We demonstrated that a bolus of lithium administration robustly reduced infarct size in a model of distal MCAo with transient bilateral CCAo. This result suggests that lithium treatment can reduce brain injury induced by focal ischemia with partial reperfusion, which is a common phenomenon in stroke patients. Therefore, our study provides novel data that supports the potential of lithium as a treatment for patients who experience stroke with partial reperfusion. The protective effect of lithium was further confirmed when lithium treatment was shown to abolish TUNEL-positive staining in the ischemic penumbra, but not in the ischemic core. However, although the Akt pathway has been shown to be critical for neuronal survival, lithium did not increase Akt activity in our study. This suggests that the neuroprotective effects of lithium following stroke do not necessarily involve Akt activity.
The Akt pathway is known to support neuronal survival after stroke [14
], implying that Akt activity is necessary for neuroprotective effects. Previous studies, including our own, have demonstrated that ischemia/reperfusion results in transient increases in Akt phosphorylation at ser473. Most of these studies used the quantity of p-Akt as a marker for Akt activity. However, we found increased p-Akt protein levels early after stroke onset yet decreased Akt activity at the same time point. The quantity of p-Akt, therefore, does not represent true Akt activity [17
]. We repeated this finding in hypothermic, ischemic preconditioning and ischemic postconditioning models [16
]. Whether lithium alters Akt kinase activity has not been previously reported. In this study we measured Akt activity using an Akt kinase assay rather than the level of p-Akt. We found decreased Akt activity after stroke and lithium did not increase Akt activity. This differs from our previous studies where all neuroprotectants, hypothermia, preconditioning, and postconditioning increased Akt activity independently of p-Akt levels [16
]. Our current study suggests that Akt activity is not always involved in how certain neuroprotectants exert their effects.
Lithium may not alter Akt activity because it is not a direct Akt activator. Rather, it may inhibit neuronal death by inhibiting GSK3β activity, a protein downstream of Akt, as reported in previous studies [15
]. Although our results showed that lithium had no effect on p-GSK3β or β-catenin protein levels, we cannot exclude the possibility that lithium did inhibit GSK3β activity. Previous studies have shown that lithium directly inhibits GSK3β activity as a competitive inhibitor of Mg2+
, since GSK-3β catalyzes the phosphorylation of many protein substrates in the presence of Mg2+
]. Lithium may also indirectly inhibit GSK3β activity by promoting GSK3β phosphorylation, however, chronic lithium treatment is required to promote GSK3β phosphorylation and alter mRNA levels of β-catenin [15
]. A single bolus of lithium injection in our study may not be able to improve GSK3β phosphorylation or β-catenin protein levels.
Our current study has some limitations. First, although we showed that lithium did not alter Akt activity, disputing the importance of Akt in the protective effects of lithium against stroke, we do not know which cell signaling pathways were involved in its protective effects. Second, one dose of lithium was used in the current study, and the therapeutic time windows were not explored. Third, only acute effects of lithium as early as 48 h after stroke were studied. Other studies have shown that lithium inhibits caspase-mediated apoptosis [9
], blocks p53 activity [5
], promotes Bcl-2 proteins [5
], recovers heat shock proteins (HSP) 70 and 90β [11
], and attenuates inflammatory factors, such as c-jun and c-fos [9
]. Thus, lithium injection may reduce infarct size through cell-signaling pathways other than the Akt pathway. To fully understand the protective effects and mechanisms of lithium, future experiments should include a series of experiments with multiple pharmacological and genetic approaches using in vivo
and in vitro
models. In addition, long term protective effects as well as neurological scores should be evaluated.
In summary, lithium treatment provided strong protection against focal cerebral ischemia with partial reperfusion in a model that mimics a large portion of human stroke cases. Our data offer novel evidence for the potential clinical translation of lithium for the treatment of stroke, and that upregulation of Akt activity. However, whether lithium truly inhibits GSK3β activity in this setting requires further study.