed the chow supplemented with an additional 150 IU vitamin A/g diet. Rats were prefed for 5 days: on Day 6 under ether anesthesia the colon was divided l-in. distal to the ileocecal junction and then reanastomosed. The rats were maintained on the above diets for 5 days and killed on the sixth postoperative day with ether and the segment of colon containing the anastomosis was resected. In 15 rats of each group, the breaking strength of the anastomosis was measured. In the remaining 10 rats of each group, the bursting strength of the anastomotic site and a segment of normal distal colon was measured. Samples of colon from the anastomotic site and the normal segment were analyzed for hydroxyproline. There was a significant decrease in hydroxyproline content at the anastomotic site when compared to the normal distal colon segment in each group of rats (P c 0.01). The hydroxyprohne content of both normal colon and the anastomotic site was significantly higher in the vitamin A-supplemented rats than in the control diet rats (P < 0.01). There was also a significant increase in bursting strength in the vitamin A-supplemented rats both of the anastomotic site (P < 0.01) and of the normal colon segment (P < 0.01). The anastomotic breaking strength peak values did not differ significantly between the two groups, but the area under the breaking strength curve (an expression of the energy required to break the anastomosis) was three times greater in the vitamin A-supplemented rats than in the controls (P i 0.001). It is concluded that vitamin A supplementation has a beneficial effect on the healing of colon anastomoses in rats.
INTRODUCTION
Ehrlich and Hunt [7] have shown that supplemental vitamin A antagonizes some of the inhibitory effects of cortisone on wound healing and Seifter et al. [ 171 and Herrmann et al. [ 1 l] have shown that when supplemental vitamin A is given to normal rats, it increases the rate at which reparative collagen accumulates within subcutaneously implanted polyvinyl alcohol sponges. Seifter and his associates [ 171 showed also that local and systemic administration of supplemental vitamin ’ Supported in part by grants from the Army Medical Research and Development Command (DAMD- I7-82C-209 1); National Institutes of Health (5 K06 GM 14208, Research Career Award, S.M.L.); and Research Funds of the Karolinska Institutet, Stockholm, Sweden.
0022-4804184 $1.50 Copyright 0 1984 by Academic Fres. Inc. All rights of reproduction in any form reserved.
A results in increased wound breaking strength in normal rats and in rats with femoral fractures. In another series of experiments, Demetriou et al. [4] have shown that oral administration of supplemental vitamin A to mice enhances peritoneal adhesion formation. It was shown by Lee and Tong [ 121 that application of retinoic acid to the healing open wound antagonizes the wound healing inhibitory effects of salicylates as well as of the glucocorticoids, prednisone and hydrocortisone. Ehrlich and his associates [8] reported that supplemental vitamin E inhibited wound healing and that this inhibitory effect could be reversed by intramuscular vitamin A. In view of these findings, we postulated that supplemental vitamin A would likely accelerate the healing of colon anastomoses in rats. 470
BARK
ET AL.: VITAMIN
A AND
COLON
ANASTOMOSIS
471
care and the breaking strength of the anastomosis was measuredwith a tensiometer [ 131. Both the peak values and areas under the curves, the latter a reflection of the energy MATERIALS AND METHODS required to break the anastomosis [9], were Fifty male Sprague-Dawley rats (Charles measured. The colon anastomosis and a norRiver Farms, Wilmington, Mass.) were placed mal segment of colon immediately distal to in individual two-mesh stainless-steel cages the anastomosiswere resectedin the remaining 10 rats of each group. Bursting strength was and acclimatized to our laboratory conditions for 1 week prior to onset of the experiment. measured in these segmentsby controlled inTemperature and relative humidity were con- sufflation of air into the segment which was trolled (26 + 1“C and about 40%, respectively) ligated at one end and a catheter introduced as was the day:night cycle (12 hr light, 12 hr into the other end and held in place by a tightdark). At that time, their mean weight was ened suture. The colon segment was kept un325 g and they were divided by closely paired der 0.9% saline in a beaker and inflated with weights into two groups, 25 rats each. One air at a rate increasing the pressure within the group was continued on a commercial labo- segment by 1 mm Hg/sec. The pressure was ratory chow (Purina regular lab chow No. measured by a sphygmomanometer and the 5001, Ralston Purina, St. Louis, MO.) con- bursting pressure was that pressure at which taining 15 IU vitamin A and 6.5 ppm P-car- air first leaked (bubbled) from the segment. otene/g diet, or about three times the National Colon collagen content was estimated by Research Council Recommended Daily Al- measurement of the hydroxyproline content lowance of vitamin A for normal rats. The using the method of Woessner [ 191.Both norother group received the same chow supple- mal colon and anastomotic site were studied. mented with 150 IU vitamin A palmitate/g The statistical calculations were done using diet; this resulted in a chow containing about Student’s t test for unpaired samples. twenty times the National Research Council Recommended Daily Allowance for normal RESULTS rats. The vitamin A palmitate was dissolved in 70% ethanol and mixed with the chow for There was moderate and similar colon dis90 min. All rats ate and drank tap water ad tension proximal to the anastomosis in most rats with slight narrowing of the lumen of the libitum throughout the experiment. On Day 6, the rats were anesthetized with anastomosis,without evidence of obstruction. ether and underwent a laparotomy. The colon There were no gross differences in the degree was divided 1 in. from the ileocecal junction of perianastomotic inflammatory reaction beand then reanastomosed with continuous 4- tween the two groups of rats. There was no statistically significant differ0 catgut (15 rats/group) or interrupted 4-O silk sutures (10 rats/group). Rats were main- encein breaking strength peak values between tained, respectively, on the above diets ad li- control and vitamin A supplemented rats (Tabitum postoperatively and killed with ether ble 1). However, there was a significant (threeon the sixth postoperative day. The segment fold) increase in the energy needed to break of colon (approximately 2 in. in length) con- the anastomoses in the vitamin A-suppletaining the anastomosis was resected. In the mented group of animals (P < 0.001) (Table 15 rats from each group in which the anas- 1). The breaking strength curve of the anastomosis was carried out with running catgut tomosis of the control group showed a sharp, suture, the resected colon was incised along narrow peak, whereas that of vitamin A-supthe mesenteric border and outfolded to a plemented rats showed a wide, blunt curve quadrangular specimen; the visible residue of (Fig. 1).There wasalso a statistically significant the catgut sutures were removed with great increase in bursting strength in the vitamin Experiments to test this postulate form the basis of this paper.
472
JOURNAL TABLE
OF SURGICAL
RESEARCH:
VOL. 36, NO. 5, MAY
1984
1
inal abscessand likely enhancement of the immune response. The data of the present study demonstrate Peak value Curve area that supplemental dietary vitamin A had a Group (EC) (cm7 beneficial effect on the healing of colon anastomoses, evidenced by- increases in colonic Basal control diet 15.3 + 1.5 25.2 + 3.0 bursting and breaking strengths and hydroxyVitamin A 78.0 f 11.5 supplementation 88.8 + 6.6 proline content at the anastomotic site. Dietary supplementation with vitamin A also NS P < 0.001 resulted in an increased hydroxyproline cona Mean values f SEM. tent in the colon away from the anastomosis. The increase in collagen seenin both the normal colon and the anastomotic site of animals A-supplemented group both in the anasto- on a vitamin A-supplemented diet may be mosed colon (P < 0.02), and in the normal due to increased collagen synthesis [ 161, decolon (P < 0.01) (Table 2). Similarly, the hy- creased collagenolytic activity [2], or both. droxyproline contents were significantly higher Brinkerhoff and associates[2] have shown that both at the anastomotic site (P < 0.01) and vitamin A inhibits collagenase production of in the normal colon segment (P < 0.01) (Ta- rheumatoid synovial cells. On the other hand, ble 3). in another series of experiments carried out in our laboratory, Demetriou et al. [6] demonstrated that addition of vitamin A to an in DISCUSSION vitro fibroblast tissue culture system can inIn previous studies in our laboratory [5], duce cell differentiation manifested by morDemetriou et al. have shown that dietary sup phologic changes,decreasein cell growth rate, plementation with vitamin A has a beneficial and increase in collagen content. It should be emphasized that the control effect in animals with intraabdominal sepsis due to cecal ligation and perforation. This ef- rats were not on a vitamin A-deficient diet. fect is manifested by improved survival and In fact, the control diet contains about three is due to better walling off of the intraabdom- times the National Research Council RecBREAKINGSTRENGTHOFCOLONANASTOMOSES'
STANDARDS
CONTROL
VITAMIN A SUPPLEMENT
A
FIG. 1. Typical curves of colon anastomosis breaking strength measurements. Two specimens from the control group and one from the vitamin A-supplemented group.
BARK ET AL.: VITAMIN
A AND COLON ANASTOMOSIS
ommended Daily Allowance for vitamin A for normal rats and supports normal growth, reproduction, lactation, and longevity of normal rats. The dietary vitamin A supplement used (150 IU/g diet), is not associated with any signs or symptoms of vitamin A toxicity in animals eating the supplemented chow for over a year [ 181. Our results in bursting strength changesare consistent with earlier studies demonstrating the correlation between increased bursting strength and increased collagen content in colon [3, lo]. It is of interest that the bursting strength of normal colon in the control group was about the sameas that of the anastomosed segment in the vitamin A-supplemented group, although the segmentsare not exactly comparable in location. As mentioned, we found that the peak values of the breaking strength of the colon anastomosis did not differ significantly in the two groups of rats but the shape of and the area under the breaking strength curve, an expression of the energy neededto break the healing anastomosis, showed a highly significant difference (P < O.OOl), the area being much greater in the vitamin A-supplemented rats. The differences in the shapesand areas of the breaking strength curves suggesta more “elastic” property of the colon anastomotic sites of the vitamin A-supplemented rats. There is little information about elastin accumulation in wounds during the early healing period. We did not chemically analyze for or look histologically for elastin in our study. Five days is too short a time to expect any substantial
473
TABLE 3 HYDROXYPROLINECONTENT(rg/mg TISSUE)” Group
Normal colon
Anastomosis
Basal control diet Vitamin A sunnlementation ..
2.8 f 0.2
1.6 f 0.1
4.1 k 0.4
2.3 + 0.2
P < 0.01
P < 0.01
’ Mean values f SEM.
increase in elastin formation in the tissue but it should be noted that the vitamin A supplement had been given for 5 days preoperatively as well. Mecham and associates [ 141 have reported tissue culture studieswhich have shown soluble elastin formation by fetal bovine ligamenturn nuchae fibroblasts evident by the fifth day, when cell proliferation began to be density inhibited and the cells became quiescent. Oakes et al. [ 151have found in primary cultures of neonatal rat aortic smooth muscle cell insoluble elastin formation after 10 days. The insoluble elastin first appeared as small particles which then formed elastic fibres in vitro. Also, the “elasticity” of a tissue depends not only on the quantity but also on the quality of elastin [I] and on the presence of other intercellular substances. In summary, our data show that dietary vitamin A supplementation increasescollagen (hydroxyproline) content of normal colon and of the healing anastomotic segment. There is also a dramatic change in “elasticity” of the anastomotic site tissue, and a significant increasein bursting strength of the anastomosis.
TABLE 2 BURSTINGSTRENGTH(mm Hg) OF COLONANASTOMOSES" Group
Normal colon
Anastomosis
Basal control diet Vitamin A supplementation
196 f 1.6
172 f 4.6
216 + 6.9
197 f 6.1
P < 0.01
’ Mean values f SEM.
P < 0.02
REFERENCES 1. Banga,I. Structure andfinction
of e&in and collagen. Akademiai Kiadb. Budapest, Hungary. 1966. 2. Brinkerhoff, C., McMillan, R. M., Dwyer, J. M., and Harris, E. Inhibition by retinoic acid of collagenase production in rheumatoid synovial cells. N. kg/. J. Med. 303: 432, 1980. 3. Cronin, K., Jackson, D. S., and Dunphy, J. E. Changing bursting strength and collagen content of the healing colon. Surg. Gynecol. Obstet. 126: 747, 1968. 4. Demetriou, A. A., Seifter, E., and Levenson, S. M.
474
JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 5, MAY 1984
Effect of vitamin A and citral on peritoneal adhesion formation. J. Surg. Res. 17: 325, 1974. 5. Demetriou, A. A., France, I., Bark, S., Rettura, G., Seiher, E., and Levenson, S. M. Effects of vitamin A and beta carotene on intraabdominal sepsis. Arch. Surg. 119: 161, 1984.
6. Demetriou, A. A., Seifter, E., and Levenson, S. M. Effect of vitamin A on fibroblast proliferation and collagen accumulation in vitro, unpublished. 7. Ehrlich, H. P., and Hunt, T. K. Effect of cortisone and vitamin A on wound healing. Ann. Surg. 167: 324, 1968. 8. Ehrlich, H. P., Tarver, H., and Hunt, T. K. Inhibitory effectsof vitamin E on collagen synthesis and wound repair. Ann. Surg. 175: 235, 1972. 9. Gottrup, F. Healing of incisional wounds in stomach and duodenum. Amer. J. Surg. 140: 296, 1980. 10. Hawley, P. R., Faulk, W. P., Hunt, T. K., and Dunphy, J. E. Collagenaseactivity in the gastrointestinal tract. Brit. J. Surg. 57: 896, 1970. 11. Herrmann, J. B., and Woodward, S. C. An experimental study on wound healing accelerators. Amer. Surg. 38: 26, 1972. 12. Lee, K. H., and Tong, T. G. Studieson the mechanism of action of salicylatesIV: Effect of topical application of retinoic acid on wound healing retardation action of salicylic acid. J Pharmacol. Sci. 58: 773, 1969. 13. Levenson, S. M., Crowley,k. V., Geever, E. F., Rosen, H., and Berard, C. W. Some studieson wound healing:
Experimental methods, effect of ascorbic acid, and effect of deuterium oxide. J. Trauma 4: 543, 1964. 14. Mecham, R. P., Lange, G., Madaras, J., and Stancher, B. Elastin synthesisby ligamentum nuchae fibroblasts: Effects of culture conditions and extracellular matrix on elastin production. J. Cell Biol. 90: 332, 1981. 15. Oakes, B. W., Batty, A. C., Handley, C. J., and Sandberg, L. B. The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study. Eur. J. Cell Biol. 27: 34, 1982. 16. Patt, L. M., Itaya, K., and Hakamori, S. Retinol induces density-dependent growth inhibition and changes in glycolipids and LETS. Nature (London) 273: 379, 1978. 17. Seifter, E., Crowley, L. V., Rettura, G., Nakao, K., Gruber, C., Kan, D., and Levenson, S. M. Influence of vitamin A on wound healing in rats with femoral fracture. Ann. Surg. 181: 836, 1975. 18. Seifter, E., Rettura, G., Padawer, J., Stratford, F., Goodwin, P., and Levenson, S. M. Regression of C3HBA mousetumor due to X-my therapy combined with supplemental beta carotene or vitamin A. J. Nat. Cancer Inst. 71: 409, 1983.
19. Woessner,J. F. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93: 440, 1961.
OF SURGICAL
RESEARCH
Effect of Supplemental
36,470-474
Vitamin
(1984)
A on the Healing
of Colon Anastomosis’
STAFFAN BARK, M.D.,* GIUSEPPE RETTURA,PH.D.,**~ DANIELGOLDMAN,M.D.,* ELI SEW-I-ER,PH.D.,*,~ STANLEY M.LEvENsoN,M.D.,* AND ACHILLES A. DEMETRIOU,M.D., PH.D.* Combined *Department of Surgery, Albert Einstein College of Medicine, and Montefrore Hospital and Medical Center, and TDepartment of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 Presented at the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 The effect of dietary supplementation with vitamin A on the healing of colon anastomoses was studied. Fifty adult male Sprague-Dawley rats were divided into two groups: (1) rats fed a standard chow which contains the equivalent of about 15 IU vitamin A/g diet: (2) rats fed the chow supplemented with an additional 150 IU vitamin A/g diet. Rats were prefed for 5 days: on Day 6 under ether anesthesia the colon was divided l-in. distal to the ileocecal junction and then reanastomosed. The rats were maintained on the above diets for 5 days and killed on the sixth postoperative day with ether and the segment of colon containing the anastomosis was resected. In 15 rats of each group, the breaking strength of the anastomosis was measured. In the remaining 10 rats of each group, the bursting strength of the anastomotic site and a segment of normal distal colon was measured. Samples of colon from the anastomotic site and the normal segment were analyzed for hydroxyproline. There was a significant decrease in hydroxyproline content at the anastomotic site when compared to the normal distal colon segment in each group of rats (P c 0.01). The hydroxyprohne content of both normal colon and the anastomotic site was significantly higher in the vitamin A-supplemented rats than in the control diet rats (P < 0.01). There was also a significant increase in bursting strength in the vitamin A-supplemented rats both of the anastomotic site (P < 0.01) and of the normal colon segment (P < 0.01). The anastomotic breaking strength peak values did not differ significantly between the two groups, but the area under the breaking strength curve (an expression of the energy required to break the anastomosis) was three times greater in the vitamin A-supplemented rats than in the controls (P i 0.001). It is concluded that vitamin A supplementation has a beneficial effect on the healing of colon anastomoses in rats.
INTRODUCTION
Ehrlich and Hunt [7] have shown that supplemental vitamin A antagonizes some of the inhibitory effects of cortisone on wound healing and Seifter et al. [ 171 and Herrmann et al. [ 1 l] have shown that when supplemental vitamin A is given to normal rats, it increases the rate at which reparative collagen accumulates within subcutaneously implanted polyvinyl alcohol sponges. Seifter and his associates [ 171 showed also that local and systemic administration of supplemental vitamin ’ Supported in part by grants from the Army Medical Research and Development Command (DAMD- I7-82C-209 1); National Institutes of Health (5 K06 GM 14208, Research Career Award, S.M.L.); and Research Funds of the Karolinska Institutet, Stockholm, Sweden.
0022-4804184 $1.50 Copyright 0 1984 by Academic Fres. Inc. All rights of reproduction in any form reserved.
A results in increased wound breaking strength in normal rats and in rats with femoral fractures. In another series of experiments, Demetriou et al. [4] have shown that oral administration of supplemental vitamin A to mice enhances peritoneal adhesion formation. It was shown by Lee and Tong [ 121 that application of retinoic acid to the healing open wound antagonizes the wound healing inhibitory effects of salicylates as well as of the glucocorticoids, prednisone and hydrocortisone. Ehrlich and his associates [8] reported that supplemental vitamin E inhibited wound healing and that this inhibitory effect could be reversed by intramuscular vitamin A. In view of these findings, we postulated that supplemental vitamin A would likely accelerate the healing of colon anastomoses in rats. 470
BARK
ET AL.: VITAMIN
A AND
COLON
ANASTOMOSIS
471
care and the breaking strength of the anastomosis was measuredwith a tensiometer [ 131. Both the peak values and areas under the curves, the latter a reflection of the energy MATERIALS AND METHODS required to break the anastomosis [9], were Fifty male Sprague-Dawley rats (Charles measured. The colon anastomosis and a norRiver Farms, Wilmington, Mass.) were placed mal segment of colon immediately distal to in individual two-mesh stainless-steel cages the anastomosiswere resectedin the remaining 10 rats of each group. Bursting strength was and acclimatized to our laboratory conditions for 1 week prior to onset of the experiment. measured in these segmentsby controlled inTemperature and relative humidity were con- sufflation of air into the segment which was trolled (26 + 1“C and about 40%, respectively) ligated at one end and a catheter introduced as was the day:night cycle (12 hr light, 12 hr into the other end and held in place by a tightdark). At that time, their mean weight was ened suture. The colon segment was kept un325 g and they were divided by closely paired der 0.9% saline in a beaker and inflated with weights into two groups, 25 rats each. One air at a rate increasing the pressure within the group was continued on a commercial labo- segment by 1 mm Hg/sec. The pressure was ratory chow (Purina regular lab chow No. measured by a sphygmomanometer and the 5001, Ralston Purina, St. Louis, MO.) con- bursting pressure was that pressure at which taining 15 IU vitamin A and 6.5 ppm P-car- air first leaked (bubbled) from the segment. otene/g diet, or about three times the National Colon collagen content was estimated by Research Council Recommended Daily Al- measurement of the hydroxyproline content lowance of vitamin A for normal rats. The using the method of Woessner [ 191.Both norother group received the same chow supple- mal colon and anastomotic site were studied. mented with 150 IU vitamin A palmitate/g The statistical calculations were done using diet; this resulted in a chow containing about Student’s t test for unpaired samples. twenty times the National Research Council Recommended Daily Allowance for normal RESULTS rats. The vitamin A palmitate was dissolved in 70% ethanol and mixed with the chow for There was moderate and similar colon dis90 min. All rats ate and drank tap water ad tension proximal to the anastomosis in most rats with slight narrowing of the lumen of the libitum throughout the experiment. On Day 6, the rats were anesthetized with anastomosis,without evidence of obstruction. ether and underwent a laparotomy. The colon There were no gross differences in the degree was divided 1 in. from the ileocecal junction of perianastomotic inflammatory reaction beand then reanastomosed with continuous 4- tween the two groups of rats. There was no statistically significant differ0 catgut (15 rats/group) or interrupted 4-O silk sutures (10 rats/group). Rats were main- encein breaking strength peak values between tained, respectively, on the above diets ad li- control and vitamin A supplemented rats (Tabitum postoperatively and killed with ether ble 1). However, there was a significant (threeon the sixth postoperative day. The segment fold) increase in the energy needed to break of colon (approximately 2 in. in length) con- the anastomoses in the vitamin A-suppletaining the anastomosis was resected. In the mented group of animals (P < 0.001) (Table 15 rats from each group in which the anas- 1). The breaking strength curve of the anastomosis was carried out with running catgut tomosis of the control group showed a sharp, suture, the resected colon was incised along narrow peak, whereas that of vitamin A-supthe mesenteric border and outfolded to a plemented rats showed a wide, blunt curve quadrangular specimen; the visible residue of (Fig. 1).There wasalso a statistically significant the catgut sutures were removed with great increase in bursting strength in the vitamin Experiments to test this postulate form the basis of this paper.
472
JOURNAL TABLE
OF SURGICAL
RESEARCH:
VOL. 36, NO. 5, MAY
1984
1
inal abscessand likely enhancement of the immune response. The data of the present study demonstrate Peak value Curve area that supplemental dietary vitamin A had a Group (EC) (cm7 beneficial effect on the healing of colon anastomoses, evidenced by- increases in colonic Basal control diet 15.3 + 1.5 25.2 + 3.0 bursting and breaking strengths and hydroxyVitamin A 78.0 f 11.5 supplementation 88.8 + 6.6 proline content at the anastomotic site. Dietary supplementation with vitamin A also NS P < 0.001 resulted in an increased hydroxyproline cona Mean values f SEM. tent in the colon away from the anastomosis. The increase in collagen seenin both the normal colon and the anastomotic site of animals A-supplemented group both in the anasto- on a vitamin A-supplemented diet may be mosed colon (P < 0.02), and in the normal due to increased collagen synthesis [ 161, decolon (P < 0.01) (Table 2). Similarly, the hy- creased collagenolytic activity [2], or both. droxyproline contents were significantly higher Brinkerhoff and associates[2] have shown that both at the anastomotic site (P < 0.01) and vitamin A inhibits collagenase production of in the normal colon segment (P < 0.01) (Ta- rheumatoid synovial cells. On the other hand, ble 3). in another series of experiments carried out in our laboratory, Demetriou et al. [6] demonstrated that addition of vitamin A to an in DISCUSSION vitro fibroblast tissue culture system can inIn previous studies in our laboratory [5], duce cell differentiation manifested by morDemetriou et al. have shown that dietary sup phologic changes,decreasein cell growth rate, plementation with vitamin A has a beneficial and increase in collagen content. It should be emphasized that the control effect in animals with intraabdominal sepsis due to cecal ligation and perforation. This ef- rats were not on a vitamin A-deficient diet. fect is manifested by improved survival and In fact, the control diet contains about three is due to better walling off of the intraabdom- times the National Research Council RecBREAKINGSTRENGTHOFCOLONANASTOMOSES'
STANDARDS
CONTROL
VITAMIN A SUPPLEMENT
A
FIG. 1. Typical curves of colon anastomosis breaking strength measurements. Two specimens from the control group and one from the vitamin A-supplemented group.
BARK ET AL.: VITAMIN
A AND COLON ANASTOMOSIS
ommended Daily Allowance for vitamin A for normal rats and supports normal growth, reproduction, lactation, and longevity of normal rats. The dietary vitamin A supplement used (150 IU/g diet), is not associated with any signs or symptoms of vitamin A toxicity in animals eating the supplemented chow for over a year [ 181. Our results in bursting strength changesare consistent with earlier studies demonstrating the correlation between increased bursting strength and increased collagen content in colon [3, lo]. It is of interest that the bursting strength of normal colon in the control group was about the sameas that of the anastomosed segment in the vitamin A-supplemented group, although the segmentsare not exactly comparable in location. As mentioned, we found that the peak values of the breaking strength of the colon anastomosis did not differ significantly in the two groups of rats but the shape of and the area under the breaking strength curve, an expression of the energy neededto break the healing anastomosis, showed a highly significant difference (P < O.OOl), the area being much greater in the vitamin A-supplemented rats. The differences in the shapesand areas of the breaking strength curves suggesta more “elastic” property of the colon anastomotic sites of the vitamin A-supplemented rats. There is little information about elastin accumulation in wounds during the early healing period. We did not chemically analyze for or look histologically for elastin in our study. Five days is too short a time to expect any substantial
473
TABLE 3 HYDROXYPROLINECONTENT(rg/mg TISSUE)” Group
Normal colon
Anastomosis
Basal control diet Vitamin A sunnlementation ..
2.8 f 0.2
1.6 f 0.1
4.1 k 0.4
2.3 + 0.2
P < 0.01
P < 0.01
’ Mean values f SEM.
increase in elastin formation in the tissue but it should be noted that the vitamin A supplement had been given for 5 days preoperatively as well. Mecham and associates [ 141 have reported tissue culture studieswhich have shown soluble elastin formation by fetal bovine ligamenturn nuchae fibroblasts evident by the fifth day, when cell proliferation began to be density inhibited and the cells became quiescent. Oakes et al. [ 151have found in primary cultures of neonatal rat aortic smooth muscle cell insoluble elastin formation after 10 days. The insoluble elastin first appeared as small particles which then formed elastic fibres in vitro. Also, the “elasticity” of a tissue depends not only on the quantity but also on the quality of elastin [I] and on the presence of other intercellular substances. In summary, our data show that dietary vitamin A supplementation increasescollagen (hydroxyproline) content of normal colon and of the healing anastomotic segment. There is also a dramatic change in “elasticity” of the anastomotic site tissue, and a significant increasein bursting strength of the anastomosis.
TABLE 2 BURSTINGSTRENGTH(mm Hg) OF COLONANASTOMOSES" Group
Normal colon
Anastomosis
Basal control diet Vitamin A supplementation
196 f 1.6
172 f 4.6
216 + 6.9
197 f 6.1
P < 0.01
’ Mean values f SEM.
P < 0.02
REFERENCES 1. Banga,I. Structure andfinction
of e&in and collagen. Akademiai Kiadb. Budapest, Hungary. 1966. 2. Brinkerhoff, C., McMillan, R. M., Dwyer, J. M., and Harris, E. Inhibition by retinoic acid of collagenase production in rheumatoid synovial cells. N. kg/. J. Med. 303: 432, 1980. 3. Cronin, K., Jackson, D. S., and Dunphy, J. E. Changing bursting strength and collagen content of the healing colon. Surg. Gynecol. Obstet. 126: 747, 1968. 4. Demetriou, A. A., Seifter, E., and Levenson, S. M.
474
JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 5, MAY 1984
Effect of vitamin A and citral on peritoneal adhesion formation. J. Surg. Res. 17: 325, 1974. 5. Demetriou, A. A., France, I., Bark, S., Rettura, G., Seiher, E., and Levenson, S. M. Effects of vitamin A and beta carotene on intraabdominal sepsis. Arch. Surg. 119: 161, 1984.
6. Demetriou, A. A., Seifter, E., and Levenson, S. M. Effect of vitamin A on fibroblast proliferation and collagen accumulation in vitro, unpublished. 7. Ehrlich, H. P., and Hunt, T. K. Effect of cortisone and vitamin A on wound healing. Ann. Surg. 167: 324, 1968. 8. Ehrlich, H. P., Tarver, H., and Hunt, T. K. Inhibitory effectsof vitamin E on collagen synthesis and wound repair. Ann. Surg. 175: 235, 1972. 9. Gottrup, F. Healing of incisional wounds in stomach and duodenum. Amer. J. Surg. 140: 296, 1980. 10. Hawley, P. R., Faulk, W. P., Hunt, T. K., and Dunphy, J. E. Collagenaseactivity in the gastrointestinal tract. Brit. J. Surg. 57: 896, 1970. 11. Herrmann, J. B., and Woodward, S. C. An experimental study on wound healing accelerators. Amer. Surg. 38: 26, 1972. 12. Lee, K. H., and Tong, T. G. Studieson the mechanism of action of salicylatesIV: Effect of topical application of retinoic acid on wound healing retardation action of salicylic acid. J Pharmacol. Sci. 58: 773, 1969. 13. Levenson, S. M., Crowley,k. V., Geever, E. F., Rosen, H., and Berard, C. W. Some studieson wound healing:
Experimental methods, effect of ascorbic acid, and effect of deuterium oxide. J. Trauma 4: 543, 1964. 14. Mecham, R. P., Lange, G., Madaras, J., and Stancher, B. Elastin synthesisby ligamentum nuchae fibroblasts: Effects of culture conditions and extracellular matrix on elastin production. J. Cell Biol. 90: 332, 1981. 15. Oakes, B. W., Batty, A. C., Handley, C. J., and Sandberg, L. B. The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study. Eur. J. Cell Biol. 27: 34, 1982. 16. Patt, L. M., Itaya, K., and Hakamori, S. Retinol induces density-dependent growth inhibition and changes in glycolipids and LETS. Nature (London) 273: 379, 1978. 17. Seifter, E., Crowley, L. V., Rettura, G., Nakao, K., Gruber, C., Kan, D., and Levenson, S. M. Influence of vitamin A on wound healing in rats with femoral fracture. Ann. Surg. 181: 836, 1975. 18. Seifter, E., Rettura, G., Padawer, J., Stratford, F., Goodwin, P., and Levenson, S. M. Regression of C3HBA mousetumor due to X-my therapy combined with supplemental beta carotene or vitamin A. J. Nat. Cancer Inst. 71: 409, 1983.
19. Woessner,J. F. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93: 440, 1961.