Cyclosporine and skin allografts for the treatment of thermal injury. II. Development of an experimental massive third-degree burn model demonstrating extensive graft survival.

We have previously reported the successful treatment and apparent development of skin allograft tolerance in a patient sustaining massive burns, utilizing skin allografts and cyclosporine. We now report the experimental correlate via successful achievement of a 75% body surface area (BSA) scald burn cyclosporine-skin allograft model in Lewis (LEW) rats. Cyclosporine (8 mg/kg/day) was given to the experimental animals daily for the first 20 days and then three times a week thereafter. Two experimental groups were studied: one received standard posttrauma care and the second critical posttrauma care. Controls (n = 22) and experimental groups 1 (n = 28) and 2 (n = 4) had average survival times of 13.8 +/- 12.8 days, 44.2 +/- 132.5 days, and 172.0 +/- 19.4 days, respectively. The allografts on the surviving experimental animals appeared normal and healthy and had nearly perfect hair growth. These results indicate that the model follows the clinical burn wound course, and treatment of massive burns with primary excision, skin allografts, and low doses of cyclosporine could provide immediate and complete functional repair of the burn wound.

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model.

The hypothesis tested in the present and accompanying study is that an effective treatment for severe burns involves early excision of necrotic tissue followed by skin allografting and cyclosporine (CsA) immunosuppressive therapy. LEW (RT1) rats served as recipients of thermal injury and/or skin allografts. BN x LEW F1 (LBN, RT1(l+n)) rats served as skin donors. LEW burn recipients received a hot water (90 degrees C for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated significant graft prolongation compared with controls (P less than 0.005). Skin graft survival was similarly prolonged in LEW recipients undergoing burn injury, primary wound excision, and CsA administration compared with burn-skin allograft controls (P less than 0.001). Mortality was not increased in the thermal injury-CsA-treated recipients compared with burn controls. A final experiment was initiated to investigate how low-level long-term (greater than 100 days) maintenance CsA treatment influenced skin allograft survival for possible future consideration in burn trauma. Recipients receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day thereafter) did not reject their grafts. However, a possible early sign of rejection (a single small ulcerative lesion) was noted in five of these long-term CsA-treated animals at a mean of 34 +/- 11 (SD) days. The lesion in these animals did not progress any further during CsA administration. Histopathologic study of selected animals removed from the CsA maintenance regimen for greater than 50 days following long-term administration revealed a number of interesting chronic lesions similar to those previously reported in the skin component of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts.

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.

Histochemical properties of muscle allografts enhanced via cyclosporine.

Until recently, the transplantation of skeletal muscle across a major histocompatibility barrier has proved difficult. However, with the advent of cyclosporine (CsA), it has become possible to achieve extended survival across such histocompatibility barriers. To date, very little is known about the histochemical, biochemical, immunological or contractile properties of long-term-surviving muscle allografts. Consequently, it was the focus of this study to histochemically examine muscle allografts prolonged with CsA and determine the cross-sectional area of fast glycolytic muscle fibers. Measurements of cross-sectional area were made because they are an important correlate to the amount of tension a muscle can generate. Animals were assigned randomly to one of three groups: control (normal) (n = 5), syngeneic (n = 4), and allogeneic (n = 4). Muscle allografts were performed by transplanting the gastrocnemius of an ACI rat (RT1a) hindlimb into the hindlimb of a Lewis rat (LEW;RT1(1]. The syngeneic model consisted of an ACI-to-ACI transplant. Animals in the allograft group were given CsA (8 mg/kg/day) until the time of sacrifice. At approximately 100 days following transplantation, both syngeneic and allogeneic muscles were removed from the recipient, and quickly frozen in isopentane cooled by liquid nitrogen. Muscle fibers were classified as slow-oxidative (SO), fast-oxidative-glycolytic (FOG), or fast-glycolytic (FG) based upon their staining for myofibrillar ATPase and NADH-dehydrogenase. From each muscle, the cross-sectional area of approximately 175 FG muscle fibers was determined. The fast-glycolytic muscle fibers of both the syngeneic and allogeneic grafts demonstrated a substantial decrease in cross-sectional area. The mean value (+/- SD) for the fibers of the allografted muscle was 1165 +/- 533 microns 2. The mean (+/- SD) fiber cross-sectional area for the fibers of the syngeneic muscle was 973 +/- 421 microns 2. These values contrast with a mean (+/- SD) value of 3552 +/- 601 microns 2 for fibers from age-matched control animals. The differences between the syngeneic and allogeneic muscles were not significant (P greater than 0.05). However, both exhibited significant (P less than 0.01) atrophy compared with the control muscle.

Composite tissue (limb) allografts in rats. I. Dose-dependent increase in survival with cyclosporine.

The dose-response effect of cyclosporine on rat limb transplant prolongation was investigated across the LBN-to-LEW histocompatibility barrier. This composite tissue allograft model has been shown to represent a strong transplantation barrier. Median limb allograft survival times increased in a dose-dependent manner with low cyclosporine doses, and then reached a plateau at higher levels. The cyclosporine dose that produced half-maximal survival based on a 20-day treatment was only 3.7 mg/kg/day. Histopathology revealed that the rejection process was distinctly different in limb allografts treated with cyclosporine compared with non-cyclosporine-treated controls. Rejection appeared to be delayed or partly arrested in certain areas of cyclosporine-treated limb allografts. These studies represent an initial step in laying the experimental foundation for clinical transplantation of composite tissue allografts using cyclosporine-induced immune suppression.

Composite tissue (limb) allografts in rats. II. Indefinite survival using low-dose cyclosporine.

Cyclosporine has reawakened interest in transplantation of peripheral composite tissue allografts (CTA) of skin, muscle, bone, vessel, and nerves. The purpose of this study was to examine whether cyclosporine could produce indefinite survival of CTA. Two groups of LEW recipients of LBN limb transplants were given different long-term treatments of cyclosporine. Tolerance was achieved in many of the animals. Several possibilities for the mechanism of this tolerance are discussed.

Composite tissue (limb) allografts in rats. III. Development of donor-host lymphoid chimeras in long-term survivors.

Eight LEW rat recipients possessing long-term-surviving (206-701 days) LBN vascularized hind limb allografts (CTAs) were tested for donor-host lymphoid chimerism. The recipients received various cyclosporine (CsA) treatment protocols in order to induce indefinite CTA acceptance. Histological examination of long-term-surviving CTAs demonstrated normal-appearing bone marrow in the donor limb. Lymphocytes isolated from host hemopoietic tissues (peripheral blood and/or spleen) by ficoll-hypaque density gradient centrifugation were tested against LEW-anti-BN antisera. Comparisons were made to standard curves employing various known concentrations of LBN and LEW cell combinations. The level of lymphocyte agglutination (dependent variable) showed a significant (P less than 0.025-0.005) linear relationship to the concentration of LBN donor cells (independent variable) present. Lymphocyte suspensions isolated from long-term CTA host peripheral blood and/or spleen showed a mean of 19.7% (+/- 9.7-95% confidence interval) donor LBN mononuclear cells present. Thus, it appeared that lymphoid cells originated from, and/or were released from LBN donor bone marrow into the circulation, resulting in chimeric repopulation of hemopoietic tissues. The presence of donor immunocytes in these limb allograft recipients may have been beneficial, and thus could have helped contribute to the long-term CTA survival observed.

Gluteal thigh flap in reconstruction of complex pelvic wounds.

Nine flaps for reconstructing pelvic wounds were centered on the posterior portion of the thigh over the inferior gluteal artery and posterior cutaneous nerve of the thighs of seven patients. No flap necrosis occurred and all wounds healed (one hematoma and one partial wound separation). Three patients had radiation wounds and three had immediate reconstruction following tumor resection. This flap represents an important advance in pelvic reconstruction and should be considered the flap of choice for this area. Contributions to the procedure from this series include (1) use in vaginal reconstruction, (2) length of flap extended to the popliteal fossa, and (3) use of an air-fluidized bed for postoperative management.

Free microvascular flaps for defects of limbs, head, and neck.

Twenty-three free microvascular flaps were transferred during the period September 1975 to December 1978. These included skin flaps, osteocutaneous flaps, and flaps of omentum and jejunum. These flaps were used for a variety of conditions ranging from life-threatening bleeding from a tumor to facial contouring in congenital deformities. In selected patients a microvascular free flap has significant advantages over multi-staged conventional flaps.

The myocutaneous flap: a versatile approach to major reconstructive problems.

Our experience with 50 myocutaneous flaps shows that the remarkable flexibility in design and execution of these flaps has opened up a new frontier in plastic and reconstructive surgery. The complication rate was 20 percent. Only one complication, partial flap necrosis, caused serious compromise of the reconstruction. Not only could the flaps be transposed in any direction locally, but they could be transferred to distant sites with microvascular anastomoses, and muscle function could be restored by incorporating nerve repairs into the reconstruction. Myocutaneous flaps permit us now to solve reconstructive problems that were heretofore unsolvable.

Genesis of a replantation program.

Development of a replantation program entails establishment of microsurgical skills in the laboratory, training in clinical microvascular technics, supervision, if possible, in the first microvascular free flaps, and close liaison with supporting personnel who transport patients, care for them acutely, and carry out their rehabilitation. Of twenty-six replantations or revascularizations (14 incomplete and 12 complete), twenty-three were successful. The three failures (1 complete and 2 incomplete procedures) were all digits, and each was part of a multiple digit injury.

Microvascular reconstruction of the mandible.

Five microvascular reconstructions of the mandible were performed in the past 2 years. One dorsalis pedis osteocutaneous flap was used to reconstruct the alveolar ridge and four groin osteocutaneous flaps were used for various defects of the mandible. Free microvascular bone grafts were found useful in previously irradiated fields, in anterior arch reconstruction, and in patients with massive soft tissue and bone loss.

Burn wound pathophysiology and care.

The pathophysiology of the burn injury and treatment factors that influence burn depth are explained. Details of burn wound care, including indications for early excision, topical antimicrobials, and the most recent advances in skin substitutes, are described. Burn wound sepsis, the most common and serious complication, is discussed in regard to diagnosis and treatment.

Reconstructing the burned face.

The priorities, timing, techniques, and philosophies of reconstruction of the burned face are outlined in this article. Each anatomic area is analyzed, and the appropriate procedure is described.

Acute and reconstructive management of the burned eyelid.

Introduced in 1875, the use of full-thickness skin graft for release of a lower eyelid ectropion secondary to burn contractures remains a mainstay of current burn treatment. The authors address issues such as acute care, the exposed cornea, scar management, and surgical management of eyelid deformities.

Vascular lesions.

Most cutaneous vascular abnormalities can be successfully, and in fact preferentially, treated with lasers. The field is evolving rapidly. It is important that the appropriate laser be used. In addition, many of the early references in this field are quite out of date. We are treating lesions at a much earlier age and our expectations are much higher. We expect the result to be virtually normal-looking skin with complete resolution of the problem. Capillary vascular malformations (port wine stain) are the most frequent indication for laser treatment in both children and adults. With the advent of the flashlamp pumped pulsed dye laser, these lesions can be treated in infancy and early childhood. This represents a significant breakthrough, because unlike with previous lasers, scarring is a rare side effect of treatment with yellow light lasers. The psychologic trauma of growing up with a facial deformity can be minimized with early treatment. It is hoped that the hypertrophy and permanent deformity associated with these lesions can be mitigated. The various lasers currently available to the plastic surgeon provide treatment options not previously available. Lasers are expected to play an increasing role in cutaneous abnormalities in the field of plastic surgery.

Carbon dioxide and ER:YAG laser resurfacing. Results.

Laser resurfacing is exciting "futuristic" surgery. The CO2 laser resurfaces using different parameters from the Er:YAG laser. When the surgeon understands these parameters, each laser can be used as a powerful tool for specific clinical applications. The Er:YAG laser was initially thought to be for the patient who has minimal skin laxity, but who desires skin resurfacing and needs a speedy return to social life. The CO2 laser has typically been thought to work best for skin laxity as well as rhytids, at the price of a longer recovery period. As the hardware and techniques continue to evolve, the differences between the clinical scope addressed by each laser diminishes. Both lasers deserve a place in the plastic surgeon's armamentarium. This new combination CO2/Er:YAG technique is intriguing and deserves further in-depth investigation. Laser resurfacing is not a cureall, but, when applied appropriately, it is an excellent tool that the plastic surgeon can use for skin rejuvenation.

Immunosurgery.

After being leaders in the field of transplantation, plastic surgeons became inactive in this field. Interest is reviving with the advent of the new immunosuppressant drug cyclosporine, as well as new knowledge of the immune mechanism. New generations of immunosuppressive drugs may allow allografting in patients with massive burns, limb transplants, and possibly even allografts of facial structures.

Lymphocyte chimerism in a full allogeneic composite tissue (rat-limb) allograft model prolonged with cyclosporine.

LEW recipients of ACI vascularized hind limb allografts were analyzed for lymphoid chimerism by a complement-dependent cytotoxicity assay using antisera produced across this strain combination. In assessing the technique, two LEW recipients of sublethal irradiation (400 rad), ACI bone-marrow allografts, and CsA exhibited mixed lymphoid chimerism 23 days posttransplant. Short-term CsA-treated CTA recipients that were assayed at various times following transplantation and underwent subacute rejection did not demonstrate any significant mixed lymphoid chimerism. Long-term CsA-treated CTA recipients that were assayed at various times prior to 100 days posttransplant also did not demonstrate any significant mixed lymphoid chimerism. However, following extensive CTA survival (greater than 100 days) significant mixed donor-host lymphocyte chimerism became evident in the peripheral blood, and in one recipient a large quantity of donor bone marrow remained viable in the ACI limb allograft at necropsy (greater than 200 days posttransplant). The development of donor-host lymphocyte chimerism and a wasting syndrome that followed long-term CTA survival was suggestive of GVHD.

Vascularized bone marrow transplantation (VBMT): induction of stable mixed T-cell chimerism and transplantation tolerance in unmodified recipients.

In this preliminary report, our model of VBMT across a semiallogeneic barrier consistently brings about antigen-specific host tolerance with absence of GVHD in the majority of recipients. No immunologic or radiologic intervention was utilized. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention.