Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL, and embolic MCAO, murine models of stroke

Abstract

Background and purposeThe role of autophagy in response to ischemic stroke has been confusing with reports that both enhancement and inhibition of autophagy decrease infarct size and improve post-stroke outcomes. We sought to clarify this by comparing pharmacologic modulation of autophagy in two clinically relevant murine models of stroke.MethodsWe used rapamycin to induce autophagy, and chloroquine to block completion of autophagy, by treating mice immediately after stroke and at 24 hours post-stroke in two different models; permanent Middle Cerebral Artery Ligation (MCAL), which does not allow for reperfusion of distal trunk of middle cerebral artery, and Embolic Clot Middle Cerebral Artery Occlusion (eMCAO) which allows for a slow reperfusion similar to that seen in most human stroke patients. Outcome measures at 48 hours post-stroke included infarct size analysis, behavioral assessment using Bederson neurological scoring, and survival.ResultsChloroquine treatment reduced the lesion size by approximately 30% and was significant only in the eMCAO model, where it also improved the neurological score, but did not increase survival. Rapamycin reduced lesion size by 44% and 50% in the MCAL and eMCAO models, respectively. Rapamycin also improved the neurological score to a greater degree than chloroquine and improved survival.ConclusionsWhile both inhibition and enhancement of autophagy by pharmacological intervention decreased lesion size and improved neurological scores, the enhancement with rapamycin showed a greater degree of improvement in outcomes as well as in survival. The protective action seen with chloroquine may be in part due to off-target effects on apoptosis separate from blocking lysosomal activity in autophagy. We conclude pharmacologic induction of autophagy is more advantageous than its blockade in physiologically-relevant permanent and slow reperfusion stroke models.

Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles

“…Similarly, inhibition of autophagy through deletion of Prkaa2 (which encodes AMP-activated, α2 catalytic subunit) or sirtuin 1 ( Sirt1 ), or by downregulation of ATG7 or TSC1, aggravated the cytotoxicity of oxygen glucose deprivation (OGD) in primary mouse 75 or rat 74,76 cortical neurons. In addition, autophagy activation (with rapamycin) or inhibition (with 3-MA, BafA1 or AMPK inhibitors) improved or worsened, respectively, disease outcome in rodents that were experiencing transient middle carotid artery occlusion (tMCAO) or pMCAO 76,77 , as well as neuroprotection in multiple models of <m>ischaemic preconditioning</m> in vivo 78–82 . Stable downregulation of Becn1 in the rat brain by stereotactic injection of a lentiviral vector limited the neurotoxic effects of tMCAO 83 .…”

Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging

“…Autophagic structures and/or markers are consistently observed in brain tissue derived from rodent models of acute hypoxic/ischemic brain injury [40,41]. However, controversy exists as to whether autophagy plays a protective or toxic role since both the positive [42,43] and the negative [41,43] modulation of autophagy has been reported to promote neuroprotection using in vivo models of brain ischemia. For instance, genetic silencing of Tuberous Sclerosis Complex 1 (TSC-1), which results in MTORC1 activation and the inhibition of autophagy, exacerbates neuronal injury using in vitro and in vivo models of transient global ischemia [44].…”

The Ketone Body, β-Hydroxybutyrate Stimulates the Autophagic Flux and Prevents Neuronal Death Induced by Glucose Deprivation in Cortical Cultured Neurons

“…The activation of autophagy under different conditions of cellular stress is well known in the nervous system, and its role in either neuronal survival [28][29][30] or neuronal death [31,32] has been suggested. The role of autophagy in hypoglycemia-and GD-induced neuronal damage has not been well characterized, but a recent study suggests that the disruption of the autophagic flux during glucose reperfusion is involved in the death of neurons exposed to glucose starvation [33].…”