Olivia Dan

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(fl) 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 × (Size) + 0.901 × (VAFlow) + 0.613 × (Muscle) + 1.040 × (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.

Induction of tolerance in a rat model of laryngeal transplantation

Background. 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. Materials and Methods. 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 &agr;&bgr; 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. Results. 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. Conclusions. In this rat laryngeal-transplantation model, functional tolerance was induced under combined tacrolimus and &agr;&bgr; TCR protocol. Mechanisms responsible for this tolerance induction require future elucidation.

Pulsed immunosuppression with everolimus and anti-αβ T-cell receptor : Laryngeal allograft preservation at six months

Objectives: Laryngeal transplantation can restore the voice in patients who have undergone laryngectomy. However, the prospect of lifelong immunosuppression is a drawback to this procedure. We present data from a study aimed at minimizing the need for immunosuppression while maintaining graft viability through a novel pulsed-dosing protocol. Methods: Larynges were transplanted from Lewis–brown Norway (RT11+n, F1) rats to Lewis (RT11) recipients. All recipients received 7 days of treatment with everolimus and mouse anti-rat αβ T-cell receptor (anti-TCR) monoclonal antibodies beginning the day before transplantation. At 90 days after transplantation, all recipients received a pulse of the same treatment combination for 5 days. From 90 to 180 days after transplantation, the rats received no treatment (group 1, n = 5), 2.5 mg/kg everolimus per day (group 2, n = 5), or 1.0 mg/kg everolimus per day (group 3, n = 5). Results: Histologic analysis of rats that received everolimus as pulse therapy evidenced no signs of rejection, whereas animals that were untreated after 90 days had normal to mild chronic rejection. T-cell reconstitution occurred 65 days after perioperative immunosuppressive treatment, but less rapidly after pulse therapy. Also, peripheral chimerism was generated in all 3 groups. Conclusions: In the rat laryngeal transplantation model, short-term perioperative therapy with everolimus and anti-TCR followed by pulsing is a viable alternative to the concerns associated with continuous, lifelong immunosuppression.

Gastric bypass surgery is protective from high-fat diet-induced non-alcoholic fatty liver disease and hepatic endoplasmic reticulum stress.

High‐fat diets are known to contribute to the development of obesity and related co‐morbidities including non‐alcoholic fatty liver disease (NAFLD). The accumulation of hepatic lipid may increase endoplasmic reticulum (ER) stress and contribute to non‐alcoholic steatohepatitis and metabolic disease. We hypothesized that bariatric surgery would counter the effects of a high‐fat diet (HFD) on obesity‐associated NAFLD.

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.

Effects of adding steroids, in vitro irradiation, or both to cyclosporine immunosuppression in the murine laryngeal transplantation model.

Adding oral prednisone or in vitro organ irradiation to an immunosuppressive regimen for laryngeal transplantation may allow cyclosporine dosage to be decreased without compromising organ rejection rates. Using an established rat laryngeal transplant model, we sought to determine whether combined immunosuppressive regimens decreased rejection rates. Twenty-two treatment groups of 10 animals each were studied. Cyclosporine was administered at 2 different historically low dosages (1.5 and 2 mg/kg per day) alone, in combination with prednisone, and with in vitro irradiation of the harvested larynx. At 15 days after transplantation, cyclosporine combined with prednisone did not significantly alter the mean severity of rejection. At 30 days after transplantation, rats treated with a combination of cyclosporine 2 mg/kg and prednisone 1 mg/kg showed the least amount of rejection. Rejection rates were significantly higher with 2 mg/kg cyclosporine alone (p < .00024). Irradiation did not significantly decrease the severity of rejection. These results demonstrate that at 1 month after transplantation, a combined steroid and cyclosporine regimen reduces the severity of rejection after laryngeal transplantation in an animal model. Our findings suggest that laryngeal transplant patients may be treated with low cyclosporine doses without experiencing organ rejection if steroids are added to the immunosuppressive regimen.

New mouse model for studying laryngeal transplantation

Objectives Laryngeal transplantation research has been studied in various animal models. For in-depth, immunology-based transplantation research, however, a thoroughly studied animal model must exist. The purpose of this study was to develop a reliable surgical technique in mice to serve as a model for further study of laryngeal transplantation. Methods Heterotopic laryngeal transplantation was attempted in 15 immunocompetent mice by use of modifications of previously described techniques established in rats. Results Various microvascular techniques were used that led to 8 successful transplants (of 15) with patent vascularity at the time of sacrifice. The first 7 attempts at transplantation were unsuccessful because of technical difficulties related to vessel size, soft tissue traumatic injury, and venous congestion. Subsequently, 8 transplantation procedures were successfully performed after modifications of the surgical technique. Conclusions This pilot study describes the reproducible surgical techniques performed in using mice for studying laryngeal transplantation. Mice are cost-effective and immunologically well studied, and are thus ideal for further laryngeal transplantation research.

Decoy NF-κB fortified immature dendritic cells maintain laryngeal allograft integrity and provide enhancement of regulatory T cells†

The increased risk of malignancy associated with post‐transplant immunosuppression limits the potential of laryngeal transplantation as a reconstructive option. This risk may be mitigated by utilizing decoy nuclear factor kappa B (NF‐κB) immature dendritic cells (iDC) to provide donor‐specific tolerance. The purpose of this study was to explore whether tolerogenic properties of iDC can be applied to composite tissue transplantation.

Postallograft Donor and Recipient Dendritic Cell Trafficking in the Rat Larynx

Objectives/Hypothesis: Dendritic cells (DC) are potent antigen‐presenting cells that instigate allograft rejection. Their migration kinetics vary depending on the type of organ transplanted. The timing of donor and recipient DC trafficking in laryngeal transplants is unknown.

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 specimens CSA concentration and the RG. Significant pathological allograft rejection correlated with CSA concentrations below 250 ng/mL.