Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

Objective: To evaluate the effects of different surface treatment methods and simulated aging on the roughness, microhardness, and color stability of lithium disilicate glass ceramics(LDC). Methods: The LDC were grouped and performed 5 kinds of surface treatments: control group, polishing group, polishing+polishing paste group, glazing group, polishing+glazing group. The roughness and microhardness of the specimen were measured. Then the specimens were divided into two subgroupswhich were treated with water and staining solution. The specimens were measured color parameters before and after processing. The above data was analyzed. Results: The LDC had changes in surface roughness and microhardness after different surface treatments. The polishing+polishing paste group had the lowest surface roughness, and the surface microhardness of the LDC decreased after glazing. After simulating aging, the difference of color and transparency of the polishing+polishing paste group, glazing group, and polishing+glazing group were the smallest. Conclusion Different surface treatments and simulated aging have a certain impact on the roughness, microhardness, and color stability of LDC. Fine polishing with polishing paste has a comparable anti staining effect to glazing on LDC.

The Treatise on Cold Pathogenic and Miscellaneous Diseases(Shanghan Zabing Lun)records a unique system of pulse diagnosis,pioneering the"site for taking wrist pulse(Cunkou)-Anterior tibial(Fuyang)-Shaoyin"three-part pulse diagnosis method.This system emphasizes the application of the wrist pulse site,classifying pulse conditions into two major categories,yin and yang,thereby laying a solid foundation for the development of traditional Chinese medicine diagnostics.Studying the pulse theory of Shanghai Zabing Lun is of great significance for understanding,inherting,and developing ZHANG Zhongjing's medicine and clinical diagnosis.However,owing to the passage of time,the precise meaning of certain pulse conditions described by ZHANG Zhongjing,such as the"shang guanshang"pulse,remains unclear.This article explores the location and clinical significance of the"shangguan shang"pulse as referenced in Treatise on Cold Pathogenic and Miscellaneous Diseases and Synopsis of Golden Chamber(Jingui Yaolue).Through textual research,it investigates this pulse and related ones based on medical texts before the Tang Dynasty,such as Inner Canon of Huangdi(Huangdi Neijing)and Classic of Questioning(Nanjing).The findings indicate that the"shangguan shang"pulse is positioned one division before the Guan pulse site.Further analysis of the clinical significance suggests the existence of a pulse-taking method involving three body regions and nine pulse-taking sites,subdividing the Cun,Guan,and Chi positions into three distinct sections.This pulse diagnostic method enriches the ways of pulse diagnosis,but further exploration and verfication are needed.

Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.

Objective:To analyze the condition of subclinical atherosclerosis (SCA) in patients with psoriatic arthritis (PsA) and to provide a reference for better management of the associated cardiovascular risk in patients with PsA.Methods:Based on the cohort of PsA patients (PKUPsA) in the Department of Rheumatism and Immunology, Peking University First Hospital, 240 PsA patients without previous clinical atherosclerotic disease between July 2018 and June 2024 were included. The demographic data traditional cardiovascular disease risk factors, PsA related indicators and medications were collected when all patients were entered into the cohort. Increased intima-media thickness and/or arterial plaque formation in bilateral carotid arteries examined by ultrasonography are defined as the presence of SCA. Based on this, patients were divided into SCA and no SCA groups, and the two groups were compared and analyzed. Statistics were performed using the Mann-Whitney U test, independent sample t test, χ2 test and Logistic regression analysis. Results:Eighty-five of 240 patients (35.4%) had SCA, including 55 (22.9%) with cIMT thickening and 51 (21.2%) with carotid plaque. Compared with the PsA patients without SCA, patients with SCA were older [55.0 (42.0, 62.5) vs. 42.0(35.0, 53.0) year of age, Z=-4.90, P<0.001], had longer disease course of arthritis [4.6 (1.0, 10.1) vs. 3.0(1.0, 6.1) years, Z=-1.98, P=0.048], more patients with combined hypertension [34.1%(29/85) vs. 15.5%(24/155), χ2=11.08, P<0.001], hyperlipidemia [47.1%(40/85) vs. 27.1%(42/155), χ2=1.22, P=0.002] and the taking of statins [14.1%(12/85) vs. 5.8%(9/155), χ2=4.75 , P=0.029], hypoglycemic agents [10.6%(9/85) vs. 3.9%(6/155), χ2=4.23, P=0.040] and antihypertensive drugs [17.6%(15/85) vs 6.5%(10/155), χ2=7.37, P=0.007]. They also had a higher blood glucose level[5.37 (5.17, 6.09)mmol/L vs. 5.26(4.97, 5.67)mmol/L, Z=-2.82 , P=0.005], low-density lipoprotein [(3.05± 0.76)mmol/L vs. (2.78±0.75)mmol/L, t=2.60, P=0.010] and blood uric acid level[351 (312, 412)μmol/L vs. 333(279, 408)μmol/L, Z=-2.10, P=0.036]. Multivariate analysis showed that older [ OR (95% CI) =1.059 (1.033, 1.086), P<0.001], increased low density lipoprotein [ OR (95% CI) =1.519 (1.018, 2.267), P=0.041] and increased blood uric acid levels [ OR (95% CI)=1.004 (1.001, 1.007), P=0.017] were an independent risk of SCA in PsA patients. Conclusion:More than 1/3 of PsA patients with SCA without past history of clinical atherosclerosis with SCA, advanced age, increased blood low density lipoprotein level, and elevated uric acid level are independent risk factors for PsA with SCA, so attention should be paid to the assessment and management of cardiovascular-related risk. Early intervention can help to improve patient prognosis.

Objective:Treat-to-target strategies for psoriatic arthritis (PsA) have been proposed for several years, however, the status of target and goal achievement in China is unknown. Therefore, we aimed to investigate the target achievement of PsA patients, and differences of treatment goals were further analyzed.Methods:A total of 360 PsA patients from Peking University First Hospital PsA patient cohort (PKUPsA) between January 2016 and March 2024 were included. We retrospectively analyzed the disease activity of patients at their enrollment. Minimal disease activity (MDA), disease activity index for PsA (DAPSA), clinical DAPSA (cDAPSA) and disease activity score based on 28 joint counts (DAS28) were evaluated. Interclass correlation coefficient (ICC) was used to test the consistency of all these assessments.Results:Three hundred and sixty patients were included in this study, with 149 females (41.4%), median age 47 (36, 57) years and duration of PsA for 2.0(1.0, 6.8) years. 129 (35.8%) patients reported family history of PsO. The most common comorbidities were hyperlipidemia (101, 28.1%) and hypertension (78, 21.7%). There were 217 (60.3%), 75 (20.8%) and 18 (5.0%) patients treated with conventional synthetic DMARDs, biologics and JAK inhibitors respectively. Forty-nine (13.6%) patients ever received intra-articular injection of glucocorticoid. Based on the different definitions, the rates of target achievement were 33.9% (MDA), 56.1% (DAPSA), 60.8% (DAS28-ESR), 63.9% (DAS28-CRP) and 64.2% (cDAPSA). The main limiting factors for MDA attainment among those who had achieved DAPSA or DAS-defined targets were pain VAS, Psoriasis Area and Severity Index (PASI), tender/swollen joint count, patient global assessment, HAQ, and number of enthesitis, based on leeds Enthesitis Index(LEI). The ICCs of these evaluation methods were 0.489~0.819 ( P<0.001). The consistency was the best between DAPSA and cDAPSA, medium between DAS28-CRP and DAS28-ESR, and worst between MDA and DAS28-CRP. Moreover, DAPSA was more consistent with DAS28 than cDASPA. MDA showed moderate consistency with all other assessments. Conclusion:33.9%~64.2% of PsA patients achieved targets based on different definitions. MDA was the most stringent target, while cDAPSA was the most loose one. In general, MDA was not well consistent with other assessments.

Objective:To explore the differences in the CVD risk and lipid profiles in psoriatic arthritis (PsA) patients with different disease activity and investigate lipid management status in Chinese patients with PsA.Methods:Patients were enrolled from PKUPsA-PC cohort in Peking University First Hospital from January 2016 to January 2024. Data were collected at their first visit, including disease activity score, lipid profiles and treatment. Two modified CVD risk prediction models (modified China-PAR and modified FRS-CVD) were applied to predict the CVD risk over 10 years. All enrolled patients were subsequently stratified into low, intermediate and high-risk groups. The status of lipid target achievement was assessed based on lipid management recommendations proposed by prediction models. In addition, DAPSA was used to stratify PsA patients into remission, low, moderate and high-disease activity groups, and the differences in CVD risk and lipid profiles among PsA patients with different disease activity status were explored. The t test was used for comparison between 2 groups for measures that conformed to normal distribution; the Mann-Whitney U test was used for comparison between 2 groups for measures that were skewed; and the chi-square test was used for comparison between 2 groups for categorical data.Results:Three hundred and seven PsA patients were included in this study. They were aged 47 (36, 57) years with 121 (39.4%) female, disease duration of skin lesions of 14 (7, 23) years and disease duration of PsA for 3 (1, 8) years. There were 148 (48.2%) patients with dyslipidemia, and 38 (25.7%) of them were receiving lipid lowering drugs. By the modified China-PAR model, there were 174 (56.7%), 76 (24.8%) and 57 (18.5%) in the low-, moderate- and high-risk groups. By the modified FRS-CVD model, there were 173 (56.4%), 58 (18.9%) and 76 (24.7%) patients in the corresponding groups. According to the recommendations for lipid management based on FRS-CVD model, 80 (26.1%) patients did not achieve the target of lipid profile, including 9/174 (5.2%) in the modified model low-risk group, 20/76 (26.3%) in the intermediate-risk group, and 51/57 (89.5%) in the high-risk group, but there were only 12 (15.0%) patients receiving statin therapy. Compared with the remission and low disease activity groups, patients in the moderate-to-high disease activity group were older [50 (37, 60) years vs. 43 (35, 55) years, Z=-2.42, P=0.016]; had a higher proportion of hypertension (30.3% vs. 15.0%, χ2=9.60, P=0.002); and had lower HDL-C levels [1.1 (0.9, 1.3) mmol/L vs. 1.2 (1.0, 1.4) mmol/L, Z=-3.18, P=0.001]. Under the modified China-PAR and modified FRS-CVD risk prediction models, a higher proportion of patients with high disease activity in PsA were stratified at high 10-year CVD risk compared with the remission and low disease activity groups (29.5% vs. 19.6%, χ2=3.81, P=0.005) and (23.5% vs. 12.4%, χ2=6.00, P=0.014). Conclusion:Nearly half of the PsA patients are at medium-high risk to CVD. CVD risk is significantly higher in patients with moderate to high disease activity than in patients with remission and low disease activity. HDL-c levels are lower in patients with high disease activity. Nevertheless a quarter of patients did not achieve the target of lipid profile, and few patients are receiving statin. More attention should be paid to CVD risk evaluation and lipid management as a part of treat-to-target strategy to improve the prognosis.

Objective:To explore the distribution variation of ultrasound-detected inflammatory lesions with clinical signs in patients with psoriatic arthritis (PsA).Methods:This was based on the Peking University First Hospital Psoriatic Arthritis (PKUPsA) cohort. Patients enrolled from January 2019 to June 2024 were inchuded, patients with complete data of physical examination and ultrasonographic evaluations of 62 joints in the hand and foot. The ultrasound-detected inflammatory lesions including synovitis, tenosynovitis, enthesitis, and soft tissue inflammation were compared with joint tenderness/swelling. The χ2 test was employed to analyze differences between groups. Results:A total of 7 440 joints in 120 PsA patients were included. Overall, the proportion of joints with clinical signs (tenderness or swelling) was higher than those with ultrasound-detected inflammatory lesions [9.14%(680/7 440) vs. 7.93%(590/7 440), χ2=1 245.928, P<0.001], with more tenderness joints than swelling joints [7.72%(574/7 440) vs. 6.14%(457/7 440), χ2=3 264.45, P<0.001]. Clinical signs were primarily observed in hand proximal interphalangeal (PIP), distal interphalangeal (DIP), wrist and ankle joints, mostly in DIP2 joints [19.58%(47/240)]. Ultrasound-detected inflammatory lesions were predominantly found in metatarsophalangeal (MTP), wrist, and ankle joints, mostly in MTP2 joints (18.75%, 45/240). Clinical signs were more prevalent than ultrasound-detected inflammatory lesions in hand PIP1-3, PIP5, DIP2, and DIP5 joints ( P<0.05), whereas more frequent ultrasound-detected inflammatory lesions than clinical tenderness/swelling were in MTP1-4 joints ( P<0.05). Among ultrasound-detected inflammatory lesions, synovitis in MTP2 joints (18.75%, 45/240), tenosynovitis in ankle joints (10.00%, 24/240), enthesitis in hand DIP2 joints (8.75%, 21/240), and soft tissue inflammation in MTP4 joints (2.50%, 6/240) most commonly observed. Dactylitis was more frequently observed in toes than in fingers, with the fourth toe most commonly affected(16.67%, 40/240). Ultrasound-detected inflammatory lesions were observed in 72.37%(55/240) of fingers/toes with clinical dactylitis, mainly presenting as synovitis, tenosynovitis, or combinations of these. Conclusion:PsA exhibits significant heterogeneity in the inflammatory lesions across different joints and lesion types. The discrepancies between clinical findings and ultrasonic inflammatory changes highlight the limitations of physical examination in fully capturing the pathological features of PsA. As a critical tool for PsA evaluation, ultrasonography offers distinct advantages in detecting subclinical inflammation and differentiating inflammatory from non-inflammatory lesions.

Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.