I. Introduction
Allotransplantation of submandibular salivary glands (SMGs) may provide an alternative treatment option for managing xerostomia, or for cases where autologous SMG transplantation is not feasible for managing severe xerophthalmia.
Challenges in allotransplantation include finding a less toxic immunosuppression method. Recently, SMG allotransplantation was incorporated in the treatment of severe xerophthalmia in two patients1. Both patients presented with complete functional loss of their SMGs and lacrimal glands due to the onset of graft-versus-host disease (GvHD) following stem cell transplantation. The SMG allotransplantations in these cases were performed without immunosuppression because both patients had complete donor chimerism following stem cell transplantation. Postoperative clinical assessments of the patients revealed primary success of the allotransplanted glands with an improvement in ocular surface lubrication and a reduction in inflammatory findings. Nevertheless, long-term sialoscintigraphy follow-up revealed a lower tracer activity than expected and a decreased level of secretion of saliva and tears. As such, further treatment with initial and minimal immunosuppressive therapy was recommended1.
The immunosuppressant FK506 (tacrolimus) is a powerful and selective anti–T-lymphocyte agent. It has a similar action mechanism to cyclosporine but is more potent2. It is a reliable candidate for immunosuppression in SMG transplantation because of its potency and ability to enhance nerve regeneration, which could boost the spontaneous autonomic innervations of transplanted glands3,4.
Two different FK506 doses were considered for our planned SMG allotransplantation based on a rabbit animal model. A dose of 0.08 mg/kg FK506 was effective and less toxic for rabbit lower limb allotransplantation procedures and a 0.16 mg/kg FK506 dose was applied for SMG allotransplantation in dogs5,6. Given this information, we conducted the present study to compare the outcomes of two doses (0.08 mg/kg and 0.16 mg/kg) of FK506 in SMG allotransplantation.
II. Materials and Methods
1. Animals and groups
This study was approved by the Institutional Animal Care and Use Committee (SNU-160720-6-2). SMG allotransplantations were completed in the right side of New Zealand white female rabbits (donors, n=18) and New Zealand white male rabbits (recipients, n=18) weighing 3 to 4 kg. All animals were nonrelated and were observed for two weeks before the terminal experiment. For this study, the rabbits were randomly divided into the following three groups, each containing six animals: (1) allograft rejection control group (Allo-Ctrl), (2) maintenance immunosuppression with low-dose FK506 (0.08 mg/kg) (FK506-L) group, and (3) maintenance immunosuppression with high-dose FK506 (0.16 mg/kg) (FK506-H) group.
2. Surgical procedures
The animal surgeries and experimental procedures were conducted in a manner previously described for SMG replantation7. Briefly, general anesthesia was maintained with isoflurane (1.5%-2.5%) via a rabbit supraglottic V-gel tube (Docsinnovent, London, UK). Dissection was performed to identify the linguofacial vein and common carotid artery. The SMG was then separated and all of the common carotid artery branches were transected and ligated while preserving the facial artery and its glandular branch. Similarly, the lingual and facial veins were ligated while preserving the linguofacial vein trunk and the glandular vein. Wharton’s duct was identified and a silicone tube measuring 0.5 mm in diameter (Beaver-Visitec, Waltham, MA, USA) was introduced into the duct. The receiver rabbit was then anesthetized and its right SMG was removed. The vessels were prepared in the same manner as for the donor. The vascular pedicles were connected by vascular microanastomoses of the linguofacial vein and common carotid artery to their corresponding proximal ends in an end-to-end fashion using 10-0 nylon (Ethicon, Somerville, NJ, USA) under a surgical microscope (Carl Zeiss, Oberkochen, Germany). The ischemia time was calculated form the time of SMG vessel detachment in the donor site to the time of blood reperfusion in the recipient site. Blood reperfusion was checked by laser Doppler (Perimed AB, Jarfalla, Sweden). Saliva flow was reconfirmed and the tube cannulated into the donor Wharton’s duct was connected to the remaining distal Wharton’s duct of the receiver. The wound was then irrigated gently with normal saline and closed using 4-0 Vicryl (Ethicon) for the platysma–fascia layer and 4-0 Dafilon (B. Braun, Melsungen, Germany) for the skin. A Penrose drain was placed for three days. Postoperatively, levofloxacin (5 mg/kg) was administered twice daily for three days, cephazolin 20 mg/kg once daily for five days, and tramadol (5 mg/kg) was administered twice daily for three days. Additionally, dextrose 5% (20 mL) and 0.9% normal saline (70 mL) were adjunctively supplied on a daily basis for three days. Assisted oral feeding with a Critical Care Formula (Oxbow Animal Health, Omaha, NE, USA) was introduced beginning on the third postoperative day in cases where the rabbit did not resume feeding spontaneously.(Fig. 1)
3. Maintenance immunosuppression
FK506 was used for immunosuppression. As previously indicated, a dose of 0.08 mg/kg was administered to the FK506-L group, while a dose of 0.16 mg/kg was administered to the FK506-H group, based on the work of Giessler et al.5 and Tanaka Isomura et al.6. Starting one day before transplantation, the drug was given intravenously, twice a day, as bolus injections over 90 minutes without monitoring of blood tacrolimus levels8,9.
4. Health condition and rejection surveillance
1) Clinical observations
The glands were gently palpated post-transplantation to detect any hardness, which is a sign of immunorejection in this context10. Body weight variations were calculated based on the weights recorded on the days of transplantation and sacrifice. Anorexia and spontaneous feeding of the animals were noted as well.
2) Interleukin-2 (IL-2) detection
Serum samples for measuring IL-2 with enzyme-linked immunosorbent assay (ELISA) (Cat. #E-EL-RB-1217; Elabscience Biotechnology, Houston, TX, USA) were taken 11 days after allotransplantation; 100 µL (duplicate) of each sample and a standard per well were added to the ELISA plate, and the ELISA plate was then incubated for 90 minutes at 37°C. Liquids were removed and a 100 µL/well of biotinylated detection antibody was added and incubated for 60 minutes at 37°C. The wells were then washed with wash buffer three times and incubated for 30 minutes at 37°C following the addition of Horseradish peroxidase-conjugate working solution. The wells were washed an additional five times and incubated for 23 minutes at 37°C after the addition of a 90 µL/well of substrate reagent. Stop solution was subsequently added to each well and the optical density of each well was determined using a microplate reader set to 450 nm.
3) Blood sampling
For the overall evaluation of the animals’ general health, blood samples were obtained on the day of sacrifice for complete blood count (CBC) analysis, including red blood cell count, white blood cell count, and hemoglobin (Hgb) and hematocrit (HCT) levels.
4) Saliva secretion
At the end of the two week experimental interval, the rabbits were anesthetized and injected with pilocarpine (1.5 mg/kg)6,11 to stimulate saliva secretion.
5) Histological evaluation
Glands were harvested and fixed in 10% formaldehyde before being divided into equal groups of specimens, embedded in paraffin, sectioned, and stained with H&E for histological evaluation. The glandular tissue was evaluated in terms of the percentage of parenchymal degeneration, acinar atrophy, and inflammatory cell infiltration. Based on the work of Kumar et al.12, the surviving glands were deemed to be those with near-normal histological glandular tissue or in which more than 50% of the glandular tissue was preserved.
5. Statistical analysis
Data analysis was conducted using the IBM SPSS Statistics (ver. 23.0; IBM Corp., Armonk, NY, USA). All data are presented as the mean with standard error of the mean. The association of nominal variables was tested using the chi-squared test. Nonparametric data comparisons were conducted with the Mann–Whitney U-test. For parameter data, the analysis of variance test followed by the post-hoc least significant difference test was used to compare data between different groups. A P-value less than 0.05 was considered to be statistically significant.
III. Results
1. Clinical findings
Intraoperatively, all glands other than one gland in the Allo-Ctrl group showed patency of anastomosed vessels and salivary flow. The ischemia time ranged between 92 and 106 minutes, with no significant differences observed among the groups. Within six to seven days, four glands of the Allo-Ctrl group and one gland of the FK506-L group were completely hard. One rabbit in the FK506-H group died on the sixth day and its allotransplanted SMG was found to be necrotic. Overall, body weight loss was higher in the FK506-H group (mean, 0.49±0.09 kg) than in the FK506-L group (mean, 0.44±0.10 kg) but without any significant difference. Anorexia was common, as seen in three rabbits in the FK506-L (50.0%) and four rabbits in the FK506-H (66.6%) groups.(Table 1)
2. Low interleukin-2 level
The expression level of IL-2 in the serum of all animals was undetectable.
3. Blood sampling results
The CBC revealed lower Hgb and HCT levels in the FK506-H group than in the F506-L group, without a statistically significant difference. The percentage of lymphocytes was similar in the two FK506 groups but higher in the control group, while the percentage of monocytes was higher in the FK506-L group than in the FK506-H group, although the result was not statistically significant. No major differences for the other hematological parameters were found between the groups.(Table 2)
4. Saliva secretion
Saliva was found in three of the FK506-H glands and in one gland in the FK506-L group.
5. Histological changes in the allotransplanted glands
Nearly normal glandular tissue structure was predominant in one gland in the FK506-H group. Partial loss of the parenchyma (not exceeding 50%) and an increase in fibrous tissue were findings in three glands in the FK506-H group and two glands in the FK506-L group. A loss of the majority of the parenchyma and an abundance of fibrous tissue were seen in one gland in the FK506-H group. Complete rejection was characterized by the complete loss of the parenchyma, except for a few remnant ducts with marked inflammatory cell infiltration, as was observed in five glands in the Allo-Ctrl group and three in the FK506-L group. The necrosis of allotransplanted glands due to failed blood perfusion was characterized by acini fragmentation with autolysis, as seen in one gland in each of the Allo-Ctrl, FK506L, and FK506-H groups.(Fig. 2)
IV. Discussion
Allotransplanted SMG can be rejected within the first few days post-transplantation. To, the best of our knowledge, Wang et al.13 is the first group to have conducted vascularized SMG allotransplantation. In their study, SMG allotransplantation was performed in 40 rabbits without immunosuppression. The glands survived for five days and were rejected after seven days. The authors concluded that, without using immunosuppressants, acute rejection occurs following the allotransplantation of SMGs with blood vessel anastomosis.
Different animal models and immunosuppressants have been used for SMG allotransplantation. Ge et al.10 conducted SMG allotransplantation with immunosuppression in swine animal models using high and low doses of cyclosporine, methylprednisolone, and azathioprine. In their study, SMGs transplanted into control subjects that were not given immunosuppressants were rejected within seven days. The rejected glands were hard to the touch and showed histological changes. In the low-dose immunosuppressant group, allografts were rejected on Days 15, 17, 19, and 20 after the operation, while, in the high-dose immunosuppressant group, the survival time of the allografts was considerably prolonged, with three allografts rejected on Days 35, 48, and 60 and another two allografts surviving for 100 days without signs of rejection. Later, Tanaka Isomura et al.6 reported on SMG allotransplantations in beagle dogs that were successful for 12 weeks, using 0.16 mg/kg of tacrolimus as an immunosuppressant.
The first human allogeneic SMG transplantations were conducted in two patients with complete chimerism following stem cell transplantation, with primarily successful results. However, long-term follow-up with sialoscintigraphy showed a decrease in the tracer uptake, and the authors recommended minimal and initial immunosuppression with the allotransplantation.
FK506 was selected for the present study owing to its superior potency in comparison with cyclosporine. In this study, the high and low (0.08 mg/kg and 0.16 mg/kg) doses that were administered resulted in the survival of four and two glands, respectively, out of six in each group. The side effects of body weight loss, anorexia, and changes in blood parameters revealed in the animals during this short-term study tended to be less significant in the FK506-L group than in the FK506-H group but the results were not statistically significant. The rabbits showed anorexia, particularly in the first three to five days. Later, almost half of them resumed feeding spontaneously, and the rest were compliant with assisted feeding, as needed. The amelioration of anorexia was particularly noticeable after ceasing the administration of antibiotics and reducing the surgical wound pain level. One rabbit in the FK506-H group died on the sixth day post-transplantation as a complication of diarrhea. Blood analysis showed non-significantly lower Hgb and HCT levels in the FK506-H group. However, these levels were still within the normal ranges and such an effect could be attributed to the nutrition status of the rabbits, which could be related to the FK506 dosage and anorexia level14,15. Monocytes were non-significantly higher in the FK506-L group and have been reported to be closely related to acute allograft dysfunction16,17.
Of note, some patients with Sjögren’s syndrome or GvHD may already take systemic FK506 to treat underlying causes of their disease or to alleviate dry eye symptoms18,19. Furthermore, FK506 has neuroprotective and neuroregenerative properties for neurodegenerative disorders and peripheral nerve injuries20,21. Elsewhere, FK506 has been reported to exert neurotrophic effects on the postganglionic parasympathetic cavernous nerve22. Such properties would boost SMG autonomic neural regeneration and subsequently result in a reduced latent saliva production period.