Research reportBumetanide reduce the seizure susceptibility induced by pentylenetetrazol via inhibition of aberrant hippocampal neurogenesis in neonatal rats after hypoxia-ischemia
Introduction
Neonatal hypoxia-ischemia brain damage (HIBD) remains a leading cause of severe neurological morbidity and mortality in neonates, occurring in approximately 1–3 newborns per 1000 live births (Shankaran et al., 2012). The mortality of HIBD is about 50% and 25% survivors of HIBD still have to face devastating sequelae, such as cerebral palsy, epilepsy, learning disabilities and other serious neurological diseases (Bass et al., 2004, Ferriero, 2004, Graham et al., 2008, Williams et al., 2004).
The hippocampal dentate gyrus is a late developing structure, and a majority of the granule cell neurons of the dentate gyrus are born and become synaptically integrated after birth (Altman and Das, 1965, M. Sanchez, 2012, Rakic and Nowakowski, 1981, Wenzel et al., 1981). Dentate gyrus reorganization have long been postulated to facilitate hyperexcitability and/or generate spontaneous recurrent seizures (SRS) by sprouting of mossy fibers, formation of hilar basal dendrites (HBDs), and aberrant synaptogenesis in rats seizure models induced by pilocarpine and kainic acid (Austin and Buckmaster, 2004, Morgan and Soltesz, 2008, Ribak et al., 2000, Scharfman et al., 2000, Thind et al., 2008, Toni et al., 2008). In individuals with temporal lobe epilepsy (TLE) and corresponding animal models, ectopically located granule cells are found in the dentate hilus (ectopic granule cells, EGCs) (Scharfman et al., 2007). Ectopic granule cells are abnormally incorporated into excitatory hippocampal networks (Scharfman et al., 2007, Scharfman et al., 2000, Scharfman et al., 2002). Pathological events including ischemia, fluid percussion injury, viral infection, hypoxia, inflammatory demyelination and traumatic brain injury have been reported to result in aberrant hippocampal neurogenesis, including increased proliferation of neural progenitors, mossy fiber sprouting (MFS), production of EGCs, neuronal hypertrophy and persistence of hilar basal dendrites (Chirumamilla et al., 2002, Dash et al., 2001, Lichtenwalner and Parent, 2006, Parent et al., 1997, Zhu et al., 2005), which may contributes to epilepsy in adults (Cho et al., 2015, Pun et al., 2012, Williams et al., 2004). Previous studies suggested that inhibiting adult hippocampal neurogenesis by nonspecific pharmacological agents reduced seizures (Jung et al., 2004, Jung et al., 2006). For example, rapamycin treatment successfully inhibited MFS but produced controversial results in regards to the development of epilepsy (Buckmaster and Lew, 2011, Zeng et al., 2009). In addition, granule neurons generating after an epileptic stimuli show variable levels of excitability (Cameron et al., 2011, Jakubs et al., 2006, Myers et al., 2013, Scharfman et al., 2000, Thind et al., 2008, Zhan et al., 2010). Hilar EGCs receive increase hippocampal excitability and more excitatory input (Cameron et al., 2011, Myers et al., 2013, Scharfman et al., 2000, Zhan et al., 2010), whereas adult-generated neurons in the granule cell layer are reported either to receive excessive excitatory input (Noebels et al., 2010, Ribak et al., 2012, Thind et al., 2008) or show decreased excitability (Jakubs et al., 2006). In spite of these controversial findings, recent studies showed that conditional deletion of tensin homologue and phosphatase in as little as 9% of postnatally generated granule neurons was sufficient to cause spontaneous seizures in mice (Pun et al., 2012) and the ablation of adult neurogenesis before pilocarpine-induced acute seizures in mice leads to a reduction in chronic seizure frequency(Cho et al., 2015). However, it remains unclear whether newly generated neurons play an essential or contributory role in the development of epilepsy.
Na-K-Cl cotransporter isoform 1 (NKCC1) is highly expressed in immature neurons (Sun and Murali, 1999, Yamada et al., 2004). Several studies have demonstrated that hippocampal NKCC1 would be significantly increased after central injury (Dai et al., 2005, Lu et al., 2008, Wang et al., 2014, Ye et al., 2012). To date, NKCC1 has been found to be involved with neurogenesis (Lu et al., 2014). In a traumatic brain injury (TBI) model, NKCC1 upregulation leads to significant neurogenesis proved to be mediated by the Raf/MEK/ERK pathway (Lu et al., 2014). In addition, NKCC1 knockdown decreases neuron production through GABAA-regulated neural progenitor proliferation resulting in decreased neuronal density (Young et al., 2012), and reduces the migration speed of neuroblasts in a GABAA-independent manner (Mejia-Gervacio et al., 2011).
Bumetanide is a FDA-approved inhibitor of NKCC1. It has been demonstrated that bumetanide plays a critical protective role on neonatal seizure activity in human neonates (Kahle et al., 2009) and rodent models of neonatal seizures (Dzhala et al., 2005, Puskarjov et al., 2014). In the present study, we evaluated whether NKCC1 was a relevant therapeutic target in immature brain injury induced aberrant hippocampal neurogenesis, and tested the preclinical efficacy of bumetanide on hypoxia-ischemia (HI) induced neuronal proliferation and migration. We sought to determine whether bumetanide treatment after HI could reduce the seizure susceptibility and associated cognitive function by rescuing the aberrant hippocampal neurogenesis after HI on neonatal rats. We show that bumetanide treatment after HI reduces the seizures frequency induced by PTZ and restores cognitive function. These findings highlight the major role of aberrant hippocampal neurogenesis in increased susceptibility to epilepsy and associated cognitive decline on rats suffered from HI in neonatal period. These results also support the idea that aberrant hippocampal neurogenesis is one of the many contributing factors to epilepsy and aberrant neurogenesis suppression maybe a promising strategy for reducing seizures.
Section snippets
Experimental animals
Sprague-Dawley rats were obtained at postnatal day 7 of age from the Animal Biosafety Level 3 Laboratory (ABSL-3, Wuhan University, China). All rats (a total of 124 animals) were grouped randomly and housed with their cages and had access to water and food ad libitum under a 12 h light and 12 h dark cycle at 25 ± 2 °C, and in a relative humidity of 60%-80%.
Neonatal hypoxia-ischemia model
All procedures of the neonatal HI model in this study were based on the Rice-Vanucci (Min et al., 2015, Rice et al., 1981). Briefly, unsexed
Increased NKCC1 expression after hypoxia-ischemia
The mRNA and protein expression of NKCC1 after HI were detected by qPCR and western blot. Compared with sham group, the data from the analysis of qPCR showed a significant increase in hippocampus at 24 h (sham: 1.000; HI: 2.447 ± 0.4673; *P < 0.05; Fig. 1A) and 48 h (sham: 1.000; HI: 2.066 ± 0.6353; *P < 0.05; Fig. 1A) after HI, and returned to normal level at 72 h (sham: 1.000; HI: 1.072 ± 0.09957; P > 0.05; Fig. 1A). Consistent with these findings, NKCC1 protein expression detected by western blot also
Discussion
In the present study, we confirmed that the expression of NKCC1 both in mRNA and protein was elevated in early period after HI in neonatal hippocampus. Post-HI bumetanide treatment can decrease PTZ-induced seizure susceptibility. Furthermore, our data revealed that bumetanide effectively act on reduction of aberrant hippocampal neurogenesis. The behavior test further confirms that bumetanide treatment during early stage after HI can alleviate hippocampal memory impairment.
A majority of relevant
Conflict of interest
None of the authors has any conflict of interest.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 81370737, 81571481 and 81371422), the authors confirm that they have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References (67)
- et al.
A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy
Am. J. Obstet. Gynecol.
(2008) - et al.
Ion transporter NKCC1, modulator of neurogenesis in murine olfactory neurons
J. Biol. Chem.
(2015) - et al.
Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability
Neuron
(2006) - et al.
Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal neurogenesis with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus
Neurobiol. Dis.
(2006) - et al.
Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments
Neuropharmacology
(2013) - et al.
Vitexin reduces hypoxia-ischemia neonatal brain injury by the inhibition of HIF-1alpha in a rat pup model
Neuropharmacology
(2015) - et al.
Seizure-induced disinhibition of the HPA axis increases seizure susceptibility
Epilepsy Res.
(2014) - et al.
Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy
Neuron
(2012) - et al.
Spontaneous recurrent seizures after pilocarpine-induced status epilepticus activate calbindin-immunoreactive hilar cells of the rat dentate gyrus
Neuroscience
(2002) - et al.
Bumetanide is not capable of terminating status epilepticus but enhances phenobarbital efficacy in different rat models
Eur. J. Pharmacol.
(2015)