Using a widely accepted in vivo SCI model, this study demonstrates the rapid and sustained loss of two astrocytic proteins believed to be critical in providing neuroprotection in the CNS, the glutamate transporter, GLT-1 and the K+ channel, Kir4.1. These proteins work hand-in-hand to clear the extracellular space of neuronally released K+ and glutamate. While Kir4.1 can function independently, the activity of GLT-1 depends on the negative membrane potential provided by Kir4.1 establishing a sufficient electrochemical gradient for glutamate import. These results would predict a loss of ion and transmitter homeostasis in the injured cord, albeit the compromised extracellular space at the lesion after injury precluded us to examine changes in K+ and glutamate concentrations directly. Instead, we used changes in protein expression as a surrogate marker. In doing so, we were surprised by the spatial extent to which these proteins were lost, extended nearly 10 mm of the spinal cord, and the time course of loss showed essentially no recovery after 4 weeks. These important and unexpected findings are consistent with the irreversible loss of spinal cord function and sustained paralysis typical of patients with SCI. However, this does not imply that loss of astrocytic support is mechanistically involved in the primary injury phase. Instead, we suggest that loss of these astrocytic homeostatic functions may be detrimental to the prolonged secondary injury phase where ion and neurotransmitter homeostasis dysregulation are unopposed by glial support mechanisms that would normally be beneficial.
This is the first study examining Kir4.1 changes in SCI in vivo
. However, other studies have demonstrated changes in these proteins in acute and chronic neurological conditions (for review see Olsen and Sontheimer, 2008
). Acute CNS injuries, epilepsy and most neurodegenerative diseases demonstrate reactive gliosis, a conspicuous morphological change, characterized by cell body hypertrophy, thickened cell processes and increased GFAP expression (Pekny and Pekna, 2004
). Following mechanical SCI, GFAP expression levels have been shown to increase within 1 h and remain elevated chronically (Dusart and Schwab, 1994
). In the spinal cord, gliotic regions may provide structural stability to lesioned tissue, however gliosis also forms persistent barriers obstructing axonal re-growth (Profyris et al.
; Rolls et al.
Numerous studies have reported that gliosis alters astrocytic biophysical properties, notably reverting to those seen in immature astrocytes, characterized by depolarized resting membrane potentials, high input resistances and little Kir mediated currents (for review see Olsen and Sontheimer, 2008
). These changes can be readily induced in vitro
by inflicting a mechanical scar (MacFarlane and Sontheimer, 1997
). These biophysical alterations may involve blood-borne signals. Focal blood–brain barrier disruption in vivo
or exposure of brain slices to albumin, resulted in down-regulation of Kir4.1 (Ivens et al.
), that increased K+
accumulation, neuronal hyperexcitability and epileptiform activity. Because SCI includes grey matter haemorrhage at the lesion centre, blood-born agents may also be responsible for the observed loss of Kir4.1. In spinal cord we would expect see a comparable dysregulation of K+
following the loss of Kir4.1. Additionally, the loss of Kir4.1 in spinal cord astrocytes after injury may markedly alter inflammatory responses as this channel has been shown to prevent glial process swelling under osmotic stress in acute spinal cord slices (Dibaj et al.
). Clearly, these studies indicate Kir4.1 plays an important role in spinal cord astrocytes however, Kir4.1 regulation following injury requires further study.
While our experiments showed a massive and sustained loss of Kir4.1 channel protein expression, our electrophysiological recordings paint a more nuanced picture, suggesting that not all astrocytes near the injury lose Kir4.1. We found that in normal and injured slices obtaining recordings was difficult particularly since myelin density obscured cell visibility and morphologic identity. Thus it was unrealistic to record from a reasonable number of animals so we limit our data interpretation and simply suggest that some astrocytic subpopulations lose Kir4.1 while others retain the channel. Furthermore, because we identified the cells we recorded visually and microglia and oligodendrocytes precursor cells exist in the control and injured spinal cord, we can not rule out the possibility that some of the cells included in our study were not astrocytes. However, since the biochemical data indicate a massive loss of Kir4.1, we would argue that most of the injury-affected astrocytes escaped our electrophysiological recordings. Our immunohistochemistry data suggest that the majority of the Kir4.1 protein is located in grey matter astrocytes, however spinal cord oligodendrocytes also express Kir4.1, and therefore some Kir4.1 in western blots may also be attributable to a loss in oligodendrocytes. It is also possible that some of the astrocytes that displayed significant Kir currents after injury did so by a compensatory up-regulation of other Kir genes, a question that deserves further examination in the future. The weak rectification, high Ba2+ sensitivity, and inactivation at very negative potentials of the recorded currents however, are most consistent with the properties of Kir4.1, suggesting that a subpopulation of astrocytes retained functional Kir4.1 channels.
17β-oestradiol effects on Kir4.1 following injury
Another interesting finding pertains to the partial recovery of Kir4.1 expression in oestrogen treated animals and that astrocytic Kir4.1 expression is directly regulated by oestrogen receptors. Numerous studies have reported on neuroprotective benefits conferred by 17β-oestradiol in brain and spinal cord. In spinal cord specifically, female rats tend to have improved hind-limb motor function compared to age-matched male counterparts following injury (Farooque et al.
). Similarly, exogenous 17β-oestradiol treatment in pre- and post-menopausal ovariectomized female rats significantly increased hind-limb locomotion, white matter sparing and decreased apoptosis (Chaovipoch et al.
). Sribinick and colleagues found that post-injury administration of 17β-oestradiol following SCI to male rats reduced lesion volume, decreased oedema and decreased markers of inflammation and apoptosis (Sribnick et al.
, 2006). And a recent study using the same injury and treatment paradigm used in this investigation demonstrated increased neuronal survival and improved hind-limb function in 17β-oestradiol-treated animals (Kachadroka et al., 2010
). Interestingly, another recent study demonstrated a progressive loss of Kir4.1 that proceeded clinical symptoms in the ventral horn in a mouse model of amyotrophic lateral sclerosis (Kaiser et al.
). These authors used cultured spinal motor neurons to demonstrate that increasing [K+
was toxic in a concentration and time dependent manner. The above studies suggest that the increased neuronal survival and resulting improvements in locomotor skills may be in part due to improved K+
homeostasis following 17β-oestradiol treatment. Our results also indicate that the increase in Kir4.1 we observed at 7 days was transient and by 4 weeks post-injury as Kir4.1 levels in oestrogen treated injured animals were similar to animals treated with a placebo. It is possible the increased Kir4.1 levels during lesion stabilization and thus increased K+
homeostasis lead to increased neuronal survival and improved functional recovery.
Several recent studies have indicated that astrocytes are targets of oestrogen receptor signalling. Acute treatment with 300 nM 17β-oestradiol decreased calcium rises and wave propagation in mechanically stimulated astrocytes (Rao and Sikdar, 2007). Importantly, reactive gliosis, as assessed by GFAP immunoreactivity, is attenuated to control levels in ovariectomized females treated with the 17β-oestradiol, in contrast to ovariectomized females treated with a placebo which demonstrate significantly higher levels of GFAP immunoreactivity (Martinez and de Lacalle, 2007
). These studies suggest that a potential effect of 17β-oestradiol may be the attenuation of the astrogliosis, which may include maintaining astrocytes in a non-reactive state. This could account for a preservation of Kir4.1 and GLT-1 expression. Although we can not say which cell type is expressing 17β-oestradiol receptors or what receptor is the target of 17β-oestradiol in these studies, our data demonstrate that oestrogen receptor-α and oestrogen receptor-β are present in the spinal cord of these adult male animals. Also, others have demonstrated that spinal cord astrocytes express both receptors in vivo
(Platania et al.
) and in vitro
(Platania et al.
The regulation of Kir4.1 expression by oestrogen receptor signalling had not been previously studied. Since our in vivo data show that Kir4.1 is preserved in the presence of 17β-oestradiol, we used primary cultures of rat spinal cord astrocytes to examine direct effects of 17β-oestradiol on Kir4.1. Both biochemical and biophysical data suggest that Kir4.1 is transcriptionally regulated by oestrogen receptor inhibition, which causes a marked reduction in Kir4.1 protein and current. It is important to note that these experiments were performed in the presence of 10% serum, which contains 17β-oestradiol, and may explain why we did not observe increases in channel activity or a significant increase in Kir4.1 protein. It is therefore possible that we are underestimating the 17β-oestradiol effects on our spinal cord astrocyte cultures. However, in a follow-up series of experiments (data not shown) we injured cultured spinal cord astrocytes in the presence or absence of 17β-oestradiol and or a 17β-oestradiol receptor antagonist. For these studies the medium was charcoal-filtered to remove serum oestrogen. As in the data in , 17β-oestradiol had little influence on Kir currents, yet the 17β-oestradiol antagonist significantly reduced Kir expression. Hence, taken together, these data suggest that Kir4.1 expression is under the control of 17β-oestradiol receptor signalling.
Expression of GLT-1 after injury and the effects of 17β-oestradiol
While the major objective of our study was to examine changes in Kir4.1 following SCI, we also paid attention to changes in the astrocytic glutamate transporter GLT-1. This is in part due to the generally held notion that these two proteins cooperate functionally, whereby the activity of Kir4.1 assures a sufficient electrochemical potential to drive glutamate uptake via GLT-1. Hence it would not be unexpected to see a functional loss of GLT-1 if Kir4.1 is compromised. Indeed, our study suggests that both proteins are similarly affected by injury and are indeed similarly rescued by 17β-oestradiol. While the here reported oestrogen effects on Kir4.1 are entirely novel, several groups have looked at oestrogen regulation of astrocyte specific glutamate transporters. For example, cultured astrocytes treated with 17β-oestradiol demonstrate significant up-regulation of GLT-1 and glutamate aspartate transporter mitochondrial RNA and protein. And glutamate uptake was reversed with ICI 182 780 treatment (Pawlak et al.
). In a second study, human astrocytes derived from cortex of patients with Alzheimer’s disease were treated with 100 nM 17β-oestradiol for 48 h, which resulted in increased glutamate uptake and protein expression of glutamate aspartate transporter and GLT-1, although no changes were observed in astrocytes cultured from non-demented brain (Liang et al.
). A third study demonstrated that after 24 h of treatment, higher doses of 17β-oestradiol (1 and 100 μM) led to decreased glutamate clearance in primary cultured cortical astrocytes (Sato et al.
). Spinal cord astrocytes were never investigated, yet our own data indicate prominent expression of both receptors in spinal cord in vivo
when compared to a positive control. GLT-1 expression both in vivo
and in vitro
, were markedly increased following 17β-oestradiol treatment. Importantly, in our in vitro
cultured astrocytes we were able to demonstrate that expression could be modulated with the oestrogen receptor antagonist ICI 182 780. Hence at least in the spinal cord, astrocytic expression of Kir4.1 and GLT-1 are both regulated via oestrogen receptor signalling.