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Updated: Jul 15, 2026
Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke
Published on: January 3, 2025
Expression of signaling molecules associated with apoptosis in human ischemic stroke tissue
Nicholas Mitsios1, John Gaffney, Jerzy Krupinski
1Department of Biological Sciences, Manchester Metropolitan University, Chester St, Manchester M1 5GD, UK.
Abstract:
There is growing evidence that, because of the highly significant differences in gene activation/protein expression between animal models of stroke and stroke patients, the current treatment strategies based on animal stroke models have been unsuccessful. Therefore, it is imperative that the pathobiology of human stroke be studied. As a first step here, Western blotting and immunohistochemistry were employed to examine expression and tissue localization of key apoptotic proteins in infarct and peri-infarcted (penumbra) from grey and white matter in human postmortem tissue of 18 patients who died between 2 and 37 d after stroke caused by large vessel disease. The contralateral hemisphere was used as a control. JNK1, JNK2, and p53 were upregulated in the majority of samples, whereas Bcl-2, caspase-3, active caspase-3, phosphorylated p53 (p-p53), phosphorylated JNK1 (p-JNK1), and phosphorylated JNK2 (p-JNK2) were upregulated in approximately half of the samples. JNK1 expression was positively correlated with JNK2 expression in grey and white matter infarct and penumbra, whereas active caspase-3 levels were positively correlated with p-JNK2 levels in grey and white matter infarct. Using indirect immunoperoxidase staining of paraffin-embedded sections, active caspase-3 was found in infarcted neurons that co-localized with TUNEL-positive cells. p-JNK localization in the nuclei of TUNELpositive cells with the morphological appearance of neurons from infarct and penumbra was also demonstrated. The use of Kaplan Meier survival data demonstrated that the presence of Bcl-2 in penumbra of grey matter correlated significantly with shorter survival (p = 0.006). In conclusion, the present study has identified significantly altered expression of apoptotic proteins in human stroke tissue and shown that the presence of Bcl-2 in penumbra of grey matter has prognostic value. It is tempting to suggest that further studies of apoptotic proteins in human stroke may lead to identification of novel targets for drug discovery.
Insights
Human stroke research is crucial due to animal model limitations. This study found altered apoptotic protein expression in human stroke tissue, with Bcl-2 in the penumbra indicating poorer survival, suggesting new drug targets.
Area of Science:
- Neuroscience
- Molecular Biology
- Pathology
Background:
- Current stroke treatments fail due to differences between animal models and human patients.
- Understanding human stroke pathobiology is essential for developing effective therapies.
Purpose of the Study:
- To investigate the expression and localization of apoptotic proteins in human stroke tissue.
- To identify potential prognostic markers and therapeutic targets for stroke.
Main Methods:
- Western blotting and immunohistochemistry on human postmortem brain tissue from stroke patients.
- Analysis of infarct and penumbra regions in grey and white matter.
- Kaplan Meier survival analysis was used to assess prognostic value.
Main Results:
- Upregulation of JNK1, JNK2, and p53 observed in most samples.
- Altered expression of Bcl-2, caspase-3, and phosphorylated proteins noted in approximately half of samples.
- Bcl-2 presence in grey matter penumbra correlated significantly with shorter survival.
Conclusions:
- Human stroke tissue exhibits significantly altered apoptotic protein expression.
- Bcl-2 in the penumbra of grey matter serves as a prognostic indicator for survival.
- Further research into apoptotic proteins may reveal novel drug discovery targets for stroke.
