Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

Abstract

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.

Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

“…Electron microscopic analysis has revealed that autophagosomes and autolysosomes are significantly increased in neuronal cytoplasm in the ischemic cortex after pMCAO in rats. Western blot analysis also has shown that the expression of LC3-II and cathepsin B (a main lysosomal protease of the brain parenchyma) are increased in the ischemic cortex [17] . These observations are also supported by Rami et al, who showed that a dramatic elevation in Beclin1 and LC3 levels in the penumbra of rats challenged by tMCAO [15] , and by Puyal et al, who indicated that lysosomal and autophagic activities are increased in the ischemic neurons after transient focal cerebral ischemia [16] .…”

“…In addition, autophagy is induced under starvation, differentiation, and normal growth control to maintain homeostasis and survival [1][2][3] ; however, it is also involved in neurodegenerative disorders and can trigger a form of programmed cell death (Type II death) distinct from apoptosis in neurons [4][5][6][7] . Accumulating evidence indicates that autophagy is activated and may be involved in the regulation of neuronal death in different animal models of ischemic brain injury, including hypoxiaischemia (HI) and global and focal ischemia [7][8][9][10][11][12][13][14][15][16][17] . Recently, using a permanent focal cerebral ischemia model of stroke and an oxygen and glucose deprivation (OGD) model in primary cultured astrocytes, the authors show that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death [18] .…”

The neuroprotective mechanism of brain ischemic preconditioning

“…In an earlier study, we found profound activation of autophagy and lysosomes after permanent middle cerebral artery occlusion (pMCAO) and an autophagic mechanism may contribute to ischemic neuronal injury [86] .…”

Identification of autophagy signaling network that contributes to stroke in the ischemic rodent brain via gene expression

“…Recent research has shown that ischemic insult activates autophagy, and an autophagic mechanism may contribute to ischemic neuronal injury [11] . Autophagy can be induced by pattern-recognition receptors and stresses such as nutrient depletion, closed head injury, or focal cerebral ischemia.…”

“…It has also been reported that global ischemia increases the autophagosomes via decreasing autophagosome degradation [16] . The activation of autophagic and lysosomal pathways has been implicated in neuronal injury in a rat model of permanent focal cerebral ischemia [11] . Furthermore, by regulating the TSC2-mTOR-S6K1 signaling pathway, autophagy is induced and further promotes neuronal survival during cerebral ischemia [20] .…”