The beneficial effect of cyclosporin-A on the no-reflow phenomenon in rat skin island flaps
SL’IUMA R IT. The no-reflow phenomenon is one of the factors that increase morbidity in flap and replantation surgery. Prevention and treatment of the phenomenon is an area of intense current research. This study investigated the possible effect of cyclosporin administered systemically on survival of skin flaps subjected to ischaemiareperfusion injury. Cyclosporin treated flaps showed a statistically significant increase in survival areas regardless of the time of infusion (p < 0.01). These findings suggest that cyclosporin could be valuable in preventing or treating no-reflow in critical flaps. Possible mechanisms of action are discussed.
Prolonged periods of ischaemia induce functional and morphologic alterations in the microvasculature of various tissues, including skin (Pang, 1990). Upon reperfusion, cellular and biochemical interactions between blood elements with the ischaemic tissue can aggravate any ischaemic injury already established. This ischaemia-evoked progressive phenomenon known as reperfusion injury is characterised by the slowing of blood flow in ischaemic tissues and may lead to a no-reflow state in the microcirculation (May et (11..1978). Multiple factors have been implicated to explain the pathogenesis. including alterations in prostaglandin metabolism and oxygen free radical generation (Suzuki et al.. 1989: Feng CJ~~1.. 1989; Pang. 1990). The complex nature of this phenomenon continues to be a significant cause of morbidity in microvascular flap and replantation surgery (Zdeblick clt l/l.. 1985). Recently. several reports have shown that cyclosporin-A (Sandimmune-Sandoz”. Switzerland). a potent immunosuppressant, is effective in protecting the liver from ischaemic injury (Hayashi c>t[I/.. 1988. 1989; Kwano et trl.. 1989; Goto pt LI/.. 1990). This prompted us to test the possibility that cyclosporin-A (CsA) might have beneficial effects in treating or preventing ischaemia-reperfusion injury in skin flaps. Our study was designed to determine whether commercially available CsA would ameliorate ischaemia-reperfusion injury in island skin flaps in rats. Materials and methods Male rats (Sprague-Dawley) weighing 330 360 g were obtained from the Uludag University Animal Care Center for Research, housed for a week in individual cages and assigned to experimental groups randomly. All experiments were conducted under the guidelines set forth by the Animal Care Centre, Uludag University. Faculty of Medicine. Bursa. Turkey. The protocol consisted of a nonischaemic group (Group I ), ischaemic controls (Group 2) and three
(Group 3, Group 4, and Group 5) CsA treated groups (Table 1). Rats were anaesthetised with a single intraperitoneal injection of pentobarbitone (31% 40 mg/kg). The left external jugular vein was cannulated with a polyethylene catheter for intravenous drug infusion. After depilation of the abdominal skin. a neurovascular axial island flap (4 x 3 cm) was constructed, based on the left inferior epigastric neurovascular bundle. The femoral neurovascular bundle feeding the flap pedicle was also dissected free. All other vessels distal to the femoral ligament were divided and ligated. Ischaemia was induced by placing a microvascular clamp across the femoral pedicle in groups 2-5. Group I served as a nonischaemic control. Flaps were sutured back to their donor bed with continuous 5-O nylon sutures and the rats recovered in individual cages. Prior to completion of the ischaemic period ( I I h). the rats were again anaesthetised (pentobarbitone. 1530 mg/kg/ip). the flaps were reexplored and the microvascular clamps were released. Arterial pulsation and venous patency were observed for 15 min before the skin was secured in place with 5-O nylon sutures. The rats were then fitted with custom made vests made of X-ray film for protection from autocannibalisation. The duration of ischaemia was selected as I I h since IO-12 his reportedly the time needed for the no-reflow phenomenon to become established in skin island flaps in rabbits (May et trl.. 1978). All the rats were given 0.5 ml intravenous solution of either injectable CsA diluted in normal saline (IO mg/kg) or normal saline via the polyethylene Table 1
Experimental protocol C;roup I
Non-whaemx Control Group 2 Ischaemic Control Group 3 Prcischaemlc Cyclosporin-A Group 4 Postlschaemic Cyclosporin-A Group 5 Pre and Postischarmic Cyclosporin-A Duration
of Ischaomia
: II
hours. Cyclosporln-A.
19 mgjkp.
i\
**
T
1
0.005). .4pplication of the t-te\t to p,lircd groups contirmed the differences hetwoen the control and treatment group means. In all the CsA treated groups (Group\ 3 5). mean sur\:i\al \?;Ls significantly higher as compared to the ischaemic control (p < 0.01. Group 3 LS Grclup 2 and Group 5 vs Group 2. p < 0.001. Group 4 vs Group 1). There were 170 significant differences between the CsA treatment groups 3.4 and 5 (p > 0. I ). These re\ults are displayed in graphic form in Figure I.
Discussion
-Y
E 2 E
n=13
R OUP
atheter prior to the application (preischaemic-group 3 I and/or prior to the removal (postischaemic-group 4) of the clamp. In group 5. half of the CsA dose (5 mg/kg in 0.5 ml normal saline) was administered both prior :o the application and prior to the removal of the cla111p. The rat; were then observed every day for 7 days. At one week postnperati\ely, they were killed by a lethal dose of ]prntobarbitnne (75 mg/kg/ip). Planimetry tracings were made on transparent acetate paper and \ur\i\al arca for each flap was analysed in relation to the 7th day contracted ffap area using a digitiser with the aid of ;I personal computer.
Krusskall-Wallis one way analysis of variance and the r-test acre employed for statistical evaluation. Data \verc expressed as group mean k standard error of the mean. P Y 0.05 was considered signiticant.
Kesults
III of the Haps In the nonischaemic control goup I ) sur\ ibed completely. Group 2 had 13.1 % i_ -1.7”,, bur\ ival. In the preischaemic group (Group 3). the mean \ur\ iv;11 area was 47.4 96 & X.7 ‘!,O.The mean zurvi\ill are;ls were 56.7 “i, + 9.2 (‘0 and 49.0 90 k IO.5 ‘IO in the postischaemic (Group 4) and pre and po\tischacmic (Group 5) groups respectively The ditTerenccs between the group means were signil?eant (K russkall-Wallis. chi-Square = 11.9 I. p < I Group
The nonischaemic control study revealed that \urvival in our flap model was consistent \vhen ra-l>ed on a pedicle consisting of inferior epigastric artery. Lcin and nerve. This allowed us to differentiate confidentI> betwzen clamp induced and other GIUSCX ~1‘ necrosis. The no-retlou phenomenon in \kin flaps ~1;~s first described by May and coworkers (197s). This work and subsequent studies have led to ;I belief that interactions between leukocytes and enclothelial cells constitute ~1 central issue in the pathogcnesis of reperfusion injury (Schmid-Schonbein. 1987: Pang. 1990). Polymorphonuclear (PMN) as \\ell L~‘rendothelial cells are active participanta ill .IIIS injury process. The mechanism by which leukoc!.tc\ migrate to and adhere to endothelium and the chemoattractants involved are not known for certain. Hltwever. it is apparent from other studies that both leukocytes and cndothelial cells produce c> tokine and proadhesive molecules that modulate inflammatory response (Pober and Cotran. 1990). When PMN adhere to the rndothelium, a microenvironment is formed such that PMN-derived proteascs and oxygenderived free radicals act on the cndothelial ccllh. leading to tissue injury (Weis. 19X9). Prostacyclin. endothelial derived relaxing factor and platelet aggregating factor are each thought to pIa\ ;L role in this predominantly vascular component -in the inflammatory response (Pober and Cotran. IQOO). Other local mediators such as interleukin- I tumour necrosis factor and inter&kin-g probably aid in sustaining the inflammatory response. leading to further increases in leukocyte-endothelial cell interaction\ (Poher and Cotran, 1990). It seems reasonable tL\ supgest that some of these local mediators of‘inllamn~ati,on are also involved In the pathogenesis of repcrfuyion injur!. We have no direct explanation for oul- observed findings. There is a body of evidence I‘rom tran\plantation studies to suggest that C\A inhibit5 neutrc)phi1 chemotaxis (Pigatto t’t (ii.. I%%) and reduces ouysen-derived free radical production (G~ldin and K&sari, 1989). It is possible that CsA ma! interfere with leukocyte responses generally during repcrfuxion: it was recently reported that reducing leukocyte availability to ischaemic areas during rcperl‘usion and inhibition of leukocyte adhesivenehs to cndothelium proved valuable in preventing tissue damage and improved survival in skin flaps (Vcdder c’t 01.. I99 I ). It has also been shown that C\A inhibit\ the action and/or synthesis of many of the cytokinr and other mediators of inflammation (Belsito t’f tri.. I Wcl; Daniel
514
British
McKenna et 01.. 1989). CsA, by modulating cytokine and other local mediators of inflammation. may delay or inhibit the amplification of vascular-inflammatory responses. thereby reducing tissue damage. Although reference has been made to its possible role in preventing ischaemic injury in the liver (Hayashi rt cd., 1988. 1989; Kwano et al.. 1989: Goto et al.. 1990) this represents the first documentation of the beneficial effect of CsA on the no-reflow phenomenon in skin flaps. This finding may be important, since reperfusion injury and consequent perfusion failure is a major determinant in the success or failure of free flaps and the amputated limbs in replantation surgery. In the present study. postischaemic administration was effective in increasing flap survival. so CsA could be useful in clinical practice. CsA has been an approved immunosuppressant drug for more than a decade. which makes it a potential candidate for use in areas where reperfusion injury constitutes a relevant clinical problem. such as cerebral and myocardial ischaemia. However. further studies are needed to elucidate the mechanisms by which CsA exerts its effect. ct 111.. 1989:
References Belsito. D. V., Epstein, S. P., Schultz, J. M., Baen, R. C. and Thorbecke. C. J. (I 989).Enhancement by various cytokines on 2-
bets-mercaptoethanol of la antigen expression on langerhans ccII> in shin from norm;LI aged and young mice. Effect of cyclosporine-A. Juwr~ul of I/llnl2rnolo,~!. 145, 1530. Daniel. G.. Remick, D. T., Nguyen, M. K., Eskandri, R. M. and Strieter, S. L. (1989). Cyclosporine-A inhibits TNF production
without decreasing TNF and RNA levels. Biwhcwwtrl Rc.\cw).d~ C’o/rr~,~irnr~~tr/ir,,l\. I61 55 I Feng, 1. J.. Berger,
B. E., Lysz, T. W.
Bioyl~j,.ri~wl
and Shaw, W. W.
N., Kwano.
K. and Kobayashi,
Hayashi, T., Nagasue, N.. Kohno, H., Chang, Y. and Nakamura. (19Xx). The beneficial effect of cvclosporine pretreatment
ischemic damage
protects Iysosomal membrane phfrrlior2, 47. 924. Kwano, K., Kim, Y., Kaketomi,
in liver &hernia
Trtrr~-
(19X9).The in rats. Twrr.c-
K. and Kobayashi,
beneficial effect of cyclosporine plunlariorl. 48. 759. McKenna.
in dogs. xl.
on liver ischemia
R. M., Szturm. K., Jeffery. J. K. and Rush, D. N. ( I YXY).
Inhibition plunttrtion.
of cytokine 41. 333.
production
by cyclosporine
A-G.
Trtr,~.\-
J. W., Jr., Chait, L. A. and O’Brien. B. M. ( 197X). The noreflow phenomenon in experimental free Ilaps. P/rr.\rlc, rrrfd Rrcon.Ffrlrc,tir,e Surgcv~~. 61. 256. Pang, C. Y. (I 990). Ischcmia-induced repcrfuslon illjury in muscle flaps: pathogenesis and major source of fret mdicals. Rcow .srrwiiw ,2~ic,rl,.vu~~cr~,.6. 17.
May,
Pigatto, P. D., Mozzanica, N., Polengghi, M. VI., Altomare, G. F. and Finzi, A. F. (198X). Cyclosporine-A inhibits polymor-
phonuclear to. 91.
leukocyte
chemotaxis
in viva
rrcifl.g~/tnl/ Pr~~
SL’IUMA R IT. The no-reflow phenomenon is one of the factors that increase morbidity in flap and replantation surgery. Prevention and treatment of the phenomenon is an area of intense current research. This study investigated the possible effect of cyclosporin administered systemically on survival of skin flaps subjected to ischaemiareperfusion injury. Cyclosporin treated flaps showed a statistically significant increase in survival areas regardless of the time of infusion (p < 0.01). These findings suggest that cyclosporin could be valuable in preventing or treating no-reflow in critical flaps. Possible mechanisms of action are discussed.
Prolonged periods of ischaemia induce functional and morphologic alterations in the microvasculature of various tissues, including skin (Pang, 1990). Upon reperfusion, cellular and biochemical interactions between blood elements with the ischaemic tissue can aggravate any ischaemic injury already established. This ischaemia-evoked progressive phenomenon known as reperfusion injury is characterised by the slowing of blood flow in ischaemic tissues and may lead to a no-reflow state in the microcirculation (May et (11..1978). Multiple factors have been implicated to explain the pathogenesis. including alterations in prostaglandin metabolism and oxygen free radical generation (Suzuki et al.. 1989: Feng CJ~~1.. 1989; Pang. 1990). The complex nature of this phenomenon continues to be a significant cause of morbidity in microvascular flap and replantation surgery (Zdeblick clt l/l.. 1985). Recently. several reports have shown that cyclosporin-A (Sandimmune-Sandoz”. Switzerland). a potent immunosuppressant, is effective in protecting the liver from ischaemic injury (Hayashi c>t[I/.. 1988. 1989; Kwano et trl.. 1989; Goto pt LI/.. 1990). This prompted us to test the possibility that cyclosporin-A (CsA) might have beneficial effects in treating or preventing ischaemia-reperfusion injury in skin flaps. Our study was designed to determine whether commercially available CsA would ameliorate ischaemia-reperfusion injury in island skin flaps in rats. Materials and methods Male rats (Sprague-Dawley) weighing 330 360 g were obtained from the Uludag University Animal Care Center for Research, housed for a week in individual cages and assigned to experimental groups randomly. All experiments were conducted under the guidelines set forth by the Animal Care Centre, Uludag University. Faculty of Medicine. Bursa. Turkey. The protocol consisted of a nonischaemic group (Group I ), ischaemic controls (Group 2) and three
(Group 3, Group 4, and Group 5) CsA treated groups (Table 1). Rats were anaesthetised with a single intraperitoneal injection of pentobarbitone (31% 40 mg/kg). The left external jugular vein was cannulated with a polyethylene catheter for intravenous drug infusion. After depilation of the abdominal skin. a neurovascular axial island flap (4 x 3 cm) was constructed, based on the left inferior epigastric neurovascular bundle. The femoral neurovascular bundle feeding the flap pedicle was also dissected free. All other vessels distal to the femoral ligament were divided and ligated. Ischaemia was induced by placing a microvascular clamp across the femoral pedicle in groups 2-5. Group I served as a nonischaemic control. Flaps were sutured back to their donor bed with continuous 5-O nylon sutures and the rats recovered in individual cages. Prior to completion of the ischaemic period ( I I h). the rats were again anaesthetised (pentobarbitone. 1530 mg/kg/ip). the flaps were reexplored and the microvascular clamps were released. Arterial pulsation and venous patency were observed for 15 min before the skin was secured in place with 5-O nylon sutures. The rats were then fitted with custom made vests made of X-ray film for protection from autocannibalisation. The duration of ischaemia was selected as I I h since IO-12 his reportedly the time needed for the no-reflow phenomenon to become established in skin island flaps in rabbits (May et trl.. 1978). All the rats were given 0.5 ml intravenous solution of either injectable CsA diluted in normal saline (IO mg/kg) or normal saline via the polyethylene Table 1
Experimental protocol C;roup I
Non-whaemx Control Group 2 Ischaemic Control Group 3 Prcischaemlc Cyclosporin-A Group 4 Postlschaemic Cyclosporin-A Group 5 Pre and Postischarmic Cyclosporin-A Duration
of Ischaomia
: II
hours. Cyclosporln-A.
19 mgjkp.
i\
**
T
1
0.005). .4pplication of the t-te\t to p,lircd groups contirmed the differences hetwoen the control and treatment group means. In all the CsA treated groups (Group\ 3 5). mean sur\:i\al \?;Ls significantly higher as compared to the ischaemic control (p < 0.01. Group 3 LS Grclup 2 and Group 5 vs Group 2. p < 0.001. Group 4 vs Group 1). There were 170 significant differences between the CsA treatment groups 3.4 and 5 (p > 0. I ). These re\ults are displayed in graphic form in Figure I.
Discussion
-Y
E 2 E
n=13
R OUP
atheter prior to the application (preischaemic-group 3 I and/or prior to the removal (postischaemic-group 4) of the clamp. In group 5. half of the CsA dose (5 mg/kg in 0.5 ml normal saline) was administered both prior :o the application and prior to the removal of the cla111p. The rat; were then observed every day for 7 days. At one week postnperati\ely, they were killed by a lethal dose of ]prntobarbitnne (75 mg/kg/ip). Planimetry tracings were made on transparent acetate paper and \ur\i\al arca for each flap was analysed in relation to the 7th day contracted ffap area using a digitiser with the aid of ;I personal computer.
Krusskall-Wallis one way analysis of variance and the r-test acre employed for statistical evaluation. Data \verc expressed as group mean k standard error of the mean. P Y 0.05 was considered signiticant.
Kesults
III of the Haps In the nonischaemic control goup I ) sur\ ibed completely. Group 2 had 13.1 % i_ -1.7”,, bur\ ival. In the preischaemic group (Group 3). the mean \ur\ iv;11 area was 47.4 96 & X.7 ‘!,O.The mean zurvi\ill are;ls were 56.7 “i, + 9.2 (‘0 and 49.0 90 k IO.5 ‘IO in the postischaemic (Group 4) and pre and po\tischacmic (Group 5) groups respectively The ditTerenccs between the group means were signil?eant (K russkall-Wallis. chi-Square = 11.9 I. p < I Group
The nonischaemic control study revealed that \urvival in our flap model was consistent \vhen ra-l>ed on a pedicle consisting of inferior epigastric artery. Lcin and nerve. This allowed us to differentiate confidentI> betwzen clamp induced and other GIUSCX ~1‘ necrosis. The no-retlou phenomenon in \kin flaps ~1;~s first described by May and coworkers (197s). This work and subsequent studies have led to ;I belief that interactions between leukocytes and enclothelial cells constitute ~1 central issue in the pathogcnesis of reperfusion injury (Schmid-Schonbein. 1987: Pang. 1990). Polymorphonuclear (PMN) as \\ell L~‘rendothelial cells are active participanta ill .IIIS injury process. The mechanism by which leukoc!.tc\ migrate to and adhere to endothelium and the chemoattractants involved are not known for certain. Hltwever. it is apparent from other studies that both leukocytes and cndothelial cells produce c> tokine and proadhesive molecules that modulate inflammatory response (Pober and Cotran. 1990). When PMN adhere to the rndothelium, a microenvironment is formed such that PMN-derived proteascs and oxygenderived free radicals act on the cndothelial ccllh. leading to tissue injury (Weis. 19X9). Prostacyclin. endothelial derived relaxing factor and platelet aggregating factor are each thought to pIa\ ;L role in this predominantly vascular component -in the inflammatory response (Pober and Cotran. IQOO). Other local mediators such as interleukin- I tumour necrosis factor and inter&kin-g probably aid in sustaining the inflammatory response. leading to further increases in leukocyte-endothelial cell interaction\ (Poher and Cotran, 1990). It seems reasonable tL\ supgest that some of these local mediators of‘inllamn~ati,on are also involved In the pathogenesis of repcrfuyion injur!. We have no direct explanation for oul- observed findings. There is a body of evidence I‘rom tran\plantation studies to suggest that C\A inhibit5 neutrc)phi1 chemotaxis (Pigatto t’t (ii.. I%%) and reduces ouysen-derived free radical production (G~ldin and K&sari, 1989). It is possible that CsA ma! interfere with leukocyte responses generally during repcrfuxion: it was recently reported that reducing leukocyte availability to ischaemic areas during rcperl‘usion and inhibition of leukocyte adhesivenehs to cndothelium proved valuable in preventing tissue damage and improved survival in skin flaps (Vcdder c’t 01.. I99 I ). It has also been shown that C\A inhibit\ the action and/or synthesis of many of the cytokinr and other mediators of inflammation (Belsito t’f tri.. I Wcl; Daniel
514
British
McKenna et 01.. 1989). CsA, by modulating cytokine and other local mediators of inflammation. may delay or inhibit the amplification of vascular-inflammatory responses. thereby reducing tissue damage. Although reference has been made to its possible role in preventing ischaemic injury in the liver (Hayashi rt cd., 1988. 1989; Kwano et al.. 1989: Goto et al.. 1990) this represents the first documentation of the beneficial effect of CsA on the no-reflow phenomenon in skin flaps. This finding may be important, since reperfusion injury and consequent perfusion failure is a major determinant in the success or failure of free flaps and the amputated limbs in replantation surgery. In the present study. postischaemic administration was effective in increasing flap survival. so CsA could be useful in clinical practice. CsA has been an approved immunosuppressant drug for more than a decade. which makes it a potential candidate for use in areas where reperfusion injury constitutes a relevant clinical problem. such as cerebral and myocardial ischaemia. However. further studies are needed to elucidate the mechanisms by which CsA exerts its effect. ct 111.. 1989:
References Belsito. D. V., Epstein, S. P., Schultz, J. M., Baen, R. C. and Thorbecke. C. J. (I 989).Enhancement by various cytokines on 2-
bets-mercaptoethanol of la antigen expression on langerhans ccII> in shin from norm;LI aged and young mice. Effect of cyclosporine-A. Juwr~ul of I/llnl2rnolo,~!. 145, 1530. Daniel. G.. Remick, D. T., Nguyen, M. K., Eskandri, R. M. and Strieter, S. L. (1989). Cyclosporine-A inhibits TNF production
without decreasing TNF and RNA levels. Biwhcwwtrl Rc.\cw).d~ C’o/rr~,~irnr~~tr/ir,,l\. I61 55 I Feng, 1. J.. Berger,
B. E., Lysz, T. W.
Bioyl~j,.ri~wl
and Shaw, W. W.
N., Kwano.
K. and Kobayashi,
Hayashi, T., Nagasue, N.. Kohno, H., Chang, Y. and Nakamura. (19Xx). The beneficial effect of cvclosporine pretreatment
ischemic damage
protects Iysosomal membrane phfrrlior2, 47. 924. Kwano, K., Kim, Y., Kaketomi,
in liver &hernia
Trtrr~-
(19X9).The in rats. Twrr.c-
K. and Kobayashi,
beneficial effect of cyclosporine plunlariorl. 48. 759. McKenna.
in dogs. xl.
on liver ischemia
R. M., Szturm. K., Jeffery. J. K. and Rush, D. N. ( I YXY).
Inhibition plunttrtion.
of cytokine 41. 333.
production
by cyclosporine
A-G.
Trtr,~.\-
J. W., Jr., Chait, L. A. and O’Brien. B. M. ( 197X). The noreflow phenomenon in experimental free Ilaps. P/rr.\rlc, rrrfd Rrcon.Ffrlrc,tir,e Surgcv~~. 61. 256. Pang, C. Y. (I 990). Ischcmia-induced repcrfuslon illjury in muscle flaps: pathogenesis and major source of fret mdicals. Rcow .srrwiiw ,2~ic,rl,.vu~~cr~,.6. 17.
May,
Pigatto, P. D., Mozzanica, N., Polengghi, M. VI., Altomare, G. F. and Finzi, A. F. (198X). Cyclosporine-A inhibits polymor-
phonuclear to. 91.
leukocyte
chemotaxis
in viva
rrcifl.g~/tnl/ Pr~~
