This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

Background and Purpose— Traditionally, cell death after cerebral ischemia was considered to be exclusively necrotic in nature, but research over the past decade has revealed that after a stroke, many neurons in the ischemic penumbra will undergo apoptosis. Summary of Review— This brief review provides a general overview and update of various signaling pathways in the development of apoptosis in ischemic lesions. Cerebral ischemia triggers two general pathways of apoptosis: the intrinsic pathway, originating from mitochondrial release of cytochrome c and associated stimulation of caspase-3; and the extrinsic pathway, originating from the activation of cell surface death receptors, resulting in the stimulation of caspase-8. Although many of the key apoptotic proteins have been identified, our understanding of the complex underlying mechanisms remains poor and hence treatment of stroke patients by manipulating apoptotic pathways remains a daunting task. However, recent advances in the field have helped broad...

Apoptotic Mechanisms After Cerebral Ischemia

We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.

https://www.jneurosci.org/content/jneuro/18/10/3659.full.pdf

Activation and Cleavage of Caspase-3 in Apoptosis Induced by Experimental Cerebral Ischemia

The interleukin 1β converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1β (IL-1β) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1β levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1β levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-d-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.

Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage.

Inflammation and glial responses in ischemic brain lesions

We measured the temporal profile and anatomic distribution of cells exhibiting DNA fragmentation at various durations of reperfusion after middle cerebral artery (MCA) occlusion in the rat. Focal cerebral ischemia was induced in male Wistar rats (n = 62) using an intraluminal monofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different durations of reperfusion (0.5, 3, 6, 9, and 12 h and 1, 2, 4, 7, 14, 21, and 28 days, n = 4 per time point). Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls. Coronal brain sections (5 microns) were analyzed, using an in situ ApopTag kit, hematoxylin and eosin, and immunohistochemical double-staining methods. Six rats subjected to 2 h of MCA occlusion were killed at 24 h for measurement of DNA fragmentation by gel electrophoresis. Our data indicate that within a coronal section, DNA fragmentation was present in zero to three cells in each hemisphere of normal and sham-operated rats as well as in the contralateral hemisphere of ischemic rats. The number of cells exhibiting DNA fragmentation increased as early as 0.5 h (8 +/- 6), peaked at 24-48 h (213 +/- 59), and persisted for 4 weeks (10 +/- 2) after onset of reperfusion (p 95% neurons) were located primarily in the inner boundary zone of the infarct. With use of gel electrophoresis, purified DNA obtained from the ischemic tissue exhibited the characteristic nucleosome ladder pattern associated with apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

https://journals.sagepub.com/doi/pdf/10.1038/jcbfm.1995.49

Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat.

Fig. 7 (corrected). TXNIP knockdown prevents lysosome enlargement in ARPE-19 cells. (A) In ScrRNA control ARPE-19 cells, HG (5 days) induces lysosome enlargement (LAMP1-mCherry) compared to LG, which is reduced by Amlx (1 μM) and NAC (5 mM). Ad-CMV-LAMP1-mCherry was transduced for 3 days while Amlx and NAC were added 24 h before taking the images. Arrows show enlarged lysosomes under HG. (B) Quantitation of lysosome sizes in scRNA ARPE-19 cells with HG in the absence or presence of Amlx and NAC. Significant lysosome size increase is seen with HG but not in the presence of Amlx or NAC. (C,D) The effect of HG on lysosome enlargement is absent in shTXNIP3+4 ARPE-19 cells, indicating a role for TXNIP in HG-induced lysosome enlargement in ARPE-19 cells. A representative image of n=3 is shown.

Fayi Yao

Papers

Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat.

Correction: TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells