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The study of fetal rat model of intra-amniotic isoproterenol injection induced heart dysfunction and phenotypic switch of contractile proteins.

Li Y, Fang J, Hua Y, Wang C, Mu D, Zhou K - Biomed Res Int (2014)

Bottom Line: Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults.Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate.ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Cardiovascular Disease, West China Second University Hospital, Sichuan University, No. 20, 3rd Section, South Renmin Road, Chengdu, Sichuan 610041, China.

ABSTRACT
To establish a reliable isoproterenol induced heart dysfunction fetal rat model and understand the switches of contractile proteins, 45 pregnant rats were divided into 15 mg/kg-once, 15 mg/kg-twice, sham-operated once, sham-operated twice, and control groups. And 18 adult rats were divided into isoproterenol-treated and control groups. H&E staining, Masson staining, and transmission electron microscope were performed. Apoptotic rate assessed by TUNEL analysis and expressions of ANP, BNP, MMP-2, and CTGF of hearts were measured. Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults. Then echocardiography was performed with M-mode view assessment on E18.5 and 6 weeks later, respectively. Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate. ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF. The isoforms transition of troponin I and myosin heavy chain of fetal heart dysfunction were opposite to adult procedure. The administration of intra-amniotic isoproterenol to fetal rats could induce heart dysfunction and the regulation of contractile proteins of fetuses was different from adult procedure.

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Myocardial apoptotic cell number determined with TUNEL staining in control, sham-operated, and ISO-treated hearts. (a) Presented TUNEL staining (with DAPI costaining) in control and ISO-treated hearts. Nuclei are stained blue. TUNEL positive nuclei are green/cyan ×200. (b) Demonstrated apoptotic cell number per microscopic field. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001. n = 4/group.
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fig4: Myocardial apoptotic cell number determined with TUNEL staining in control, sham-operated, and ISO-treated hearts. (a) Presented TUNEL staining (with DAPI costaining) in control and ISO-treated hearts. Nuclei are stained blue. TUNEL positive nuclei are green/cyan ×200. (b) Demonstrated apoptotic cell number per microscopic field. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001. n = 4/group.

Mentions: On the day of E18.5, the interstitial fibrosis rates were, respectively, 13.0 ± 0.9% in Iso1 group, 21.7 ± 2.6% in Iso2 group, 12.3 ± 1.2% in Sham1 group, 11.3 ± 1.8% in Sham2 group, and 10.6 ± 1.4% in Con group (Figures 2(a) and 2(b)). We also measured the morphology of cardiomyocytes and myocardium of whole ventricular area among five experimental groups using H&E staining (Figure 3). In ISO-treated hearts, cardiomyocytes were hypertrophic and nonuniform in shape with disorganized arrangement. In control hearts, cardiomyocytes were of a uniform size and shape, and interstitial space was clearly visible as dark, variegated bands throughout the myocardium. Figure 4(a) demonstrates representative TUNEL stains from control to ISO-treated hearts and the overall apoptotic cells number per microscopic field in any area of fetal heart as they were quite a small tissue. As compared to control group, ISO treatment enhanced cardiomyocytes apoptosis in hearts (apoptotic cells number per microscopic field of Iso1, Iso2, Sham1, Sham2, and Con was 21.63 ± 1.27, 30.10 ± 3.11, 9.30 ± 3.01, 5.30 ± 0.95, and 5.51 ± 0.76, respectively; Figure 4(b)). This increase was statistically significant in the Iso2 group (P < 0.0001).


The study of fetal rat model of intra-amniotic isoproterenol injection induced heart dysfunction and phenotypic switch of contractile proteins.

Li Y, Fang J, Hua Y, Wang C, Mu D, Zhou K - Biomed Res Int (2014)

Myocardial apoptotic cell number determined with TUNEL staining in control, sham-operated, and ISO-treated hearts. (a) Presented TUNEL staining (with DAPI costaining) in control and ISO-treated hearts. Nuclei are stained blue. TUNEL positive nuclei are green/cyan ×200. (b) Demonstrated apoptotic cell number per microscopic field. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001. n = 4/group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4127273&req=5

fig4: Myocardial apoptotic cell number determined with TUNEL staining in control, sham-operated, and ISO-treated hearts. (a) Presented TUNEL staining (with DAPI costaining) in control and ISO-treated hearts. Nuclei are stained blue. TUNEL positive nuclei are green/cyan ×200. (b) Demonstrated apoptotic cell number per microscopic field. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001. n = 4/group.
Mentions: On the day of E18.5, the interstitial fibrosis rates were, respectively, 13.0 ± 0.9% in Iso1 group, 21.7 ± 2.6% in Iso2 group, 12.3 ± 1.2% in Sham1 group, 11.3 ± 1.8% in Sham2 group, and 10.6 ± 1.4% in Con group (Figures 2(a) and 2(b)). We also measured the morphology of cardiomyocytes and myocardium of whole ventricular area among five experimental groups using H&E staining (Figure 3). In ISO-treated hearts, cardiomyocytes were hypertrophic and nonuniform in shape with disorganized arrangement. In control hearts, cardiomyocytes were of a uniform size and shape, and interstitial space was clearly visible as dark, variegated bands throughout the myocardium. Figure 4(a) demonstrates representative TUNEL stains from control to ISO-treated hearts and the overall apoptotic cells number per microscopic field in any area of fetal heart as they were quite a small tissue. As compared to control group, ISO treatment enhanced cardiomyocytes apoptosis in hearts (apoptotic cells number per microscopic field of Iso1, Iso2, Sham1, Sham2, and Con was 21.63 ± 1.27, 30.10 ± 3.11, 9.30 ± 3.01, 5.30 ± 0.95, and 5.51 ± 0.76, respectively; Figure 4(b)). This increase was statistically significant in the Iso2 group (P < 0.0001).

Bottom Line: Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults.Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate.ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Cardiovascular Disease, West China Second University Hospital, Sichuan University, No. 20, 3rd Section, South Renmin Road, Chengdu, Sichuan 610041, China.

ABSTRACT
To establish a reliable isoproterenol induced heart dysfunction fetal rat model and understand the switches of contractile proteins, 45 pregnant rats were divided into 15 mg/kg-once, 15 mg/kg-twice, sham-operated once, sham-operated twice, and control groups. And 18 adult rats were divided into isoproterenol-treated and control groups. H&E staining, Masson staining, and transmission electron microscope were performed. Apoptotic rate assessed by TUNEL analysis and expressions of ANP, BNP, MMP-2, and CTGF of hearts were measured. Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults. Then echocardiography was performed with M-mode view assessment on E18.5 and 6 weeks later, respectively. Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate. ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF. The isoforms transition of troponin I and myosin heavy chain of fetal heart dysfunction were opposite to adult procedure. The administration of intra-amniotic isoproterenol to fetal rats could induce heart dysfunction and the regulation of contractile proteins of fetuses was different from adult procedure.

Show MeSH
Related in: MedlinePlus