The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber.

UNLABELLED: The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation. METHODS: Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2 x 10(6) rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n = 6), 7 (n = 6), 14 (n = 4) or 28 (n = 4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO2 < 10 mmHg. RESULTS: At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge > 0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole. CONCLUSION: Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers < or = 7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7-28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.

Vascularisation of tissue-engineered grafts: the regulation of angiogenesis in reconstructive surgery and in disease states.

Angiogenesis (the formation of new blood vessels) is essential for the growth of new tissue, tissue repair and wound healing. Tissue engineering, the construction of new tissue and organs for reparative purposes, relies on angiogenesis for the vascularisation of these new grafts. In tissue engineering, the emphasis to date has been on vascularisation of newly constructed tissue grafts by an extrinsic blood supply, and relatively little attention has been given to the possibility of building these grafts around an intrinsic blood supply. However, there are many disease processes, notably tumour growth, where excess angiogenesis can be a major problem. The purposes of this review are, first, to examine various methods of vascularising tissue-engineered grafts, and, second, to compare the role of angiogenesis in tissue engineering, where stimulation of angiogenesis is paramount, with pathological states, such as tumour growth, where angiogenesis needs to be inhibited.

Formation of new tissue from an arteriovenous loop in the absence of added extracellular matrix.

A major requirement for the microsurgical repair of contour defects of the skin, for example, following removal of a skin cancer on the face, is a mass of vascularised subcutaneous tissue. Such tissue can be generated in vivo using basic tissue engineering principles. In previous studies in our laboratory, we have used a model comprising an arteriovenous (AV) shunt loop sandwiched in artificial dermis, placed in a cylindrical plastic growth chamber, and inserted subcutaneously to grow new connective tissue progressively up to 4 weeks. To learn more about the basic growth characteristics with this model, the same AV shunt loop within a chamber without added extracellular matrix was inserted subcutaneously into the groins of rats for 2, 4, or 12 weeks (n = 5 per group). There was a progressive increase in the mass and volume of tissue such that the chamber was two-thirds full after 12 weeks. Histological examination showed that at 2 weeks there was evidence of fibroblast and vascular outgrowth from the AV shunt, with the formation of granulation tissue, surrounded by a mass of coagulated exudate. At 4 weeks the connective tissue deposition was more extensive, with a mass of more mature granulation tissue containing considerable collagen. By 12 weeks there was an extensive, well vascularized mass of mature fibrous tissue. The blood vessels and residual adventitia of the AV shunt were the likely source of growth factors and of the cells which populated the chamber with new maturing connective tissue. A patent AV shunt in an isolated chamber appears to be the minimal requirement for the generation of new vascularized tissue that is potentially suitable for microsurgical transplantation.

Cold storage of rat skeletal muscle free flaps and pre-ischemic perfusion with modified UW solution.

We used the rat medial gastrocnemius free flap, based on a pedicle of the femoral artery and vein, in order to test the tolerance of skeletal muscle to cold ischemia-reperfusion (IR) injury, and to determine whether tolerance can be enhanced by pre-ischemic perfusion with tissue/organ preservation solutions. Muscle flaps (n = 6 per group) were subjected to variable periods of cold storage (0, 1, 2, 3, or 4 days) and 24-h normothermic reperfusion. Muscle viability, as determined by nitroblue tetrazolium (NBT) histochemical staining of viable mitochondria and supported by histological examination, was 100%, 26%, 11%, 4%, and 1%, respectively. Using 24-h cold storage/24-h reperfusion as the experimental conditions, groups of muscle flaps (n = 5 per group) were perfused immediately before cold storage with either modified, colloid-free University of Wisconsin (UW) solution, a solution described by Kohout et al. (Br J Plast Surg 1995;48:132-144) or normal saline. Perfusion with modified UW solution or Kohout's solution increased survival to 33% (P < 0.05) and 28% (not statistically significant), respectively, compared with the 19% viability of separate groups of nonperfused or saline-perfused controls. These findings indicate that cold-stored skeletal muscle is highly susceptible to cold IR injury and that the viability can be increased by prior perfusion with a tissue preservation solution such as modified UW solution.

Clinical associations and characterisation of antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein and azurocidin.

Bactericidal/permeability-increasing protein (BPI) and azurocidin (AZ) are recently described target antigens of antineutrophil cytoplasmic antibodies (ANCA). In this study, BPI-ANCA were demonstrated most often in patients with ulcerative colitis (36/92, 39%), Crohn's disease (17/66, 26%) and cystic fibrosis (11/14, 79%), but also in patients with rheumatoid arthritis (8/40, 20%), systemic lupus erythematosus (SLE) (111/65, 17%) and mixed connective tissue disease (4/18, 22%). BPI-ANCA were also common in sera containing antinuclear (ANA) (9/43, 21%) or antidouble-stranded (ds) DNA (7/28, 25%) antibodies. There was no increased frequency of abnormal alpha1-antitrypsin (alphal1AT) phenotypes in patients with BPI-ANCA, and BPI-ANCA were not more common in individuals with an abnormal phenotype. The predominant IgG subclasses were IgG1 and IgG3; IgA but not IgM was present. Both IgG and IgA BPI-ANCA were high affinity antibodies, and the affinity of IgG antibodies did not change with time in the sera tested. Four of the five sera (80%) containing BPI-ANCA did not bind to denatured, reduced BPI, suggesting that most BPI-ANCA recognised conformational epitopes. AZ-ANCA were demonstrated in 2/11 patients (18%) with Wegener's granulomatosis, 3/12 (25%) with cystic fibrosis and 3/14 (21%) with chronic active hepatitis. AZ-ANCA were present in 5/25 sera (25%) with ANA, but the levels were only marginally elevated. AZ-ANCA were uncommon in patients with inflammatory bowel and rheumatological diseases, and in sera containing other autoantibodies. Again, there was no association with abnormal alpha1-AT phenotypes. BPI represents a major ANCA target antigen in patients with rheumatological as well as inflammatory bowel disease and cystic fibrosis, but AZ-ANCA are uncommon.

Neutrophil-independent protective effect of r-metHuG-CSF in ischaemia-reperfusion injury in rat skeletal muscle.

The aim of this study was to investigate the effect of the cytokine, r-metHuG-CSF, in a rat model of ischaemia-reperfusion (IR) injury and the pathophysiological mechanism involved. The administration of r-metHuG-CSF (20 (g/kg, s.c.) 4 h prior to either 100 min or 2 h of tourniquet ischaemia to the upper thigh significantly improved the viability of skeletal muscle after 24 h reperfusion compared with saline-treated rats (P < 0.05). Administration of r-metHuG-CSF earlier (24 h before ischaemia) or later (immediately before ischaemia) had no protective effect. At the dose used, r-metHuG-CSF caused a three-fold increase in the level of circulating blood neutrophils and a modest but significant increase in the neutrophil content of ischaemic muscle after 24 h reperfusion. Reduction of neutrophils to 1.4% of normal levels by cyclophosphamide (150 mg/kg, i.p.) prior to injury had no significant effect on the survival of muscle subjected to 2 h ischaemia and 24 h reperfusion or on the protective effect of r-metHuG-CSF. IR injury to skeletal muscle was accompanied by a time-dependent increase in plasma TNFalpha levels during the first 8 h of reperfusion and the increase was reduced significantly by pretreatment with r-metHuG-CSF. However, a similar time-dependent increase in plasma nitrite/nitrate levels was unaffected by pretreatment with r-metHuG-CSF. These findings suggest that the protective effect of r-metHuG-CSF may be mediated by the attenuated release of TNFalpha and indicate that the level of neutrophils in either blood or injured tissue does not influence significantly the viability of rat muscle after IR injury.

The beneficial effect of heparin in preischemic perfusion solutions for cold-stored skin flaps.

Storage of skin flaps in the cold before replantation increases their tolerance to ischemic damage. Rat epigastric skin flaps were perfused immediately before 2 days of cold ischemia with 3 ml of normal saline containing either 10 U per milliliter of heparin (group 1, N = 11) or normal saline (group 2, N = 10), or stored without perfusion (group 3, N = 6), and replanted. Flap viability was assessed 7 days later. The mean flap survival in groups 1, 2, and 3 was 73% (p<0.01 compared with group 2), 10%, and 33% respectively. Intravascular fibrin deposits were detected histochemically 5 minutes before reperfusion in nonperfused flaps and 5 minutes after reperfusion in saline-perfused flaps, but not in flaps perfused with heparinized saline. Angiography revealed evidence of no reflow in the first 5 minutes of reperfusion in saline-perfused flaps, but normal blood flow in heparinized saline-perfused flaps. Tissue water content, myeloperoxidase activity, and hydroperoxide levels after 1 and 24 hours of reperfusion were not significantly different in flaps perfused with heparinized saline and normal saline. These findings indicate that in skin flaps perfused before ischemia, flaps perfused with heparinized saline survive significantly better than flaps perfused with normal saline. They also survive better than nonperfused flaps but the improvement is not significant.

Muscle cells become necrotic rather than apoptotic during reperfusion of ischaemic skeletal muscle.

While necrosis is known as a major mechanism for the loss of viability of skeletal muscle following ischaemia and reperfusion, much less is known of the role of apoptosis. In this study rat hind limbs were subjected to 2 h of tourniquet ischaemia, then reperfused for either 0, 0.25, 0.5, 1, 3, 8, 16 or 24 h (n = 6 per group). Mean viability of muscle, assessed by tetrazolium dye reduction, after 2 h ischaemia and 24 h reperfusion was 17%. Histological examination revealed disrupted, necrotic muscle fibres from 30 min to 24 h reperfusion. Apoptotic nuclei were identified by haematoxylin staining and TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labelling. No TUNEL-positive cells were observed at the end of the ischaemic period, but a small number of TUNEL-positive endothelial and smooth muscle cells were found at 30 min reperfusion, with a progressive increase in their number up to 24 h reperfusion. Apoptotic neutrophils were detected after 8-24 h reperfusion. At no stage was apoptosis seen in the nuclei of skeletal muscle fibres. It appears that apoptosis plays no role in the death of muscle fibres after ischaemia-reperfusion injury to skeletal muscle.

Dexamethasone treatment prior to reperfusion improves the survival of skin flaps subjected to secondary venous ischaemia.

The potential use of the anti-inflammatory glucocorticoid, dexamethasone, to treat ischaemic skin flaps prior to reperfusion was investigated. Island flaps were raised in rats, subjected to arteriovenous ischaemia for 2 h, normal blood flow for 24 h, secondary venous ischaemia for 4 h and secondary reperfusion for 7 days. This sequence mimics the clinical sequence of free flap transfer followed by a subsequent venous thrombosis. Groups of 10 rats were administered with an intraperitoneal dose of either saline (controls) or dexamethasone (2.5 mg/kg) 30 min before the end of the venous ischaemia. Compared with viability of 70.0% in controls, dexamethasone treatment increased viability significantly to 92.0% (P < 0.01). In skin flap tissue harvested at 24 h reperfusion, dexamethasone treatment resulted in significant attenuation of tissue water content, tissue myeloperoxidase activity and tissue hydroperoxide levels. Thus the protective effect of this agent may be explained by the combined reduction of oedema formation, neutrophil infiltration and free radical production, respectively. We conclude that a single systemic dose of dexamethasone prior to reperfusion may be beneficial in treating skin flaps that have undergone secondary venous ischaemia.

The proteinase-antiproteinase theory of emphysema: a speculative analysis of recent advances into the pathogenesis of emphysema.

This review concerns the reasons why only an estimated 10-15% of patients with alpha-1-antitrypsin (A1AT) deficiency develop the destructive lung disease known as emphysema. The arguments presented revolve around the proteinase-antiproteinase balance in the 'microenvironment' of the epithelial space of the lung. Attention is focused on the balance between destructive enzymes such as neutrophil elastase and protective proteins such as A1AT, secretory leucocyte proteinase inhibitor (SLPI), human elastase inhibitor (HEI) and elafin. When neutrophil elastase is already attached to the elastin fibres the smaller molecules SLPI and elafin appear to be better inhibitors of this enzyme than larger inhibitors such as A1AT and HEI. Furthermore, SLPI and elafin may provide the first line of defence against proteinase attack from neutrophil elastase. In trying to explain the variability in the clinical expression of A1AT-deficiency and the development of emphysema, the importance of changes to A1AT, SLPI and elafin molecules induced by smoking and/or oxygen free radicals has been considered. It is possible that emphysema only develops in patients who have SLPI/elafin deficiency as well as A1AT deficiency.

Influence of postischemic administration of oxyradical antagonists on ischemic injury to rabbit skeletal muscle.

The aim of this study was to determine whether the administration of free radical antagonists, immediately before and during the early minutes of reperfusion, improves muscle survival 24 hr after a period of ischemia. Rabbit rectus femoris muscles were isolated, made ischemic for 3 1/2 hr and treated with either desferrioxamine (DFX), an Fe3+ chelator, superoxide dismutase and catalase (SOD & CAT), which quench superoxide and hydrogen peroxide, or allopurinol, an inhibitor of xanthine oxidase (XO). After 24 hr reperfusion, muscle viability (+/-s.e.m.), measured by the nitro blue tetrazolium (NBT) vital staining technique, was 41.6 +/- 11.3% for saline-treated ischemic controls, 30.6 +/- 7.6% for DFX-treated, 46.7 +/- 10.3% for SOD & CAT-treated, and 43.3 +/- 9.5% for allopurinol-treated muscles. None of the treated groups differed significantly from the ischemic control group. Tissue myeloperoxidase, ATP and reduced glutathione levels, and plasma lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels were increased by ischemia and reperfusion in all groups, but the changes did not differ between the treatment groups. Levels of XO in the rabbit muscle were determined and found to be very low in both normal and postischemic muscle. As XO is the target enzyme of allopurinol, its absence provides a basis for the lack of effect of this agent. However, it is not clear why DFX and SOD & CAT had no protective effect.

Phosphoenolpyruvate/adenosine triphosphate enhances post-ischemic survival of skeletal muscle.

This study examined whether ischemia-reperfusion injury to skeletal muscle could be reduced by post-ischemic infusion of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP). The rectus femoris muscle of 54 rabbits was rendered ischemic for 3.5 hr. Eighteen rabbits received no further treatment. Thirty-six were infused intra-arterially at the end of ischemia, 18 with vehicle alone, and 18 with a mixture of PEP (80 mumol/kg) and ATP (2.6 mumol/kg). Six rabbits from each group were explored after 24 hr reperfusion and the muscles assessed for viability (by nitro blue tetrazolium), ATP (by luciferin-luciferase chemiluminescence), malonyldialdehyde (MDA) (thiobarbituric acid method), and water content. The remaining muscles in each group were examined histologically after either 1 hr or 4 days of reperfusion. At 24 hr the viability of the PEP/ATP infused muscles (78.9 +/- 15.4 percent) was significantly greater than that of untreated (41.4 +/- 27.3 percent) or vehicle-infused groups (34.0 +/- 32.7 percent). ATP stores were significantly higher and MDA (indicative of free radical activity) and water content significantly lower in the PEP/ATP treated group. At 24 hr and 4 days, muscles infused with PEP/ATP showed less necrosis and fewer infiltrating neutrophils than the untreated groups. Studies with isolated rabbit neutrophils showed that ATP alone significantly inhibited superoxide anion production by stimulated neutrophils. However, when combined with PEP at concentrations similar to those achieved in vivo, ATP did not significantly affect superoxide production. The findings indicate that post-ischemic infusion of PEP/ATP significantly reduces ischemia-reperfusion injury in rabbit skeletal muscle. The protective effect of PEP/ATP is more likely to be due to supplementation of intracellular ATP stores than to the inhibition of superoxide production by infiltrating neutrophils.

Platelet-activating factor (PAF) receptor antagonism by WEB 2170 improves the survival of ischaemic skeletal muscle.

The platelet-activating factor (PAF) receptor antagonist, WEB 2170, significantly improved the survival of ischaemic rabbit skeletal muscle from 42% in saline-infused muscle to 65% in WEB 2170-infused muscle at 24 hours post reperfusion. Evidence is presented which suggests that WEB 2170 inhibits neutrophil infiltration during reperfusion. In addition, tissue lipid peroxide levels and the release into blood of the enzyme creatine kinase were inhibited by local infusion of WEB 2170. In contrast, the level of oedema in muscles receiving an infusion of WEB 2170 was not different from that in saline controls. It is concluded that infusion of a PAF antagonist into ischaemic skeletal muscle at the time of reperfusion inhibits neutrophil recruitment and activation. These results provide an impetus for PAF receptor antagonism as a means of reducing reperfusion injury in limb replantation surgery.

The effect of regular sunscreen use on vitamin D levels in an Australian population. Results of a randomized controlled trial.

BACKGROUND AND DESIGN: Studies published have suggested a possibility that regular use of sunscreen to prevent skin cancer may put the population, particularly elderly people, at risk of vitamin D deficiency. We aimed to determine whether regular use of sunscreens in the normal adult population, as recommended by public health authorities for the prevention of skin cancer, may put individuals at risk of vitamin D deficiency. A randomized double-blind control trial of the daily use of a broad-spectrum sunscreen (sun protection factor [SPF] 17) vs placebo cream over a summer period in Australia was conducted in 113 people aged 40 years and over, with sampling stratified by age. All participants had at least one solar keratosis. Serum samples taken at the beginning and at the end of the study were analyzed for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3. RESULTS: Mean levels of 25-hydroxyvitamin D3 rose significantly by the same amount in both groups over the period of the study (placebo, +12.8 mmol/L; sunscreen, +11.8 mmol/L). Mean levels of 1,25-dihydroxyvitamin D3 increased significantly in the placebo group only (placebo, +10.8 pmol/L; sunscreen, +1.3 pmol/L), but for no subject in either group was the level of 1,25-dihydroxyvitamin D3 outside the reference range either at the start or at the end of the study. There were no significant differences by age, sex, and skin type in the change in 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 over the study period. CONCLUSIONS: No person, including those aged 70 years and over, developed any vitamin D levels outside the normal reference range during the period of the study. The data suggest that over an Australian summer sufficient sunlight is received, probably through both the sunscreen itself and the lack of total skin cover at all times, to allow adequate vitamin D production in people who are recommended to use sunscreens regularly. More work is required to elucidate the relationship between 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3, particularly during the different seasons of the year.

Cool perfusion solutions for skin flaps: a new mixture of pharmacological agents which improves skin flap viability.

This study tested the hypotheses that perfusion of cooled skin flaps with established organ preservation solutions improves their viability and that this improvement can be further enhanced by pharmacological manipulation. Rabbit epigastric skin flaps were perfused with different solutions before explantation and stored at 8 degrees C for 6 days. In the first part of the experiment, flap viability was assessed 7 days after reperfusion of the flap via microvascular anastomoses. The different solutions were heparinised blood, University of Wisconsin solution, two of its modifications, EuroCollins solution and a pharmacological mixture containing phosphoenolpyruvate, desferrioxamine, nitrendipine, dextran 70 and a platelet-aggregating factor receptor antagonist (WEB 2170). In the second part, biochemical parameters of skin were measured at various reperfusion times. Adenosine triphosphate (ATP), reduced glutathione (GSH), myeloperoxidase (MPO) and tissue water were assayed at 0, 1, and 24 h after reperfusion. In addition, plasma thromboxane (TXB2) was measured at 0, 30 and 60 minutes after reperfusion. The viability of flaps perfused with the mixture (81%) was significantly higher than that of any of the other groups (39% for controls, 38% for EuroCollins, 13% for UW solution, 27% and 31% for its modifications). ATP levels after reperfusion were higher in the mixture group than in UW-perfused group. GSH levels in the mixture group were also higher than in the UW group, indicating higher level of protection against oxidative stress during reperfusion. There were no differences in MPO levels. Thromboxane levels associated with UW-perfused flaps were significantly higher than those associated with any other perfusion solution. In conclusion, perfusion of a mixture of pharmacological agents targeting specific aspects of ischaemia/reperfusion injury improved the viability of skin flaps stored in the cold for 6 days, whereas standard organ preservation solutions failed to affect significantly skin flap survival.

Autologous lymphocyte therapy for experimental canine lymphoedema: a pilot study.

Obstructive lymphoedema, an accumulation of protein-rich fluid in interstitial spaces, was created in five dogs by a combination of the irradiation of one groin and subsequent surgical ablation of any remaining lymphatics. The lymphoedema was stable for up to 2 years. The aim was to test the efficacy of intra-arterial injection of autologous lymphocytes as a therapy for lymphoedema. The hypothesis was that cytokines produced by lymphocytes mediate proteolysis by macrophage proteinases in the lymphoedematous limb to remove the excess protein and relieve the oedema. A concentrated lymphocyte-rich preparation was isolated from blood by the Ficoll-Paque method. These preparations were injected into the femoral artery four times at approximately 4 weekly intervals. Three months after the first injection of lymphocytes, lymphoedematous limbs showed a marked 69% reduction in the mean excess circumferences compared with opposite control limbs. After treatment, skin thickness and hydroxyproline content (both measures of fibrosis) as well as water content (a measure of oedema) had reduced significantly. In specimens of interstitial fluid and in skin homogenates acidic proteinase activity increased and the protein concentration decreased significantly compared with controls. It is concluded that increased proteolysis, possibly due to activated macrophages recruited to the lymphoedematous limb, may partly explain these results.

Drug mixture which improves survival of ischemic rabbit epigastric skin flaps.

The chief aim of this study was to maximize flap survival by counteracting the pathophysiological changes occurring during ischemia-reperfusion. Rabbit epigastric skin flaps given 21 hours of ischemia were infused intra-arterially with selected drugs at the start of reperfusion. Compared with control infused ischemic flaps, which had a 33% survival rate on day 7 post-ischemia, significant improvement was found with vasodilators nitrendipine (61%) and prostacyclin (65%) and the thrombolytic agent urokinase (65%); marginal improvement with the free radical scavenger desferrioxamine (53%); but no change with streptokinase (44%), heparin (21%), and ATP-MgCl2 (35%). A drug mixture comprising all of these agents except streptokinase and urokinase produced 87% survival, suggesting an additive effect. Biochemical assays on skin homogenates and blood implicated oxygen free radicals, neutrophil infiltration, and thromboxane in flap failure. These results imply that multiple factors are responsible for ischemic flap failure and that a mixture of drugs needs to be infused to counteract all of the detrimental changes.

Review of postoperative pharmacological infusions in ischemic skin flaps.

No single drug has yet been found which can overcome the inflammatory and microvascular changes which occur in a skin flap after ischemia-reperfusion. Nevertheless, the continued failure of approximately 7% of all free flap transfers clinically suggests that there may be a place for pharmacological intervention at the time of threatened flap failure. To date, plastic and reconstructive microsurgeons have been reluctant to use drugs because of the mass of conflicting evidence emanating from the plastic surgery literature. However, scientists and surgeons now have a clearer understanding of the problems arising in ischemia-reperfusion. Multi-acting drugs which can inhibit most of the important inflammatory changes would be the ideal. This review considers some of the historical developments in the pharmacological treatment of ischemic flaps in the past decade and looks to the future when pharmacological infusions may be part of the routine for salvaging failing skin flaps.