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How does sepsis promote thrombosis in microvascular anastomoses?
0007-1226/89/0042-0314/$10.00
British Journal ofPIasfic Surgery (1989), 42, 314-3 17 0 1989 The Trustees of British Association of Plastic Surgeons
How does sepsis promote thrombosis anastomoses?
in microvascular
N. R. MCLEAN and H. ELLIS Department of Plastic, Reconstructive end Burns Surgery, Queen Mary’s Hospital, Roehampton, and the Academic Department of Surgery, Page Street Wing, Westminster Hospital, London Summary-This study on 100 rats, divided into five equal groups, has shown that chronic distant sepsis promotes occlusion of microvascular anastomoses to a significantly greater extent than aseptic inflammation, transient bacteraemia and even local infection. This may be related to the significant increase in the number of platelets in the group with distant sepsis.
In the rat experimental model, sepsis promotes occlusion of microvascular anastomoses even when the anastomotic site is not directly involved (McLean and Ellis, 1988). Carrel in 1924, showed that distant aseptic inflammation could adversely influence the healing of skin and this effect was further demonstrated in our laboratory in experimental abdominal, gastric and peritoneal wounds (DeHaan et al., 1974). Howe (1968, 1969) demonstrated that a clean surgical wound could become colonised by organisms in the circulation and a distant bacterial focus of infection can inhibit the healing of stomach, muscle, skin and peritoneum (DeHaan et al., 1974). In the latter study with Pseudomonas aerugirwsa, over 50% of the animals with a distant abscess had colonisation of the abdominal wound by the organism and it was postulated that the infection had been blood-borne. To study possible mechanisms by which distant sepsis causes thrombosis of microvascular anastomoses, the role of distant septic inflammation, distant sterile inflammation, local wound sepsis and a transient sublethal bacteraemia (induced at the time of anastomosis) have been investigated.
bacteraemia. These were respectively 2 x lo’, 2 x lo6 and 2 x lo5 organisms. It was also shown that 2 ml of sterile turpentine oil gave an acute aseptic inflammatory mass. One hundred female Sprague-Dawley rats, weighing between250 and 350grams, wererandomised to give 5 groups of 20 animals, prior to end-toend anastomosis of both femoral arteries. Group 1: A 2 ml suspension
Group 2.
Group 3
Group 4 :
Materials and methods Because it had been shown that our laboratory strain of Pseudomonas aeruginosa had increased in virulence since 1974 (De Haan et al., 1974), a preliminary study using ex-breeder female SpragueDawley rats, weighing between 300 and 400 grams, was performed to assess the dose of P. aeruginosa required to produce a distant pyogenic abscess, a local wound infection and a transient sublethal
Group 5 :
of P. aeruginosa (containing 2 x lo5 organisms) was injected into the external iliac vein immediately prior to the release of microvascular clamps, to produce a transient sublethal bacteraemia. A 2 ml suspension of P. aeruginosa (containing 2 x lo6 organisms) was applied topically into the groin wounds to produce a local wound infection. A 2 ml suspension of P. aeruginosa (containing 2 x 10’ organisms) was injected into the right axilla one week prior to anastomosis, to produce a distant pyogenic abscess (McLean and Ellis, 1988). ! ml of sterile turpentine oil was injected into the right axilla one week prior to mastomosis, to produce a remote sterile abscess (McLean and Ellis, 1988). ?ml of sterile saline was injected into the ight axilla as controls (McLean and Ellis, 1988).
The details of the operative technique, animal husbandry and methods of collecting and staining the specimens were fully discussed in our previous communication (McLean and Ellis, 1988).
314
HOW DOES SEPSIS PROMOTE
THROMBOSIS
IN MICROVASCULAR
Seven animals in group 1 (transient bacteraemia) and eight in group 2 (local sepsis) died before the microsurgical anastomoses could be examined at 7 days. They were removed from the study and replaced with new animals. The deaths in groups 3, 4 and 5 have been previously described (McLean and Ellis, 1988). There were thus 20 animals (with 40 femoral artery anastomoses) in each of the 5 groups for study. Results were analysed for statistical significance using the chi-square test. In a separate study, 20 further animals were randomised either to a control or to a distant pyogenic abscess group. Thirty minutes after microsurgical anastomosis of the right femoral artery, a blood sample was taken from the inferior vena cava and a full haematological investigation performed. In this part of study, results were analysed using the Kruskal-Wallis H test across the samples and the Mann-Whitney U two-tailed test between pairs of groups. Results
The results of groups 3,4 and 5 have already been published in our previous communication (McLean and Ellis, 1988). One week postoperatively, 39 of the 40 control anastomoses were patent. This was not significantly influenced by either a transient sublethal bacteraemia or a distant sterile abscess, where 35 anastomoses were patent in each (chi*= 1.62, p=O.203). Thrombosis was markedly increased in those animals with a local wound infection, 13/40 (chi2 = 10.48, p=O.OOl), but was greatest of all in the distant pyogenic abscess group 20140 (chi* = 20.92, p < 0.001) (Table 1). The histological appearances of the anastomosed arteries in control animals and the group with a distant abscess have previously been described (McLean and Ellis, 1988). The occluded vessels in the animals with a distant sterile abscess and a transient sublethal bacteraemia were similar to
Table 1
315
ANASTOMOSES?
those seen in the control group. Those with local wound sepsis had an intense vasculitis with a heavy polymorphonuclear leucocyte infiltration both in the vessel wall and extending into the occluding thrombus (Fig. 1). In the patent vessels in this group there was some evidence of vasculitis with clumping of polymorphonuclear leucocytes on the luminal surface of the intima (Fig. 2). The inflammatory response to the locally induced infection was of two types. In one, there was a thick fibrinous exudate, sometimes associated with enlargement of the inguinal lymph nodes, and in the other there was a copious watery inflammatory exudate accompanied by intense hyperaemia in the soft tissues around the femoral neurovascular bundle. The occlusion rate was similar in each. In five of the locally infected groins, the femoral vein had undergone spontaneous thrombosis. In all the animals with local sepsis, Pseudomonas aeruginosa was recovered from the groin wound. In none of the other groups was the local presence of this organism demonstrated. In five of the ten animals with distant septic abscess which underwent post-mortem examination, multiple abscesses were found in the liver, spleen, kidneys and lungs. Histological sections of the spleens confirmed the presence of extensive extra-medullary haemopoiesis. The results of the haematological investigations are shown in Table 2. A distant septic abscess significantly increased the platelet count when compared to the control animals (U = 13.00, p =O.OOS). The white cell count was also significantly increased and the differential was reversed, with a much higher percentage of polymorphonuclear leucocytes in the infected group (Table 3). Table 2
Haematological
results (median)
Parameter
Control
Dhtant
Haemoglobin (gm!di) Haematocrit (“A) White cell count ( x lO’/L) Platelet count ( x Io”/L)
15.60 44.20 4.95 975
13.25(p=O.218) 36.85 (p = 0.029) 10.85 (p
ohscr.w
Postoperative patency rates Table 3
Group
Number patent
Control Distant sterile abscess Transient bacteraemia Local wound sepsis Distant septic abscess
39/40 35/40 35140 27140 20140
p=O.203 p=O.203 p=O.OOl p
Median differential
white cell counts (“,)
Parameter
Control
Distant abscess
Polymorphonuclear leucocytes Lymphocytes Monocytes Eosinophilic leucocytes
14 82 3
6X(p
I
I
316
BRITISH JOURNAL
Fig. 1
OF PLASTIC SURGERY
Fig. 2
Figure I-Thrombosed
anastomosis in animal with local wound sepsis stained with haematoxylin and eosin (x 32). Note the vasculitis with presence of polymorphonuclear leucocytes both in the vessel wall and the occluding thrombus. Figure Z-Patent anastomosis in animal with local wound sepsis stained with haematoxylin and eosin ( x 32). The lumen is patent and there is some polymorphonuclear leucocyte clumping on ihe intimal surface.
Discussion Local staphylococcal infection induced at the time of surgery does not significantly lower the patency of femoral micro-anastomoses in the rat (Luk and Chow, 1985) but if surgery is performed in the presence of established infection then 75% of the anastomoses are occluded at 2 weeks (Luk et al., 1987). Interestingly, the corrected patency rate in the infected group (at the time of surgery) of 70% (Luk and Chow, 1985) is very similar to that of 67.5% for our group locally infected with pseudomonas. The discrepancy in significance arises because of their lower overall patency rates in the control, non-infected animals. Our previous study using Pseudomonas aeruginosa (McLean and Ellis, 1988) showed that distant infection has a significant effect on the patency of small vessel anastomoses. The first part of this investigation, which ran concurrently using in part the same animals, set out to see if this result was due to blood stream contamination leading to a transient bacteraemia resulting in colonisation of the anastomotic site, or by enhancement of thrombosis. The role of aseptic inflammation was also studied as this also inhibits the early healing of wounds (De Haan et al., 1974).
Our results showed that only distant and local sepsis significantly influenced the occlusion of microvascular anastomoses. As we were unable to culture P. aeruginosa from the groin wounds and anastomotic sites in the animals with distant septic inflammation, it is unlikely that the enhanced thrombosis was due to colonisation of the wound. Similarly, as a transient sublethal bacteraemia had an insignificant effect, this is unlikely to be the sole reason, although distant sepsis could cause repeated bacteraemia episodes which might influence the tendency to thrombosis. In five of the rats with distant sepsis, there were multiple abscesses found at post-mortem examination in a variety of organs, suggesting the repeated presence of organisms in the circulation. As there were significant signs of extra-medullary haemopoiesis found in the spleens of the animals with a distant abscess (much more so than would be expected in normal rats of this age and size), it was postulated that chronic sustained sepsis promoted hypercoagulability. Our results have shown a significant increase in the number of platelets in the infected group and further work is under way in this laboratory to assess the role of humoral factors in the enhancement of the coagulation in the infected animals.
HOW DOES SEPSIS PROMOTE
THROMBOSIS
IN MICROVASCULAR
In conclusion, chronic sustained sepsis causes occlusion of microvascular anastomoses, more so than either aseptic inflammation, transient bacteraemia or even local infection. This effect is mediated in part by a significant increase in the number of cir&lati& platelets. Acknowledgements We wish to thank the Departments of Medical Photography at the Westminster and Queen Mary’s Hospitals, and the Department of Medical Computing at the Westminster Hospital for the statistical analysis. This work was supported by a locally organised grant from the South West Thames Regional Health Authority.
References Carrel, A. (1924). Effet d’un abces a distance sur la cicatrisation dune plaie aseptique. Comptes Rendus des Seances de la SociktP de Biologie et de ses Filiales (Paris), 90, 333. DeHaan, B. B., Ellis, H. and Wilks, M. (1974). The role of infection on wound healing. Surgery, Gynecology and Obstetrics, 138, 693. Howe, C. W. (1968). Experimental wound sepsis from transient bacteremia. Surgery. Gynecology and Obstetrics, 126, 1066.
317
ANASTOMOSES?
Howe, C. W. (1969). Experimental wound sepsis from transient Escherichia coli bacteremia. Surgery, 66,570. Luk, K. D. K. and Chow, S. P. (1985). An experimental study of the effect of infection on microvascular anastomosis. Microsurgery. 6, 113. Luk, K. D. K., Zhou, L. R. and Chow, S. P. (1987). The effect of established infection on microvascular surgery. Plastic and Reconstructive Suraerv. 80.423. McLean, N. R. and T&i~, H. (1988). Does remote sepsis influence the patency of microvascular anastomoses? British Journal qf Plastic Surgery. 41. 395.
The Authors Neil R. McLean, BSe, FRCS, Consultant Plastic Surgeon, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 8BE; formerly Senior Registrar in Plastic Surgery, Queen Mary’s University Hospital, Roehampton, London, Harold Ellis, CBE, DM, MCh, FRCS, Professor of Surgery, Academic Department of Surgery, Page Street Wing, Westminster Hospital, London. Requests
for reprints
to Mr McLean
at Newcastle
upon Tyne.
Paper received 2 February 1988. Accepted 10 October 1988 after revision, This paper was presented to the British Association Surgeons, Summer Meeting, Edinburgh, July 1987.
of Plastic
British Journal ofPIasfic Surgery (1989), 42, 314-3 17 0 1989 The Trustees of British Association of Plastic Surgeons
How does sepsis promote thrombosis anastomoses?
in microvascular
N. R. MCLEAN and H. ELLIS Department of Plastic, Reconstructive end Burns Surgery, Queen Mary’s Hospital, Roehampton, and the Academic Department of Surgery, Page Street Wing, Westminster Hospital, London Summary-This study on 100 rats, divided into five equal groups, has shown that chronic distant sepsis promotes occlusion of microvascular anastomoses to a significantly greater extent than aseptic inflammation, transient bacteraemia and even local infection. This may be related to the significant increase in the number of platelets in the group with distant sepsis.
In the rat experimental model, sepsis promotes occlusion of microvascular anastomoses even when the anastomotic site is not directly involved (McLean and Ellis, 1988). Carrel in 1924, showed that distant aseptic inflammation could adversely influence the healing of skin and this effect was further demonstrated in our laboratory in experimental abdominal, gastric and peritoneal wounds (DeHaan et al., 1974). Howe (1968, 1969) demonstrated that a clean surgical wound could become colonised by organisms in the circulation and a distant bacterial focus of infection can inhibit the healing of stomach, muscle, skin and peritoneum (DeHaan et al., 1974). In the latter study with Pseudomonas aerugirwsa, over 50% of the animals with a distant abscess had colonisation of the abdominal wound by the organism and it was postulated that the infection had been blood-borne. To study possible mechanisms by which distant sepsis causes thrombosis of microvascular anastomoses, the role of distant septic inflammation, distant sterile inflammation, local wound sepsis and a transient sublethal bacteraemia (induced at the time of anastomosis) have been investigated.
bacteraemia. These were respectively 2 x lo’, 2 x lo6 and 2 x lo5 organisms. It was also shown that 2 ml of sterile turpentine oil gave an acute aseptic inflammatory mass. One hundred female Sprague-Dawley rats, weighing between250 and 350grams, wererandomised to give 5 groups of 20 animals, prior to end-toend anastomosis of both femoral arteries. Group 1: A 2 ml suspension
Group 2.
Group 3
Group 4 :
Materials and methods Because it had been shown that our laboratory strain of Pseudomonas aeruginosa had increased in virulence since 1974 (De Haan et al., 1974), a preliminary study using ex-breeder female SpragueDawley rats, weighing between 300 and 400 grams, was performed to assess the dose of P. aeruginosa required to produce a distant pyogenic abscess, a local wound infection and a transient sublethal
Group 5 :
of P. aeruginosa (containing 2 x lo5 organisms) was injected into the external iliac vein immediately prior to the release of microvascular clamps, to produce a transient sublethal bacteraemia. A 2 ml suspension of P. aeruginosa (containing 2 x lo6 organisms) was applied topically into the groin wounds to produce a local wound infection. A 2 ml suspension of P. aeruginosa (containing 2 x 10’ organisms) was injected into the right axilla one week prior to anastomosis, to produce a distant pyogenic abscess (McLean and Ellis, 1988). ! ml of sterile turpentine oil was injected into the right axilla one week prior to mastomosis, to produce a remote sterile abscess (McLean and Ellis, 1988). ?ml of sterile saline was injected into the ight axilla as controls (McLean and Ellis, 1988).
The details of the operative technique, animal husbandry and methods of collecting and staining the specimens were fully discussed in our previous communication (McLean and Ellis, 1988).
314
HOW DOES SEPSIS PROMOTE
THROMBOSIS
IN MICROVASCULAR
Seven animals in group 1 (transient bacteraemia) and eight in group 2 (local sepsis) died before the microsurgical anastomoses could be examined at 7 days. They were removed from the study and replaced with new animals. The deaths in groups 3, 4 and 5 have been previously described (McLean and Ellis, 1988). There were thus 20 animals (with 40 femoral artery anastomoses) in each of the 5 groups for study. Results were analysed for statistical significance using the chi-square test. In a separate study, 20 further animals were randomised either to a control or to a distant pyogenic abscess group. Thirty minutes after microsurgical anastomosis of the right femoral artery, a blood sample was taken from the inferior vena cava and a full haematological investigation performed. In this part of study, results were analysed using the Kruskal-Wallis H test across the samples and the Mann-Whitney U two-tailed test between pairs of groups. Results
The results of groups 3,4 and 5 have already been published in our previous communication (McLean and Ellis, 1988). One week postoperatively, 39 of the 40 control anastomoses were patent. This was not significantly influenced by either a transient sublethal bacteraemia or a distant sterile abscess, where 35 anastomoses were patent in each (chi*= 1.62, p=O.203). Thrombosis was markedly increased in those animals with a local wound infection, 13/40 (chi2 = 10.48, p=O.OOl), but was greatest of all in the distant pyogenic abscess group 20140 (chi* = 20.92, p < 0.001) (Table 1). The histological appearances of the anastomosed arteries in control animals and the group with a distant abscess have previously been described (McLean and Ellis, 1988). The occluded vessels in the animals with a distant sterile abscess and a transient sublethal bacteraemia were similar to
Table 1
315
ANASTOMOSES?
those seen in the control group. Those with local wound sepsis had an intense vasculitis with a heavy polymorphonuclear leucocyte infiltration both in the vessel wall and extending into the occluding thrombus (Fig. 1). In the patent vessels in this group there was some evidence of vasculitis with clumping of polymorphonuclear leucocytes on the luminal surface of the intima (Fig. 2). The inflammatory response to the locally induced infection was of two types. In one, there was a thick fibrinous exudate, sometimes associated with enlargement of the inguinal lymph nodes, and in the other there was a copious watery inflammatory exudate accompanied by intense hyperaemia in the soft tissues around the femoral neurovascular bundle. The occlusion rate was similar in each. In five of the locally infected groins, the femoral vein had undergone spontaneous thrombosis. In all the animals with local sepsis, Pseudomonas aeruginosa was recovered from the groin wound. In none of the other groups was the local presence of this organism demonstrated. In five of the ten animals with distant septic abscess which underwent post-mortem examination, multiple abscesses were found in the liver, spleen, kidneys and lungs. Histological sections of the spleens confirmed the presence of extensive extra-medullary haemopoiesis. The results of the haematological investigations are shown in Table 2. A distant septic abscess significantly increased the platelet count when compared to the control animals (U = 13.00, p =O.OOS). The white cell count was also significantly increased and the differential was reversed, with a much higher percentage of polymorphonuclear leucocytes in the infected group (Table 3). Table 2
Haematological
results (median)
Parameter
Control
Dhtant
Haemoglobin (gm!di) Haematocrit (“A) White cell count ( x lO’/L) Platelet count ( x Io”/L)
15.60 44.20 4.95 975
13.25(p=O.218) 36.85 (p = 0.029) 10.85 (p
ohscr.w
Postoperative patency rates Table 3
Group
Number patent
Control Distant sterile abscess Transient bacteraemia Local wound sepsis Distant septic abscess
39/40 35/40 35140 27140 20140
p=O.203 p=O.203 p=O.OOl p
Median differential
white cell counts (“,)
Parameter
Control
Distant abscess
Polymorphonuclear leucocytes Lymphocytes Monocytes Eosinophilic leucocytes
14 82 3
6X(p
I
I
316
BRITISH JOURNAL
Fig. 1
OF PLASTIC SURGERY
Fig. 2
Figure I-Thrombosed
anastomosis in animal with local wound sepsis stained with haematoxylin and eosin (x 32). Note the vasculitis with presence of polymorphonuclear leucocytes both in the vessel wall and the occluding thrombus. Figure Z-Patent anastomosis in animal with local wound sepsis stained with haematoxylin and eosin ( x 32). The lumen is patent and there is some polymorphonuclear leucocyte clumping on ihe intimal surface.
Discussion Local staphylococcal infection induced at the time of surgery does not significantly lower the patency of femoral micro-anastomoses in the rat (Luk and Chow, 1985) but if surgery is performed in the presence of established infection then 75% of the anastomoses are occluded at 2 weeks (Luk et al., 1987). Interestingly, the corrected patency rate in the infected group (at the time of surgery) of 70% (Luk and Chow, 1985) is very similar to that of 67.5% for our group locally infected with pseudomonas. The discrepancy in significance arises because of their lower overall patency rates in the control, non-infected animals. Our previous study using Pseudomonas aeruginosa (McLean and Ellis, 1988) showed that distant infection has a significant effect on the patency of small vessel anastomoses. The first part of this investigation, which ran concurrently using in part the same animals, set out to see if this result was due to blood stream contamination leading to a transient bacteraemia resulting in colonisation of the anastomotic site, or by enhancement of thrombosis. The role of aseptic inflammation was also studied as this also inhibits the early healing of wounds (De Haan et al., 1974).
Our results showed that only distant and local sepsis significantly influenced the occlusion of microvascular anastomoses. As we were unable to culture P. aeruginosa from the groin wounds and anastomotic sites in the animals with distant septic inflammation, it is unlikely that the enhanced thrombosis was due to colonisation of the wound. Similarly, as a transient sublethal bacteraemia had an insignificant effect, this is unlikely to be the sole reason, although distant sepsis could cause repeated bacteraemia episodes which might influence the tendency to thrombosis. In five of the rats with distant sepsis, there were multiple abscesses found at post-mortem examination in a variety of organs, suggesting the repeated presence of organisms in the circulation. As there were significant signs of extra-medullary haemopoiesis found in the spleens of the animals with a distant abscess (much more so than would be expected in normal rats of this age and size), it was postulated that chronic sustained sepsis promoted hypercoagulability. Our results have shown a significant increase in the number of platelets in the infected group and further work is under way in this laboratory to assess the role of humoral factors in the enhancement of the coagulation in the infected animals.
HOW DOES SEPSIS PROMOTE
THROMBOSIS
IN MICROVASCULAR
In conclusion, chronic sustained sepsis causes occlusion of microvascular anastomoses, more so than either aseptic inflammation, transient bacteraemia or even local infection. This effect is mediated in part by a significant increase in the number of cir&lati& platelets. Acknowledgements We wish to thank the Departments of Medical Photography at the Westminster and Queen Mary’s Hospitals, and the Department of Medical Computing at the Westminster Hospital for the statistical analysis. This work was supported by a locally organised grant from the South West Thames Regional Health Authority.
References Carrel, A. (1924). Effet d’un abces a distance sur la cicatrisation dune plaie aseptique. Comptes Rendus des Seances de la SociktP de Biologie et de ses Filiales (Paris), 90, 333. DeHaan, B. B., Ellis, H. and Wilks, M. (1974). The role of infection on wound healing. Surgery, Gynecology and Obstetrics, 138, 693. Howe, C. W. (1968). Experimental wound sepsis from transient bacteremia. Surgery. Gynecology and Obstetrics, 126, 1066.
317
ANASTOMOSES?
Howe, C. W. (1969). Experimental wound sepsis from transient Escherichia coli bacteremia. Surgery, 66,570. Luk, K. D. K. and Chow, S. P. (1985). An experimental study of the effect of infection on microvascular anastomosis. Microsurgery. 6, 113. Luk, K. D. K., Zhou, L. R. and Chow, S. P. (1987). The effect of established infection on microvascular surgery. Plastic and Reconstructive Suraerv. 80.423. McLean, N. R. and T&i~, H. (1988). Does remote sepsis influence the patency of microvascular anastomoses? British Journal qf Plastic Surgery. 41. 395.
The Authors Neil R. McLean, BSe, FRCS, Consultant Plastic Surgeon, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 8BE; formerly Senior Registrar in Plastic Surgery, Queen Mary’s University Hospital, Roehampton, London, Harold Ellis, CBE, DM, MCh, FRCS, Professor of Surgery, Academic Department of Surgery, Page Street Wing, Westminster Hospital, London. Requests
for reprints
to Mr McLean
at Newcastle
upon Tyne.
Paper received 2 February 1988. Accepted 10 October 1988 after revision, This paper was presented to the British Association Surgeons, Summer Meeting, Edinburgh, July 1987.
of Plastic