Orthotopic forelimb transplantation in a Yucatan minipig model: Anatomic and in vivo study.

INTRODUCTION: Above elbow transplants represent 19% of the upper extremity transplants. Previous large-animal models have been too distal or heterotopic, did not use immunosuppression and had short survival. We hypothesize that an orthotopic forelimb transplant model, under standard immunosuppression, is feasible and can be used to address questions on peri-transplant ischemia reperfusion injury, and post-transplantation vascular, immunologic, infectious, and functional outcomes. MATERIALS AND METHODS: Four forelimbs were used for anatomical studies. Four mock transplants were performed to establish technique/level of muscle/tendon repairs. Four donor and four recipient female Yucatan minipigs were utilized for in-vivo transplants (endpoint 90-days). Forelimbs were amputated at the midarm and preserved through ex vivo normothermic perfusion (EVNP) utilizing an RBC-based perfusate. Hourly perfusate fluid-dynamics, gases, electrolytes were recorded. Contractility during EVNLP was graded hourly using the Medical Research Council scale. EVNP termination criteria included systolic arterial pressure ≥115 mmHg, compartment pressure ≥30 mmHg (at EVNP endpoint), oxygen saturation reduction of 20%, and weight change ≥2%. Indocyanine green (ICG) angiography was performed after revascularization. Limb rejection was evaluated clinically (rash, edema, temperature), and histologically (BANFF classification) collecting per cause and protocol biopsies (POD 1, 7, 30, 60 and endpoint). Systemic infections were assessed by blood culture and tissue histology. CT scan was used to confirm bone bridging at endpoint. RESULTS: Animals 2, 4 reached endpoint with grade 0-I rejection. Limbs 1, 3 presented grade III rejection on days 6, 61. CsA troughs averaged 461 ± 189 ng/mL. EVNLP averaged 4.3 ± 0.52 h. Perfusate lactate, PO2 , and pH were 5.6 ± 0.9 mmol/L, 557 ± 72 mmHg and 7.5 ± 0.1, respectively. Muscle contractions were 4 [1] during EVNLP. Transplants 2, 3, 4 showed bone bridging on CT. CONCLUSION: We present preliminary evidence supporting the feasibility of an orthotopic, mid-humeral forelimb allotransplantation model under standard immunosuppression regimen. Further research should validate the immunological, infectious, and functional outcomes of this model.

Histomorphometry in Peripheral Nerve Regeneration: Comparison of Different Axon Counting Methods.

BACKGROUND: Histomorphometry quantitatively evaluates nerve regeneration. Total myelinated fiber count (TMFC) is most accurately obtained manually across full nerve cross-sections, but most researchers opt for automated, sampled analysis. Few of the numerous techniques available have been validated. The goal of this study was to compare common histomorphometric methods (full manual [FM], sampled manual [SM], and sampled automatic [SA]) to determine their reliability and consistency. MATERIAL AND METHODS: Twenty-four rats underwent sciatic nerve (SN) repair with 20mm isografts; SNs distal to the graft were analyzed. TMFC was manually determined in each full cross-section. Counts were also extrapolated from sampled fields, both manually and automatically with ImageJ software. Myelinated fiber diameter, axon diameter, and myelin sheath thickness were measured manually in full and sampled fields; G-ratio was calculated. Repeated-measures MANOVA, Spearman correlation, and Wilcoxon signed-rank tests were performed. A systematic review of histomorphometry in rat SN repair was performed to analyze the variability of techniques in the literature. RESULTS: FM TMFC was 13,506 ± 4,217. Both sampled methods yielded significantly different TMFCs (SM:14.4 ± 13.4%, P< 0.001; SA:21.8 ± 44.7%, P = 0.037). All three methods strongly correlated with each other, especially FM and SM (rs = 0.912, P< 0.001). FM fiber diameter, axon diameter, and myelin sheath thickness did not differ from SM (P = 0.493, 0.209, and 0.331, respectively). 65% of papers used sampling; 78% utilized automated or semi-automated analysis. Software, sampling, and histomorphometric parameters varied widely. CONCLUSION: SM and SA analysis are reliable with standardized, systematic sampling. Transparency is essential to allow comparison of data; meanwhile, researchers must be cognizant of the wide variety of methodologies in the literature.

Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier Perfusate.

INTRODUCTION: Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability better than standard cold storage. Perfusates containing packed red blood cells (pRBC) improve outcomes when compared to acellular perfusates. Limitations of pRBC-based perfusion include limited availability, need for cross match, mechanical hemolysis, and activation of pro-inflammatory proteins. Hemoglobin-based oxygen carrier (HBOC)-201 (Hemopure) is a solution of polymerized bovine hemoglobin, characterized by low immunogenicity, no risk of hemolytic reaction, and enhanced convective and diffusive oxygen delivery. This is a preliminary study on the feasibility of EVNLP using HBOC-201 as an oxygen carrier. MATERIALS AND METHODS: Three porcine forelimb perfusions were performed using an established EVNLP model and an HBOC-201-based perfusate. The perfusion circuit included a roller pump, oxygenator, heat exchanger, and reservoir. Electrolytes, limb temperature, weight, compartment pressure, nerve conduction, and perfusion indicated by indocyanine green angiography and infra-red thermography were monitored. Histological evaluation was performed with hematoxylin and eosin and electron microscopy. RESULTS: Three limbs were perfused for 21.3 ± 2.1 hours. Muscle contractility was preserved for 10.6 ± 2.4 hours. Better preservation of the mitochondrial ultrastructure was evident at 12 hours in contrast to crystallization and destruction features in the cold-storage controls. CONCLUSIONS: An HBOC-201-EVNLP produced outcomes similar to RBC-EVNLP with preservation of muscle contractility and mitochondrial structure.