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Updated: Apr 24, 2026

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke
Published on: January 3, 2025
The role of autophagic and lysosomal pathways in ischemic brain injury
Zhaohua Gu1, Yinyi Sun2, Kangyong Liu3
1Department of Neurology, Shanghai Pudong New Area Zhoupu Hospital, Shanghai 234318, China.
Abstract:
Autophagy is involved in neural cell death after cerebral ischemia. Our previous studies showed that rapamycin-induced autophagy decreased the rate of apoptosis, but the rate of apoptosis was creased after the autophagy inhibitor, 3-methyladenine, was used. In this study, a suture-occluded method was performed to generate a rat model of brain ischemia. Under a transmission electron microscope, autophagic bodies and autophagy lysosomes were markedly accumulated in neurons at 4 hours post brain ischemic injury, with their numbers gradually reducing over time. Western blotting demonstrated that protein levels of light chain 3-II and cathepsin B were significantly increased within 4 hours of ischemic injury, but these levels were not persistently upregulated over time. Confocal microscopy showed that autophagy was mainly found in neurons with positive light chain 3 signal. Injection of rapamycin via tail vein promoted the occurrence of autophagy in rat brain tissue after cerebral ischemia and elevated light chain 3 and cathepsin B expression. However, injection of 3-methyladenine significantly diminished light chain 3-II and cathepsin B expression. Results verified that autophagic and lysosomal activity is increased in ischemic neurons. Abnormal components in cells can be eliminated through upregulating cell autophagy or inhibiting autophagy after ischemic brain injury, resulting in a dynamic balance of substances in cells. Moreover, drugs that interfere with autophagy may be potential therapies for the treatment of brain injury.
Insights
Autophagy, a cellular process, is elevated in brain cells following ischemic injury. Modulating autophagy with drugs like rapamycin or 3-methyladenine impacts neural cell death, suggesting therapeutic potential for brain injury.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Autophagy plays a role in neural cell death after cerebral ischemia.
- Previous studies indicated rapamycin (autophagy inducer) decreased apoptosis, while 3-methyladenine (autophagy inhibitor) increased it.
Purpose of the Study:
- To investigate the role and dynamics of autophagy in a rat model of cerebral ischemia.
- To examine the effects of autophagy modulation on ischemic brain injury.
Main Methods:
- A suture-occluded method was used to induce cerebral ischemia in rats.
- Transmission electron microscopy, Western blotting, and confocal microscopy were employed.
- Rapamycin and 3-methyladenine were administered to modulate autophagy.
Main Results:
- Autophagic bodies and lysosomes accumulated in neurons within 4 hours post-ischemia, decreasing over time.
- Levels of light chain 3-II and cathepsin B increased early post-injury but were not sustained.
- Rapamycin promoted autophagy and increased protein expression, while 3-methyladenine diminished them.
- Autophagy was primarily observed in neurons positive for light chain 3.
Conclusions:
- Autophagic and lysosomal activity are upregulated in ischemic neurons.
- Modulating autophagy (either upregulating or inhibiting) influences cellular balance after ischemic brain injury.
- Drugs targeting autophagy pathways show potential for treating brain injury.
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