The Bio-directional Effects And Mechanisms Of Adenosine A2A Receptors In Traumatic Brain Injury

Traumatic brain injury (TBI) is a severe human injury condition with high incidence, disability and fatality in modern society, which is known to result from neurological deficits through both primary and secondary (delayed) events. While primary damage to the brain cells and tissues is irreversible and incurable, secondary damage results from a potentially reversible process, and the extent of secondary damage closely correlates with clinical prognosis. Three possible mechanisms have been proposed for such secondary brain injury, including glutamate release, cytokine production and Ca2+ overload. Interfering with any of these three pathological processes may reduce TBI-induced brain damage. During brain injury, adenosine levels increase rapidly and markedly to make adenosine receptors active. Recent studies have showed that activation of the adenosine A2A receptors (A2ARs) exert both attenuation and aggravation with regulation of glutamate release, inflammation and Ca2+ influx in a lot of brain injury models, such as Parkinson's disease, ischemic brain injury and excitotoxicity and experimental allergic encephalitis (EAE). However, the role of A2ARs in TBI is unknown.In this study, to investigate the effects and mechanisms of A2ARs in TBI, we constructed a cortical impact injury model of TBI in mice by weight-dropped method and firstly investigated the effect of A2ARs on the injury at 24h post-TBI by use of the selective agonist CGS21680 and antagonist ZM241385 at 15min, 3h, 6h and 12h after TBI, respectively. Together with the assay of glutamate levels in cerebral spinal fluid (CSF) at the medicine treatment time described above, we analyzed the relationship of the effects of A2ARs-activation and the concentration of glutamate in internal environment in TBI. Secondly, we assayed the effect of CGS21680 on LPS-induced NOS activity of cultured microglia cells and LPS-induced TNF-αmRNA expression of peripheral blood polymorphonuclear neutrophils (PMN) in the presence of increased concentrations of glutamate (0, 0.1, 0.5 and 5.0 mmol/L) and examined the signal transduction of PKA and PKC pathway associated differential regulation of inflammation by A2ARs. We further observed the effect of A2ARs activation after inhibition of glutamate release in mouse TBI model to explore the possible mechanisms of the bi-directional effects of A2ARs regulated by glutamate. Finally, to prove whether the A2ARs on bone marrow-derived cells (BMDCs) were the main targets of glutamate regulation, we constructed two transgenic mouse models: selective inactivation and reconstruction of A2ARs on BMDCs by bone marrow transplantation. Then we investigated their effects in TBI model, which was a injury with high level of glutamate in internal surrounding and in acute lung injury (ALI) model, which was a injury without glutamate in circumstance.The main results and conclusions are summarized as follows:1. In TBI model of mouse, activation A2ARs by CGS21680 at 3h post-TBI , which time the glutamate level in CSF was relative low, attenuated the neurological deficits, cerebral edema, glutamate release as well as cytokine expressions of mice at 24h post-TBI significantly; while inactivation of A2ARs by ZM241385 performed aggravation. However, CGS21680 treatment at 15min, 6h post-TBI, which time the glutamate level in CSF was relative high, significantly accelerated the brain damage with upregulation of glutamate concentration and cytokine expressions of mice at 24h post-TBI, while ZM241385 showed protection. In addition, administration of CGS21680 at 12h post-TBI which the glutamate level was much higher than that of 15min, 6h post-TBI, even caused mice dead and ZM241385 didn't show significant protective effect. These results prove that A2ARs also have bi-directional effects in TBI which is connected with the concentration of glutamate in internal environment.2. In primarily cultured microglia cells from wild type mice, CGS21680 inhibited LPS-induced NOS production significantly in parallel with increased level of cAMP in the glutamate level at 0 and 0.1mM when compared with the LPS alone group. This effect was completely abolished by the PKA inhibitor H-89. In the presence of 0.5mM and 5.0 mM of glutamate, LPS-induced NOS production was enhanced rather than inhibited by CGS21680. This enhancement effect was not inhibited by associated with the cAPM level and not affected by H-89, but was blocked by the PKC inhibitor GF109203X. The differential modulation of CGS21680 on NO activity in the increased concentration of glutamate is mediated by the A2AR since CGS21680 failed to inhibit LPS-induced NOS production and cAMP levels (in the presence or absence of glutamate) in microglial cells derived from A2AR-deficient mice. And the different role of A2AR on inflammation regulated by glutamate were confirmed in LPS-induced neutrophil TNF-αexpression These results demonstrate that, increased extracellular glutamate switches the cAMP-mediated inhibition by adenosine A2ARs agonist to the PKC-mediated potentiation on inflammation in cultured microglia and neutrophil.3. At 15min post-TBI, CGS21680 treatment accelerated brain damage of mice at 24hs post-TBI as described above. However, after treating the mice with antagonist of glutamate release (s)-4c3HPG at 30 min before TBI, CGS21680 treatment at 15min post-TBI attenuated brain injury, glutamate level in CSF and cytokine mRNA expressions of mice at 24h post-TBI. This result is consistent with previously cell experimental results and confirms that high level of glutamate switches the protective role to the damaging effect of A2ARs activation.4. Mice models of selective inactivation and reconstruction of A2ARs on BMDCs were constructed and applied to different injury conditions with or without glutamate in circumstance. In TBI model, which was a condition with high level of glutamate, both selective inactivation and reconstruction of A2ARs on BMDCs played protective role, just like global A2ARs inactivation. All of the three could reduce the concentration of glutamate in CSF of mice at 24h post-TBI, but the effect on inhibition of cytokine expressions by selective inactivation A2ARs was much better than the other two. However, in ALI model, which was a injury without glutamate in circumstance, selective inactivation of A2ARs on BMDCs and global A2ARs inactivation promoted lung damage by promotion of inflammatory cytokine expressions, while selective reconstruction of A2ARs on BMDCs didn't show any significant difference compared with injured wild type mice. These results showthat high level of glutamate may switch the effect of A2ARs on BMDCs from suppression inflammation to promotion inflammation, and suggest that A2ARs on BMDCs regulation by glutamate may be the important cell mechanism of A2ARs'bi-directional in TBI.In summary, we demonstrated the bi-directional effects of A2ARs in the mouse TBI model and that high level of glutamate in internal enviroment might switch the effects of A2ARs, mainly on BMDCs, on inflammation and glutamate release from inhibition to promotion in accompany with the signal pathway switch from PKA to PKC activation. These findings may provide some experimental evidence and a new strategy for clinical treatment by regulation of A2ARs.