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A reproducible protocol for neonatal ischemic injury and cardiac regeneration in neonatal mice.

ABSTRACT: Cardiac regeneration is one of the prime visions in cardiovascular research. The mouse neonatal apical resection and left anterior descending artery (LAD) ligation model introduced novel in vivo mammalian assays to study cardiac regeneration. However, recent reports and editorials discussed and critically questioned the value and technical reproducibility of the mouse neonatal myocardial infarction approach, making it paramount to develop and use a reproducible model system. We established a mouse neonatal myocardial infarction model by visually confirmed ligation of the LAD using microsurgery. TdT-mediated dUTP nick-end labeling (TUNEL) proved reproducible massive myocardial infarctions in a defined region of the apex and anterior wall of neonatal and 7-day-old mice. Whereas hearts ligated on postnatal day 7 displayed chronic injury, cardiac samples ligated immediately after birth always showed complete structural regeneration after long-term follow-up. Cardiac regeneration was observed in all mouse stains (C57BL/6J, ICR, and mixed background C57BL/6JxSv129) tested so far. We present a detailed in vivo protocol to study complex mechanisms of complete cardiac repair following ischemic cardiac damage. Neonatal LAD ligation surgery is feasible, and results in reproducible myocardial infarctions 24 h after ligation, and no structural myocardial defects are detectable following long-term follow-up. We encourage the cardiovascular community to use our protocol and teaching video to answer key scientific questions in the field of cardiac regeneration.

SUBMITTER: Haubner BJ

PROVIDER: S-EPMC5035663 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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A reproducible protocol for neonatal ischemic injury and cardiac regeneration in neonatal mice.

Haubner Bernhard J BJ Schuetz Thomas T Penninger Josef M JM

Basic research in cardiology 20160924 6

Cardiac regeneration is one of the prime visions in cardiovascular research. The mouse neonatal apical resection and left anterior descending artery (LAD) ligation model introduced novel in vivo mammalian assays to study cardiac regeneration. However, recent reports and editorials discussed and critically questioned the value and technical reproducibility of the mouse neonatal myocardial infarction approach, making it paramount to develop and use a reproducible model system. We established a mou ...[more]

PMID: 27665606

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Project description:Hypoxic-ischemic injury to the developing brain remains a major cause of significant long-term morbidity and mortality. Emerging evidence from neonatal brain injury models suggests a detrimental role for peripheral lymphocytes. The immunomodulatory substance FTY720, a sphingosine-1-phosphate receptor agonist, was shown to reduce adult ischemia-induced neurodegeneration through its lymphopenic mode of action. In the present study, we hypothesized that FTY720 promotes neuroprotection by reducing peripheral lymphocytes and their infiltration into the injured neonatal brain. Term-born equivalent postnatal day 9 C57BL/6 mice were exposed to hypoxia ischemia (HI) followed by a single injection of 1 mg/kg FTY720 or vehicle (0.9% sodium chloride). Brain injury, microglia, and endothelial activation were assessed 7 days post HI using histology and western blot. Peripheral and cerebral leukocyte subsets were analyzed by multichannel flow cytometry. Whether FTY720s' effects could be attributed to its lymphopenic mode of action was determined in T cell-depleted mice. In contrast to our hypothesis, FTY720 exacerbated HI-induced neuropathology including loss of gray and white matter structures. While microglia and endothelial activation remained unchanged, FTY720 induced a strong and sustained depletion of peripheral T cells resulting in significantly reduced cerebral infiltration of CD4 T cells. CD4 T cell subset analysis revealed that circulating regulatory and effector T cells counts were similarly decreased after FTY720 treatment. However, since neonatal HI per se induces a selective infiltration of Foxp3 positive regulatory T cells compared to Foxp3 negative effector T cells effects of FTY720 on cerebral regulatory T cell infiltration were more pronounced than on effector T cells. Reductions in T lymphocytes, and particularly regulatory T cells coincided with an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Importantly anti-CD3-mediated T cell depletion resulted in a similar exacerbation of brain injury, which was not further enhanced by an additional FTY720 treatment. In summary, peripheral T cell depletion by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T cell infiltration and exacerbation HI-induced brain injury. This study indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention.

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A reproducible protocol for neonatal ischemic injury and cardiac regeneration in neonatal mice.

Publications

A reproducible protocol for neonatal ischemic injury and cardiac regeneration in neonatal mice.

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