Objective To explore the feasibility and reference value of allogeneic lung transplantation and postoperative monitoring in miniature pigs for lung transplantation research. Methods Two miniature pigs (R1 and R2) underwent left lung allogeneic transplantation. Complement-dependent cytotoxicity tests and blood cross-matching were performed before surgery. The main operative times and partial pressure of arterial oxygen (PaO₂) after opening the pulmonary artery were recorded during surgery. Postoperatively, routine blood tests, biochemical blood indicators and inflammatory factors were detected, and pathological examinations of multiple organs were conducted. Results The complement-dependent cytotoxicity test showed that the survival rate of lymphocytes between donors and recipients was 42.5%-47.3%, and no agglutination reaction occurred in the cross-matching. The first warm ischemia times of D1 and D2 were 17 min and 10 min, respectively, and the cold ischemia times were 246 min and 216 min, respectively. Ultimately, R1 and R2 survived for 1.5 h and 104 h, respectively. Postoperatively, in R1, albumin (ALB) and globulin (GLB) decreased, and alanine aminotransferase increased; in R2, ALB, GLB and aspartate aminotransferase all increased. Urea nitrogen and serum creatinine increased in both recipients. Pathological results showed that in R1, the transplanted lung had partial consolidation with inflammatory cell infiltration, and multiple organs were congested and damaged. In R2, the transplanted lung had severe necrosis with fibrosis, and multiple organs had mild to moderate damage. The expression levels of interleukin-1β and interleukin-6 increased in the transplanted lungs. Conclusions The allogeneic lung transplantation model in miniature pigs may systematically evaluate immunological compatibility, intraoperative function and postoperative organ damage. The data obtained may provide technical references for subsequent lung transplantation research.

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective To compare the hemodynamic characteristics in internal carotid artery models, which were obtained by multi-scale unidirectional and bidirectional coupling models, so as to provide references for selecting models in further studies. Methods Based on the nuclear magnetic resonance image of one patient with mild stenosis of internal carotid artery, the lumped parameter model of the circle of Willis and the three-dimensional model of internal carotid artery were constructed. Those two different multi-scale models were constructed by unidirectional and bidirectional coupling. Results With the increase of stenosis degree, the inlet and outlet blood pressure and the outlet blood flow of internal carotid artery all decreased under two kinds of coupling method. The distribution of low time average wall shear stress (TAWSS) and high oscillatory shear index (OSI) of the internal carotid artery both increased with the increase of stenosis degree under two kinds of coupling method in general. The anterior cerebral artery segment showed lower shear stress and higher OSI with bidirectional coupling in 70% stenosis, and the blood flow direction of posterior communicating artery was changed, which was significantly different from unidirectional coupling results. Conclusions At a low degree of stenosis, the result of those two kinds of coupling method were consistent in general, but there was a significant difference in 70% stenosis, and the result of bidirectional coupling was closer to physiological parameters. The research findings can be better applied to the hemodynamic study of cerebrovascular diseases.

Objective To assess whether asymmetric spin echo version of echo planar imaging (ASE EPI) sequence can reflect cerebral oxygen metabolism by means of the changes in oxygen extraction fraction values before and after surgical operations in the canine model of ischimia. Methods A total of 12 healthy crossbreed dogs were enrolled in this study. All canines underwent cerebral MR imaging including coronal T2WI, DWI, MRA, ASE EPI and 3D PCASL. The bilateral carotid arteries (CA) were separated by surgical operations. The bilateral CA were ligated in 6 dogs, and the other 6 dogs had embolization in ligated bilateral CA. The cerebral MR imaging with the above protocol was repeated at 1 hour after surgical operation for each dog. All dogs were executed when they finished the MR examinations. Then the whole brains of the dogs were taken out for cutting coronal sections and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The ASE EPI data were processed by the Functool software in the workstation to generate OEF maps. For measuring the OEF values before and 1 hour after surgical operations, one and the same region of interest (ROI) was respectively placed in the left and right hemispheres on the OEF maps for each dog. 3D PCASL data were processed by the Functool software in the workstation to yield CBF maps. The method for measuring the CBF values before and 1 hour after surgical operations on CBF maps was same as that for measuring on OEF maps. The changes of measurements before and after surgical operations were compared between the dogs with ligated bilateral CA and the dogs with embolization in ligated bilateral CA by using an independent samples t test. The measurements before and after surgical operations were respectively compared in the dogs with ligated bilateral CA and the dogs with embolization in ligated bilateral CA by using a paired t-test. The cutting slices with TTC staining were analysed for determining ischimia. Results The CBF values of the dogs with ligated bilateral CA were (59.81±23.59) and (38.56± 12.98) ml/(min · 100 g) before and after surgical operations, respectively. The CBF values of the dogs with embolization in ligated bilateral CA were (58.94±18.35) and (28.01±11.41) ml/(min·100 g) before and after surgical operations, respectively. The CBF values were significantly lower before surgical operations than after surgical operations in the dogs with ligated bilateral CA and the dogs with embolization in ligated bilateral CA, respectively (t=8.92 and 13.42, respectively; P=0.00, respectively). The CBF values were decreased more significantly in the dogs with embolization in ligated bilateral CA than in the dogs with ligated bilateral CA (t=2.92, P=0.00). The OEF values of the dogs with ligated bilateral CA were 0.29±0.02 and 0.32 ± 0.06 before and after surgical operations, respectively. The OEF values of the dogs with embolization in ligated bilateral CA were 0.29 ± 0.02 and 0.35 ± 0.06 before and after surgical operations, respectively. The OEF values were significantly higher before surgical operations than after surgical operations in the dogs with ligated bilateral CA and the dogs with embolization in ligated bilateral CA, respectively (t=-7.21 and-4.43, respectively;P=0.00, respectively). The OEF values were increased more significantly in the dogs with embolization in ligated bilateral CA than in the dogs with ligated bilateral CA (t=2.18, P=0.03). The pathological results showed that the dogs with embolization in ligated bilateral CA had swollen cortex with focal infarction which was matched with the hyperintensity areas on the OEF and DWI images. However, the dogs with ligated bilateral CA only had swollen cortex whereas didn't have focal infarction. Conclusions ASE EPI sequence can reliably reflect cerebral oxygen metabolism in ischemia. This sequence has an important role for assessing hemodynamic state in patients with cerebral vascular diseases.