Research ArticleLocal Injection of Endothelin-1 in the Early Neonatal Rat Brain Models Ischemic Damage Associated with Motor Impairment and Diffuse Loss in Brain Volume
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.
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Ischemic Injury Does Not Stimulate Striatal Neuron Replacement Even during Periods of Active Striatal Neurogenesis
2020, iScienceCitation Excerpt :Injection of ET-1 into the striatum in neonatal rats produced an ischemic injury manifesting at 4 and 12 weeks as striatal atrophy with concomitant hydrocephaly as well as malformation of the overlying cortex. We have recently shown that this model captures various elements relevant for pre-term brain injury leading to cerebral palsy, such as white matter damage and motor deficit (Wright et al., 2018). To assess the impact of striatal injury on neurogenesis during this early postnatal period, birth-dating was performed over the first week after ET-1 injection and histology was performed at both 4 and 12 weeks to allow for the possibility of a delayed or protracted response.
Focal ischemic injury to the early neonatal rat brain models cognitive and motor deficits with associated histopathological outcomes relevant to human neonatal brain injury
2021, International Journal of Molecular Sciences