Laryngeal Transplantation Research Articles

The larynx as an immunological organ: immunological architecture in the pig as a large animal model.

The larynx is a mucosal organ positioned at the divergence of the respiratory and digestive tracts. It is exposed to a wide variety of environmental components, including foreign antigens, tobacco smoke, laryngopharyngeal reflux and pollutants. The mucosal immune system generates either active immune responses or tolerance, depending on the nature of the antigen and we hypothesize that the larynx is important organ for immunological decision-making in the airway. Because the pig is an ideal large animal model in which to explore laryngological research questions, such as those relating to laryngeal transplantation, we investigated the normal mucosal immunology of the porcine larynx. Pig larynges and tracheae were processed and prepared for bright-field microscopy and quantitative, multiple-colour immunofluorescence histology using pig-specific monoclonal antibodies. There was an abundance of immunologically active cells within the mucosa of the larynx and trachea of both the newborn and adult animal. Specifically, major histocompatibility complex class II (MHC class II+) cells, CD4+ and CD8+ cells were identified, although regional differences in numbers were apparent: specifically, the supraglottis contained fewer immunologically relevant cells than other sites sampled. There was a significant correlation between the numbers of MHC class II+ and CD4+ cells indicating co-ordinate regulation and therefore functional local interactions. The presence of such an immunological structure suggests that the larynx may have important functions in respiratory immunology and that it may trigger strong alloresponses after laryngeal transplantation.

Laryngeal nerve function after total laryngeal transplantation

The first successful composite human laryngeal transplantation was performed by a team led by the senior author on January 4, 1998. The recipient was a 40-year-old male who had sustained a crush injury to his larynx 20 years prior, rendering him aphonic. Multiple previous attempts for reconstruction at an outside hospital were unsuccessful. The donor was a 40-year-old male who had died from a ruptured cerebral aneurysm. The specifics of the procedure have been detailed elsewhere. 1 Throughout the patient’s postoperative course, serial fiberoptic evaluations and voice testing were performed to evaluate laryngeal reinnervation reflected in phonatory function. We herein report the results of these exams, as well as the results of electromyographic recordings of the laryngeal musculature 4 years posttransplantation.

Intrinsic muscles and distribution of the recurrent laryngeal nerve in the pig larynx.

To use the pig larynx in studies of laryngeal reinnervation, it is essential to have a clear understanding of its anatomy. We aimed to define the macroscopic anatomy of the intrinsic muscles and the course of the recurrent laryngeal nerve (RLN) in the pig larynx. Twelve large white pig larynges were used. Five larynges were preserved in formalin, then dissected to study the anatomy of the intrinsic muscles. Seven larynges were stained using the modified Sihler's staining technique, which results in nerves being stained dark purple while the remainder of the larynx is rendered translucent. The intrinsic muscles of the pig larynx were similar to those in the human. The RLN gives off a branch that enters the posterior cricoarytenoid muscle (PCA) on its deep surface and supplies the entire muscle, although the branching pattern of the nerve within the muscle varies considerably. These results facilitate detailed reinnervation studies in the pig laryngeal transplant model.

Total lymphoid irradiation as an immunomodulator in laryngeal transplantation

Problem: Transplant research continues to seek immunosuppressive regimens that minimize recipient morbidity and do not promote tumor growth. Several animal studies have demonstrated long-term donor-specific allograft tolerance after posttransplant total lymphoid irradiation (TLI) when used in conjunction with anti-lymphocyte globulin (ALG) and donor-specific transfusion (DST). The goals of the current study were as follows: (1) to assess the ability of posttransplant TLI to induce allograft tolerance in an established murine laryngeal transplant model, (2) to compare postoperative TLI regimens with varying doses of radiation and anti-lymphocyte globulin, and (3) to evaluate the ability of sirolimus to augment tolerance when used in combination with a posttransplant TLI regimen. Methods: Using 8 treatment arms containing 10 animals each, recipient rats received 1 of 4 postoperative regimens: (1) 1,000 rads of TLI in 5 fractions, ALG and DST, (2) 2,400 rads of TLI in 10 fractions, ALG and DST, (3) 2,400 rads of TLI in 10 fractions, ALG, DST, and intraperitoneal sirolimus, and (4) ALG only. Recipient groups were assessed at 15 or 30 days after the completion of the assigned treatment regimen. Laryngeal allografts were graded for histologic rejection level using an established scale. Results: Both 1,000 and 2,400 rads of TLI were well tolerated by recipient animals. Laryngeal allograft preservation was significantly better using 2,400 rads of TLI when compared to 1,000-rad regimens. The addition of sirolimus did not augment graft preservation. All 2,400-rad TLI regimens were superior to ALG alone, with only mild to moderate rejection observed on average at 30 days after treatment. No treatment regimen yielded consistent allograft preservation on histologic analysis. Conclusion: Posttransplant total lymphoid irradiation regimens significantly reduce the severity of allograft rejection at 15 and 30 days. However, consistent histologic evidence of allograft tolerance was not observed. Significance: At this point, TLI has limited applicability to human laryngeal transplantation. Support: This study was funded by the Cleveland Clinic Department of Otolaryngology and Communicative Disorders and a matching Cleveland Clinic Research Programs Council grant.

Short-term therapy with TCR and FK-506 leads to long-term laryngeal transplant function

Problem: A mouse anti-rat T-cell receptor monoclonal antibody (TCR) has great potential in the development of long-term immunologic tolerance. Combination therapy with FK-506 and TCR may enable the establishment of long-term immunotolerance while reducing dose-related toxicities. The use of combined TCR and FK-506 in a completely allogeneic model permits a rigorous evaluation of potential synergism. Methods: Nineteen Brown Norway larynges were transplanted to Lewis recipients. Treatment consisted of 7 days of FK-506 (1.2 mg/kg/day) alone and in combination with TCR (0.5 mL/day). Control groups consisted of untreated animals, as well as 11 Lewis-Brown Norway larynges transplanted to Lewis recipients and treated with FK-506 monotherapy. Each group consisted of 6–11 rats. Median duration of engraftment was 44 days (range, 14–106 days). Graft histopathology was graded according to an established 31-point scale in a blinded fashion. Long-term grafts (>75 days) were followed with serum PTH levels. Results: Untreated controls experienced almost complete rejection (mean score, 27, SD 0). Allogeneic transplants treated with FK-506 monotherapy experienced near-complete rejection (mean score, 25.2, SD 2.1). Allogeneic transplants treated with combination therapy and followed for a median duration of 100 days demonstrated significantly better rejection scores than controls (mean score, 11.1, SD 1.7; P = 0.003). Combination therapy was also significantly more effective in preventing acute rejection than monotherapy with FK-506 in a semi-allogeneic model (mean score, 22.1, SD 4.6; P < 0.04). Conclusion: Short-term combination therapy with FK-506 and TCR prevents rejection in an allogeneic model for up to 100 days. This regimen was associated with significantly better histopathologic rejection scores than monotherapy with either agent, or monotherapy with FK-506 in a semi-allogeneic model. Significance: The blockade of primary antigen recognition with TCR may prevent the need for daily immunosuppressive therapy, thereby limiting treatment-associated toxicities. Pulsing protocols may also provide a mechanism for intermittent immunomodulation with this regimen. Support: None reported.

Laser doppler fluxmetry and the larynx

Problem: The study has three aims: (1) to determine whether a laser Doppler flux meter is a reliable and reproducible measure of laryngeal blood flux. (2) to determine whether blood flux, as measured, correlates with microvessel density as a less invasive, reliable method of quantifying angiogenesis. (3) to measure the effect of sequential division of nerves and vessels on laryngeal blood flow using the aforementioned specially designed laser Doppler probe. Methods: Using a laser Doppler monitor we measured the response to a known vasoconstrictor in the animal experiments. Reproducibility was tested and LDF readings obtained were compared to conventionally obtained quantification of microvessel density. Patients undergoing laryngectomies had continuous LDF measurements and the effect of sequential division of nerves and vessels was assessed. Results: In an animal model we demonstrated that the laser Doppler flux meter was capable of detecting change when one was expected ( P = 0.01). We also demonstrated good reproducibility of laser Doppler measurements in laryngeal mucosa both in the human and animal models (correlation coefficients of 0.956 and 0.947, respectively; with P = 0.01). Division of the superior laryngeal nerve caused a marked drop in ipsilateral laryngeal blood flux. Laser Doppler fluxmetry did not correlate with micro-vessel density as measured conventionally using immuno-histochemistry. Conclusion: Laser Doppler fluxmetry is a reliable and reproducible method of estimating blood flux in the larynx. We were able to show that division of the superior laryngeal nerve causes a significant fall in mucosal blood flux and therefore should be preserved whenever possible. However, it would seem that blood flux and microvessel density were not directly comparable parameters. Significance: It would appear from our study that LDF cannot be directly used to quantify angiogenesis. An improved understanding of laryngeal innervation and blood supply will contribute to the complex knowledge base that underpins the future of laryngeal transplantation. Support: This study was made possible by funds kindly donated from the estate of the late Thomas Green Esquire, and managed by the Medway Head and Neck Cancer Research Fund.

Facial transplantation: a working party report from the Royal College of Surgeons of England.

We are publishing this working party report on facial transplantation from the Royal College of Surgeons, England, in order to get opinions from the transplant community as to whether it is appropriate or not to perform this procedure at this point in time. There is no question that technically this can be done, but do the risks of failure, both technical and immunological, and the long-term risks of immunosuppression outweigh the potential benefits to the recipient who undoubtedly would be a severely burnt patient with a very scarred immobile face? The problems are not primarily technical but are immunological, psychological, and legal.

Concerns on Clinical Application of Composite Tissue Allotransplantation

Composite tissue allograft has become a clinical reality: hands, vascularized femoral diaphyses, abdominal walls, a larynx have all been transplanted throughout the world. Conventional immunosuppressive protocol has shown to be sufficient and effective. Rejection has been prevented in most cases and when it did occur it was successfully reversed. Skin has been confirmed as the principal target of acute and chronic rejection. There has been no mortality or early graft losses and, particularly in hand transplantation, the survival graft rate is 91% with a follow-up period ranging from 6 months to 61 months. The side effects of immunosuppression are limited and include primarily transient hyperglycemia, an increase in creatinine values and some opportunistic infections (i.e. cytomegalovirus infection). Nerve regeneration and cortical reorganization have been demonstrated in hand transplantation. Functional results have been encouraging particularly for hand and larynx transplantation. Appropriate indications and patient selection, based particularly on patient motivation and compliance, are essential requirements for composite tissue allograft success.

Induction of tolerance in a rat model of laryngeal transplantation.

The major limitation preventing expansion of laryngeal transplantation as a therapeutic modality is the necessity of lifelong immunosuppression. In this report, we describe an immunomodulatory strategy for tolerance induction in laryngeal allotransplantation that permits escape from chronic immunosuppression. Larynges were transplanted from Lewis-Brown-Norway (RT1l/n, F1) donors to Lewis (RT1l) recipients. Recipients received 7 days of treatment with tacrolimus and mouse anti-rat alphabeta T-cell-receptor (TCR) monoclonal antibodies. Histology, mixed lymphocyte reaction (MLR), skin grafting, and flow cytometry assessed functional tolerance, efficacy of immunodepletion, and donor-specific chimerism. All 10 recipients survived until sacrifice at 100 days. Histology suggested functional allograft tolerance. Skin grafting, MLR, and flow cytometry revealed that tolerance is neither donor-specific nor related to systemic immunocompromise. In this rat laryngeal-transplantation model, functional tolerance was induced under combined tacrolimus and alphabeta TCR protocol. Mechanisms responsible for this tolerance induction require future elucidation.

Tacrolimus and Mycophenolate Mofetil Provide Effective Immunosuppression in Rat Laryngeal Transplantation

Tacrolimus is efficacious in several transplantation settings. Some studies have demonstrated improved results using combination therapy with mycophenolate mofetil. Our primary objective was to evaluate the efficacy and optimal dosing of tacrolimus in preventing rejection, using an established rat model of laryngeal transplantation. Further, the ability of mycophenolate to allow lower dosing of tacrolimus while achieving equivalent immunosuppression was investigated. A dosage efficacy study with 10 experimental arms was conducted. Dosage groups were 0.1, 0.2, 0.3, and 0.6 mg/kg tacrolimus alone and 0.1 mg/kg tacrolimus combined with 15 mg/kg mycophenolate mofetil, 0.1 mg/kg tacrolimus combined with 30 mg/kg mycophenolate mofetil, 0.1 mg/kg tacrolimus combined with 40 mg/kg mycophenolate mofetil, 0.2 mg/kg tacrolimus combined with 15 mg/kg mycophenolate mofetil, 0.2 mg/kg tacrolimus combined with 30 mg/kg mycophenolate mofetil (30 d only), and 0.2 mg/kg tacrolimus combined with 40 mg/kg mycophenolate mofetil. Each group contained 8 to 10 rats. Grafts were harvested for histopathological analysis on day 15 or 30 after transplantation. Histopathological appearance of the graft was blindly graded according to an established scale. Dosage groups were compared on rejection score using Wilcoxon's rank sum test and the Jonckheere-Terpstra test for trend. There was a significant association between increasing dose of tacrolimus and decreasing rejection score at both 15 and 30 days (P <.001). In the groups treated with 0.1 mg/kg T, an increasing dose of mycophenolate was associated with lower rejection scores at both 15 and 30 days (P =.001). In the group treated with 0.2 mg/kg T, there was no evidence that the addition of mycophenolate resulted in lower rejection at 15 days. However, at 30 days, combination therapy with increasing doses of mycophenolate was associated with decreasing rejection score (P =.002). Tacrolimus is an effective immunosuppressive agent for laryngeal transplantation. Mycophenolate mofetil allows lower doses of tacrolimus to be used while preserving graft viability in the early post-transplantation period.

Cyclosporine dose, serum trough levels, and allograft preservation in a rat model of laryngeal transplantation.

Using a rat model of laryngeal transplantation, we sought to define the relationships between acute laryngeal rejection grade (RG) and cyclosporin A (CSA) concentration and CSA dosage. Five recipient Lewis rat groups (N = 10 per group) were administered intramuscular CSA doses of 1.0 (group 1), 2.5 (group 2), 5.0 (group 3), 7.5 (group 4), and 10 mg/kg per day (group 5) for 14 days. Immediately before sacrifice, 5 mL of whole blood was obtained to assay CSA trough levels by high-performance liquid chromatography. The specimens were graded microscopically by blinded reviewers by day of RG, 0 to 14 days after transplantation, as described in earlier reports. Despite high intragroup variability in CSA levels, significantly different mean CSA concentrations were achieved among all CSA dosage groups: 1, 2, 3, 4, and 5 (.0001 < p < .02). The mean laryngeal RGs did not test significantly different from each other with groups 3, 4, and 5 (RG, 2.3 +/- 1.3 versus 1.9 +/- 1.1 versus 1.7 +/- 0.3, respectively, .2 < p < .6). The RG for group 1 was significantly greater than those for groups 2 through 5 (p < .001), and the group 2 RG was greater (p < .02) than those for groups 3, 4, and 5. Polynomial fitting was used to determine the continuous relationship between each individual specimen's CSA concentration and the RG. Significant pathological allograft rejection correlated with CSA concentrations below 250 ng/mL.

Surgical anatomy of the recurrent laryngeal nerve: implications for laryngeal reinnervation.

Functional laryngeal reinnervation depends upon the precise reinnervation of the laryngeal abductor and adductor muscle groups. While simple end-to-end anastomosis of the recurrent laryngeal nerve (RLN) main trunk results in synkinesis, functional reinnervation can be achieved by selective anastomosis of the abductor and adductor RLN divisions. Few previous studies have examined the intralaryngeal anatomy of the RLN to ascertain the characteristics that may lend themselves to laryngeal reinnervation. Ten human larynges without known laryngeal disorders were obtained from human cadavers for RLN microdissection. The bilateral intralaryngeal RLN branching patterns were determined, and the diameters and lengths of the abductor and adductor divisions were measured. The mean diameters of the abductor and adductor divisions were 0.8 and 0.7 mm, while their mean lengths were 5.7 and 6.1 mm, respectively. The abductor division usually consisted of one branch to the posterior cricoarytenoid muscle; however, in cases in which multiple branches were seen, at least one dominant branch could usually be identified. We conclude that the abductor and adductor divisions of the human RLN can be readily identified by an extralaryngeal approach. Several key landmarks aid in the identification of the branches to individual muscles. These data also indicate the feasibility of selective laryngeal reinnervation in patients who might be candidates for laryngeal transplantation after total laryngectomy.

Anthropological variations in the anatomy of the human thyroid arteries.

Knowledge of anatomic variability of the superior (STA), inferior (ITA), and lowest accessory (IMA) thyroid arteries may be helpful in certain clinical conditions. However, details of this variability have not been thoroughly described. Specifically, whether the presence and site of origin of STA, ITA, and IMA are influenced by the anthropological group, to what extent their origin is symmetric or asymmetric, and the role played by this variability in visualizing each thyroid artery by nonselective thyroid angiography is not known. To clarify this we conducted a meta-analytical study on Caucasian and Asian subjects, the latter including only Japanese and Koreans. In Caucasians and Asians the presence of superior vessels compared to inferior vessels was more frequent and the probability of symmetric or asymmetric arterial origin for STA were equivalent. However, better recognition of inferior rather than superior vessels was achieved by nonselective angiography in Caucasians. Finally, different frequencies of presence and site of origin for each artery were identified in Caucasians compared to Asians. Our results suggest that the higher frequency of IMA in Asians than in Caucasians should result in a search for an IMA-dependent feeding artery of inferior parathyroid adenomas, primarily the mediastinal ones, especially in Asians both by imaging and transcatheter ablative approaches. In addition, we have found that a small percentage of Caucasian subjects lack an STA on the left side. Therefore, anatomic arterial compatibility should be carefully evaluated in the preoperative stage of laryngeal transplantation maintaining in situ the donor's thyroid by terminal anastomoses between donor and recipient STAs. Finally, the lack of any individual thyroid artery in either Caucasians or Asians might influence the distribution of autonomic supply that runs with thyroid vessels to the thyroid parenchyma. This appears functionally relevant in cases of traumatic or surgical lesions of the cervical sympathetic chain involving thyroid nerves. In fact, a restricted local autonomic control of thyroid activity might be related to individual rami of thyroid nerves.

Rat model of laryngeal transplantation with normal circulation maintained by combination with the tongue.

Recent research on laryngeal transplantation used a rat model with physiologically abnormal circulation, because the common carotid artery was used for venous drainage. Since a model with normal circulation is better for accurate assessment, we developed a vascularized laryngeal graft, using the common carotid artery as the arterial supply and the external jugular vein as the venous drainage. The transplant included other tributaries from the tongue and the pharynx by combination with the tongue, because the tributaries from the larynx were very small. Ten transplants were performed. The transplanted organs were examined daily. Seven days after transplantation, the composite grafts were evaluated histologically. The mortality rate was 10%. All composite grafts were fully viable, and their structure was maintained in the remaining nine rats. Our model is appropriate for experiments on laryngeal transplantation.

Composite Tissue Allotransplantation and Reconstructive Surgery

To review the first clinical cases of composite tissue allotransplantation (CTA) for reconstructive surgery and to discuss the outcome of and indications for these procedures in the context of chronic immunosuppression. The first human hand transplant was performed in 1998. This procedure, as well as other composite tissue transplants, offers the potential for correcting untreatable large tissue defects. However, concerns remain regarding obligatory chronic immunosuppression and long-term functional results. All the CTAs performed in humans that have been published or documented were reviewed. The preexisting clinical conditions and surgical procedures and the immunosuppressive therapy are described. The functional results and the complications or side effects of the treatment are detailed. Vascularized tendons (two cases), vascularized femoral diaphyses (three cases), knees (five cases), hands (four bilateral and seven unilateral cases), larynx (one case), and nonvascularized peripheral nerves (seven cases) have been transplanted in humans in the past decade. Rejection was prevented in most cases without difficulty. Early results are encouraging, particularly for hand and larynx transplants, but will need to be evaluated in the long term and in a larger number of patients. CTA holds great potential for reconstructive surgery but is at present restricted by the risks of chronic immunosuppression and uncertain long-term results.

Model for experimental revascularized laryngeal allotransplantation.

Although a human laryngeal transplant has been undertaken successfully, important questions remain that require a suitable animal model. A pig model for allotransplantation has been developed. Organ perfusion was studied in nine animals before four transplants were performed in congenic (unrecovered) animals and eight in unmatched (recovered) animals. Larynges were regularly examined endoscopically until death at 14 days. Immunosuppression included the use of tacrolimus. Revascularization was achieved by anastomosing the donor right cervical vascular tree to the recipient common carotid. In recovered animals, four allografts were placed orthotopically and four heterotopically. The pig larynx was perfused adequately via the right cervical vascular tree and congenic grafts were well tolerated. Of eight allografts, seven were well tolerated and remained healthy for the duration of the study (14 days). One allograft became infected between days 4 and 7 after operation. Median operating time was 6 h, with a median cold ischaemia time of 3 h. Revascularized allotransplants of the larynx can be undertaken reliably in pigs and this provides a preclinical model for studies of laryngeal transplantation.

Rat laryngeal transplant model: technical advancements and a redefined rejection grading system.

The rat laryngeal transplant model, introduced in 1992, laid the basic science foundation that contributed to the first successful human larynx transplant in 1998. Over 1,500 rat transplants later, numerous modifications have improved the model, increasing the initially reported evaluability rate of 50% to almost 100%. The observed histologic rejection process has been altered, as well. We report the technique modifications, as well as the results of a new study using nonimmunosuppressed, allogenic transplantations, in order to define a new rejection grading system. Using the updated model, we performed 50 transplantations between LBN(f1) donor rats and Lewis recipients. Larynges from 8 groups of 5 to 10 animals were harvested at intervals between 1 and 20 days after transplantation. The larynges were examined grossly and microscopically in a blinded manner for evidence of rejection. A multivariable linear regression model was used to define a new rejection grading scale. All 50 animals survived their assigned posttransplantation period. No animals exhibited vascular thromboses, for an evaluability rate of 100%. Histologic criteria in 7 categories and gross criteria in 5 categories demonstrated increased rejection proportional to the amount of time after transplantation. The equation [Group = -2.209 + 0.465 x (Size) + 0.901 x (VA Flow) + 0.613 x (Muscle) + 1.040 x (Thyroid)] reproducibly grades rejection on the basis of gross and histologic findings. New modifications to the rat laryngeal transplant model have conferred greatly improved animal survival rates and anastomotic patency rates. Additionally, the observed rates of rejection have been reduced in comparison to those of initial studies. This updated rejection staging system will be used to compare immunosuppressive regimens in future rat laryngeal transplant studies.

Special feature: Current state of laryngeal transplantation

Human laryngeal transplantation has been contemplated by otolaryngologists for at least 35 years. The authors recently reported their success with the first total human laryngeal transplant, which included the thyroid gland, the parathyroid glands, the pharynx, and five rings of trachea. The lessons gleaned from this experience regarding reinnervation, immunosuppression, swallowing, vocal function, and impact on quality of life have been invaluable. The authors review their human experience and the recent advances in animal research in both immunosuppressive regimens and reinnervation techniques.

Laryngotracheal reconstruction update

Laryngotracheal reconstruction has become the standard therapeutic option for the management of different types of laryngotracheal abnormalities such as stenosis or tumor invasion. The surgical reconstruction of the laryngotracheal airway is an effective method for achieving decannulation, facilitating the normal development of communication skills, and producing normal quality voice. Various grafting materials, such as autogenous nasal, costal, auricular, or thyroid cartilage; homograft cartilages; hyoid bone; perichondrium; hydroxyapatite; or titanium have been used for airway lumen augmentation. Rotary door flap, muscle pedicle graft of hyoid bone, free flaps with bone or cartilage from iliac crest or rib, and segmental resection with primary anastomosis have been practiced in laryngotracheal reconstruction with different success rates. None of these techniques and graft materials, however, fulfills the basic requirements for establishing internal lining, support, and vascularization. Human laryngeal transplantation is another future option for severe or alaryngeal cases when the immunologic mechanism is completely understood and the host reaction problem is solved. Still, laryngotracheal reconstruction techniques require refinement, especially in graft selection, so future studies are needed.

Effect of in vitro irradiation of donor larynges on cyclosporine requirements and rejection rates in rat laryngeal transplantation.

Total lymphoid irradiation is an acknowledged adjunctive immunosuppressant in whole organ transplantation in humans and animals. Local irradiation administered for a similar purpose is at best controversial. We evaluated in vitro donor larynx irradiation immediately preceding laryngeal transplantation as an immunomodulator. Each donor larynx was pretreated with 7.34 Gy of radiation in vitro. After transplantation, cyclosporine was administered in doses of 5 mg/kg per day, 2.5 mg/kg per day, and 1 mg/kg per day for trial lengths of 15 days and 30 days. Each of these 6 groups consisted of 10 rats per group. Earlier data have shown cyclosporine dosed at 5 mg/kg per day, without irradiation, administered for 1 month to have varied efficacy. Established histologic criteria were used to determine rejection patterns. All recipient rats survived the 15-day and 30-day trials. In all 10 rats receiving 5 mg/kg per day of cyclosporine for 15 days, the harvested transplanted larynges were viable without evidence of meaningful rejection (mild rejection). In 9 of the 10 rats receiving 5 mg/kg per day of cyclosporine for 30 days, the transplanted larynges displayed no meaningful rejection (mild rejection). In 9 of the 10 rats receiving 2.5 mg/kg per day of cyclosporine for 15 days, the transplanted larynges displayed no meaningful rejection (mild rejection). One rat receiving 2.5 mg/kg per day of cyclosporine for 15 days had a transplanted larynx that displayed moderate rejection. In all 10 rats receiving 2.5 mg/kg per day of cyclosporine for 30 days, the transplanted larynges displayed no meaningful rejection (mild rejection). At 15 days, 5 rats treated with 1 mg/kg per day of cyclosporine displayed mild rejection, 2 displayed moderate rejection, 2 displayed advanced to moderate rejection, and 1 displayed severe rejection. At 30 days, 4 rats treated with 1 mg/kg per day of cyclosporine displayed moderate rejection, 2 displayed advanced to moderate rejection, and 4 displayed severe rejection. We conclude that pretransplantation in vitro irradiation of donor larynges has immunomodulatory effects, allowing reduced cyclosporine immunosuppression with less rejection.