Br. J. Surg. Vol. 62 (1975) 642444
A rabbit model of perforated appendicitis with peritonitis D . W. K I N G , J. F . G U R R Y , R . B . ELLIS-PEGLER A N D B . N . B R O O K E * SUMMARY
No satisfactory animal model simulating acute perforating appendicitis followed by peritonitis in man has hitherto been achieved. A technique has been developed in New Zealand White rabbits to fulfil these desiderata. The bacterial flora associated with the ensuing peritonitis is similar to that found in man both as regards aerobes and anaerobes and their concentrations. The method effectively produces the required model in 70 per cent of rabbits subjected to the experiment. Satisfactory postoperative survival is achieved enabling therapeutic studies.
PRODUCTION of peritonitis experimentally, similar to that in humans, has always been difficult. We have attempted to produce an animal model of peritonitis akin to the human situation.
Fig. 2. Appearance of an appendix a t the second operation. The upper pair of forceps is inserted through the perforation and the lower pair is holding the base tie.
Mean :/o original weight Materials and methods Thirty-two New Zealand White rabbits weighing approximately 3000 g were anaesthetized using ' O 0 1 Sham pentobarbitone induction and maintained by gaseous 95 agents (Hynd, 1975). Through a lower midline incision the base of the appendix was tied with black silk thread, leaving the , blood supply intact (Fig. 1). Using the same anaes- 90 thetic technique and the same incision, further ," ----- Perforated appendicitis laparotomy was performed 18 hours after the first 8. followed by appendicectomy (8 rabbits) operation. If perforation had not occurred sponta- 85 3 Laparotomies at t h e same neously the appendix was incised near the base, intervals (4 rabbits) allowing the contents to enter the peritoneal cavity. * v I I i The wound was then closed. Six hours later, using a 80' I 2 3 4 5 6 7 8 9 1011I21314 gaseous anaesthetic only, appendicectomy was perDays after operation formed through the same incision. The rabbits were weighed immediately before Fig. 3. Postoperative weight changes of the experimental operation and at the same time each subsequent day. animals compared with those of the controls.
--.-.
-
I
sYbowe'
Fig. 1. Diagram showing the method of producing
appendicitis.
642
I
I
I
1
I
I
I
I
Results Twenty-eight of the 32 rabbits (87.5 per cent) survived the series of operations and for 36 hours postoperatively; 71 per cent (20 out of 28) of the appendices in the surviving rabbits had perforated spontaneously by the time of the second operation (Fig. 2). Of the 28 surviving rabbits, 19 were used in a study which is the subject of another report; 89 per cent (8 out of 9) of the remaining surviving rabbits lived for at least 14 days and were killed during the third week. Post-mortem examination showed a diffuse purulent peritonitis with many adhesions resulting in multiple abscesses throughout the peritoneal cavity, particularly
* Department of Surgery and Communicable Disease Unit, St George's Hospital, London. Present address of D. W. King: St Mark's Hospital, London.
*
Perforated appendicitis with peritonitis Aerobes
r-
En t e ro-
jtaphylo-
bacteria
COCCI
-
8-
I
Streptococci
e
Bifidobacteria
B a a II i
-
o
7-
6Log,, 5organisms ml 4-
e
0 8
0 0
;
3-
2-
o
0
0
0
ee
0
0
*
o
0
3
8
10 -
e
o 0
2
12
6
I2 -
8 Human
0 Rabbit
I2
Anaerobes
Bacteroides
3
0
Clostridia
0
8
0 0
0
0
Veillonella
0
)O
80
0 0
0 )OC
3 -
3 -
Fig. 4. Comparison of the organisms isolated from 1 3 rabbits with perforated appendicitis with those in pus found in 14 patients at operation for perforated appendicitis. The number a t the base of each column is the number of specimens with a concentration of less than 100 organisms/ml.
the caudal half. Post-mortem examination of the rabbit dying on the third postoperative day showed purulent peritonitis. Three rabbits in which only a base tie of the appendix was performed had died by 30 hours postoperatively. Tying less of the appendix produced less predictable results ; if insufficient was tied, only a localized abscess, without mortality, resulted. Four control rabbits had laparotomies performed at the same intervals, and at each operation the appendix was brought out on to the abdominal wall for a short time and then replaced. All 4 survived for at least 14 days and lost less weight than the rabbits with peritonitis (Fig. 3 ) ; no abnormality was found when they were sacrificed. The rabbits with perforated appendicitis followed by appendicectomy lost weight for 4-5 days, then regained weight to reach preoperative levels in the third postoperative week (Fig. 3).
Bacteriology In 13 rabbits, 9 with spontaneous perforations and 4 with artificial perforations, culture of peritoneal fluid present at the third laparotomy was begun within 5 minutes of removal of the sample from the peritoneal cavity. Since the bacterial groups expected in the human situation include enterobacteria, staphylococci, streptococci, bacilli, lactobacilli, yeasts, bacteroides, bifidobacteria, clostridia and veillonella, these organisms were sought in the samples. The viable counts of the bacteria were expressed as log,, organisms/ml of peritoneal fluid; the lower limit of detection of bacteria with this method was 102organisms/ml of fluid (Ellis-Pegler et al., 1975). A comparison has been made of cultures from 13 animals with those in pus taken at operation for perforated appendicitis with purulent peritonitis in
Fig. 5. Histological section of a rabbit appendix showing features similar to those of acute appendicitis in man.
14 patients (Pickersgill and Ellis-Pegler, 1975) (Fig. 4). The distribution was found to be similar; veillonella is an organism not found in the rabbit gut and was only grown from 1 human case. Histology Histologically, the appearances were similar in all the specimens examined and had many features in common with those seen in acute appendicitis in man (Fig. 5). However, in the rabbit suppuration was generally less pronounced and less evenly distributed throughout the wall and there was a zone of suppurative necrosis extending from the mucosa into the superficial parts of the lymphoid follicles. The latter is not seen normally in the human where the process seems to start from a focus rather than diffusely. There was also more lymphoid tissue in the rabbit appendices. 643
D. W. King et al. Discussion Induction of peritonitis in animals by intraperitoneal injection of bacterial suspension alone has proved to be difficult (Browne and Stoller, 1970). Furthermore, rats and mice are relatively resistant to induction of Gram-negative peritonitis (Burnett et al., 1957 ; Browne and Stoller, 1970). In 1964 Schumer et al. used an injection of a known amount of Escherichiu coli into the peritoneal cavity of guinea-pigs. Artz et al. in 1962 used pooled filtered faecal suspension, producing at least an 80 per cent mortality rate at 24 hours in dogs; variations on this method have been used often Filler and Sleeman, 1967; Lavigne et al., 1974). Burnett et al. (1957) removed a 3-mm ellipse from the colon of guinea-pigs and found that this routinely produced a severe diffuse peritonitis with an 85 per cent mortality rate. Zintel et al. (1950) induced peritonitis by ligating the appendiceal vessels and the base of the appendix and contaminating the peritoneal cavity with the appendix opened longitudinally; the abdomen was then closed. In 1938 Bower et al. described the technique of isolating and then tying the base of the caecal pouch of dogs, allowing the appendix to perforate spontaneously to produce a spreading peritonitis which in a later series produced a mortality of 78 per cent in untreated animals (Crook et al., 1968). The method was modified by Silvani et al. (1947) to include crushing of the wall of the appendix. The methods used in injection of known bacterial suspension or perforating the bowel release organisms suddenly into a normal peritoneal cavity, aod do not simulate the situation of acute obstructive appendicitis with perforation in humans. The method of Bower et al. (1938) and its subsequent variation (Silvani et al., 1947) involve the production of gangrene of the appendix by tying the blood vessels and do not include removal of the appendix. Neither of these reflect the human situation. To overcome these objections a model has been created akin to the disease process in man. It is recognized that the faecal and colonic flora of rabbits and man are not identical. Nevertheless, with isolation and identification techniques widely used in human studies the same bacterial groups have been isolated from rabbit and human faeces, although in different concentrations and with some exceptions (WilliamsSmith, 1965; Hawksworth et al., 1971). Perforation of the appendix is followed by peritonitis similar in bacteriological flora and degree of contamination to the situation found in humans. Moreover, the immediate effect in the animal is not so overwhelming as to produce rapid death, and this permits assessment of the postoperative course; the degree of peritonitis appears to be reflected in weight loss. Since perforation of the appendix is achieved in 70 per cent of animals the rate of reproducibility of the model is acceptable. If a high mortality is required for study, ligation of the appendix alone without subsequent removal may be undertaken; in this circumstance therapeutic measures under trial should be instituted after 24 hours. 644
Acknowledgements We would like to thank Dr J. P. O’Sullivan for reviewing the histology, Mr John Hynd for carrying out the excellent anaesthesia, the Clinical Laboratory staff for their help and the St George’s Hospital Research Fund for financial support. R. B. Ellis-Pegler is in receipt of a research grant from the Wellcome Trust. References ARTZ c. P., BARNETT w. 0. and GROGAN J. B. (1962) Further studies concerning the pathogenesis and treatment of peritonitis. Ann. Surg. 155, 756766. BOWER J. o., BURNS J. c. and MENGLE H. A. (1938) Induced spreading peritonitis complicating acute perforative appendicitis. Surg. Gynecol. Obstet. 66, 947-961. BROWNE M. K. and STOLLER J. L. (1970) Intraperitoneal noxythiolin in faecal peritonitis. Br. J. Surg. 57, 525-5 29. BURNETT W. E., BROWNE G. R., ROSEMOND G. P., CASEWELL H. T., BUCHER R. B. and TYSON R. R.
(1957) The treatment of peritonitis using peritoneal lavage. Ann. Surg. 145,657-681. CROOK J. N., COTLAR A. M., BORNSIDE G. H. and COHN I. (1968) Intraperitoneal cephalothin in the treatment of experimental appendiceal peritonitis. Am. Surg. 34, 736-739. ELLIS-PEGLER R. B., CRABTREE c. and LAMBERT H. P. (1975) To be published. FILLER R. M. and SLEEMAN H. K. (1967) Pathogenesis of peritonitis. 1. The effect of Escherichiu coli and hemoglobin on peritoneal absorption. Surgery 61, 385-392. HAWKSWORTH G., DRASER B. s. and HILL M. J. (1971) Intestinal bacteria and the hydrolysis of glycosidic bonds. J. Med. Microbiol. 4, 451-459. HYND J. (1975) To be published. LAVIGNE J. E., BROWN C. S., MACHIEDO G. W., BLACKWOOD J. M. and RUSH B. F. (1974) The treatment
of experimental peritonitis with intraperitoneal Betadine solution. J. Szirg. Res. 16, 307-31 1 . PICKERSGILL J. and ELLIS-PEGLER R. B. (1975) To be published. SCHUMER w., LEE D. K. and JONES B. (1964) Peritoneal lavage in postoperative therapy of ‘late peritoneal’ sepsis. Preliminary report. Surgery 55, 841845. SILVANI H. L., ROTHENBERG S., WARMER H., AMLUXEN J. and MCCORKLE H. J. (1947) Laboratory and clinical
experiences with Streptomycin therapy in the management of infections of intestinal origin. Surg. Gynecol. Obstet. 85, 721-726. WILLIAMS-SMITH H. (1965) Observations on the flora of the alimentary tract of animals and factors affecting its composition. J. Puthol. Bucteviol. 89, 95-122. ZINTEL H. A., ZINSSER H. H., RUNYON W., JACKSON G. L., NICHOLS A. c., WILEY M. M. and WILLIAMS w. J.
(1950) Influence of antibiotics and sulfonamides on the mortality and bacteria of experimental peritonitis. Surg. Gynecol. Obstet. 91, 742-750.
A rabbit model of perforated appendicitis with peritonitis D . W. K I N G , J. F . G U R R Y , R . B . ELLIS-PEGLER A N D B . N . B R O O K E * SUMMARY
No satisfactory animal model simulating acute perforating appendicitis followed by peritonitis in man has hitherto been achieved. A technique has been developed in New Zealand White rabbits to fulfil these desiderata. The bacterial flora associated with the ensuing peritonitis is similar to that found in man both as regards aerobes and anaerobes and their concentrations. The method effectively produces the required model in 70 per cent of rabbits subjected to the experiment. Satisfactory postoperative survival is achieved enabling therapeutic studies.
PRODUCTION of peritonitis experimentally, similar to that in humans, has always been difficult. We have attempted to produce an animal model of peritonitis akin to the human situation.
Fig. 2. Appearance of an appendix a t the second operation. The upper pair of forceps is inserted through the perforation and the lower pair is holding the base tie.
Mean :/o original weight Materials and methods Thirty-two New Zealand White rabbits weighing approximately 3000 g were anaesthetized using ' O 0 1 Sham pentobarbitone induction and maintained by gaseous 95 agents (Hynd, 1975). Through a lower midline incision the base of the appendix was tied with black silk thread, leaving the , blood supply intact (Fig. 1). Using the same anaes- 90 thetic technique and the same incision, further ," ----- Perforated appendicitis laparotomy was performed 18 hours after the first 8. followed by appendicectomy (8 rabbits) operation. If perforation had not occurred sponta- 85 3 Laparotomies at t h e same neously the appendix was incised near the base, intervals (4 rabbits) allowing the contents to enter the peritoneal cavity. * v I I i The wound was then closed. Six hours later, using a 80' I 2 3 4 5 6 7 8 9 1011I21314 gaseous anaesthetic only, appendicectomy was perDays after operation formed through the same incision. The rabbits were weighed immediately before Fig. 3. Postoperative weight changes of the experimental operation and at the same time each subsequent day. animals compared with those of the controls.
--.-.
-
I
sYbowe'
Fig. 1. Diagram showing the method of producing
appendicitis.
642
I
I
I
1
I
I
I
I
Results Twenty-eight of the 32 rabbits (87.5 per cent) survived the series of operations and for 36 hours postoperatively; 71 per cent (20 out of 28) of the appendices in the surviving rabbits had perforated spontaneously by the time of the second operation (Fig. 2). Of the 28 surviving rabbits, 19 were used in a study which is the subject of another report; 89 per cent (8 out of 9) of the remaining surviving rabbits lived for at least 14 days and were killed during the third week. Post-mortem examination showed a diffuse purulent peritonitis with many adhesions resulting in multiple abscesses throughout the peritoneal cavity, particularly
* Department of Surgery and Communicable Disease Unit, St George's Hospital, London. Present address of D. W. King: St Mark's Hospital, London.
*
Perforated appendicitis with peritonitis Aerobes
r-
En t e ro-
jtaphylo-
bacteria
COCCI
-
8-
I
Streptococci
e
Bifidobacteria
B a a II i
-
o
7-
6Log,, 5organisms ml 4-
e
0 8
0 0
;
3-
2-
o
0
0
0
ee
0
0
*
o
0
3
8
10 -
e
o 0
2
12
6
I2 -
8 Human
0 Rabbit
I2
Anaerobes
Bacteroides
3
0
Clostridia
0
8
0 0
0
0
Veillonella
0
)O
80
0 0
0 )OC
3 -
3 -
Fig. 4. Comparison of the organisms isolated from 1 3 rabbits with perforated appendicitis with those in pus found in 14 patients at operation for perforated appendicitis. The number a t the base of each column is the number of specimens with a concentration of less than 100 organisms/ml.
the caudal half. Post-mortem examination of the rabbit dying on the third postoperative day showed purulent peritonitis. Three rabbits in which only a base tie of the appendix was performed had died by 30 hours postoperatively. Tying less of the appendix produced less predictable results ; if insufficient was tied, only a localized abscess, without mortality, resulted. Four control rabbits had laparotomies performed at the same intervals, and at each operation the appendix was brought out on to the abdominal wall for a short time and then replaced. All 4 survived for at least 14 days and lost less weight than the rabbits with peritonitis (Fig. 3 ) ; no abnormality was found when they were sacrificed. The rabbits with perforated appendicitis followed by appendicectomy lost weight for 4-5 days, then regained weight to reach preoperative levels in the third postoperative week (Fig. 3).
Bacteriology In 13 rabbits, 9 with spontaneous perforations and 4 with artificial perforations, culture of peritoneal fluid present at the third laparotomy was begun within 5 minutes of removal of the sample from the peritoneal cavity. Since the bacterial groups expected in the human situation include enterobacteria, staphylococci, streptococci, bacilli, lactobacilli, yeasts, bacteroides, bifidobacteria, clostridia and veillonella, these organisms were sought in the samples. The viable counts of the bacteria were expressed as log,, organisms/ml of peritoneal fluid; the lower limit of detection of bacteria with this method was 102organisms/ml of fluid (Ellis-Pegler et al., 1975). A comparison has been made of cultures from 13 animals with those in pus taken at operation for perforated appendicitis with purulent peritonitis in
Fig. 5. Histological section of a rabbit appendix showing features similar to those of acute appendicitis in man.
14 patients (Pickersgill and Ellis-Pegler, 1975) (Fig. 4). The distribution was found to be similar; veillonella is an organism not found in the rabbit gut and was only grown from 1 human case. Histology Histologically, the appearances were similar in all the specimens examined and had many features in common with those seen in acute appendicitis in man (Fig. 5). However, in the rabbit suppuration was generally less pronounced and less evenly distributed throughout the wall and there was a zone of suppurative necrosis extending from the mucosa into the superficial parts of the lymphoid follicles. The latter is not seen normally in the human where the process seems to start from a focus rather than diffusely. There was also more lymphoid tissue in the rabbit appendices. 643
D. W. King et al. Discussion Induction of peritonitis in animals by intraperitoneal injection of bacterial suspension alone has proved to be difficult (Browne and Stoller, 1970). Furthermore, rats and mice are relatively resistant to induction of Gram-negative peritonitis (Burnett et al., 1957 ; Browne and Stoller, 1970). In 1964 Schumer et al. used an injection of a known amount of Escherichiu coli into the peritoneal cavity of guinea-pigs. Artz et al. in 1962 used pooled filtered faecal suspension, producing at least an 80 per cent mortality rate at 24 hours in dogs; variations on this method have been used often Filler and Sleeman, 1967; Lavigne et al., 1974). Burnett et al. (1957) removed a 3-mm ellipse from the colon of guinea-pigs and found that this routinely produced a severe diffuse peritonitis with an 85 per cent mortality rate. Zintel et al. (1950) induced peritonitis by ligating the appendiceal vessels and the base of the appendix and contaminating the peritoneal cavity with the appendix opened longitudinally; the abdomen was then closed. In 1938 Bower et al. described the technique of isolating and then tying the base of the caecal pouch of dogs, allowing the appendix to perforate spontaneously to produce a spreading peritonitis which in a later series produced a mortality of 78 per cent in untreated animals (Crook et al., 1968). The method was modified by Silvani et al. (1947) to include crushing of the wall of the appendix. The methods used in injection of known bacterial suspension or perforating the bowel release organisms suddenly into a normal peritoneal cavity, aod do not simulate the situation of acute obstructive appendicitis with perforation in humans. The method of Bower et al. (1938) and its subsequent variation (Silvani et al., 1947) involve the production of gangrene of the appendix by tying the blood vessels and do not include removal of the appendix. Neither of these reflect the human situation. To overcome these objections a model has been created akin to the disease process in man. It is recognized that the faecal and colonic flora of rabbits and man are not identical. Nevertheless, with isolation and identification techniques widely used in human studies the same bacterial groups have been isolated from rabbit and human faeces, although in different concentrations and with some exceptions (WilliamsSmith, 1965; Hawksworth et al., 1971). Perforation of the appendix is followed by peritonitis similar in bacteriological flora and degree of contamination to the situation found in humans. Moreover, the immediate effect in the animal is not so overwhelming as to produce rapid death, and this permits assessment of the postoperative course; the degree of peritonitis appears to be reflected in weight loss. Since perforation of the appendix is achieved in 70 per cent of animals the rate of reproducibility of the model is acceptable. If a high mortality is required for study, ligation of the appendix alone without subsequent removal may be undertaken; in this circumstance therapeutic measures under trial should be instituted after 24 hours. 644
Acknowledgements We would like to thank Dr J. P. O’Sullivan for reviewing the histology, Mr John Hynd for carrying out the excellent anaesthesia, the Clinical Laboratory staff for their help and the St George’s Hospital Research Fund for financial support. R. B. Ellis-Pegler is in receipt of a research grant from the Wellcome Trust. References ARTZ c. P., BARNETT w. 0. and GROGAN J. B. (1962) Further studies concerning the pathogenesis and treatment of peritonitis. Ann. Surg. 155, 756766. BOWER J. o., BURNS J. c. and MENGLE H. A. (1938) Induced spreading peritonitis complicating acute perforative appendicitis. Surg. Gynecol. Obstet. 66, 947-961. BROWNE M. K. and STOLLER J. L. (1970) Intraperitoneal noxythiolin in faecal peritonitis. Br. J. Surg. 57, 525-5 29. BURNETT W. E., BROWNE G. R., ROSEMOND G. P., CASEWELL H. T., BUCHER R. B. and TYSON R. R.
(1957) The treatment of peritonitis using peritoneal lavage. Ann. Surg. 145,657-681. CROOK J. N., COTLAR A. M., BORNSIDE G. H. and COHN I. (1968) Intraperitoneal cephalothin in the treatment of experimental appendiceal peritonitis. Am. Surg. 34, 736-739. ELLIS-PEGLER R. B., CRABTREE c. and LAMBERT H. P. (1975) To be published. FILLER R. M. and SLEEMAN H. K. (1967) Pathogenesis of peritonitis. 1. The effect of Escherichiu coli and hemoglobin on peritoneal absorption. Surgery 61, 385-392. HAWKSWORTH G., DRASER B. s. and HILL M. J. (1971) Intestinal bacteria and the hydrolysis of glycosidic bonds. J. Med. Microbiol. 4, 451-459. HYND J. (1975) To be published. LAVIGNE J. E., BROWN C. S., MACHIEDO G. W., BLACKWOOD J. M. and RUSH B. F. (1974) The treatment
of experimental peritonitis with intraperitoneal Betadine solution. J. Szirg. Res. 16, 307-31 1 . PICKERSGILL J. and ELLIS-PEGLER R. B. (1975) To be published. SCHUMER w., LEE D. K. and JONES B. (1964) Peritoneal lavage in postoperative therapy of ‘late peritoneal’ sepsis. Preliminary report. Surgery 55, 841845. SILVANI H. L., ROTHENBERG S., WARMER H., AMLUXEN J. and MCCORKLE H. J. (1947) Laboratory and clinical
experiences with Streptomycin therapy in the management of infections of intestinal origin. Surg. Gynecol. Obstet. 85, 721-726. WILLIAMS-SMITH H. (1965) Observations on the flora of the alimentary tract of animals and factors affecting its composition. J. Puthol. Bucteviol. 89, 95-122. ZINTEL H. A., ZINSSER H. H., RUNYON W., JACKSON G. L., NICHOLS A. c., WILEY M. M. and WILLIAMS w. J.
(1950) Influence of antibiotics and sulfonamides on the mortality and bacteria of experimental peritonitis. Surg. Gynecol. Obstet. 91, 742-750.
