Establishment of an acute renal failure animal model by renal transitory ischemia

AIM: To establish an animal model of acute renal failure (ARF) according to the clinical manifestations by reducing renal blood flow.METHODS: Renal ischemia in rabbits was induced by the ligation of bilateral renal arteries incompletely for 20 min and then the ligation was released. Before and after incomplete ligation of bilateral renal arteries, and after resuming renal blood flow, the urine volume, serum creatinine, electrolytes and blood gas analysis in the experimental animals were determined. One kidney of each rabbit was harvested at different stages for pathological examination.RESULTS: After reducing renal blood flow, the urine volume of the rabbit decreased (P<0.05) and the serum levels of creatinine increased significantly (P<0.01). At the same time, the level of serum K⁺ appeared an elevated trend. pH and HCO^-₃ in the arterial blood decreased, and the negative value of BE increased markedly, indicating that metabolic acidosis developed. After resuming the kidney perfusion, the urine volume of the rabbits increased to normal, the level of serum creatinine also decreased to normal as the renal reflow recovery for 1 h, but metabolic acidosis remained continuously. The level of serum K⁺ decreased for a while and elevated again. No obvious pathological changes of the kidney were observed under microscope at the stage of ischemia for 20 min, reflow for 1 h and 5 h.CONCLUSION: Incomplete ligation of bilateral renal arteries develops multiple clinical manifestations and characteristics of functional ARF. This animal model of ARF is helpful for relevant purposes of teaching and research.     

Effects of glucosidorum tripterygii tororum on cytokine productions in graft-versus-host disease mice

AIM:To observe the effect of glucosidorum tripterygii tororum (GTT) on cytokine productions in acute graft-versus-host disease (aGVHD) mice. METHODS: C₅₇BL/6 mice were exposed to radiation delivered by a linear accelerator. To establish a aGVHD model, the cell suspensions, which were obtained from bone marrow and spleen of the BALB/C mice, were transplanted to the radiated C₅₇BL/6 mice. The recipients were treated with GTT, GTT+CsA and CsA+MTX. The serum concentrations of IL-2, TNF-α, IL-4 and IL-10 were determined by ELISA. RESULTS:The survival rate on day 11 in GTT group (9/10) was higher than in allogeneic bone marrow transplatation (allo-BMT) group (8/19). The concentrations of IL-2 and TNF-α in GTT group were significantly lower, but the concentration of IL-10 was remarkably higher than that in allo-BMT group (P＜0.05). However, the concentration of IL-4 showed no changed in all groups (P＞0.05). CONCLUSION:GTT inhibited aGVHD development by regulating the production of cytokines in the host.     

An animal model of acute lung injury induced by left ventricular ischemia-reperfusion

AIM: To establish a method of making acute lung injury model induced by left ventricular ischemia-reperfusion.METHODS: Forty New Zealand rabbits were randomly divided into model group (MG) and control group (CG). The left ventricular ischemia-reperfusion was established by ligaturing and loosing the anterior descending branch of the left coronary artery in the rabbits in MG group. The electrocardiogram, the phrenic nerve discharge curve, the ultrastructure and histological analysis of the lung tissues were compared between MG group and CG group.RESULTS: The myocardial ischemia-reperfusion injury resulted in acute lung injury in the rabbits of MG group, and the duration and amplitude of the phrenic nerve discharge curve were reduced. The ultrastructure and histological analysis of the lung tissues in MG group showed acute injury. These situations were not appeared in CG group. The correlation between myocardial ischemia-reperfusion injury and acute lung injury were significant in MG group. CONCLUSION: The animal model of acute lung injury induced by left ventricular ischemia-reperfusion is reliable and feasible.     

Establishment of insulinoma animal models and observation of tumor characteristics

AIM：To establish the animal model of insulinoma and to analyze the properties of insulinoma for further study. METHODS：The hormone-releasing ability of rat insulinoma cell line INS-1 was detected in vitro. INS-1 cells were transplanted into the left kidney capsule of nude mice. The islets of the animals were destroyed by intraperitoneal injection of streptozocin (STZ) 3 d before transplantation or 2 weeks after transplantation. The venous blood was sampled, and the level of blood glucose less than 2.8 mmol/L was defined as successful establishment of insulinoma model. Different irritants were given to the model animals, and the changes of blood glucose and insulin content in serum were observed. The pancreatic tissues and the renal tissues in the injecting sites were taken from all mice for detecting insulin and glucagon by immunohistochemical staining. RESULTS：Insulinoma cells expressed insulin and glucagon at the same time. The blood glucose was less than 2.8 mmol/L 3 to 4 weeks after inoculation of INS-1 cells. Apparent tumor formed in the left kidneys where INS-1 cells were transplanted and the tumor diameters were more than 1 cm. The level of blood glucose transiently increased to higher than the normal level in the mice with tumor cell transplantation after intraperitoneal injection of STZ, and then decreased gradually and returned to less than 2.8 mmol/L after 2 weeks. The level of blood glucose in the normal nude mice after administration of STZ increased significantly. After transplantation of INS-1 cells, the level of blood glucose decreased gradually, and returned to less than 2.8 mmol/L after 4 weeks. After stimulated with high glucose, the blood glucose levels in the mice with 3 methods to establish the insulinoma models showed lower glucose peaks than that in the normal control mice. After stimulated with high glucose plus arginine or acetylcholine in the normal animals, the blood glucose peak was lower than that in the normal animals only stimulated with high glucose, and rapidly recovered to the normal level. However, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models under the same stimulations were significantly higher than that in the mice only stimulated with high glucose. After stimulated with high glucose plus norepinephrine, the blood glucose peak time in the normal animals delayed, and the blood glucose level declined slowly. After stimulated with high glucose plus norepinephrine, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models increased as compared with that in the mice only stimulated with high glucose. Compared with normal control group, serum insulin in insulinoma mice increased significantly. CONCLUSION：The insulinoma animal model is successfully established by transplantation of INS-1 cells into the renal capsule of nude mice. The insulinoma cells express both insulin and glucagon, and are not easily damaged by STZ.     

Activation of Akt signal pathway cascades in kidney tissue in murine chronic graft-versus-host disease lupus nephritis and its regulation by prednisone

AIM:To examine whether Akt signal pathway proteins, including Akt, NF-κB and IκBα, are activated in kidney tissue of murine chronic graft-versus-host disease (GvHD) lupus nephritis in vivo, and whether prednisone suppresses activation of them.METHODS:Akt activity and phosphorylated IκBα were detected by Western-blot. Activation of NF-κB was detected by electropheretic mobility shift assay (EMSA). RESULTS:Activity of Akt, NF-κB and phosphorylated IκBα were significantly increased in kidney tissue of murine chronic graft-versus-host disease (GvHD) in 8th week and 12th week after monocell injection, respectively. However, they were no significant elevation in 16th week, when compared with controls. Prednisone treatment significantly prevented the increase in serum anti-dsDNA antibody level, urinary protein excretion and glomerular cell proliferation in GvHD mice, indicating the beneficial effects of prednisone on this model. Prednisone also significantly suppressed the increase in the activities of glomerular Akt, NF-κB and phosphorylated IκBα. CONCLUSION:This study provides the first evidence of marked increase in glomerular Akt-NF-κB signal pathway activities in murine chronic graft-versus-host disease lupus nephritis. The beneficial effect of prednisone on this lupus nephritis model may be partially mediated by the suppression of abnormal Akt- NF-κB activation.     

Establishment of intrauterine hypoxic-ischemic brain damage model in near term fetal rabbits

AIM: To establish intrauterine hypoxic-ischemic brain damage (HIBD) model in near term fetal rabbits at 29 d gestation age for the investigation of the pathogenesis and treatment of newborn HIBD. METHODS: Twenty-four pregnant New Zealand white rabbits at 29th gestational day were chosen for this project. Under combined general anesthesia and spinal anesthesia, a 4F Fogarty arterial embolectomy catheter was introduced into the left femoral artery. The blood supply of uterus in experiment group was blocked by inflating the catheter balloon with 0.3 mL saline for 20 min, 25 min, 28 min, 30 min and 40 min (n=4 for each experimental time group). The catheter balloon was not inflated in control group (n=4). All pregnant rabbits were subject to cesarean section 24 h after the experimental procedure to induce hypoxia-ischemia to the fetus. The general conditions of the newborn rabbits were recorded, and the neurobehavioral damage and histology of the brain tissue were assessed. RESULTS: During the entire procedure, the pregnant rabbits had stable vital signs, no hypoxia happened,and had a good tolerance to the anesthesia program. When the balloon was inflated, the pulses of right femoral artery disappeared and the right leg blood pressure became non-detectable in experimental groups. In contrast, no fluctuation of the right leg blood pressure in control group (P>0.05) was observed. Intrauterine hypoxia-ischemia caused neonatal and fetal rabbit death, neurobehavioral damage and brain cell death. When the balloon was inflated for 20 min, all fetal rabbits were alive and had no obvious neurologic damage. For 25~28 min, the stillbirth rates were 12.9% and 40.6%, respectively, while the live neonatal rabbits manifested neurobehavioral damage, edema neural cells, activated microglia cells and apoptotic brain cells. When blocking time beyond 30 min, above 80% fetal rabbits died. CONCLUSION: Continuous blockage of uterine blood supply in pregnant rabbits causes neonatal rabbit death, neurobehavioral damage and brain cell death. Different blocking time arouses different levels of brain damage. Continuous blockage of uterine blood supply for 25-28 min can establish fetal generalize hypoxic-ischemic brain damage rabbit model, which is a good animal model for the investigation of newborn HIBD.     

Establishment and evaluation of a hyperbilirubinemia and kernicterus model in neonatal rats

AIM：To establish and evaluate a hyperbilirubinemia and kernicterus model in neonatal SD rats. METHODS：Three-day-old SD rats were randomly divided to 7 experimental groups by litter and body weight, and were intraperitoneally injected with physiological saline (control group), and 6.25 μg/g (T1), 12.5 μg/g (T2), 25 μg/g (T3), 50 μg/g (T4), 100 μg/g (T5) and 200 μg/g (T6) bilirubin, respectively, twice every day for 3 d. All rats were photographed, weighed and killed 12 h after the last injection. The contents of the stomach were drawn and weighed, and the index was calculated. The liver/body weight ratio was determined, the total and unconjugated bilirubin in the serum and total bilirubin in the brain were calculated, and the contents of ATP and water in the brain were measured. HE and Nissl staining were used to observe the pathological changes. RESULTS：Along with the increase in bilirubin, gradual exacerbation of the general performance of the rats, and yellowish discoloration of the skin and mucous membranes were observed.The degree of the activity gradually reduced, and the weight gain was suppressed. The weight of T6 group showed negative growth, and the 72 h mortality rate was close to 100%. The mortality rate in T4 and T5 groups continued to rise 1 week after injection. Compared with control group, the weight of stomach contents and stomach content index in T3~T5 groups significantly decreased (P<0.01). The liver/body weight ratio in T5 group was significantly higher (P<0.05). The concentrations of serum total and unconjugated bilirubin and brain bilirubin levels in T1~T5 groups were gradually increased, while the brain water content had no difference among groups. The brain ATP content in T1~T5 groups increased at the beginning and reached its peak in T3 group, but compared with control group, that in T4 group and T5 group significantly reduced (P<0.05). HE results showed that, with the increase in bilirubin concentration, the number of the neurons in the cerebral cortex of the rats decreased. In T4 group and T5 group, the neuronal structural disorder, cell swelling, nuclear pyknosis, fragmentation and dissolution, increase in non-homogeneous structure of the material dyed red, and disappearance of nuclear staining were observed. Nissl staining showed that, compared with control group, in T1 group and T2 group, the cortical neurons became smaller, Nissl bodies decreased, and cytoplasmic staining changed little. The cortical neuronal tigroid body color became light gradually, neuron cells become small, and Nissl bodies decreased obviously in T3, T4 and T5 groups. The T4 and T5 rat ce-rebral cortical neurons dissolved or even disappeared. CONCLUSION：Newborn 3-day-old SD rats receiving intraperitoneal injection of bilirubin at doses of 12.5, 25, 50 and 100 μg/g, 2 times a day, can induce hyperbilirubinemia, and 50 and 100 μg/g can cause bilirubin encephalopathy.     

Function of dendritic cells in cord blood

AIM: To explore the function of dendritic cells in cord blood.METHODS: Dendritic cell precursor subsets pDC/pDC(CD11c+CD123- /CD11c-CD123+) in cord blood and adult peripheral blood were analyzed gated by CD34-Lin- HLA-DR+ cells and the levels of IL-12p40,IL-10,IFN-γ,IL-4 in the serums were tested by ELISA.RESULTS: The level of IL-12p40 in cord blood serum was higher than that in peripheral blood.pDC1/pDC2,IL-10,IFN-γ,IL-4 in cord blood were similar to that in peripheral blood.CONCLUSION: Function development of dendritic cells in cord blood may be consummate.     

Effect of G-CSF on dendritic cell subsets in donors mobilized peripheral blood cells

AIM: To explore the effect of granulocyte colony stimulating factor (G-CSF) on donor’s dendritic cell （DC）subsets in peripheral blood cells (PBC). METHODS: The subsets of dendritic cells in PBC were analyzed by CD34^-Lin^- HLA-DR⁺ cells and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in the serum were tested by ELISA before (25 cases) or after G-CSF mobiling. The relationship between the ratio of DC₁/DC₂ (CD11c⁺CD123^-/CD11c^-CD123⁺) and CD34⁺/MNC was explored. RESULTS: CD34⁺/MNC in PBC harvests was above 0.4% in 23 cases, and the ratio of DC₁/DC₂ was lower, the HLA-DR expression of DC₂ was enhanced after G-CSF mobiling than before, but the levels of IL-12p40, IL-10, IFN-γ, IL-4 in donors serum and CD83 expression of DC₂ were not changed by G-CSF. CONCLUSION: DC₂ increased accompanied by the increase in CD34⁺ cells in the PBC harvests. Although the expression of HLA-DR in DC₂ was up-regulated by G-CSF, these DC₂ did not regulate Th2 cells to excrete inhibitor cell factors and kept on the precursor characters.     

Establishment of a human chronic myeloid leukemia-like mouse model

AIM：To establish a new model of chronic myeloid leukemia (CML) for further studying the pathogenesis mechanisms and discovering new therapeutic targets of this disease. METHODS：The retroviral packaging technique was improved by increasing retroviral titer with higher-quality plasmid, better cell state and appropriate cell seeding density for further studying. Donor BALB/c mice were pretreated with 5-fluorouracil (5-FU), and their bone marrow cells were infected with p210 BCR/ABL retroviral supernatant (BCR/ABL group) or empty retroviral vector (MigR1) supernatant (MigR1 group). Infected bone marrow cells were transplanted into lethally irradiated recipient BALB/c mice via tail vein. The activities of the mice were observed after bone marrow transplantation (BMT). The morphology of peripheral blood cells and bone marrow cells, and the pathological changes of the liver and spleen in dying mice were also determined. RESULTS：Retroviral packaging efficiency was improved by optimizing the experimental conditions with higher-quality plasmid, better cell state and appropriate cell seeding density. All mice in BCR/ABL group died at 19 to 25 d after BMT. Their myelocytes, metamyelocytes and mature granulocytes in peripheral blood and bone marrow were abundant. Hepatosplenomegaly and granulocyte infiltration in the liver and spleen were also observed. All mice in MigR1 group were normal. CONCLUSION: We have successfully set up a mouse model of CML by increasing retroviral titer with improved retroviral packaging technique, transfecting BCR/ABL into mouse bone marrow cells in vitro and transplanting the cells to the recipients of the same lineage.     

Role of NK cells in mouse allogeneic bone marrow transplantation

AIM: To study the effect of natural killer(NK)-cells on graft-versus-host disease(GVHD), graft rejection, engraftment and reconstituting of hematopoiesis in mouse allogeneic bone marrow transplantation. METHODS: Lethally irradiated BALB/c(H-2^d) mice were transplanted with C57/6j(H-2^b) bone marrow containing donor peripheral T cells and/or NK cells. Recipients CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were detected by flow cytometry, peripheral white blood cell(WBC) was detected by auto-cytometry, and the recipients survival rates, GVHD, engraftment and hematopoiesis recovery were then observed. RESULTS: In the group of transplanted with NK cells infusion, the incidence of GVHD was evidently lessened, the survival rates, WBC and CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were significantly high than that without NK cells infusion. CONCLUSION: In mouse allogeneic bone marrow transplantation, alloreactivity NK cells prevents GVHD, reduces graft rejection, and promotes engraftment and reconstituting of hematopoiesis.     

Establishment of human pancreatic tumor xenograft mouse model for evaluating tumor-homing and gene-silencing effects of siRNA-loading nanoparticles

AIM：To establish an effective and rapid method to develop transplanted subcutaneous pancreatic carcinoma by inducing PANC-1 cells into nude mice, and then use this mouse model to evaluate the tumor-homing and gene-silencing effects of siRNA-loading nanoparticles in vivo. METHODS：Different numbers of PANC-1 cells in 100 μL or 300 μL PBS were inoculated subcutaneously into the right flank of BALB/c (nu/nu) mice. When the tumor volume reached 100 mm3, siRNACY5.5 nanoparticles were injected through the mouse tail vein to perform in vivo imaging assay. Besides, the mice were randomly divided into 3 treatment groups treated with PBS, scrambled control RNA nanoparticles and siKras nanoparticles, respectively. The protein expression of Kras was detected by Western blotting and immunohistochemical staining. RESULTS：After inoculated with 1×107 PANC-1 cells in 300 μL PBS, all mice developed tumors within 2 weeks. The in vivo results showed that siRNA-loading nanoparticles accumulated in the tumor tissues and exerted gene silencing effect. CONCLUSION:In the present study, an effective and rapid method was established for PANC-1 cells to induce transplanted subcutaneous pancreatic carcinoma in nude mice within 2 weeks, which is suitable for in vivo imaging and treatment evaluations as a reproducible and reliable way for the further experiments.