We studied the process of mechanotransmission in astrocytes to understand whether there were broad signals transmitted through the brain during and after trauma that could influence neuronal survival and cognitive deficits after traumatic brain injury. We found mechanical injury causes the release of ATP from astroglia and initiates widespread intercellular calcium waves through astrocytes in vitro as well as in vivo. Increases in astrocyte intracellular calcium influence neural activity in regions remote from the mechanical injury epicentre, and selectively attenuating intracellular calcium elevation within astrocytes reduces cell death in the CA3 region of organotypic slice cultures exposed to traumatic mechanical injury. The broad importance of purinergic signalling was also evident after in vivo traumatic brain injury, as P2Y1 purinergic receptor antagonism reduced post-traumatic cognitive deficits in mice. Together, these results show that mechanically triggered signalling through astrocytes significantly contributes to the pathobiology of traumatic brain injury. Consequently, targeting receptors on astrocytes and neurons that are activated by gliotransmitters provides a new therapeutic target for treating traumatic brain injury.
Traumatic brain injury is a complex and evolving disease, and some treatments have progressed recently to human clinical trials (Maas et al., 2010
). Remarkably, however, few studies have targeted gliocentric mechanisms to develop new interventions for traumatic brain injury. Although our approach focuses on the events triggered in astrocytes during and after injury, we expect that the broad manipulation of purinergic signalling will affect both neuronal and glial networks in the recovering brain. Presynaptically, ATP inhibits the release of glutamate (Mendoza-Fernandez et al., 2000
), although high doses of ATP will facilitate glutamate release through P2X receptor activation (Rodrigues et al., 2005
). The likely rapid enzymatic breakdown of ATP into adenosine also suggests a potential role for the activation of adenosine receptors, which will inhibit presynaptic release through a reduction in presynaptic calcium currents (Wu and Saggau, 1994
). Even with a more targeted inhibition of P2Y1 receptors, the improvement in functional outcome may be targeted to both neuronal and glial receptors. Inhibiting P2Y1 receptors on astrocytes can limit vesicular glutamate release from astrocytes (Domercq et al., 2006
), reducing the activation of extrasynaptic N
-aspartate receptors on neurons (Bezzi et al., 2004
; Jourdain et al., 2007
), and therefore, suppress signalling of a pathway commonly associated with neuronal death through the mitochondria (Hardingham et al., 2002
; Starkov et al., 2004
). Our data at least partly support this potential protective mechanism, as the neuroprotection afforded by purinergic antagonism was similar to broad-spectrum N
-aspartate receptor antagonism.
Purinergic (P2Y1) receptors are also found at neuronal synapses (Rodrigues et al., 2005
; Tonazzini et al., 2007
). Activation of P2Y1 receptors on hippocampal interneurons increases synaptic inhibition (Bowser and Khakh, 2004
; Kawamura et al., 2004
). Additionally, activation of presynaptic P2Y1 receptors will inhibit presynaptic glutamate release in hippocampal neurons (Mendoza-Fernandez et al., 2000
). Therefore, blocking neuronal P2Y1 receptors leads to a general reduction in the inhibitory tone of the hippocampal circuitry, providing an enhancement of synaptic signalling through the excitatory networks. Neuroprotection through reduced synaptic inhibition initially seems counterintuitive. Evidence in the literature, however, suggests that modest synaptic stimulation triggers pro-survival pathways and antioxidant signalling (Hardingham and Bading, 2002
). Therefore, it seems possible that the neuroprotection provided by P2Y1 antagonists has two components: the reduction in the release of glutamate from astrocytes, and the restoration of pro-survival synaptic signalling.
It remains unclear, however, what the optimal level of synaptic signalling is that supports neuronal survival without triggering deleterious excitotoxic pathways. Interestingly, we noticed a slight reduction in nucleoli counts in MRS 2179 infusion control mice compared with artificial CSF infusion control mice (L). Conversely, we observed a slight increase in cell counts in the contralateral hemisphere after controlled cortical impact (N). Although these differences were not detected as statistically significantly, we propose that by modulating the level of synaptic activity, MRS 2179 could be deleterious in a healthy brain by overstimulating synapses. In the injured brain, this enhanced activity may compensate for the lost pro-survival signalling of disrupted synaptic connections. Hence, purinergic receptor antagonists may provide different mechanisms of neuroprotection through neurons and glia, but these mechanisms seem to work together to improve outcome after traumatic mechanical injury.
We focused on the hippocampus because some of these synapses are ensheathed by glia, and consequently, we hypothesized that the interruption of glial mechanotransmission during injury might be beneficial. We cannot rule out, however, that other brain regions could be affected by purinergic antagonism. We did not observe obvious histological differences in the thalamus or striatum. In our controlled cortical impact model at a moderate severity, these areas do not exhibit the same stark loss of cells as in the CA3 pyramidal cell layer, but subtler changes, such as compensatory alterations in receptor expression, may have been present.
The neuroprotective effects of purinergic antagonism in the hippocampus did not seem to extend to the haemorrhagic lesion in the cortex. A recent study in a rat thoracic spinal cord injury model also did not find a neuroprotective effect of purinergic antagonism at the lesion epicentre (Rodriguez-Zayas et al., 2012
). Instead, purinergic antagonism seemed to reduce the astrogliotic response that normally acts to contain lesion growth (Myer et al., 2006
). Our data suggest that purinergic antagonism may have clearer neuroprotective effects in the penumbra of the injury (, and ) rather than at the lesion epicentre where overt cellular disruption—exacerbated by haemorrhage—and the need for local astrogliosis may supersede any protection afforded by interrupting long-distance glial mechanotransmission. We attribute the cognitive improvements observed to hippocampal sparing. In contrast, functional improvements in thoracic spinal cord injury are dominated by sparing of ascending and descending long axonal tracts rather than rostrocaudal sparing in the grey matter (Hedel and Curt, 2006
). Any neuroprotection afforded by purinergic antagonism of glial and synaptic receptors in the penumbra of the thoracic spinal cord lesion could be difficult to detect if these white matter tracts are already compromised at the lesion epicentre. It would be important in the future to determine the relevant volume of the injury penumbra, and correspondingly, which structures might be most amenable to neuroprotection by purinergic antagonism. An interesting observation from our in vitro
studies is that purinergic antagonism may be beneficial over a broader spectrum of injury severities (); hence, it may be a more robust therapeutic avenue.
Pre-administration of MRS 2179 enabled us to assess the maximal effect of P2Y1 inhibition, but pre-administration of purinergic antagonist is not a clinically viable neuroprotection strategy. A consideration for future studies is the delivery method, which is complicated by the cardiovascular effects of P2Y1 antagonism if the drug is delivered systemically (Kunapuli et al., 2003
). For this reason, we chose direct pharmacological administration through the intraventricular infusion pump. A disadvantage of our current delivery method is the thinning of the CA3 layer that occurs in vehicle-control mice, which indicates chronic implantation of the cannula has some adverse effects, and that treatment efficacy after controlled cortical impact should be interpreted relative to this baseline. Interestingly, we observed a trend in the statistical interaction between MRS 2179 infusion with and without injury contralateral to the lesion epicentre (N). The trend suggests that purinergic antagonism is beneficial in the presence of controlled cortical impact, whereas chronic MRS 2179 administration in uninjured tissue tends to reduce nucleoli counts. Hence, post-injury administration of MRS 2179 may have fewer side effects than the chronic antagonism modelled here. Defining the therapeutic window as well as the dosing regimen for this approach would be a key next step. A recent study found MRS 2179 reduced infarct volume when administered as late as 24 h after cerebral ischaemia (Kuboyama et al., 2011
). Although ischaemia mechanisms differ from the astrocyte mechanotransmission, we analysed here, encouragingly, we also observed that post-injury treatment of organotypic slices with MRS 2179 blocked cell death (C–E), thereby raising the possibility that a P2Y1-based approach has translational utility.
The results presented here add to the complex role of astrocytes in neurotrauma. Although post-traumatic reactive astrogliosis forms a physical and biochemical barrier that inhibits axonal regeneration (Yiu and He, 2006
), the early traumatic response of astroglia is thought to be neuroprotective, as astrocyte ablation worsens traumatic brain injury (Faulkner et al., 2004
; Myer et al., 2006
). Here, we report that during the primary-mechanical trauma, mechanotransmission through astrocytes may play a leading role in mediating neuronal fate and impairment after traumatic brain injury. As such, this work could highlight an important therapeutic direction for improving outcome after traumatic brain injury.