Establishing the Feasibility of Face Transplantation in Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA; formerly Wegener's granulomatosis) is a rare vasculitis that commonly starts in the craniofacial region. We report a case that was masked by prior facial trauma and associated with pyoderma gangrenosum (PG). Disease progression and aggressive debridements led to severe facial tissue loss. The decision to perform a face transplant was controversial because of the risk of disease relapse on the facial allograft. We reviewed renal transplant outcomes in GPA for possible relevance. A PubMed search retrieved 29 studies. Patient and graft survival, relapse, morbidity, mortality, rejection and immunosuppression were assessed. Ten-year patient survival and graft survival were 84.4% and 72.6%, respectively. GPA relapse occurred in 31.5%, and upper airway/ocular relapse occurred in 17.8% (resolved in 76.9%). Mortality was 12.3%. Acute and chronic rejection rates were 14.9% and 6.8%, respectively. Traditional posttransplant immunosuppression was effective. Our review suggests that GPA renal transplant outcomes are comparable to general renal transplant cohorts. Furthermore, transplanted GPA patients exhibit lower disease relapse secondary to lifelong immunosuppression. This supported our decision to perform a face transplant in this patient, which has been successful up to the present time (1-year posttransplantation). Untreated GPA and PG are potential causes of worse surgical outcomes in the craniofacial region.

Composite tissue allotransplantation: a proposed classification system based on relative complexity.

Numerous achievements have been made encompassing a wide array of composite tissue allograft (CTA) subtypes. We sought to develop a simple, reproducible CTA classification system for the purpose of comparing clinical investigation. Each CTA subtype differs in relative complexity and can therefore be theoretically classified based on its unique combination of multiple factors. Eight complexity factors (CFs) are hypothesized: anatomic detail, psychological obstacles, rejection risk, required rehabilitation, relative antigenicity, functionality/cosmesis, skin ratio, and salvageability. A distribution of total complexity scores, ranging from 8 to 24, is classified into 3 ordered categories representing varying degrees of complexity. In conclusion, we have created a new classification system so that ongoing research and future data may be compared in a type-specific fashion.

Computer-guided microsurgery: surgical evaluation of a telerobotic arm.

A new telerobotic arm was evaluated in different microsurgical procedures. The system consisted of a robotic arm bearing a microsurgical forceps controlled by a joystick-like master unit operated by the surgeon's hand. The robotic arm functioned as an operating as well as assisting instrument. Advantages of the system were its precise functioning, which was especially important when tissue or instruments had to be held for a longer period of time, as well as its ability to replace an assisting person to some extent. Deficiencies of the system were its 10-min startup, the spatial conflict with in the operative field, and the poor rotation of the robotic tip. In some procedures, the telerobotic arm could replace the standard microsurgical instrument guided by the surgeon's second hand; in other maneuvers it could function as the surgeon's third hand with precision and void of tremor.

Complications of postmastectomy breast reconstructions in smokers, ex-smokers, and nonsmokers.

Smoking results in impaired wound healing and poor surgical results. In this retrospective study, we compared outcomes in 155 smokers, 76 ex-smokers, and 517 nonsmokers who received postmastectomy breast reconstructions during a 10-year period. Ex-smokers were defined as those who had quit smoking at least 3 weeks before surgery. Transverse rectus abdominis musculocutaneous (TRAM) flap surgery was performed significantly less often in smokers (24.5 percent) than in ex-smokers (30.3 percent) or nonsmokers (39.1 percent) (p < 0.001). Tissue expansion followed by implant was performed in 112 smokers (72.3 percent), 50 (65.8 percent) ex-smokers, and 304 nonsmokers (58.8 percent) (p = 0.002). The overall complication rate in smokers was 39.4 percent, compared with 25 percent in ex-smokers and 25.9 percent in nonsmokers, which is statistically significant (p = 0.002). Mastectomy flap necrosis developed in 12 smokers (7.7 percent), 2 ex-smokers (2.6 percent), and 8 nonsmokers (1.5 percent) (p < 0.001). Among patients receiving TR4AM flaps, fat necrosis developed in 10 smokers (26.3 percent), 2 ex-smokers (8.7 percent), and 17 nonsmokers (8.4 percent). Abdominal wall necrosis was more common in smokers (7.9 percent) than in ex-smokers (4.3 percent) or nonsmokers (1.0 percent). In this large series, tissue expansion was performed more often in smokers than was autogenous reconstruction. Complications were significantly more frequent in smokers. Mastectomy flap necrosis was significantly more frequent in smokers, regardless of the type of reconstruction. Breast reconstruction should be done with caution in smokers. Ex-smokers had complication rates similar to those of nonsmokers. Smokers undergoing reconstruction should be strongly urged to stop smoking at least 3 weeks before their surgery.

Relationship of obesity and specimen weight to complications in reduction mammaplasty.

Obesity and specimen weight have both been associated with a higher incidence of complications for patients undergoing reduction mammaplasty. However, obesity has been arbitrarily and inconsistently defined, and the degree of obesity has not been considered in these previous studies. Because insurance companies are increasingly demanding weight loss before authorizing surgery, the relationship of obesity and breast size to complications is of great importance. Upon critical review of the literature, a number of fundamental questions remain unanswered. If complications are more frequent in the obese patient, are these complications directly proportional to the degree of obesity? Also, if the patient is required to lose weight before surgery, is weight loss effective in reducing complication rates? In an attempt to clarify these issues, 395 patients who underwent reduction mammaplasty over a 10-year period were reviewed retrospectively. Patients were arbitrarily divided into five groups in which, depending on their degree of relative obesity, they were classified as less than 5 percent, 5 to 10 percent, 10 to 15 percent, 15 to 20 percent, or greater than 20 percent above average body weight. To evaluate the relationship of specimen weight to complications, patients were also arbitrarily divided into five groups according to weight of the breast reduction specimen, which was classified as less than 300 g, 300 to 600 g, 600 to 900 g, 900 to 1200 g, and greater than 1200 g reduction per breast. Complications were then divided into local and systemic and major and minor. When bilateral reductions alone were analyzed (n = 267), there was a statistically significant increase in complication rate in the obese (p = 0.01). However, when the obese population was further subdivided according to their degree of obesity (less than 5 percent, 5 to 10 percent, 10 to 15 percent, 15 to 20 percent, and greater than 20 percent above average body weight), no further correlation was found. However, the relationship between specimen weight per breast and complications was much stronger with a direct correlation existing between increasing specimen weight and the incidence of complications. Although this study has shown that patients who are average body weight have fewer complications than obese patients after breast reduction surgery, it has not shown an increasing incidence of complication with increasing degrees of obesity. The implications of these findings and their relationship for denying patients surgery on the basis of weight alone are discussed in detail.

[Improving microcirculation of muscle flaps by tissue plasminogen activator in the rat cremaster muscle flap model]. / Verbesserung der Mikrozirkulation von Muskellappen durch Gewebsplasminogenaktivator im Ratten-Cremaster-Muskellappenmodell.

Currently, tissue-plasminogen activator (t-PA) is the most potent and nevertheless safe fibrinolytic in clinical use. Its indications are fibrinolysis in acute myocardial infarction, ischemic stroke, deep vein thrombosis, pulmonary thromboembolism, as well as different kinds of peripheral arterial embolism. However, controlled studies on the effect of t-PA in microsurgery and free tissue transplantation are lacking. This study was designed to evaluate the effect of tissue-plasminogen activator on skeletal muscle flap perfusion after a thrombogenic stimulus. 24 male Sprague-Dawley rats were divided into four experimental groups of six animals each. In group 1, the cremaster was isolated as an end organ flap, in group 2 after cremaster isolation a semicircular inverted suture as a thrombogenic insult was performed at the ipsilateral common iliac artery. In group 3, local t-PA infusion followed the inverted suture and in group 4, vehicle was infused. After 24 hours, we measured cremaster muscle flap hemodynamics using intravital microscopy. Capillary perfusion significantly decreased after the inverted suture from 6.23 (group 1) to 1.50 (group 2) functional capillaries per visual field (medians). t-PA significantly increased capillary perfusion after the thrombogenic insult from 1.50 (group 2) and 2.50 (group 4) to 6.00 (group 3) (medians). Restoring capillary perfusion after a thrombogenic insult t-PA may increase flap survival rates.

Microcirculatory window for early detection of allograft rejection.

The purpose of this study is to introduce a technical detail on a transplantation model for in vivo evaluation of microcirculatory changes during the acute phase of allograft rejection. The cremaster muscle is incorporated and transplanted along with the hind limb to detect and study ischemia/reperfusion injury and the acute phase of allograft rejection in rats. Thirty-six animals were studied in three experimental groups of 12 animals each. Each group was divided into subgroups and microcirculatory measurements were taken at two different time periods: 24 and 72 hours. In the ischemic control group (N = 12), cremaster muscles were denervated, prepared as a tube flap, and submitted to the same interval of ischemia as the other groups but without transplantation. In the isograft group (N = 12), rat hind limb-cremaster grafts were transplanted between genetically identical Lewis rats (RT11). In the allograft group (N = 12), 12 transplantations were performed across a major histocompatibility barrier between Lewis Brown-Norway (RT-11+/-n) and Lewis (RT 11) rats. The diameters of first-, second-, and third-order arterioles and venules; red blood cell velocities; and functional capillary density were recorded at 24 and 72 hours after transplantation. Daily follow-up observations were continued until 3 days after the first clinical signs of graft rejection. The mean number of perfused capillaries in the two transplantation groups was significantly lower than in the control group at both 24 hours and 72 hours (p < 0.05). Those results were as follows: 8.2 +/- 2.1 at 24 hours, 7.7 +/- 0.85 at 72 hours in the ischemic control group; 5.4 +/- 0.9 at 24 hours, 6 +/- 0.6 at 72 hours in the isograft group; and 5 +/- 0.9 at 24 hours, 5.5 +/- 0.3 at 72 hours in the allograft group. Red blood cell velocities and vessel diameters in the main arteries were also decreased in transplant groups at 24 hours (p < 0.05) but returned to normal 72 hours after the operation (p > 0.05). The composite rat hind limb-cremaster model presented in this study introduces a reproducible in vivo approach to monitor the differences in microcirculatory hemodynamics of ischemia/reperfusion injury and acute graft rejection. The model allows the study of the timing, sequence, and correlation between clinical and hemodynamic signs during the acute phase of allograft rejection.

Cranial defect repair using e-PTFE: part I. Evaluation of bone stiffness.

Autologous bone grafts are the preferred material for craniofacial reconstruction, but such procedures lead to increased operative time and bleeding, donor site morbidity, and graft resorption. The efficacy of expanded-polytetrafluoroethylene (e-PTFE) sheets to increase bone regeneration and remodeling in cranial defects using a rabbit model was evaluated by mechanical testing. New Zealand white rabbits were divided into 3 groups and sacrificed 6 months after surgery. In the Split Table group, (n = 16), a bilateral bone defect was created on the outer table of the parietal bones. In the Full Table group, (n = 16), a bilateral defect was created through both the inner and outer table of the cranium. The control group, (n = 10) was subjected to a sham operation. Indentation testing was performed to determine the stiffness of newly formed bone in and around the defect. Near the center of the defect, Split Table defects repaired with e-PTFE resulted in significantly stiffer bone than regenerated control bone. The Full Table defects repaired with e-PTFE also resulted in bone significantly stiffer than control regenerated bone around the central region of the defect. The data supports the hypothesis that e-PTFE improves the repair of cranial defects in a rabbit model. It is surmised that the porosity of the e-PTFE provides a stable scaffold for migration of tissue regenerating cells, which may be preferentially localized near the cranial suture lines. This porosity may also provide a barrier to fibrous tissue regenerating cells.

[A model for allogenic extremity transplantation in the rat with possible microcirculatory monitoring]. / Modell zur allogenen Extremitätentransplantation der Ratte mit Möglichkeit des mikrozirkulatorischen Monitoring.

A rat model for monitoring allograft rejection of transplanted rat hindlimbs at the microcirculatory level is described. The well-established rat cremaster flap model is combined with a rat hindlimb transplantation procedure at a level proximal to the neurovascular pedicle of the cremaster muscle. The cremaster serves as a microcirculatory monitor for in vivo evaluation of graft rejection by measuring skeletal muscle perfusion. Donor animals are male Lewis Brown Norway rats and recipients are Lewis rats. The right cremaster muscle of the donor animal is dissected as a tubular island flap and preserved in a subcutaneous tunnel in the hindlimb. Afterwards, the right hindlimb including the cremaster is amputated at the mid level of the common iliac vessels and transplanted to the recipient at the level of the external iliac vessels. Over a time period of five days, the cremaster muscle of the composite transplants showed appropriate tissue quality for intravital microscopical observations. This transplantation model allows evaluation of allograft rejection in vivo at the microcirculatory level.

Effect of anti-ICAM-1 antibodies on macromolecular leakage and leukocyte activation: a study of hindlimb allografts in the rat.

We investigated the ability of anti-ICAM-1 monoclonal antibodies to reduce endothelial cell damage by assessing microvascular permeability and microcirculatory function during the acute phase of allograft rejection. The composite rat hindlimb-cremaster muscle transplantation model was employed in three experimental groups of 18 animals each. Isograft control transplantations were performed between genetically identical Lewis (LEW, RT11) rats. Allograft transplantations were performed across a major histocompatibility barrier between Lewis-Brown-Norway (LBN, RT-11+n), and Lewis (LEW, RT11) rats. In addition, a third group of animals receiving allografts was treated with 1 mg/kg/day of anti-ICAM-1 monoclonal antibody. After 24 hours, 72 hours, and 7 days, we measured microvascular permeability, leukocyte activation, functional capillary perfusion, red blood cell velocity, vessel diameters, and endothelial edema index in six animals per each follow-up period. Endothelial cell damage was assessed by measuring graft permeability to fluorescein isothiocyanate-labeled albumin (0.2 ml/100 g body weight) with computer-aided image analysis. Mean microvascular permeability was lower in the treated allograft group than in untreated controls at all follow-up times (p<0.001). In addition, anti-ICAM-1 treatment significantly reduced the activation of sticking leukocytes at 24 and 72 hours (p<0.001) and the activation of transmigrating leukocytes at 72 hours and 7 days (p<0.05). The allografts presented a characteristic microcirculatory pattern of acute rejection as early as 24 hours after transplantation. The dysfunction of the endothelial cell barrier at all time points was indicated by significant increases in the degree of allograft macromolecular permeability and in the number of activated sticking and transmigrating leukocytes. Treatment with anti-ICAM-1 antibodies significantly reduced the surge of leukocytes in the allograft transplants and protected the endothelial barrier from the acute effects of transplantation trauma.

Arterial crush injury causes decrease in tissue perfusion at the level of the microcirculation in skeletal muscle flap.

This study was designed to evaluate the effects of crush injuries to the feeding arteries of a muscle flap on microcirculatory haemodynamics. Eighteen male Sprague-Dawley rats were divided into three experimental groups for intravital microscopy of the cremaster muscle flap. Group 1 served as control. In group 2 the common iliac artery and in group 3 additionally the lower abdominal aorta was crushed with a Kocher clamp (17.4 N) over 5 min. Microcirculatory parameters (red blood cell velocity, vessel diameter, and capillary perfusion) were monitored before and 2 h after crush. In the one-level crush group, red blood cell velocities significantly decreased by 39.17% (P=0.046) in first order arterioles and by 32. 91% (P=0.0106) in second order arterioles. In capillary perfusion, a drop of 48.02% (P=0.0039) was noted. In the two-level crush group, red blood cell velocities significantly dropped over 32.06% (P=0. 0250) in first order arterioles, 35.91% (P=0.0065) in second order arterioles, and 45.69% (P=0.0782) in first order venules. Capillary perfusion was reduced by 20.16% (P=0.374). Arterial crush injuries as possible thrombogenic insults may result in a significant decrease in skeletal muscle perfusion although the blood supply through the crushed supplying vessel is maintained.

Failure in developing a model for complete vascular thrombosis in the common iliac artery in the rat.

The purpose of this study was to develop a model for complete arterial thrombosis proximal to the rat cremaster flap for subsequent fibrinolytic studies at the microcirculatory level. We divided 20 male Sprague-Dawley rats into four experimental groups of five animals each. We assigned each group to an established thrombosis model using crush and standard microsurgical anastomosis, crush and intimal abrasion, inverted arterial suture, and intravascular silk sutures combined with microsurgical anastomosis at the common iliac artery. Vessel patency was examined using the milking test 30, 60, 90, and 120 min after the thrombogenic insults. The model of perpendicular silk sutures and anastomosis caused complete arterial thrombosis in one animal over 120 min. The other models failed in all animals. In conclusion, the thrombogenic models used in this study are not capable of creating a reliable complete arterial thrombosis in the common iliac artery of the rat.

[Applicability of the robot arm for microsurgical operations]. / Verwendbarkeit eines Roboterarmes für mikrochirurgische Operationen.

In this project, we evaluated a new robotic arm, RAMS (Robot Assisted Microsurgery) Workstation, for microsurgical procedures. We assigned seven microsurgical tasks to the robotic arm to investigate its capabilities and limitations during microsurgery. The robotic arm was able to function as the primary operating tool in removal of foreign bodies and thrombi as well as in intravascular positioning and holding of needles and catheters. The robot worked with great precision and without vibration. It served as an assisting tool in vessel dissection, ligation of side branches, and microsurgical anastomosis. The main drawbacks include a long warm-up period, the large size, poor rotation of the tip of the robotic arm, and frequent unintended shut-downs. The RAMS Workstation is a precise tool and can assist the surgeon as a "second" or "third hand". It cannot entirely replace the microsurgical instruments held by the surgeon.

Tissue-plasminogen activator restores muscle flap perfusion in the rat.

This study was designed to evaluate the effect of tissue-plasminogen activator on skeletal muscle flap perfusion after a thrombogenic insult. Twenty-four male Sprague-Dawley rats were divided into 4 experimental groups of 6 animals each. In group 1 (sham), the cremaster muscle was isolated as an end-organ flap. In group 2, after cremaster muscle isolation, a semicircular inverted suture as a thrombogenic insult was performed at the ipsilateral common iliac artery. In group 3, local tissue-plasminogen activator infusion followed the inverted suture. In group 4, vehicle was infused. After 24 hours, the cremaster muscle flap hemodynamics and leukocyte-endothelial interactions were measured using intravital microscopy. Capillary perfusion significantly decreased after the inverted suture from a median of 6.23 (group 1) to 1.50 (group 2) functional capillaries per visual field. Tissue-plasminogen activator significantly increased capillary perfusion after the thrombogenic insult from a median of 1.50 (group 2) and 2.50 (group 4) to 6.00 (group 3). Tissue-plasminogen activator restored capillary perfusion after a thrombogenic insult to the main feeding artery.

Effects of 8-Gy radiation on the microcirculation of muscle flaps in the rat.

Combination of radical excision and radiation has been used as a treatment modality for cancer patients. As a result, in reconstructive surgery there is often a need to harvest flaps in the vicinity of previously irradiated tissues. Radiation has been shown to cause progressive injury to the macrocirculation and microcirculation, often jeopardizing flap survival. The purpose of this study was to examine whether radiation significantly affects the sequence of leukocyte-endothelial interactions or the hemodynamics of the muscle flap in both acute and chronic situations. Male Sprague-Dawley rats (n = 42) were divided into seven groups of six rats each. Rats in group I were not irradiated. Groups II through VII received 8-Gy radiation to the right groin and scrotum. Groups II, III, and IV were examined at 4, 24 and 72 hours, respectively, and groups V, VI, and VII were examined at 1, 2 and 12 weeks. For intravital microscopy, the cremaster muscle was dissected on its neurovascular pedicle. Vessel diameters and red blood cell velocities were measured in the central cremasteric branches and branch arterioles. Capillary perfusion was evaluated in 27 visual fields of each flap. Leukocyte-endothelial interactions were evaluated by numbers of rolling, adhering, and transmigrating leukocytes in post-capillary venules. In the same postcapillary venule, we measured the endothelial edema index (constriction index). The hemodynamics of irradiated flaps did not differ significantly from those of controls. Diameter and red blood cell velocity were increased in the first- and second-order arterioles and were highest at 72 hours and 1 week. After irradiation, third-order arterioles were constricted. Radiation reduced capillary perfusion by 4.3, percent. None of the differences were statistically significant. Neither leukocyte behavior nor the constriction indices differed among the groups. In conclusion, low-dose radiation of 8 Gy does not affect hemodynamics or leukocyte-endothelial interactions of muscle flaps in the rat. Muscle tissue with intact microvasculature can be harvested for reconstructive procedures after low-dose radiation.

Interferon-gamma improves muscle flap microcirculation in double-strand RNA-induced inflammation.

Endothelial cell (EC) activation and subsequent expression of leukocyte adhesion molecules are initial events in multiple pathological processes. Viral double-strand ribonucleic acid (dsRNA) induces EC adhesion protein expression and leukocyte adhesion in vitro. Interferon-gamma (IFN-gamma) has been demonstrated to modulate the expression of certain adhesion proteins. The purpose of this study was to measure the inflammatory response to a viral mimetic--a synthetic dsRNA, polyinosinic-polycytidylic acid (poly-I:C)-on the microcirculation of a muscle flap in a rat model and to determine whether IFN-gamma attenuated the response. Two-stage surgery to create a cremaster muscle end-organ tube flap was performed on 18 male Sprague-Dawley rats in three groups. After intra-arterial injection into the abdominal aorta, the reagents (phosphate-buffered saline-bovine serum albumin [PBS-BSA] in groups I and II, and IFN-gamma in group III) were kept for 1 hour in this end-organ system. During the second stage at 16 hours, after injection into the penile vein (PBS-BSA in group I, poly-I:C in groups II and III), the flap was prepared for intravital microscopic measurement. The following parameters were measured: red blood cell velocity; vessel diameter; number of functional capillaries; and number of rolling, sticking, and transmigrating neutrophils and lymphocytes. Wilcoxon's rank sum test was used for statistical comparison. Poly-I:C caused a 70% increase in the main artery diameter and a 7% increase in velocity. But as a consequence of dynamic activation of leukocyte interaction, a 30% drop in functional capillary perfusion was observed. Injury to the entire vascular endothelium was confirmed by a 160% increase in transmigrating leukocytes. Treatment with IFN-gamma inhibited the poly-I:C-induced inflammation, as shown by 88%, 63%, and 85% decreases in rolling, sticking, and transmigrating leukocytes respectively, and by a 28% increase in capillary perfusion. Treating the system with IFN-gamma in advance, inhibited poly-I:C-induced inflammation, shown by marked decreases in rolling, adhering, and transmigrating leukocytes, and a notable increase in perfused capillaries. These observations reflect an inhibitory effect of IFN-gamma on leukocyte adhesion molecule expression in vascular endothelium in response to dsRNA in a muscle flap at the microcirculatory level.

Muscle flaps' triphasic microcirculatory response to sympathectomy and denervation.

Whether sympathectomy and somatic denervation in muscle flaps increased microcirculatory flow in the short or long term, thus producing an effect similar to the delay phenomenon, which increases survival in transferred skin flaps, was determined. The rat cremaster muscle flap model was used for in vivo microscopy. In the left cremasters of 30 Sprague-Dawley rats, the genitofemoral nerve was divided and the proximal vessels were stripped of their adventitia. The muscle was not elevated. In each rat, the contralateral cremaster served as the control. The rats were assigned to one of five groups: no delay before observation, a 24-hour delay, a 48-hour delay, a 7-day delay, or a 14-day delay. After the delay, red blood cell velocity, vessel diameters, number of functional capillaries, and leukocyte-endothelial interactions were measured. Microvessel response to topical vasoactive substances was measured. Immediately after denervation, red blood cell velocity increased transiently (71 percent; p = 0.006). Main arterioles dilated (20 percent; p = 0.02) at 24 hours, and capillary perfusion increased 36 percent (p = 0.001) at 2 weeks. The microvessels had hyperactive responses to all vasoactive agents 2 weeks after denervation. These findings indicate that proximal sympathectomy with somatic denervation leads to a triphasic, dynamic response in the peripheral microcirculation of the cremaster muscle flap. An initial acute hyperadrenergic phase was followed by a nonadrenergic phase, with significant vasodilatation, and a sensitized phase, with increased capillary perfusion and hyperresponsiveness to vasoactive substances. This study shows that with minimal access to the cremaster muscle flap neurovascular pedicle and without changing the blood supply to the flap, significant hemodynamic improvements can be made in the peripheral microcirculation.

In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats.

Experimental and clinical studies of vascular allogenic extremity transplantation have yielded disappointing results and have not been clinically useful. With recent advances in transplantation immunology, considerable interest has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characterize the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcirculatory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograft rejection. A total of 24 transplantations were performed among the four experimental groups. In a control group, 12 rat hindlimb-cremaster grafts were transplanted between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transplantations were performed across a major histocompatibility barrier between Lewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The following parameters were evaluated to discover the leukocyte-endothelial interaction: endothelial edema index and the number of rolling, adherent, and transmigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formation on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a significant increase in the number of adherent leukocytes in allograft transplants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percent; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05). The activation of leukocyte transmigration increased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.72 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothelial edema index, a measure of endothelial swelling and cellular deposit accumulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair loss; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduces a new in vivo approach to monitor acute graft rejection using the intravital microscopy system. This is a valuable model for defining the timing, sequence, and correlation between immunologic events and clinical signs during the acute phase of allograft rejection.

Determination of hindlimb transplantation-induced vascular albumin leakage and leukocyte activation during the acute phase of rejection.

Following transplantation, the microvascular endothelium and endothelial cells play a critical role in allograft rejection, as well as response to surgical trauma. In this study, endothelial-cell damage was assessed through microvascular permeability, and the role of surgical trauma was evaluated during the acute phase of limb allograft rejection. Eighteen isograft and 18 allograft composite-tissue transplantations were performed between 72 rats. At 24-hr, 72-hr, and 7-days follow-up, microvascular permeability, leukocyte activation, functional capillary perfusion, red-blood-cell velocity, vessel diameter, and an endothelial edema index were measured. The permeability index (PI) was statistically significantly greater in the allografts at all follow-up points, compared with the isograft controls (p <0.001). The number of rolling leukocytes was significantly greater in the allografts at 24 and 72 hr; the number of sticking and transmigrating leukocytes was greater at all three follow-up points; and the number of rolling lymphocytes was greater at 7 days (p <0.05). These findings demonstrate the increased rejection phenomenon in allografts, and the increased susceptibility to ischemia and reperfusion injury, compared with isograft transplants. Increased leukocyte activation and acute destruction of endothelial-cell barrier function were demonstrated during the acute rejection period following composite limb allotransplantation.