The Role of p39 in Cerebral Ischemic Injury

Background Introduction

Stroke is an extremely serious public health problem, with current research mainly focusing on injury mechanisms and new target identification. p39, as an activator of CDK5 (Cyclin-dependent kinase 5), plays a crucial role in various diseases. This article mainly investigates the role and mechanism of p39 in cerebral ischemic injury. The study found that p39 levels significantly decreased at different time points after cerebral ischemia-reperfusion (I/R) injury. Further research showed that the short-term absence of p39 provided neuroprotection to the brain, but exacerbated behavioral dysfunction during the I/R repair phase. This may be due to demyelination induced by higher levels of p35.

Paper Information

This paper was written by Danyang Meng, Di Wu, Xiaojing Li, and Zhigang Miao. The authors are from the Second Affiliated Hospital of Soochow University, Suzhou Hospital, Nanjing University Medical School Affiliated Hospital, and Nanjing Jinling Hospital. The paper was published in the journal “Neuromolecular Medicine”, issue 26:22, DOI: https://doi.org/10.1007/s12017-024-08792-3, and was received on April 22, 2024, and accepted on May 20, 2024.

Research Process

Experimental Animals

Male C57/B6 mice aged 8-10 weeks were selected, divided into p39 wild-type (p39wt) and knockout (p39ko) groups. All animal procedures were approved by the Animal Care Committee of Soochow University.

Middle Cerebral Artery Occlusion (MCAO) Model

Following the method described by Xu et al. in 2006, a 6-0 monofilament was inserted into the vessel and reperfused after 45 minutes. During surgery, the mice’s body temperature was maintained at 36.5-37.5°C, with a success rate of 80%.

TTC Staining

24 hours after MCAO, brain tissue was sectioned and stained with 0.2% 2,3,5-triphenyltetrazolium chloride (TTC), and analyzed using AlphaEase image analysis software.

Behavioral Tests

Tests included grip strength test, rotarod test, hot plate test, and elevated plus maze, conducted before MCAO, and at 24 hours, 3 days, 7 days, and 14 days after reperfusion. Grip strength, rotation time, reaction time on the hot plate, and entry time into the elevated plus maze were tested.

Immunohistochemical Analysis

14 days after reperfusion, mice were anesthetized and perfused with 4% paraformaldehyde, and 15μm thick sections were cut. Immunofluorescence secondary antibody incubation was performed using anti-p35/25 and anti-MBP antibodies, and microscopic photos were taken using a fluorescence microscope.

Western Blot and Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Western Blot was used to detect p39 levels at different time points (6 hours, 12 hours, 24 hours, 3 days, 7 days, and 14 days), and qPCR was used to detect mRNA levels. SDS-PAGE and PVDF membrane transfer were used for electrophoresis analysis, and quantification was performed using Alpha Ease image analysis software.

Statistical Analysis

Data analysis was performed using GraphPad Prism software, with Student’s t-test used to compare differences between two groups, with significance level set at p < 0.05.

Research Results

p39 Levels Decrease After Ischemic Injury

Western Blot revealed that p39 significantly decreased at different time points after ischemic injury, reaching the lowest point at 24 hours and returning to normal at 7 days. Quantitative analysis results were consistent with qPCR results.

p39 Deficiency Reduces Infarct Volume After Ischemic Injury

Behavioral tests showed that p39ko mice performed normally in various tests before MCAO modeling. TTC staining results 24 hours later showed that p39ko mice had significantly reduced infarct volume, significantly lower neurological function scores, significantly higher body weight than p39wt mice, and higher survival rates within 3 days.

Adverse Effects of p39 Deficiency on Sustained Recovery After Ischemic Injury

When measuring behavioral function at 7 and 14 days after ischemic injury, p39ko mice showed significantly increased grip time, number of footprint errors, and forelimb asymmetry, indicating that p39 deficiency affects long-term recovery of neurological function.

Potential Mechanisms of p39 Deficiency Affecting Repair Mechanisms

Detection of p35 levels showed that p39ko mice had significantly increased p35 at 14 days after injury, but no significant changes before injury and at 1 day. Immunohistochemistry and fluorescence detection confirmed the increase in p35 and decrease in myelin production, showing that p39 deficiency leads to demyelination and poor repair effects.

Conclusions and Implications

This study found that p39 significantly decreases after ischemia-reperfusion injury, and the absence of p39 reduces cerebral infarct volume and promotes neurological function repair in the short term, but leads to neurological dysfunction in the long term, mainly due to demyelination. These findings provide a new theoretical basis for future drug development for cerebral ischemic injury.

Research Highlights

1. Research Innovation: First in-depth study of the role and mechanism of p39 in cerebral ischemic injury.
2. Methodological Diversity: Combination of various behavioral tests, immunohistochemistry, Western Blot, and qPCR.
3. Theoretical Foundation: Provides a solid theoretical basis for discovering new mechanisms of cerebral ischemic injury and future therapeutic drugs.

Other Important Information

This research was supported by the Basic Frontier Innovation Crossing Project of Suzhou Medical Research Institute (YXY2304057) and the Jiaxing Science and Technology Project (2021AY30022). The authors believe that p39 may be an important target for cerebral ischemic injury repair, with the potential to develop new therapeutic drugs by regulating p39 levels in the future.