Study on the damage of auditory path of the experimental autoimmune encephalomyelitis

AIM: To explore if there is lesion in the auditory path in the brainstem of the Wistar rat with experimental autoimmune encephalomyelitis（EAE）.METHODS: EAE was induced by guinea pig spinal cord homogenate (GPSCH),then the evoked potential in both EAE and control groups was examined.The HE staining and myelin staining of the auditory nucleus were conducted in order to corroborate the damage of the auditory path in the brainstem.Through studying the pathological changes using light microscopy and behavior changes of rats,we defined that the model was successful.RESULTS: The latency period of auditory evoked potentials of EAE rats was much longer than that in control group.The pathological pictures manifested that auditory nucleus were invaded by a great number of lymphocytes,demyelinations were discovered in it.CONCLUSION: The auditory path in the brainstem of the Wistar rat with EAE is suffered from inflammation and demyelination.     

Immunoregulatory effect of Buyang-Huanwu decoction on monocyte-macrophages in mice with experimental autoimmune encephalomyelitis

AIM: To explore the therapeutic effect of Buyang-Huanwu decoction (BYHWD) on experimental autoimmune encephalomyelitis (EAE) and its immunoregulatory effect on monocyte-macrophages.METHODS: Chronic EAE was induced by myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG_35-55) in the female C57BL/6 mice, which were randomly divided into saline group and BYHWD group. On day 3 after immunization, the mice in BYHWD group were orally administrated with BYHWD, while normal saline was given to the control mice. The clinical score and body mass were recorded every other day. At day 17 after immunization, the mice were sacrificed and spinal cords were obtained for HE staining and myelin staining. The M1 and M2 macrophage phenotypes of splenic cells were detected by flow cytometry and immunofluorescence staining. The protein expression of iNOS, TNF-α, arginase and IL-10 in the spinal cord macrophages was determined by Western blotting.RESULTS: BYHWD delayed the onset of EAE, reduced the clinical scores of EAE, inhibited the inflammatory cell infiltration and demyelination in the spinal cord, and promoted the conversion of M1 macrophages into M2 phenotype in the spinal cord and spleen.CONCLUSION: BYHWD intervention attenuates the behavioral and pathological changes in the EAE mice, and its mechanism may be related to the macrophage conversion.     

Effects of PPARγ agonist on calpain expression in model of acute experimental autoimmune encephalomyelitis

AIM: To explore the effects of peroxisome proliferator-activated receptor γ （PPARγ） agonist on calcium-activated neutral proteinase, calpain, expression in the brain of rats with acute experimental autoimmune encephalomyelitis (EAE). METHODS: EAE model was established in rats by inoculating the homogenate contained spinal cord of guinea pig and complete Freunds adjuvant. Respectively, the PPARγ agonist rosiglitazone and non-steroid anti-inflammatory drug (NSAID) ibuprofen were used to treat the EAE rats. Outcome measures (Kohs scale) were applied at baseline and after treatment to assess the improvement of clinical symptoms. Calpain expression levels were detected by RT-PCR and Western blotting. RESULTS: All the groups showed significant improvements of scales scores after treatment with rosiglitazone and ibuprofen. No significant difference of the expression of calpain mRNA was found among EAE group, rosiglitazone and ibuprofen groups (P>0.05), but the expression of calpain reduced markedly in rosiglitazone and ibuprofen groups compared with that in EAE group (P<0.05). CONCLUSION: Rosiglitazone and ibuprofen inhibit the expression of calpain and improve the clinical symptoms of EAE rats. PPARγ agonist plays a neuroprotective role in EAE rats.     

Oral administration of fasudil hydrochloride suppresses development of experimental autoimmune encephalomyelitis in mice

AIM:To explore the therapeutic effect of fasudil hydrochloride by the oral route on the development of experimental autoimmune encephalomyelitis (EAE) in mice and its possible mechanism. METHODS:The EAE model in female C57BL/6 mice was established by myelin oligodendrocyte glycoprotein 35-55 peptide(MOG35-55) immunization and the immunized mice were randomly divided into saline control group and fasudil intervention group, in which saline and fasudil were administered by the oral route once every day from post-immunization (PI) day 3 to day 27. Clinical score and body weight were recorded every other day. On PI day 28, the spinal cords were obtained for HE and myelin staining. The splenocytes were isolated and the expression of CD16/32, CD206 and interleukin (IL)-10 was analyzed by flow cytometry. The levels of IL-1β and tumor necrosis factor α (TNF-α) were detected by ELISA. RESULTS:Oral administration of fasudil delayed the onset of EAE, and attenuated the myelinoclasis of the model animals and the severity of EAE, accompanied by the phenotypic switch from M1 to M2 macrophages, the inhibition of the proinflammatory cytokine (IL-1β and TNF-α) production and the increase in IL-10 release. CONCLUSION: Oral administration of fasudil exhibits therapeutic effect on the development of EAE possibly through switching M1 macrophages to M2 phenotype and inhibiting inflammatory responses in mice.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis

AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH₂O) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining. RESULTS: In comparison with ddH₂O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01). CONCLUSION: Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect.     

Effect of deafferentation on calretinin of adult rat olfactory bulb

AIM: To investigate the effect of deafferentation on the expression level of calretinin (CR) in adult Sprague Dawley rat olfactory bulb (OB). METHODS: Adult Sprague Dawley rats were given a single intranasal irrigation with ZnSO4. A specific period (10, 20, 30 and 60 days, respectively) after the treatment, immunoreactivities of tyrosine hydroxylase (TH) and calretinin of OB were examined by using immunohistochemistry. Down-regulation of TH expression level was regarded as a mark of deafferentation. RESULTS: 10, 20, 30 and 60 days after ZnSO4 treatment, staining intensity and density of TH-immunoreactive cells of OB significantly reduced and the degree of reduction was correlated with the time of treatment before 30 days, but the result of 60 d was similar to that of 30 d. There was no obvious change in the density of CR-immunopositive cells after the same treatment. CONCLUSION: Expression of CR in adult rat OB was not affected by deafferentation.     

Effect of recombinant plasmids encoding IL-1RII and IL-1RAcP on rat experimental autoimmune myocarditis and possible mechanism

AIM To evaluate the effects of recombinant plasmids encoding interleukin-1 type II receptor （IL-1RII） and interleukin-1 receptor accessory protein （IL-1RAcP） on rat experimental autoimmune myocarditis （EAM） and the possible mechanism. METHODS The recombinant plasmids pCAGGS-IL-1RII and pCAGGS-IL-1RAcP were constructed, and pCAGGS-SP （signal peptide） served as the control plasmid. Male Lewis rats （n=29） were divided into 4 groups： control group （rats without immunization or injection, n=5）, EAM+SP group （immunized rats injected with pCAGGS-SP, n=9）, EAM+IL-1RII group （immunized rats injected with pCAGGS-IL-1RII, n=8） and EAM+IL-1RII+IL-1RAcP group （immunized rats injected with pCAGGS-IL-1RII and pCAGGS-IL-1RAcP, n=7）. The rats were immunized to induce EAM on day 0, and injected with recombinant plasmids by hydrodynamics-based delivery on day 6. Echocardiography was performed, and the rats were killed on day 17. The ratio of heart weight to body weight （HW/BW） was evaluated, and the histopathological changes of the myocardial tissues were observed by HE staining. The mRNA expression of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and inflammatory factors in the myocardial tissues was detected by RT-qPCR. Recombinant plasmids pUC19-IL-1RII-actin and pUC19-IL-1RAcP-tub were transfected into Cos7 cells, and the culture supernatants were collected and added to lipopolysaccharide （LPS）-induced H9c2 cells. The expression of inflammatory genes were detected by RT-qPCR. Recombinant plasmids pEGFP-IL-1RII-actin and pEGFP-IL-1RAcP-tub were transfected into the Cos7 cells to identify the formation of IL-1RII/IL-1RAcP heterodimer by co-immunoprecipitation （Co-IP）. RESULTS Compared with EAM+SP group, injection with plasmids effectively attenuated EAM in EAM+IL-1RII group and EAM+IL-1RII+IL-1RAcP group, as indicated by the decreases in HW/BW, left ventricular end-systolic diameter, and myocardial expression of ANP, BNP, TNF-α, IL-2, IFN-γ and TGF-β, and the increase in expression of IL-4 in the hearts. In LPS-induced H9c2 cells, compared with LPS group, the levels of TGF-β and IL-6 in the culture supernatants were significantly decreased （P<0.01）, and the level of IL-10 was significantly increased （P<0.05） in LPS+IL-1RII group and LPS+IL-1RII+IL-1RAcP group. Compared with LPS+IL-1RII group, the expression of TNF-α and IL-2 was significantly decreased （P<0.05）, and the expression of IL-13 was significantly increased in LPS+IL-1RII+IL-1RAcP group （P<0.01）. The formation of IL-1RII/IL-1RAcP heterodimer was detected by Co-IP. CONCLUSION Plasmids encoding IL-1RII and IL-1RAcP effectively attenuate EAM, and the possible mechanism may be related to the inhibition of inflammatory factor expression and the formation of IL-1RII/IL-1RAcP heterodimer.     

Establishment of experimental autoimmune encephalomyelitis model in mice induced by MOG and its effect on CD4+CD25+ T cells

AIM: To investigate the feasibility of inducing experimental autoimmune encephalomyelitis(EAE)model in C57BL/6 mice by TrxA-extracellular immunoglobulin domain of MOG(MOGIgd-TrxA)fusion protein produced by molecular cloning in our laboratory. Also to investigate the role of CD4+CD25+ T cells in the pathogenesis of EAE. METHODS: (1)The MOGIgd-TrxA fusion protein was induced and produced by molecular cloning and purified by metal chelate affinity chromatography and concentrated through ultrafiltration. The concentration of the protein was measured by Bradford method at last. (2)Animal experiment: C57BL/6 mice(12 mice in each group)were used. The mice in group MOG were immunized with MOGIgd -TrxA fusion protein. The mice in group GPSCH were received emulsion of spinal cord homogenate of guinea pigs(GPSCH), and mice in group TrxA or normal control group(group NC)were received the same volume emulsion of TrxA or saline/adjuvant, respectively. Clinical scores and histopathology were measured to value the models quality. (3)The percentages of CD4+CD25+ regulatory T cells in EAE mice were tested through flow cytometric analysis. RESULTS: (1)The purity of purified MOGIgd -TrxA fusion protein was about 98%, and its concentration was 2.3 g/L. (2)No significant difference between group MOG and group GPSCH in the clinical score was observed(P>0.05). Histologic sections of the brain and spinal cord taken from affected animals in both groups showed pathological change of different level throughout the central nervous system(CNS). (3)Percentages of CD4+CD25+ T cells in group MOG and group GPSCH were(4.71±1.61)%and(1.44±0.65)%,respectively, both of which were significantly lower than those in group NC(9.22±1.24)%and TrxA group(8.97±1.20)％(P<0.01). CONCLUSION: (1)The animal model of EAE in C57BL/6 mice induced by MOGIgd fusion protein produced through molecular cloning in our laboratory is stable and with high incidence. Thus, the author finds a good way to study the immune mechanisms of MS further and to search for the effective treatments as well. (2)The reduction of CD4+CD25+ T cells in EAE mice may have some relationship with the clinic.     

Effect of intravenous injecting plasmid encoding interleukin-19-IgG on experimental autoimmune myocarditis in rats

AIM: To evaluate the effect of intravenous injecting plasmid encoding interleukin-19-IgG on experimental autoimmune myocarditis (EAM) in rats.METHODS: Cardiac myosin was emulsified with equal volume of complete Freund's adjuvant. The animal model of EAM was established by injecting with the preparation in both footpads of the Lewis rats. The rats were intravenously injected with the plasmid encoding IL-19-IgG on day 6. Echocardiography was performed before the rats were sacrificed on day 17. The effect of IL-19-IgG plasmid injection was evaluated by measuring the heart weight/body weight, myocarditis area, relative expression levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the hearts. The mRNA expression levels of related cytokines including IL-18, IL-1β, IL-12p35 and IFN-γ were detected.RESULTS: The rats in model group showed significant myocardial damage and a decrease in the left ventricular functions. The rats in the treatment group injected with IL-19-IgG plasmid showed an improvement of the cardiac functions. The ratio of heart weight/body weight, the area of myocarditis and the mRNA levels of ANP and BNP were significantly lower in IL-19-IgG treatment group than those in model group. The mRNA levels of IL-18, IL-1β, IL-12p35 and IFN-γ were also significantly decreased in IL-19-IgG treatment group.CONCLUSION: Intravenous injection of plasmid encoding IL-19-IgG effectively prevents the development of the left ventricular remodeling and myocardial damage in EAM rats.     

Effect of mesenchymal stem cell transplantation on papain and ［60Co］ irradiation-induced rat pulmonary emphysema

AIM: To investigate the effect of bone marrow mesenchymal stem cells(MSCs) transplantation on papain and ［⁶⁰Co］ irradiation-induced rat pulmonary emphysema and the underlying mechanisms.METHODS: Female rats were randomly divided into three groups: control group, emphysema group, emphysema+MSCs transplantation group. Rats were exposed to ［⁶⁰Co］ irradiation and instilled with papain intratracheally. Bone marrow MSCs from male rats were infused through tail veins. Morphologic analysis of the lung tissue was performed. The engraftment of male bone marrow MSCs in female recipient lung was determined by PCR of Sry gene and Y chromosome fluorescence in situ hybridization (Y-FISH). Sry gene was amplified by PCR using the genomic DNA from the lungs as template. Surfactant protein C (SP-C) immunofluorescent staining and Y-FISH were performed on the same lung section to determine whether the engrafted bone marrow MSCs differentiated into type II pneumocytes. RESULTS: Destruction of alveolar walls was observed in rat lungs from emphysema group and emphysema+MSCs transplantation group. MSCs transplantation significantly ameliorated the emphysematous changes. Significant differences in the mean linear interval (MLI), the mean alveoli number (MAN) and mean alveoli area (MAA) between emphysema group and emphysema+MSCs transplantation group were observed. DNA fragment of Sry gene was amplified in the genomic DNA from the rat lungs in emphysema+MSCs transplantation group. Y chromosome positive cells were observed in the lungs tissue from emphysema+MSCs transplantation group. Some of the Y chromosome positive cells also expressed SP-C, the marker of type II pneumocytes. CONCLUSION: Bone marrow MSCs transplantation improves papain and irradiation-induced pulmonary emphysema. The underlying mechanisms may include the engraftment of bone marrow MSCs in the lungs and differentiation of MSCs into type II pneumocytes.     

Effect of autologous bone marrow mononuclear cell local transplantation on peripheral neovascularization

AIM: To investigate the effect of autologous bone marrow mononuclear cells (BM-MNCs) local transplantation (BM-LT) on a rabbit model of hindlimb ischemia. METHODS: After unilateral femoral artery and its branches were surgically ligated and excised in rabbits (n=14) 1 hour or so, autologous BM-MNCs were injected into the ischemic skeletal muscles in 7 rabbits, and phosphate buffered saline for another 7 ones as control. Hemodynamics parameters were measured at day 0, day 7 and day 28, respectively. On day 28, the animals were sacrificed in groups for pathological analysis. RESULTS: Compared with control group, color Doppler flow image in BM-LT group was recovered significantly, and its' systolic peak velocity and end-diastolic velocity were also improved (P<0.05). Resistance index and flow volume were significantly improved on day 28 (P<0.05). A higher capillary density was detected in BM-LT group than that in control group (P<0.01). Ratio of the number of capillary and the number of myocyte fiber were significant difference (P＜0.05). CONCLUSION: BM-LT improves peripheral neovascularization. It might provide a new therapeutic method for ischemic peripheral vascular disease.     

Effects of atorvastatin on the experimental autoimmune myocarditis in Lewis rats

AIM:To test the hypothesis that atorvastatin affects T cell-mediated autoimmunity through modulating the balance of Th1/Th2 and reduces the severity of EAM. METHODS:Myocarditis was induced in Lewis rats by injection of porcine cardiac myosin. High-dose (10 mg·kg^-1·d^-1) or low-dose (1 mg·kg^-1·d^-1) atorvastatin or vehicle was administered orally for 3 weeks. On day 21, echocardiography was examined and the severity of myocarditis was detected by histopathological evaluation. Levels of serum IFN-γ, IL-2, IL-4 and IL-10 were measured by ELISA. RESULTS:Cardiac function and histological severity of myocarditis were improved in the two atorvastatin-treated groups. Treatment with atorvastatin decreased the levels of Th1 cytokine (IFN-γ, IL-2) and increased the levels of Th2 cytokine (IL-4, IL-10). CONCLUSION:These results suggest that HMG-CoA reductase blockade may be a promising new strategy for the treatment of autoimmune myocarditis.     

Effect of VEGF on morphology and function of rat ovarian tissues after autologous transplantation

AIM: To evaluate the effect of vascular endothelial growth factor (VEGF) on the ovarian tissues during transplantation. METHODS: Twenty regularly cycling rats were randomly divided into 2 groups: the rats in group A received fresh autologous ovarian transplantation without VEGF administration (n=10); the rats in group B received fresh autologous ovarian transplantation with VEGF administration (n=10). Four weeks after transplantation, the ovarian tissues and sera of the rats were collected for histological examination and determination of the hormone levels. RESULTS: The serum concentration of estradiol in group B was significantly higher than that in group A (P<0.05). Four weeks after transplantation, we found that the ovarian tissues were significantly developed, and the average size of the ovaries in group B was significantly larger than that in group A (P<0.05). CONCLUSION: The ovarian tissues with VEGF administration develop better than those without VEGF administration. VEGF can increase the graft vascularity and the percentage of surviving tissues.     

Effect of bone marrow stem cell transplantation on mdx mice at different ages

AIM: To study the effect of bone marrow stem cell transplantation on mdx mice at different ages. METHODS: The bone marrow stem cells of C57BL/6 mice (4-to-weeks age) were cultured in vitro for 3 days, then injected intravenously into the 6-week and 8-week aged mdx, which were preconditioned with 7 Gy γ ray. 12 weeks after being transplanted, the mdx mice were studied for the dystrophin protein expression on the skeletal muscle membrane. RESULTS: Three months after transplanted with bone marrow stem cells, about 16% and 7% muscles cells in 6-week and 8-week mdx mice expressed dystrophin protein， respectively. CONCLUSION: 12 weeks after transplantation with bone marrow stem cells of homologous series mice, different amounts of dystrophin protein expressed on the membrane of skeletal muscle cells were observed in different aged mdx mice. Bone marrow stem cell transplantation show more benefic effect for younger mdx mice.     

Altered T cell signaling events caused by anti-CD4 monoclonal antibody in suppressing experimental autoimmune cardiomyopathy

AIM:We examined the efficacy of anti-L3T4 McAb in the T cell signaling pathway in treating experimental autoimmune cardiomyopathy in BALB/c mice, as a model of the autoimmune mechanism involved in human dilated cardiomyopathy (DCM). METHODS:ADP/ATP carrier peptides were used to induce autoimmune cardiomyopathy in BALB/c mice. After 3 months, anti-L3T4 McAb was administered to deplete CD4+ T cells in the mice. Real-time PCR were used to detect the expression of intracellular signaling molecules (p56lck, p59fyn and Zap-70) and cytokine production (IFN-γ, IL-2 and IL-4) in T cells. The expression of CD45 was determined by immunohistochemistry analysis. RESULTS:Reduced expression of p56lck, p59fyn and Zap-70 and the reduced cytokine production of IFN-γ, IL-2 and IL-4 in T cells of anti-L3T4-treated DCM mice were found. Also, the expression of CD45 in spleen T cells was significantly decreased in the anti-L3T4-treated group. In contrast, immunization with irrelevant Ab did not protect the mice, the expression of T cell signaling molecules, CD45, and cytokine were not inhibited. CONCLUSION:These studies provide direct evidence that anti-L3T4 McAb can be an effective immunomodulator to T cell signal molecules and subsequent cytokine production events in ADP/ATP carrier-induced DCM in BALB/c mice.     

Effect of experimental acute necrotizing pancreatitis on sodium and L-type calcium current in rat cardiomyocytes

AIM: To study the effect of experimental acute necrotizing pancreatitis (ANP) on sodium and L-type calcium current in rat cardiomyocytes. METHODS: I_Na and I_Ca-L were recorded using whole cell patch-clamp techniques from left ventricular myocytes in ANP model established by retrograde injection of 3.5% sodium taurocholate 2.5 mL/kg into pancreatic duct. RESULTS: Peak I_Na current density (at -30 mV) was significantly reduced in ANP [(12.45±2.26)pA/pF,n=16] compared with sham [(25.32±3.31)pA/pF,n=14], P<0.01; I_Ca-L current density (at +10 mV) was also significantly reduced in ANP [(3.63±0.65)pA/pF,n=16] compared with sham [(5.46±1.03)pA/pF,n=12], P<0.05. CONCLUSIONS: There were changes in both I_Na and I_Ca-L in cardiomyocytes of ANP. These changes may underlie the altered excitability and abnormally short transmembrane action potentials and repolarization of cardiomyocytes, thus contributing to arrhymias in ANP.     

Effect of livin-modified BM-MSCs transplantation on cardiac function following acute myocardial infarction in a rat model

AIM: To study the effect of livin gene-modified bone marrow mesenchymal stem cells(BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin, caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs. METHODS: The MSCs were obtained by the whole bone marrow culture method, and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein(EGFP) gene and livin recombinant vector(rAd-livin) were detected by flow cytometry. The expression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot. After permanent left anterior descending artery occlusion, the rats were randomized to receive intramyocardial injection of DMEM without cells(vehicle group), or containing MSCs(MSCs group), MSCs(EGFP)(rAd-control/MSCs group) or MSCs(livin)(rAd-livin/MSCs group). Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), the maximum increased rate of left ventricular pressure(-dp/dt_max) and the maximum decline rate of left ventricular pressure(+dp/dt_max) were recorded for evaluating the cardiac functions. RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs(P<0.05). Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups(P<0.05). The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group, and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved. Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups. CONCLUSION: The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significantly downregulated while the expression of livin is significantly upregulated. Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival.     

Expression of Toll-like receptor 3 in myocardium in experimental autoimmune myocarditis

AIM: To establish an animal model of experimental autoimmune myocarditis (EAM) in BALB/c mice and to investigate the expression and significance of Toll-like receptor 3 in mouse EAM. METHODS: BALB/c mice were immunized with cardiac myosin extracted from porcine ventricular myocardium covered by complete freund’s adjuvant (CFA) on 0 d and 7 d, then divided into EAM group (n=20) and poly (I∶C) group (n=24). Control mice (n=18) were immunized with CFA only. Serum and myocardium samples were collected at 14 d and 21 d after the first immunization. HE staining was used to identify the areas of inflammation. The myosin IgG antibody was examined by indirect ELISA assay. The changes of TLR3 protein and mRNA expression in myocardial tissue were measured by immunohistochemistry and real time-PCR. RESULTS: Compared to control group, immunohistochemistry results showed that there was positive expression of TLR3 in the myocardium of mice with EAM and the mRNA of TLR3 were more than 20 times increased in a time-dependent manner at 12 h after poly (I∶C) injection (P<0.05). The expression of interferon beta mRNA in EAM group was more than 14 times as many as basal expression, that of tumor necrosis factor alpha was more than 18 times (P<0.05). CONCLUSION: The expression of Toll-like receptor 3 in myocardium is up-regulated in experimental autoimmune myocarditis. The inflammatory response to cardiac myosin may associate with the TLR3 signal transduction pathway.