Objective To investigate the changes of esophageal visceral sence stimulated by esophageal distention in rabbits and the protein expressions of calcitonin gene related peptide(CGRP),P substance(SP),5-hydroxytryptamine(5-HT),Fos protein in central nervous system(CNS).Methods Twenty New Zealand rabbits were randomly divided into experimental group(n=8,received esophageal distention with 0.9 cm balloon for 30 s twice a day for 14 days),control group(n=6,received esophageal stimulation without balloon for 30 s twice a day for 14 days)and blank control grouop(n=6).The esophageal visceral sense was evaluated by animal behavior scores.The expressions of SP,CGRP,5-HT and Fos protein esophagus mucosa,spine,nucleus tractus solitari (NTS),periaqueductal gray(PAG)and thalamus were measured by immunochistochemistry.Results At the same behavior scores,the tube diameter of experimental group was significant lower than those of control group(P＜0.05).The expression of SP in esophagus mucosa,spine and NTS was significant increased in experimental group compared to two control groups(P＜0.05).The expression of CGRP and Fos in esophagus mucosa.spine,NTS,PAG and thalamus was increased in experimental group compared to two control groups(P＜0.05).The expression of 5-HT in esophagus mucosa and spine was higher in experimental group than that in control and blank control groups (esophagus: 27.67±3.27 vs 11.00±1.79 or 11. 17±1.33;spine:24.00±5.22 vs 11.33±2.94 or 11. 83±2. 48, P＜0. 01). But the expression of 5-HT in PAG was lower in experimental group( 13. 17±2.04) than that in control 17.67±2.07)and blank control (16.83±2.32) groups (P＜0. 05). There was significant correlation between CGRP and Fos, SP and Fos, CGRP and SP in spine (r=0. 813,0. 779,0. 772,P=0. 025,0.034, 0. 036, respectively). Conclusions Esophageal hypersensitivity may be induced by esophageal distention. The expresstion of SP, CGRP, 5-HT was increased in the esophageal mueosa and CNS, which indicate that these neurotransmitters and CNS may play an important role in the increase of esophageal visceral sense.

Objective:To investigate the protective effect of hypothermic antegrade machine perfusion against canine ischemic brain injury.Methods:Thirteen beagle dogs were divided into the mild hypothermia with perfusion group ( n=6) and normothermia with perfusion group ( n=7) according to the random number table. The model of ischemic brain injury was established by neck transection. After 1 hour of ischemic circulatory arrest, the perfusion fluid based on autologous blood was continuously perfused through bilateral common carotid artery for 6 hours. The temperature of the perfusion fluid was set at 33 ℃ in the mild hypothermia with perfusion group and 37℃ in the normothermia with perfusion group, respectively. Blood oxygen saturation was recorded at 0, 1, 2, 3, 4, 5 and 6 hours after the beginning of perfusion to evaluate the perfusate oxygen level. The perfusate was collected, and the levels of Na +, K +, Ca 2+ and glucose as well as the pH value of the perfusate were detected in the two groups. At the end of perfusion, the parietal brain tissues of 1 dog from each group were collected to evaluate the water contents of brain tissues. Nissl staining was used to evaluate the morphological integrity of the pyramidal neurons in the frontal cortex and hippocampus. Neuronal nuclei antigen (NeuN) was used to evaluate the structural and morphological integrity of pyramidal neurons. Immunofluorescence glial fibrillary acidic protein (GFAP) and ionic calcium binding adaptor molecule 1 (Iba1) were used to evaluate the integrity and activity of astrocytes and microglia fragments. Results:At 0, 1, 2, 3, 4, 5 and 6 hours of perfusion, there was no significant difference in the blood oxygen saturation or Na + concentrations between the two groups (all P>0.05); the K + concentrations in the mild hypothermia with perfusion group were (4.57±0.12)mmol/L, (4.67±0.14)mmol/L, (4.27±0.12)mmol/L, (4.45±0.10)mmol/L, (6.60±0.15)mmol/L, (7.37±0.18)mmol/L and (9.03±0.16)mmol/L, respectively, which were significantly lower than those in the normothermia with perfusion group [(4.84±0.10)mmol/L, (5.31±0.13)mmol/L, (5.44±0.24)mmol/L, (5.70±0.18)mmol/L, (7.79±0.18)mmol/L, (10.44±0.40)mmol/L, (10.40±0.41)mmol/L] (all P<0.01). At 0, 1, 2 and 3 hours of perfusion, the Ca 2+ concentrations in the mild hypothermia with perfusion group were (0.72±0.15)mmol/L, (1.55±0.16)mmol/L, (1.62±0.15)mmol/L and (1.88±0.15)mmol/L, respectively, being significantly higher than those in the normothermia with perfusion group [(0.41±0.13)mmol/L, (0.99±0.12)mmol/L, (1.29±0.13)mmol/L, (1.57±0.11)mmol/L] (all P<0.01), and no significant differences were found at other time points (all P>0.05). At 0, 1 and 2 hours of perfusion, the glucose concentrations in the mild hypothermia with perfusion group were (5.75±0.19)mmol/L, (5.17±0.15)mmol/L and (4.72±0.15)mmol/L, respectively, being significantly higher than those in the normothermia with perfusion group [(5.30±0.22)mmol/L, (4.89±0.20)mmol/L, (4.30±0.17)mmol/L] (all P<0.01), with no significant differences found at other time points (all P>0.05). At 2, 3, 4, 5 and 6 hours of perfusion, the pH values of the mild hypothermia with perfusion group were 7.32±0.06, 7.25±0.02, 7.23±0.02, 7.24±0.02 and 7.24±0.02, respectively, being significantly higher than those in the normothermia with perfusion group (7.26±0.01, 7.21±0.01, 7.17±0.02, 7.15±0.02, 7.08±0.02) ( P<0.05 or 0.01), with no significant differences at other time points (all P>0.05). The water content of brain tissues in the mild hypothermia with perfusion group was (74.9±0.4)%, which was significantly lower than (79.9±0.9)% in the normothermia with perfusion group ( P<0.01). Nissl staining showed that the pyramidal neurons in prefrontal cortex and dentate gyrus had good integrity in the mild hypothermia with perfusion group. NeuN immunofluorescence staining showed that the morphology and structure of pyramidal neuron cells in the mild hypothermia with perfusion group were better with clearly visible axons than those in the normothermia with perfusion group, whereas the cytosol was full and swollen with scarce axons in the normothermia with perfusion group. GFAP and Iba1 immunofluorescence staining showed that more structurally intact glial cells, more abnormally active cells, thickener axons and better axon integrity in all directions were found in the mild hypothermia with perfusion group than those in the normothermia with perfusion group. Conclusion:Compared with normal temperature antegrade mechanical perfusion, the mild hypothermia antegrade mechanical perfusion can protect canine brain tissue and alleviate ischemic brain injury by maintaining stable energy and oxygen supply, balancing ion homeostasis and perfusion fluid pH value, reducing tissue edema, and maintaining low metabolism of pyramidal neurons, astrocytes and microglia.

Objective To evaluate the preservation effect of normothermic mechanical perfusion with red blood cells from humanized genetically modified pig on severed human limbs. Methods Severed human limbs were perfused with red blood cells from humanized genetically modified pigs for 6 h. Perfusion solution was taken every hour to measure the oxygen partial pressure, Na⁺, K⁺, Ca²⁺, pH value, glucose, lactic acid and creatine kinase levels. Superficial flexor muscle was sampled to detect the changes of tumor necrosis factor (TNF)-α, interleukin (IL)-2 and IL-1 levels. At 0 and 6 h after perfusion, the superficial flexor muscles of the forearm were taken for pathological examination. Intercellular space and glycogen consumption of skeletal muscles were observed. An appropriate amount of forearm vessels was collected every 2 h to detect the apoptosis of vascular endothelial cells. X-ray angiography was performed before perfusion and 6 h after perfusion to observe the filling degree of finger-tip peripheral vessels. Results The oxygen partial pressure was observed in the normal range throughout the perfusion. Na⁺ concentration peaked at 1 h, reaching 138.7 mmol/L, and then fluctuated within the normal range. K⁺ level peaked at 2 h up to 6.08 mmol/L, then decreased and fluctuated within the normal range. Ca²⁺ concentration reached the peak at 4 h, up to 1.03 mmol/L. Glucose level was gradually decreased at the beginning of perfusion, reaching the lowest value of 17.7 mmol/L at 2 h after perfusion, and then maintained a dynamic balance. The pH value was decreased to 7.28 at 6 h after perfusion. The lactic acid level was increased to 9.6 mmol/L at 1 h after perfusion, and then gradually decreased. The creatine kinase level was increased at the start of perfusion, reached the peak at 2 h up to 20 030 U/L, then decreased and remained stable at the end of perfusion. At the end of perfusion, the morphology of muscle fibers was normal, the gap among muscle fibers was expanded slightly, and the glycogen of skeletal muscles was not significantly accumulated. At 0 h perfusion, the number of apoptotic cells in vascular endothelial cells was large, which was declined at 6 h perfusion. Evident vascular filling was observed at 0 h, and the filling degree of some finger-tip vessels was decreased at 6 h. Conclusions Normothermic mechanical perfusion of severed human limbs with red blood cells from humanized genetically modified pigs may continuously and stably supply energy and oxygen, adjust the ion pH balance of perfusion solution, maintain normal cellular metabolism and exerts certain protective effect upon severed human limbs.

Objective To investigate the establishment of a six-gene-edited pig-to-non-human primate kidney xenotransplantation model. Methods The kidney of humanized genetically-edited pig (GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM) was transplanted into a cynomolgus monkey. The survival of the recipient and kidney condition after blood perfusion were observed. The parenchymal echo, blood flow changes, and size of the kidney were monitored on a regular basis. Routine blood test, kidney function test and electrolyte assessment were carried out. Dynamic changes of urine, feces and body mass were monitored. At the end of life, the transplant kidney, heart, liver, spleen, lung, and cecum were collected for pathological examination. Results The recipient died at postoperative 7 d. After blood flow was restored, the kidney was properly perfused, the organ was soft and the color was normal. At the end of the recipient's life, a slight amount of purulent secretion was attached to the ventral side of the kidney, with evident congestion and swelling, showing the appearance of "red kidney". Postoperatively, the echo of renal parenchyma was increased, blood flow was decreased, the cortex was gradually thickened, and a slight amount of effusion surrounded the kidney and abdominal cavity over time. In the recipient, the amount of peripheral red blood cells, hemoglobin, albumin, and platelets was progressively decreased, and serum creatinine level was increased to 308 μmol/L at postoperative 7 d, whereas the K⁺ concentration did not significantly change. Light yellow urine was discharged immediately after surgery, diet and drinking water were resumed within postoperative 3 h, and light yellow and normal-shape stool was discharged. The reddish urine was gradually restored to normal color within postoperative 1 d, which were consistent with the results of the routine urine test. A large amount of brown bloody stool was discharged twice in the morning of 2 d after surgery. Omeprazole was given for acid suppression, and the stool returned to normal at postoperative 4 d. The β₂-microglobulin level was increased to 0.75 mg/L at postoperative 7 d. The body mass was increased by 1.7 kg. Autopsy pathological examination showed interstitial edema and bleeding of the transplant kidney, a large amount of infiltration of lymphocytes and macrophages, infiltration of lymphocytes in the arteriole wall and arterial cavity, accompanied by arteritis changes, lymphocyte infiltration in the cecal stroma and congestion in the spleen tissues. No significant abnormal changes were observed in other organs. Conclusions The humanized genetically-edited pig-to-non-human primate kidney xenotransplantation model is successfully established, and postoperative survival of the recipient is 1 week.

Objective To investigate the differences and the immunocompatibility of wild-type (WT), four-gene modified (TKO/hCD55) and six-gene modified (TKO/hCD55/hCD46/hTBM) pig erythrocytes with human serum. Methods The blood samples were collected from 20 volunteers with different blood groups. WT, TKO/hCD55, TKO/hCD55/hCD46/hTBM pig erythrocytes, ABO-compatible (ABO-C) and ABO-incompatible (ABO-I) human erythrocytes were exposed to human serum of different blood groups, respectively. The blood agglutination and antigen-antibody binding levels (IgG, IgM) and complement-dependent cytotoxicity were detected. The immunocompatibility of two types of genetically modified pig erythrocytes with human serum was evaluated. Results No significant blood agglutination was observed in the ABO-C group. The blood agglutination levels in the WT and ABO-I groups were higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (all P<0.001). The level of erythrocyte lysis in the WT group was higher than those in the ABO-C, TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups. The level of erythrocyte lysis in the ABO-I group was higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (both P<0.01). The pig erythrocyte binding level with IgM and IgG in the TKO/hCD55 group was lower than those in the WT and ABO-I groups. The pig erythrocyte binding level with IgG and IgM in the TKO/hCD55/hCD46/hTBM group was lower than that in the WT group and pig erythrocyte binding level with IgG was lower than that in the ABO-I group (all P<0.05). Conclusions The immunocompatibility of genetically modified pig erythrocytes is better than that of wild-type pigs and close to that of ABO-C pigs. Humanized pig erythrocytes may be considered as a blood source when blood sources are extremely scarce.

Although blood banks based on human blood can provide blood transfusions for the wounded timely and effectively, scientific research has never given up on finding new blood sources due to the restrictions of human blood sources. With the application of transgenic technology and the successful breeding of gene-edited pigs, gene-edited pig blood as a potential source of clinical transfusion has attracted wide attention. Now there are preclinical studies showing the feasibility of transfusing gene-edited pig red blood cells into primates. This paper discusses the related research and future development of xenogeneic transfusion of porcine red blood cells by gene editing.