Elsevier

Neuroscience

Volume 393, 21 November 2018, Pages 110-122
Neuroscience

Research Article
Local Injection of Endothelin-1 in the Early Neonatal Rat Brain Models Ischemic Damage Associated with Motor Impairment and Diffuse Loss in Brain Volume

https://doi.org/10.1016/j.neuroscience.2018.09.043Get rights and content

Highlights

  • Focal injection of ET-1 into the newborn brain results in robust ischemic damage.

  • Early analysis in this model revealed robust apoptosis and immune cell activation.

  • Analysis of adult rats revealed permanent cortical and white matter atrophy.

  • Adult rats exhibited gross motor deficits resulting from ET-1 ischemia at birth.

  • ET-1 delivery to newborns is a simple, effective model for early neonatal ischemia.

Abstract

Cerebral palsy is an irreversible movement disorder resulting from cerebral damage sustained during prenatal or neonatal brain development. As survival outcomes for preterm injury improve, there is increasing need to model ischemic injury at earlier neonatal time-points to better understand the subsequent pathological consequences. Here we demonstrate a novel neonatal ischemic model using focal administration of the potent vasoconstrictor peptide, endothelin-1 (ET-1), in newborn rats. The functional and histopathological outcomes compare favourably to those reported following the widely used hypoxic ischemia (HI) model. These include a robust motor deficit sustained into adulthood and recapitulation of hallmark features of preterm human brain injury, including atrophy of subcortical white matter and periventricular fiber bundles. Compared to procedures involving carotid artery manipulation and periods of hypoxia, the ET-1 ischemia model represents a rapid and technically simplified model more amenable to larger cohorts and with the potential to direct the locus of ischemic damage to specific brain areas.

Introduction

Cerebral palsy (CP) is a disorder involving motor and cognitive impairment resulting from brain damage sustained during fetal, neonatal or pediatric periods. Arterial ischemic stroke (AIS) is a major cause of neonatal brain damage (Nelson and Lynch, 2004) which can result in CP (Wu et al., 2003, Golomb et al., 2008) with newborns around birth having a higher risk of arterial infarction. Both pre-term infants (Golomb et al., 2008, Benders et al., 2009) and term infants (deVeber et al., 2000, Wu et al., 2004) are at risk of developing CP, however pathological damage resulting from neonatal ischemia can vary between the two groups of infants (Hagberg et al., 2015). A number of studies have identified pre-term infants (23–32 week of gestation), particularly extremely low-birth weight (<2500 g) infants, to be at a higher risk of developing periventricular white matter injury (WMI), including periventricular leukomalacia (PVL) compared to full-term infants (Deng et al., 2008, Volpe, 2009, Back, 2017). A number of rodent models have been developed, to better understand why the pre-term and term brains have varying susceptibility to ischemic insult and to model pathological hallmarks present in both groups.

Study of neonatal brain injury in rodents has had a predominant focus on hypoxic ischemic (HI) injury models performed at P7–P10. Specifically, this has most commonly involved the hypoxic ischemia encephalopathy (HIE) model in P7 pups originally described by Rice and Vanucci (Rice et al., 1981), which results in damage to cortical, striatal and hippocampal areas. Variations on this model may involve direct electrocoagulation of the MCA (Tsuji et al., 2013) and temporary filament occlusion of the MCA (Ashwal et al., 1995). HIE is often performed between P7 and P10 in rodents, which approximates key aspects of human brain development equivalent to near- and full-term infants (Rice et al., 1981, Vannucci and Vannucci, 1997, Semple et al., 2013), whereas younger animals between P1 and P5 have characteristic developmental hallmarks matching pre-term infants (Mallard and Vexler, 2015). Studies aimed at modeling pre-term developmental injury using the HIE approach on earlier rodent ages (<P3) have reported the younger brain to be more resistant to HIE injury and hence requiring a greater hypoxic environment for longer durations (Grafe, 1994, Back et al., 2002). In fact, a direct comparison of P1 to P7 HI induction has illustrated less overall structural atrophy in P1 compared to P7 induction – highlighting the differences of HI induction at these ages (McClure et al., 2006).

Modeling of neonatal ischemia has been less extensively studied in comparison to HIE models, possibly due to technical challenges of eliciting ischemia-only brain damage in the neonate. However, studies focused on HI induction at early neonatal time-points have identified common pathological features between early neonatal rodent damage and preterm infants (McQuillen et al., 2003, Volpe, 2009). MCA occlusion models have only been reported in P7 or older animals (Ashwal et al., 1995, Mitsufuji et al., 1996, Renolleau, 1998, Derugin et al., 1998, Wen et al., 2004, Comi et al., 2004, Bonnin et al., 2011), with early developmental ischemic models focusing on the perinatal period through embryonic intrauterine hypoperfusion models (Ohshima et al., 2016) – a technically challenging model resulting in global ischemia.

Endothelin-1 peptide (ET-1)-based stroke models have been well documented in the adult rodent (Robinson et al., 1990, Macrae et al., 1993, Sharkey et al., 1993, Vahid-Ansari et al., 2016). In this context, the soluble peptide elicits a potent vasoconstrictor action through binding to the Endothelin type A (ETA) receptor on resident vascular smooth muscle in blood vessels (Lin et al., 1991, Sokolovsky, 1992, Huggins et al., 1993, Haynes et al., 1995). Administration of ET-1 to local blood vessels in the adult brain results in robust, focal infarction due to vasoconstriction of the effected vascular territory (Glendenning et al., 2008, Mecca et al., 2011, Zgavc et al., 2012)

Here we describe local injection of ET-1 for modeling ischemic brain damage in rodents as early as P0. The approach has been used extensively in models of ischemic damage to the adult rodent brain and, compared to MCA occlusion models, offers not only a more rapid and technically simple procedure adaptable to early postnatal ages, but also the capacity for modeling focal ischemic injury to pre-defined targets. We describe here that injection of ET-1 into the striatum and overlying cortex of neonatal rats at P0 results in impairment of gross motor function persisting into adulthood, along with robust and consistent pathohistological damage characterized by cortical, striatal and subcortical white matter atrophy.

Section snippets

Surgical procedures and ethical approval

Time-mated Sprague–Dawley rats were housed under a 12-h light/dark cycle with ad libitum access to food and water. All procedures were conducted in accordance with the Australian National Health and Medical Research Council's published Code of Practice for the Use of Animals in Research. Experiments were approved by the Florey Institute of Neuroscience and Mental Health Animal Ethics Committee (Ethics no: 09-105).

ET-1 injection: For induction of ischemia using ET-1, neonatal rats were taken on

Early histopathological impact of ET-1 injection and HI in the cortex and striatum

The morphological and cyto-architectural impact of ischemia was assessed in coronal sections immunolabeled for NeuN. An initial assessment at 7 days showed hydrocephaly and observable atrophy of the cortex and striatum in the ET-1-treated animals (Fig. 1A). A conspicuous loss of NeuN+ cells in the deep cortical layers in ET-1-treated animals was not evident in the saline group, the HI group and the occlusion-alone group (Fig. 1A – black arrows on ET-1 NeuN stain), and also columns of neuronal

Discussion

These results show that local, intracerebral delivery of the vasoconstrictor ET-1 models key functional and histopathological aspects of neonatal brain injury that can lead to CP. It thus represents a technically simple yet effective neonatal ischemia model that can be readily applied to large cohorts of animals at early postnatal ages. Multiple pathological events can lead to developmental brain damage, including HI and focal arterial stroke and each present challenges for pre-clinical

Conflicting interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors thank Mong Tien for her expert technical assistance in the preparation of tissue used in this study. This work was supported NHMRC project grants #1042584 and #1102704.

References (68)

  • L.W. Fan et al.

    Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat

    Behav Brain Res

    (2005)
  • M.L. Glendenning et al.

    Protective effect of estrogen in endothelin-induced middle cerebral artery occlusion in female rats

    Neurosci Lett

    (2008)
  • M.R. Golomb et al.

    Very early arterial ischemic stroke in premature infants

    Pediatr Neurol

    (2008)
  • M.R. Grafe

    Developmental changes in the sensitivity of the neonatal rat brain to hypoxic/ischemic injury

    Brain Res

    (1994)
  • J.P. Huggins et al.

    The structure and specificity of endothelin receptors: their importance in physiology and medicine

    Pharmacol Ther

    (1993)
  • T. Ikeda et al.

    Selective and long-term learning impairment following neonatal hypoxic-ischemic brain insult in rats

    Behav Brain Res

    (2001)
  • E.M. Jansen et al.

    Long-term effects of neonatal ischemic-hypoxic brain injury on sensorimotor and locomotor tasks in rats

    Behav Brain Res

    (1996)
  • A. Lubics et al.

    Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury

    Behav Brain Res

    (2005)
  • S. Marret et al.

    Pathophysiology of cerebral palsy

    Handb Clin Neurol

    (2013)
  • M.M. McClure et al.

    Rapid auditory processing and learning deficits in rats with P1 versus P7 neonatal hypoxic-ischemic injury

    Behav Brain Res

    (2006)
  • K.B. Nelson et al.

    Stroke in newborn infants

    Lancet Neurol

    (2004)
  • M.J. Robinson et al.

    Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat

    Neurosci Lett

    (1990)
  • B.D. Semple et al.

    Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species

    Prog Neurobiol

    (2013)
  • M. Sokolovsky

    Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling

    Pharmacol Ther

    (1992)
  • L.H. Thompson et al.

    Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon

    Exp Neurol

    (2006)
  • L.H. Thompson et al.

    Non-dopaminergic neurons in ventral mesencephalic transplants make widespread axonal connections in the host brain

    Exp Neurol

    (2008)
  • M. Tsuji et al.

    A novel reproducible model of neonatal stroke in mice: comparison with a hypoxia-ischemia model

    Exp Neurol

    (2013)
  • H. Uehara et al.

    A new model of white matter injury in neonatal rats with bilateral carotid artery occlusion

    Brain Res

    (1999)
  • J.J. Volpe

    The encephalopathy of prematurity-brain injury and impaired brain development inextricably intertwined

    Semin Pediatr Neurol

    (2009)
  • T.C. Wen et al.

    A reproducible experimental model of focal cerebral ischemia in the neonatal rat

    Brain Res Brain Res Protoc

    (2004)
  • J.Y. Yager et al.

    Animal models of perinatal hypoxic-ischemic brain damage

    Pediatr Neurol

    (2009)
  • M. Antunes et al.

    The novel object recognition memory: neurobiology, test procedure, and its modifications

    Cogn Process

    (2012)
  • S.A. Back et al.

    Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury

    J Neurosci

    (2001)
  • S.A. Back et al.

    Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia

    J Neurosci

    (2002)
  • Cited by (4)

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