- Email: [email protected]
Reduction of primary postoperative adhesion formation under calcium channel blockade in the rabbit
JOURNAL
OF SURGICAL
RESEARCH
48.42-45
(1990)
Reduction of Primary Postoperative Adhesion Formation under Calcium Channel Blockade in the Rabbit’ HOVEYLAMBERT, PH.D., CAROLKAZENSKY, B.S., ALEX STEINLEITNER,M.D.,**~ IGNACIO SANCHEZ,M.D., AND CARLOS SUELDO, M.D. Departments of Obstetrics and Gynecology, *Division of Reproductive Endocrinology and Infertility, University of Southern California School of Medicine, Los Angeles, California 90033; University of California, San Francisco, California 94143; and Fresno Medical Education Program-Valley Medical Center of Fresno, Fresno, California 93702 Submitted
for publication
INTRODUCTION Adhesion re-formation following adhesiolysis remains a limiting factor in the operative treatment of pelvic factor ’ Supported by grants from the medical research committee of Valley Medical Center of Fresno and by Fresno Community Hospital, Fresno, California. ’ To whom correspondence and reprint requests should be addressed at LAC/USC Medical Center, Women’s Hospital, Room lM2, Los Angeles, CA 90033. $1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
1. 1988
infertility [ 11. Currently employed adjuvant therapies (e.g., antihistamines, corticosteroids, dextran solutions, progestins, antiprostaglandin agents) have not been shown to improve outcome of reproductive surgery, as judged by fertility rates [2]. This failure to improve outcome has prompted a continuing effort to develop more potent antiadhesion modalities. Recently, we reported the results of preliminary studies employing a hamster model for primary adhesion formation in which the adhesiopreventive potential of calcium channel blocking agents was assessed [3-51. The theoretical grounds for this investigation included the emerging role of calcium metabolism in the regulation of cellular systems contributing to the inflammatory response and peritoneal repair, and a large body of experimental evidence which suggested calcium entry blockade might favorably influence multiple facets of the adhesion formation cascade [6-131. The results of these initial experiments demonstrated the calcium channel blockers currently available on the American market (verapamil, nifedipine, diltiazem) to be extremely potent inhibitors of primary adhesion formation. The objectives of the present study were twofold. First, we sought to extend our observations to the rabbit, a standard model for adhesion research which has previously predicted the outcome of adjuvant therapy in humans. In addition, we had noted with interest recent evidence in the literature suggesting that the efficacy of adjuvant-induced adhesion prophylaxis may be related to the route of administration [14-161. Specifically, we wished to establish whether continuous intraperitoneal delivery of calcium channel blocking agents offered any advantage over systemic administration. These issues were deemed to be of essential importance for the design of subsequent clinical trials of calcium channel blockade as an adjuvant antiadhesion modality in abdominal and pelvic surgery.
Preliminary studies in a hamster model have demonstrated calcium channel blocking agents to be potent inhibitors of primary post-traumatic peritoneal adhesion formation. The present investigation was designed to extend these observations to an extensively studied model, the rabbit, and to evaluate the optimal route of administration of these drugs for intraabdominal surgery. Rabbits were subjected to a standardized traumatic lesion of the left uterine horn. Subsequently, animals were divided into the following treatment groups: subcutaneous vehicle control (n = 7), intraperitoneal (ip) vehicle control (n = 8), subcutaneous verapamil treatment (n = 6), low-dose (2.5 pg/kg/hr) ip verapamil treatment (n = lo), and high-dose ip (25 pg/kg/hr) verapamil treatment (n = 6). All animals were reexplored at 1 week postop for evaluation of adhesion formation (scale: 0 to 4+). Calcium channel blockade-treated animals formed significantly fewer adhesions (0.45) than controls (3.93) (P < 0.01). There was no significant difference between animals treated with SC with verapamil SC and those treated with low- or high-dose verapamil ip (0.33 vs 0.20 vs 1.0). These data confirm our preliminary results, suggesting that calcium channel blockade potently modulates peritoneal healing and regeneration. Furthermore, intraperitoneal delivery and systemic administration appear equipotent in this model. Further study of these agents as potential adjuvants for intraperitoneal surgery is indicated. o 1990 Academic PWSS, IN.
oozz-4804/90
November
MATERIALS
AND METHODS
Sexually mature New Zealand White rabbits were obtained from a commercial source (Simonson Animal Lab42
STEINLEITNER
ET
AL.:
ADHESION
PREVENTION
oratories, Gilroy, CA) for these experiments. Each animal was observed for health over the 2 weeks preceding surgery. Animal care was performed within the guidelines and standards of the vivarium at California State University, Fresno. A standardized model, based on the experimental lesion developed for our initial work in the hamster [3], was employed in this investigation. Anesthesia was induced by intramuscular injection of acepromazine, xylazine, and ketamine. Each animal was shaved preoperatively and prepped with a solution of povidone-iodine. Midline laparotomies utilizing clean but not sterile technique were employed to enter the abdominal cavity. Each rabbit was carefully inspected for evidence of prior pelvic adhesive disease or anomalous development of pelvic structures. The left uterine horn was then identified and brought out of the wound. In each instance the horn was devascularized by placing a ligature at the base of the left uterine artery, then opening and cauterizing the vascular arcade with an ophthalmologic cautery. Numerous burns were inflicted with the cautery across the antimesenteric surface of the horn. The right uterine horn was left untouched, and served as an internal control. Extreme care was taken to produce a consistent lesion in each animal. Prior to abdominal closure each rabbit was randomly assigned to one of five experimental groups: Group 1: Subcutaneous vehicle control (n = 7)-One milliliter of normal saline was injected SCbeginning immediately postop and continuing every 8 hr for a total of nine doses. Group 2: Continuous intraperitoneal vehicle control (n = 8)-Four Alzet miniosmotic pumps (No. 2001, Alza Corp., Palo Alto, CA) delivering a total of 2.0 ~1 of normal saline per hour over 200 hr’were sutured to the right subdiaphragmatic surface of the peritoneal cavity prior to closure. Group 3: Subcutaneous verapamil treatment (n = 6)Rabbits received injections of verapamil (Calan, Searle Pharmaceuticals, Skokie, IL) 200 pg/kg on a schedule identical to that employed for group 1. Group 4: Continuous low-dose intraperitoneal verapamil treatment (n = lO)-Rabbits received verapamil 2.5 pg/kg/hr ip over 200 hr via miniosmotic pump (as per group 2). Group 5: Continuous high-dose intraperitoneal verapamil treatment (n = 6)-Verapami125 pg/kg/hr ip over 200 hr via miniosmotic pump. Dosages of verapamil administered in this trial were based on the results of our previous studies in the hamster [5], and are consistent with the amount of drug required for modulation of electrophysiologic events in rodents and the starting dosages for the usual clinical indications for verapamil in humans. Following treatment assignment abdominal closure of each animal was performed using 3-O interrupted vicryl sutures and skin staples.
VIA
Ca CHANNEL
BLOCKADE
43
All rabbits were closely observed for health during the ensuing week. On the seventh postoperative day each animal was sacrificed by pentobarbital overdose for assessment of adhesion formation. Adhesions were scored on a scale ranging from 0 to 4+ (Table 1) by the original surgeons who were blinded to the identity of the individual animals. Analysis of variance, as performed by the Statgraphics statistical system (STSC Inc, Rockville, MD), was employed for evaluation of the data. RESULTS
As in our preliminary investigation in the hamster [3], all animals tolerated surgery, recovery, and calcium channel blockade therapy. There was no overt evidence of cardiopulmonary compromise, increased infectious morbidity, or failure of wound healing. Upon inspection of the Alzet pumps at the time of repeat laparotomy, a thick, fibrinous-opaque deposit was observed covering a majority of the pumps. This finding was consistent with that of Orita et al. [14]. The time course for development of this deposit and its influence on drug delivery by the pump are unknown. Evaluation of the right (nonlesioned) uterine horn on Postoperative Day 7 demonstrated no evidence of macroscopic adhesion formation. Control animals from both the subcutaneous and the intraperitoneal vehicle-treated groups demonstrated extensive dense, vascular adhesions connecting the adnexa to the bowel and pelvic sidewall. Of 15 control animals, 14 were judged to have 4+ adhesive disease. In contrast to control animals, rabbits receiving parenterally administered calcium blockade displayed excellent healing of pelvic structures. Adhesion scores for the various treatment groups were significantly lower than those for controls (Table 2). In 16 of 22 animals there was no evidence of macroscopic adhesive disease. Comparing the various verapamil treatment groups, there was no statistically significant difference in outcome between animals receiving systemic (subcutaneous) administration and animals treated by continuous intraperitoneal delivery of the calcium entry blocking agent (Table 2).
TABLE Adhesion 0 If 2f 3f 4+
No macroscopic Filmy intrahorn Thick, vascular Filmy adhesions sidewall Thick, vascular or pelvic sidewall
1
Scoring
adhesions adhesions intrahorn adhesions connecting uterine adhesions
connecting
Scale
horn
to bowel
uterine
horn
or pelvic to bowel
44
JOURNAL
TABLE Effect
OF SURGICAL
RESEARCH:
2
of Calcium Blockade on Primary Post-Traumatic Adhesion Formation in the Rabbit Adhesion score
Treatment Control SC Control ip Verapamil SC200 pg/kg/8 hr ip 2.5 pg/kg/hr ip 25 ~glklhr ’ Nonsignificant. * Differs from control,
Mean
0
1+
2+
3+
4+
0
0
0
0
7
4.00
0
0
0
1
7
3.88
5 9 2
0 0 2
1 1 2
0 0 0
0 0 0
0.33*-” 0.20*, a l.oo**”
P < 0.01, ANOVA.
DISCUSSION
The results of this investigation confirm our preliminary observations in the hamster model [3-51, demonstrating a potent reduction of post-traumatic primary adhesion formation in the rabbit under the influence of calcium channel blockade. A principal objective of this study was to evaluate the effect of route of administration on the efficacy of calcium blockade-mediated adhesion prophylaxis. Experimental and clinical experience has suggested that intraperitoneal approaches to adhesion prevention (e.g., Hyskon, barrier materials, heparin, tissue plasminogen activator) may be superior to current forms of systemic treatment. Failure of systemic administration to deliver sufficient quantities of medication to the site of injury has been offered as an explanation for the ineffectiveness of certain pharmacologic adjuvants (e.g., antihistamines, nonsteroidal antiinflammatory agents, progesterone) in clinical practice. This point has been aptly illustrated by the data of Orita et al. [14], who demonstrated a 600-fold reduction in the dose of ibuprofen required for adhesion prophylaxis with ip delivery (as compared to systemic therapy) in rabbits. These observations have prompted considerable research in an effort to develop a variety of intraperitoneal delivery systems for pharmacologic antiadhesion adjuvants. Whereas data from animal studies suggest promise, the use of intraperitoneal pumps or continuous-release materials/solutions is fraught with difficulties [ 151. No presently developed system is currently approved for clinical use. Furthermore, many of these systems, particularly the continuous-release solutions now being evaluated, may adversely affect peritoneal healing of their own accord. In this context, our demonstration of equipotent adhesion reduction under calcium blockade administered by either the systemic or the intraperitoneal route is of particular note. Unfortunately, the sensitivity of the model
VOL. 48, NO. 1, JANUARY
1990
employed in this study is limited by the dramatic reduction in adhesion formation induced by all modes of verapamil administration tested. This phenomenon limits our ability to make definite statements concerning the extreme limits of adhesiopreventive potency in the rabbit. Nevertheless, the results of this study strongly suggest that the quantity of calcium channel blocker delivered to the site of injury by systemic administration may be adequate to induce the desired effect on peritoneal repair. If confirmed in clinical trials, this property of calcium channel blockade would represent a significant advantage over other pharmacologic adjuvants which require an intraperitoneal means for delivery. As in the hamster, there were no evident deleterious effects of perioperative calcium blockade therapy. Specifically, the rabbits employed in the experimental groups of this experiment did not suffer from increased infectious morbidity, cardiopulmonary compromise, impaired wound healing, or failure to thrive. These results are consistent with the clinical literature which is notable for the absence of reports of significant complications in patients undergoing surgery while being maintained on calcium channel blocking agents for the usual (i.e., cardiovascular) indications. The mechanism(s) through which calcium blockade influences peritoneal healing remains uncertain. Data from the clinical and in vitro calcium blockade literature suggest that these drugs may intervene at sequential loci of the adhesion formation cascade, including protection against toxic and ischemic cell injury [6, 71, reduced vasoactive/ inflammatory mediator production during the acute inflammatory response [8, 91, inhibition of irreversible platelet aggregation [lo], protection against acute granulocyte-mediated tissue injury [ 111, reduced microvascular permeability resulting in decreased exudation of fibrinrich plasma as substrate for clot formation [12], and inhibition of fibroblast penetration into clot matrices [ 131. In summary, these data obtained in the rabbit confirm our initial observation that calcium channel blockade is a potent modulator of post-traumatic inflammation and peritoneal healing, significantly inhibiting the formation of postsurgical adhesions. Comparison of systemic vs intraperitoneal delivery suggests that they are equipotent in the rabbit model system. These results suggest that systemically delivered verapamil may be efficacious as an adjuvant therapy in abdominal and pelvic surgery. Although these preliminary animal data are provocative, we would advocate restraint in the clinical application of these drugs at this time. Studies are ongoing (rabbit) to assess the effect of calcium blockade on wound healing and to test the efficacy of these drugs in preventing adhesion reformation following microsurgical lysis of previously formed adhesions (i.e., inhibition of postsurgical adhesion reformation in contrast to primary adhesion formation as in these experiments). Given a favorable outcome of these investigations, a controlled clinical trial is contemplated. With adequate study, calcium channel
STEINLEITNER
ET AL.: ADHESION
PREVENTION
blockade would appear to hold great potential as an important constituent of future adjunctive regimens for infertility surgery.
8.
9.
REFERENCES 10. 1.
DeCherney, A. H., and Mezer, H. C. The nature of posttuboplasty pelvic adhesions as determined by early and late laparoscopy. Fertil. Steril. 41: 643, 1984.
2.
Holtz, G. Prevention and management Fertil. Steril. 41: 497, 1984.
3.
Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. The use of calcium channel blockade for prevention of postoperative adhesion formation. Fertil. Steril. 50: 818, 1988. Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. The use of diltiazem for prevention of postoperative adhesion formation. J. Reprod. Med. 33: 891, 1988.
4.
of peritoneal
11.
adhesions.
5.
Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. Reduction of adhesion formation with perioperative verapamil treatment. In Abstracts, 36th Annual Meeting of the Pacific Coast Fertility Society, Palm Springs, CA, April 1988.
6.
Schanne, F. A. X., Kane, A. B., Young, E. E., et al. Calcium dependence of toxic cell death, a final common pathway. Science 206: 700,1979.
7.
Kloner, R. A., and Braunwald, E. Effects of calcium antagonists on infarcting myocardium. Amer. J. Cardiol. 59: 84B, 1987.
12.
13.
14.
15.
16.
VIA Ca CHANNEL
BLOCKADE
45
Chand, N., Diamantis, W., Pillar, J., et al. Inhibition of allergic and nonallergic histamine secretion from rat peritoneal mast cells by calcium antagonists. Brit. J. Pharmacol. 83: 899, 1984. Escoubet, B., Griffaton, G., and Lechat, P. Verapamil depresses the synthesis of lipogenase products by hypoxic cardiac rat fibroblasts in culture. Biochem. Pharmacol. 35: 1879, 1986. Mehta, J., Mehta, P., and Ostrowski, N. Calcium channel blocker diltiazem inhibits platelet activation and stimulates vascular prostacyclin release. Amer. J. Med. Sci. 291: 20, 1986. Elferink, J. G. R., and Deierkauf, M. The effect of verapamil and other calcium antagonists on chemotaxis of polymorphonuclear leukocytes. Biochem. Pharmacol. 33: 35, 1984. Mayhan, W. G., and Joyner, W. L. The effect of altering the external calcium concentration and a calcium channel blocker verapamil, on microvascular leaky sites and dextran clearance in the hamster cheek pouch. Microuasc. Res. 28: 159, 1984. Azzarone, B., Krief, P., Soria, J., et al. Modulation of fibroblastinduced clot retraction by calcium channel blocking drugs and the monoclonal antibody ALBG. J. Cell Phystil. 126: 420,1985. Orita, H., Girgis, W., and diZerega, G. S. Prevention of postsurgical peritoneal adhesion formation by intraperitoneal administration of ibuprofen. Drug Deu. Res. 10: 97, 1987. Hockel, M., Ott, S., Siemann, U., and Kissel, T. Prevention of peritoneal adhesions in the rat with sustained intraperitoneal dexamethasone delivered by a novel therapeutic system. Ann. Chir. Gym 76: 306,1987. DeCherney, A. H. Preventing postoperative pelvic adhesions with intraperitoneal treatment. J. Reprod. Med. 29: 157, 1984.
OF SURGICAL
RESEARCH
48.42-45
(1990)
Reduction of Primary Postoperative Adhesion Formation under Calcium Channel Blockade in the Rabbit’ HOVEYLAMBERT, PH.D., CAROLKAZENSKY, B.S., ALEX STEINLEITNER,M.D.,**~ IGNACIO SANCHEZ,M.D., AND CARLOS SUELDO, M.D. Departments of Obstetrics and Gynecology, *Division of Reproductive Endocrinology and Infertility, University of Southern California School of Medicine, Los Angeles, California 90033; University of California, San Francisco, California 94143; and Fresno Medical Education Program-Valley Medical Center of Fresno, Fresno, California 93702 Submitted
for publication
INTRODUCTION Adhesion re-formation following adhesiolysis remains a limiting factor in the operative treatment of pelvic factor ’ Supported by grants from the medical research committee of Valley Medical Center of Fresno and by Fresno Community Hospital, Fresno, California. ’ To whom correspondence and reprint requests should be addressed at LAC/USC Medical Center, Women’s Hospital, Room lM2, Los Angeles, CA 90033. $1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
1. 1988
infertility [ 11. Currently employed adjuvant therapies (e.g., antihistamines, corticosteroids, dextran solutions, progestins, antiprostaglandin agents) have not been shown to improve outcome of reproductive surgery, as judged by fertility rates [2]. This failure to improve outcome has prompted a continuing effort to develop more potent antiadhesion modalities. Recently, we reported the results of preliminary studies employing a hamster model for primary adhesion formation in which the adhesiopreventive potential of calcium channel blocking agents was assessed [3-51. The theoretical grounds for this investigation included the emerging role of calcium metabolism in the regulation of cellular systems contributing to the inflammatory response and peritoneal repair, and a large body of experimental evidence which suggested calcium entry blockade might favorably influence multiple facets of the adhesion formation cascade [6-131. The results of these initial experiments demonstrated the calcium channel blockers currently available on the American market (verapamil, nifedipine, diltiazem) to be extremely potent inhibitors of primary adhesion formation. The objectives of the present study were twofold. First, we sought to extend our observations to the rabbit, a standard model for adhesion research which has previously predicted the outcome of adjuvant therapy in humans. In addition, we had noted with interest recent evidence in the literature suggesting that the efficacy of adjuvant-induced adhesion prophylaxis may be related to the route of administration [14-161. Specifically, we wished to establish whether continuous intraperitoneal delivery of calcium channel blocking agents offered any advantage over systemic administration. These issues were deemed to be of essential importance for the design of subsequent clinical trials of calcium channel blockade as an adjuvant antiadhesion modality in abdominal and pelvic surgery.
Preliminary studies in a hamster model have demonstrated calcium channel blocking agents to be potent inhibitors of primary post-traumatic peritoneal adhesion formation. The present investigation was designed to extend these observations to an extensively studied model, the rabbit, and to evaluate the optimal route of administration of these drugs for intraabdominal surgery. Rabbits were subjected to a standardized traumatic lesion of the left uterine horn. Subsequently, animals were divided into the following treatment groups: subcutaneous vehicle control (n = 7), intraperitoneal (ip) vehicle control (n = 8), subcutaneous verapamil treatment (n = 6), low-dose (2.5 pg/kg/hr) ip verapamil treatment (n = lo), and high-dose ip (25 pg/kg/hr) verapamil treatment (n = 6). All animals were reexplored at 1 week postop for evaluation of adhesion formation (scale: 0 to 4+). Calcium channel blockade-treated animals formed significantly fewer adhesions (0.45) than controls (3.93) (P < 0.01). There was no significant difference between animals treated with SC with verapamil SC and those treated with low- or high-dose verapamil ip (0.33 vs 0.20 vs 1.0). These data confirm our preliminary results, suggesting that calcium channel blockade potently modulates peritoneal healing and regeneration. Furthermore, intraperitoneal delivery and systemic administration appear equipotent in this model. Further study of these agents as potential adjuvants for intraperitoneal surgery is indicated. o 1990 Academic PWSS, IN.
oozz-4804/90
November
MATERIALS
AND METHODS
Sexually mature New Zealand White rabbits were obtained from a commercial source (Simonson Animal Lab42
STEINLEITNER
ET
AL.:
ADHESION
PREVENTION
oratories, Gilroy, CA) for these experiments. Each animal was observed for health over the 2 weeks preceding surgery. Animal care was performed within the guidelines and standards of the vivarium at California State University, Fresno. A standardized model, based on the experimental lesion developed for our initial work in the hamster [3], was employed in this investigation. Anesthesia was induced by intramuscular injection of acepromazine, xylazine, and ketamine. Each animal was shaved preoperatively and prepped with a solution of povidone-iodine. Midline laparotomies utilizing clean but not sterile technique were employed to enter the abdominal cavity. Each rabbit was carefully inspected for evidence of prior pelvic adhesive disease or anomalous development of pelvic structures. The left uterine horn was then identified and brought out of the wound. In each instance the horn was devascularized by placing a ligature at the base of the left uterine artery, then opening and cauterizing the vascular arcade with an ophthalmologic cautery. Numerous burns were inflicted with the cautery across the antimesenteric surface of the horn. The right uterine horn was left untouched, and served as an internal control. Extreme care was taken to produce a consistent lesion in each animal. Prior to abdominal closure each rabbit was randomly assigned to one of five experimental groups: Group 1: Subcutaneous vehicle control (n = 7)-One milliliter of normal saline was injected SCbeginning immediately postop and continuing every 8 hr for a total of nine doses. Group 2: Continuous intraperitoneal vehicle control (n = 8)-Four Alzet miniosmotic pumps (No. 2001, Alza Corp., Palo Alto, CA) delivering a total of 2.0 ~1 of normal saline per hour over 200 hr’were sutured to the right subdiaphragmatic surface of the peritoneal cavity prior to closure. Group 3: Subcutaneous verapamil treatment (n = 6)Rabbits received injections of verapamil (Calan, Searle Pharmaceuticals, Skokie, IL) 200 pg/kg on a schedule identical to that employed for group 1. Group 4: Continuous low-dose intraperitoneal verapamil treatment (n = lO)-Rabbits received verapamil 2.5 pg/kg/hr ip over 200 hr via miniosmotic pump (as per group 2). Group 5: Continuous high-dose intraperitoneal verapamil treatment (n = 6)-Verapami125 pg/kg/hr ip over 200 hr via miniosmotic pump. Dosages of verapamil administered in this trial were based on the results of our previous studies in the hamster [5], and are consistent with the amount of drug required for modulation of electrophysiologic events in rodents and the starting dosages for the usual clinical indications for verapamil in humans. Following treatment assignment abdominal closure of each animal was performed using 3-O interrupted vicryl sutures and skin staples.
VIA
Ca CHANNEL
BLOCKADE
43
All rabbits were closely observed for health during the ensuing week. On the seventh postoperative day each animal was sacrificed by pentobarbital overdose for assessment of adhesion formation. Adhesions were scored on a scale ranging from 0 to 4+ (Table 1) by the original surgeons who were blinded to the identity of the individual animals. Analysis of variance, as performed by the Statgraphics statistical system (STSC Inc, Rockville, MD), was employed for evaluation of the data. RESULTS
As in our preliminary investigation in the hamster [3], all animals tolerated surgery, recovery, and calcium channel blockade therapy. There was no overt evidence of cardiopulmonary compromise, increased infectious morbidity, or failure of wound healing. Upon inspection of the Alzet pumps at the time of repeat laparotomy, a thick, fibrinous-opaque deposit was observed covering a majority of the pumps. This finding was consistent with that of Orita et al. [14]. The time course for development of this deposit and its influence on drug delivery by the pump are unknown. Evaluation of the right (nonlesioned) uterine horn on Postoperative Day 7 demonstrated no evidence of macroscopic adhesion formation. Control animals from both the subcutaneous and the intraperitoneal vehicle-treated groups demonstrated extensive dense, vascular adhesions connecting the adnexa to the bowel and pelvic sidewall. Of 15 control animals, 14 were judged to have 4+ adhesive disease. In contrast to control animals, rabbits receiving parenterally administered calcium blockade displayed excellent healing of pelvic structures. Adhesion scores for the various treatment groups were significantly lower than those for controls (Table 2). In 16 of 22 animals there was no evidence of macroscopic adhesive disease. Comparing the various verapamil treatment groups, there was no statistically significant difference in outcome between animals receiving systemic (subcutaneous) administration and animals treated by continuous intraperitoneal delivery of the calcium entry blocking agent (Table 2).
TABLE Adhesion 0 If 2f 3f 4+
No macroscopic Filmy intrahorn Thick, vascular Filmy adhesions sidewall Thick, vascular or pelvic sidewall
1
Scoring
adhesions adhesions intrahorn adhesions connecting uterine adhesions
connecting
Scale
horn
to bowel
uterine
horn
or pelvic to bowel
44
JOURNAL
TABLE Effect
OF SURGICAL
RESEARCH:
2
of Calcium Blockade on Primary Post-Traumatic Adhesion Formation in the Rabbit Adhesion score
Treatment Control SC Control ip Verapamil SC200 pg/kg/8 hr ip 2.5 pg/kg/hr ip 25 ~glklhr ’ Nonsignificant. * Differs from control,
Mean
0
1+
2+
3+
4+
0
0
0
0
7
4.00
0
0
0
1
7
3.88
5 9 2
0 0 2
1 1 2
0 0 0
0 0 0
0.33*-” 0.20*, a l.oo**”
P < 0.01, ANOVA.
DISCUSSION
The results of this investigation confirm our preliminary observations in the hamster model [3-51, demonstrating a potent reduction of post-traumatic primary adhesion formation in the rabbit under the influence of calcium channel blockade. A principal objective of this study was to evaluate the effect of route of administration on the efficacy of calcium blockade-mediated adhesion prophylaxis. Experimental and clinical experience has suggested that intraperitoneal approaches to adhesion prevention (e.g., Hyskon, barrier materials, heparin, tissue plasminogen activator) may be superior to current forms of systemic treatment. Failure of systemic administration to deliver sufficient quantities of medication to the site of injury has been offered as an explanation for the ineffectiveness of certain pharmacologic adjuvants (e.g., antihistamines, nonsteroidal antiinflammatory agents, progesterone) in clinical practice. This point has been aptly illustrated by the data of Orita et al. [14], who demonstrated a 600-fold reduction in the dose of ibuprofen required for adhesion prophylaxis with ip delivery (as compared to systemic therapy) in rabbits. These observations have prompted considerable research in an effort to develop a variety of intraperitoneal delivery systems for pharmacologic antiadhesion adjuvants. Whereas data from animal studies suggest promise, the use of intraperitoneal pumps or continuous-release materials/solutions is fraught with difficulties [ 151. No presently developed system is currently approved for clinical use. Furthermore, many of these systems, particularly the continuous-release solutions now being evaluated, may adversely affect peritoneal healing of their own accord. In this context, our demonstration of equipotent adhesion reduction under calcium blockade administered by either the systemic or the intraperitoneal route is of particular note. Unfortunately, the sensitivity of the model
VOL. 48, NO. 1, JANUARY
1990
employed in this study is limited by the dramatic reduction in adhesion formation induced by all modes of verapamil administration tested. This phenomenon limits our ability to make definite statements concerning the extreme limits of adhesiopreventive potency in the rabbit. Nevertheless, the results of this study strongly suggest that the quantity of calcium channel blocker delivered to the site of injury by systemic administration may be adequate to induce the desired effect on peritoneal repair. If confirmed in clinical trials, this property of calcium channel blockade would represent a significant advantage over other pharmacologic adjuvants which require an intraperitoneal means for delivery. As in the hamster, there were no evident deleterious effects of perioperative calcium blockade therapy. Specifically, the rabbits employed in the experimental groups of this experiment did not suffer from increased infectious morbidity, cardiopulmonary compromise, impaired wound healing, or failure to thrive. These results are consistent with the clinical literature which is notable for the absence of reports of significant complications in patients undergoing surgery while being maintained on calcium channel blocking agents for the usual (i.e., cardiovascular) indications. The mechanism(s) through which calcium blockade influences peritoneal healing remains uncertain. Data from the clinical and in vitro calcium blockade literature suggest that these drugs may intervene at sequential loci of the adhesion formation cascade, including protection against toxic and ischemic cell injury [6, 71, reduced vasoactive/ inflammatory mediator production during the acute inflammatory response [8, 91, inhibition of irreversible platelet aggregation [lo], protection against acute granulocyte-mediated tissue injury [ 111, reduced microvascular permeability resulting in decreased exudation of fibrinrich plasma as substrate for clot formation [12], and inhibition of fibroblast penetration into clot matrices [ 131. In summary, these data obtained in the rabbit confirm our initial observation that calcium channel blockade is a potent modulator of post-traumatic inflammation and peritoneal healing, significantly inhibiting the formation of postsurgical adhesions. Comparison of systemic vs intraperitoneal delivery suggests that they are equipotent in the rabbit model system. These results suggest that systemically delivered verapamil may be efficacious as an adjuvant therapy in abdominal and pelvic surgery. Although these preliminary animal data are provocative, we would advocate restraint in the clinical application of these drugs at this time. Studies are ongoing (rabbit) to assess the effect of calcium blockade on wound healing and to test the efficacy of these drugs in preventing adhesion reformation following microsurgical lysis of previously formed adhesions (i.e., inhibition of postsurgical adhesion reformation in contrast to primary adhesion formation as in these experiments). Given a favorable outcome of these investigations, a controlled clinical trial is contemplated. With adequate study, calcium channel
STEINLEITNER
ET AL.: ADHESION
PREVENTION
blockade would appear to hold great potential as an important constituent of future adjunctive regimens for infertility surgery.
8.
9.
REFERENCES 10. 1.
DeCherney, A. H., and Mezer, H. C. The nature of posttuboplasty pelvic adhesions as determined by early and late laparoscopy. Fertil. Steril. 41: 643, 1984.
2.
Holtz, G. Prevention and management Fertil. Steril. 41: 497, 1984.
3.
Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. The use of calcium channel blockade for prevention of postoperative adhesion formation. Fertil. Steril. 50: 818, 1988. Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. The use of diltiazem for prevention of postoperative adhesion formation. J. Reprod. Med. 33: 891, 1988.
4.
of peritoneal
11.
adhesions.
5.
Steinleitner, A., Lambert, H., Montoro, L., Swanson, J., and Sueldo, C. Reduction of adhesion formation with perioperative verapamil treatment. In Abstracts, 36th Annual Meeting of the Pacific Coast Fertility Society, Palm Springs, CA, April 1988.
6.
Schanne, F. A. X., Kane, A. B., Young, E. E., et al. Calcium dependence of toxic cell death, a final common pathway. Science 206: 700,1979.
7.
Kloner, R. A., and Braunwald, E. Effects of calcium antagonists on infarcting myocardium. Amer. J. Cardiol. 59: 84B, 1987.
12.
13.
14.
15.
16.
VIA Ca CHANNEL
BLOCKADE
45
Chand, N., Diamantis, W., Pillar, J., et al. Inhibition of allergic and nonallergic histamine secretion from rat peritoneal mast cells by calcium antagonists. Brit. J. Pharmacol. 83: 899, 1984. Escoubet, B., Griffaton, G., and Lechat, P. Verapamil depresses the synthesis of lipogenase products by hypoxic cardiac rat fibroblasts in culture. Biochem. Pharmacol. 35: 1879, 1986. Mehta, J., Mehta, P., and Ostrowski, N. Calcium channel blocker diltiazem inhibits platelet activation and stimulates vascular prostacyclin release. Amer. J. Med. Sci. 291: 20, 1986. Elferink, J. G. R., and Deierkauf, M. The effect of verapamil and other calcium antagonists on chemotaxis of polymorphonuclear leukocytes. Biochem. Pharmacol. 33: 35, 1984. Mayhan, W. G., and Joyner, W. L. The effect of altering the external calcium concentration and a calcium channel blocker verapamil, on microvascular leaky sites and dextran clearance in the hamster cheek pouch. Microuasc. Res. 28: 159, 1984. Azzarone, B., Krief, P., Soria, J., et al. Modulation of fibroblastinduced clot retraction by calcium channel blocking drugs and the monoclonal antibody ALBG. J. Cell Phystil. 126: 420,1985. Orita, H., Girgis, W., and diZerega, G. S. Prevention of postsurgical peritoneal adhesion formation by intraperitoneal administration of ibuprofen. Drug Deu. Res. 10: 97, 1987. Hockel, M., Ott, S., Siemann, U., and Kissel, T. Prevention of peritoneal adhesions in the rat with sustained intraperitoneal dexamethasone delivered by a novel therapeutic system. Ann. Chir. Gym 76: 306,1987. DeCherney, A. H. Preventing postoperative pelvic adhesions with intraperitoneal treatment. J. Reprod. Med. 29: 157, 1984.