Added Value of Histological Evaluation of Muscle Biopsies in Porcine Vascularized Composite Allografts

Abstract

Background: Machine perfusion (MP) offers extended preservation of vascularized complex allografts (VCA), but the diagnostic value of histology using hematoxylin and eosin (H&E) in detecting ischemia-reperfusion injury (IRI) in muscle cells remains unclear. This study aims to document the application of the Histology Injury Severity Score (HISS) and to assess whether additional staining for nicotinamide adenine dinucleotide (NADH) and membrane attack complex (MAC) improves IRI detection in a porcine limb replantation model. Methods: The forelimbs of 16 Dutch Landrace pigs were amputated and preserved for 24 h using hypothermic MP (n = 8) with Histidine-Tryptophan-Ketoglutarate (HTK) or for 4 h with SCS (n = 8) before heterotopic replantation and 7 days of follow-up. Muscle damage was assessed via biochemical markers and light microscopy using H&E, NADH, and MAC at baseline, post-intervention, and post-operative day (POD) 1, 3, and 7 timepoints, using the HISS and a self-developed NADH and MAC score. Results: H&E effectively identified damaged muscle fibers and contributed to IRI assessment in porcine limbs (p < 0.05). The highest HISS was measured on POD 3 between MP (4.9) and SCS (3.5) (p = 0.029). NADH scores of both preservation groups varied over the 7-day follow-up and were statistically insignificant compared with baseline measurements (p > 0.05). MAC revealed no to minimal necrotic tissue across the different timepoints. Conclusions: This study documents the application of the HISS with H&E to detect IRI in muscle fibers. NADH and MAC showed no significant added diagnostic utility. The 24 h MP showed similar muscle alterations using the HISS compared to that of the 4 h SCS after a 7-day follow up.

Keywords: Histology Injury Severity Score; hematoxylin and eosin; ischemia–reperfusion injury; machine perfusion; membrane attack complex; nicotinamide adenine dinucleotide; static cold storage; vascularized composite allografts.

Figure 1

Color images capturing the perfused and static cold storage limbs at baseline and on post-operative days 3 and 7. One week later, assessments of limb viability (such as capillary refill times, temperature, and skin color) showed comparable results to those recorded at the baseline.

Figure 2

Mean Histology Injury Severity Scores (HISS) of cross sections of skeletal muscle biopsies preserved via static cold storage (SCS) or machine perfusion (MP) and stained with H&E. The values are shown for the baseline (MP n = 8; SCS n = 8), post-intervention (MP n = 8; SCS n = 8), and post-operative day (POD) 1 (MP n = 8; SCS n = 8), POD 3 (MP n = 8; SCS n = 8), and POD 7 (MP n = 6; SCS n = 6) timepoints. A 95% confidence interval is displayed. A Mann–Whitney U test was performed to determine the significance of changes between groups.

Figure 3

Mean sub Histology Injury Severity Scores (HISS) of cross sections of skeletal muscle biopsies preserved with static cold storage (SCS) or machine perfusion (MP) and stained with H&E on post-operative days (PODs) 3 and 7. (A) Subscores on POD 3 (MP n = 8; SCS n = 8): muscle fiber damage (p = 0.631), edema (p = 0.003), inflammation (p = 0.820), and variation in cell shape and size (p = 0.036). (B) Subscores on POD 7 (MP n = 6; SCS n = 6): muscle fiber damage (p = 0.347), edema (p = 0.001), inflammation (p = 0.319), and variation in cell shape and size (p = 0.091). Asterisks (*) indicate significance. A Mann–Whitney U test was performed to determine the significance of changes between groups.

Figure 4

Representative H&E, MAC, and NADH images of skeletal muscle biopsies, preserved with static cold storage (SCS) or machine perfusion (MP), at baseline and on post-operative days 1, 3, and 7.

Figure 5

Mean NADH scores of cross sections of skeletal muscle biopsies preserved with static cold storage (SCS) or machine perfusion (MP) and stained with NADH. The values are shown for the baseline (MP n = 8; SCS n = 8), post-intervention (MP n = 8; SCS n = 8), and on post-operative day (POD) 1 (MP n = 8; SCS n = 8), POD 3 (MP n = 8; SCS n = 8), and POD 7 (MP n = 6; SCS n = 6) timepoints. A 95% confidence interval is displayed. Only descriptive analysis was performed to present changes between groups.

Figure 6

H&E, MAC, and NADH images of skeletal muscle biopsies preserved with static cold storage (SCS) at baseline and on post-operative days 1, 3, and 7.

Figure 7

Biochemical parameters of blood of the limbs after preservation with static cold storage (SCS) or machine perfusion (MP) at baseline (MP n = 8; SCS n = 8), post-intervention (MP n = 8; SCS n = 8), post-operative day (POD) 1 (MP n = 8; SCS n = 8), POD 3 (MP n = 8; SCS n = 8), and POD 7 (MP n = 6; SCS n = 6). The mean per group is displayed with 95% confidence intervals. A Kruskal–Wallis test was performed to determine the significance of changes between groups.