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 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 explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

Objective To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

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.

Aim To investigate the effects of Naotai formula extract(NTE)on the expression of heme oxygenase-1(HO-1) and hephaestin(Heph) in hippocampus of rats after cerebral ischemia/reperfusion by Keap1-Nrf2/ARE signaling pathway.Methods Eighty rats were randomly divided into five groups as follows: sham operation group(Sham), cerebral ischemia/reperfusion group(I/R), low dose group of NTE(4.5 g·kg-1), middle dose group of NTE (9 g·kg-1) and high dose group of NTE(18 g·kg-1).Rats were pretreated by intragastric administration for three consecutive days, and then subjected to middle cerebral artery occlusion (MCAO) 2 hours before reperfusion.The rats were administered with intragastric administration for two days.After cerebral ischemia reperfusion 72 hours, the behavioral activity of rats was recorded by Zea Longa neurological score, and the infarct volume was measured by TTC staining.The expressions of Nrf2, HO-1 and Heph in hippocampus of cerebral ischemia reperfusion rats were observed by real-time quantitative PCR and Western blot, respectively.Results Compared with model group, the neurobehavioral scores significantly decreased in NTE high-dose and middle-dose groups (P<0.01);the infarct volume of NTE groups markedly decreased (P<0.01);the expression of HO-1 mRNA apparently increased (P<0.05) in NTE groups;the expression of Heph mRNA significantly increased in NTE middle-dose and high-dose groups (P<0.05);the expression of Nrf2 and Heph protein evidently increased in the NTE middle and high dose groups (P<0.05, P<0.01);and the expression of HO-1 protein also increased in NTE groups(P<0.01).Conclusions Naotai formula can relieve cerebral ischemia-reperfusion injury.The mechanism might be associated with activating Keap1-Nrf2/ARE signaling pathways, promoting HO-1 generation, advancing the expression of Heph, and then reducing brain iron deposition, to achieve the protection of neurons after cerebral ischemia-reperfusion.

Objective To observe the effects of different compatibility ratios of Astragali Radix and Angelicae Sinensis Radix on vascular intimal hyperplasia; To ascertain the effective compatibility of Astragali Radix and Angelicae Sinensis Radix for antagonizing vascular intimal hyperplasia. Methods SD rats were divided into different groups by baseline geometric design method: Astragali Radix and Angelicae Sinensis Radix different compatibility ratios groups, Astragali Radix group, Angelicae Sinensis Radixgroup, positive medicinegroup and sham-operation group. A model of intimal hyperplasia of thoracoabdominal aorta was established by balloon catheter injury in vascular endothelium of rats. Then the thoracoabdominal aorta was taken out after gavage of Astragali Radix and Angelicae Sinensis Radix with different compatibility ratios for 14 days. Bloodletting was used to take thoracoabdominal aorta. Masson staining and Morphometric methods were used to analyze the intimal hyperplasia. Results IA, IT, HRIA and HRIT increased 14 day after intimal injury. Compared with the model group, IA, IT, HRIA and HRIT in Angelicae Sinensis Radix group, Astragali Radix and Angelicae Sinensis Radix 1:2 group, Astragali Radix and Angelicae Sinensis Radix 1:5 group, Astragali Radix and Angelicae Sinensis Radix 1:1 group and Astragali Radix and Angelicae Sinensis Radix 5:1 group were lower, and the effects of Astragali Radix and Angelicae Sinensis Radix 1:1 ratio were strongest. The effects on intimal hyperplasia in Astragali Radix group and Astragali Radix and Angelicae Sinensis 2:1 group had no significant differences compared with model group. Conclusion Astragali Radix and Angelicae Sinensis can inhibit vascular intimal hyperplasia in a certain compatibility ratios, and the effects of Astragali Radix and Angelicae Sinensis Radix 1:1 on intimal hyperplasia are the best.

Objective Using gradient-echo sampling of spin-echo (GESSE) sequence to study the change of oxygen extraction fraction (OEF) in patients with unilateral cerebral vessel stenosis and the relationship between OEF and cerebral blood flow (CBF). Methods Eight normal volunteers and 16 patients with unilateral cerebral vessel stenosis were enrolled in this study. Written informed consents were obtained from all subjects. Routine MRI, GESSE and arterial spin labeling (ASL) sequences were performed for all patients. Raw data from GESSE and VE-ASL sequences were transferred to PC to conduct postprocessing. To obtain quantitative OEF and CBF of the brain parenchyma, 6 ROIs were placed respectively in the anterior, middle and posterior part of both hemispheres. The relative CBF (rCBF) was defined as the ratio of CBF of ischemic hemisphere to that of contralateral hemisphere. T test was used for statistics. Results The mean value and normal range of OEF in the volunteers were 0. 318 ± 0. 023 and 0. 272-0. 364, respectively. In the 16 patients with unilateral cerebral vessel stenosis, 8 patients had ROIs with greater OEF in unilateral hemisphere than those in contralateral hemisphere. These cases presented multiple intracranial main arterial stenoses in digital subtraction angiography (DSA) or MR angiography (MRA) examination. The other 8 patients had normal OEF in all ROIs. And they only had single arterial stenosis in DSA or MRA. Set rCBF = 0. 50 as a dividing point, the mean OEF value was 0. 397 ±0. 010 in the patients with rCBF ＜ 0. 50. In the patients with rCBF ≥ 0. 5, the mean OEF value was 0. 325 ±0. 028. The difference between the two groups was statistically significant (t = - 8. 840, P = 0. 000).Conclusion Patients with chronic cerebral ischemia may present with various hemodynamic impairment.The more CBF decreases, the more OEF increases. Those with increased OEF tended to have more than one lesion in the major intracranial arteries.

Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.