Mizhuma et al., in a ground-breaking 2004 study [
21], demonstrated high levels of LC3 isoforms, key markers of autophagy, in the brains of transgenic mice overexpressing a GFP-LC3 construct. The suggestion of high levels of LC3, and therefore autophagy, in the brain remained controversial for many years as research in neurodegenerative diseases revealed high levels of double membrane autophagosomes. This was incorrectly interpreted to conclude that the brain had a low level of autophagy, which is only up-regulated during neurodegeneration or injury [
22‐
24]. In an effort to confirm if the brain has endogenously high levels of autophagy, we collected brain, heart, lungs, liver, kidney, spleen, and skeletal muscle, metabolically active tissues that have been thought to undergo active autophagy constitutively [
25‐
27]. These were collected from 22–24 week old male C57BL6 mice and the levels of LC3I and II were assessed. We demonstrate that, relative to these tissues, brain expresses LC3I, the precursor to LC3II, and the active autophagic form, LC3II, at significantly higher levels.
In vitro autophagic flux is a more reliable indicator of autophagic activity than static snap-shots of autophagic pathway markers, as is most commonly reported in the stroke literature [
20,
28]. We attempted to assess autophagic flux using a short-term
in-vivo chloroquine treatment compared to untreated animals since there has not been a previous report of
in vivo autophagic flux for murine brain. Chloroquine raises intracellular pH including lysosomal compartments thereby inhibiting acid-dependent lysosomal proteolytic degradation of cellular components within the autolysosome. This allows accumulation of autophagosomes and their contents including autophagy marker proteins. Moreover, when exposed to short-term autophagic inhibition these levels remain high. While the chloroquine treated brains do not have a statistically significant increase in LC3II, there is a strong trend supporting high autophagic flux, and importantly this is not abrogated by flux inhibition. If flux were low, or dysfunctional, there would be no change, or a decrease in LC3II, and potentially in LC3I, too. Future studies will examine optimizing
in vivo inhibition of autophagosome turnover, both in terms of the inhibitor and its application, to assess
in vivo autophagic flux. Importantly, the higher levels of both LC3I and LC3II suggest that brain has both the capacity for the initiation of higher levels of autophagy acutely in response to stress, as well as actually having a higher background constitutive autophagic flux. This suggests an enhanced ability to support constitutively active autophagy is critical for adult brain, possibly due to the high metabolic demand of the brain and lack of storage of nutrients like glucagon, which renders the brain, and neurons in particular, vulnerable to ischemic insult [
29]. A high level of preformed autophagy constituents, (e.g. LC3I and II) ready to rapidly support autophagy, might provide an inherent degree of energy and nutrient buffering to the brain. Therefore, we hypothesized that driving autophagy is protective following stroke.
Autophagy has been well established to play a role in ischemic injury and neurodegeneration although the role(s) is complicated and not fully delineated [
6,
26,
30]. For example, sustained stress may lead to depletion of key preformed autophagy pathway or regulatory proteins needed for successful protective autophagy [
31‐
33]. Consequently, this may result in compromised or dysfunctional autophagy. While the role of autophagy in adult cerebral ischemia is still not clear, its induction appears to be protective and of potential translational value in limited slow reperfusion. However, whether autophagy is protective to neurons and other brain cells in response to ischemia may also be contextual, relative to the extent of ischemia and the presence, or type, of reperfusion. In earlier autophagy and stroke studies, tsMCAO with rapid complete reperfusion, was the most commonly used model. Importantly, the hyperemic effect of rapid and immediate complete reperfusion seen with this type of pre-clinical model is non-physiological relative to most human stroke. It can result in CBF rebounding above the pre-ischemic basal level. This effect may provide protection to both the ischemic core and penumbra when combined with pre-clinical treatments that reduce secondary injury, however, such treatments may not be protective with slow reperfusion [
10]. Therefore, it may be important to find treatments that are protective when reperfusion is limited.
Ultimately this study supports the use of rapamycin, or analogues, as well as potentially other “pro-autophagic” approaches after stroke; specifically where there is partial recanalization and delayed reperfusion. Autophagy has recently been reported to potentially have two distinct roles during “ischemia” and “reperfusion” in studies of myocardial infarction [
9]. Induction of autophagy provided protection during ischemia more than during reperfusion [
34]. However, the timing of autophagy induction might be critical to obtain benefits during stroke. Therefore, future studies are still warranted. Importantly, given that the overwhelming majority of stroke patients do not have reperfusion reversals within the short time frame seen with tsMCAO, the increased vulnerability to secondary molecular cell death effects is not an issue. Indeed, this vulnerability may even be blocked by induction of active autophagy. Therefore, autophagy inducing interventions after stroke during the “golden hour” [
10,
12] may limit the expansion of penumbra and the necrosis of ischemic core. Rapamycin is a safe drug to use, possibly even in the presence of hemorrhagic stroke [
35,
36]. Therefore, rapid induction of autophagy, allowing neurons to survive loss of nutrients and energy production by using nutrient recycling and anaerobic ATP production may prevent, or significantly reduce, the perfusion/metabolism mismatch responsible for
primary infarct expansion into the penumbra and beyond. This may allow a longer window of time for increased spontaneous recanalization based reperfusion and penumbral recovery, or even allow tPA use to be extended out in time and therefore to a larger percentage of the stroke patient population.