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A Neuronal Apoptosis Model induced by Spinal Cord Compression in Rat
Published on: June 29, 2021
MGMT-Mediated neuron Apoptosis in Injured Rat Spinal Cord
Yingjie Ni1, Jun Gu1, Jianyue Wu1
1Department of Orthopaedics, Xishan People's Hospital, Wuxi, Jiangsu, PR China.
Abstract:
Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. The aim of this study was to investigate the potential effect and mechanism of action of MGMT in strengthing neuronal apoptosis following SCI. To determine MGMT-mediated apoptosis in spinal cord injury, we performed western blot and analyzed the expression change of MGMT with different timepoints. Western blot analysis showed the upregulation of MGMT has a peak at 21 days in injured spinal cord tissues. Expression and location was observed in the neurons after SCI. Upregulation of p53, Bax, cleaved caspase3 and cleaved caspase9 and downregulation of Bcl2 were detected after SCI. Co-localization of cleaved caspase3 with MGMT indicated MGMT involved in apoptosis taking place after SCI. In addition, we carried out H2O2 stimulation to further confirm MGMT played a role in neuron apoptosis process and activated p53 signaling pathway in vitro. Finally, based above data, we packaged lenti-associated virus inhibit MGMT expression and injected into rat spinal cords after SCI model was built. LV-MGMT not only reduces the neuron apoptosis, but also increases GAP43 expression and promotes hindlimbs locomotor function recovery. Taken together, the in vivo data and the in vitro observations prove MGMT-mediated apoptosis in the injured spinal cord.
Insights
MGMT upregulation exacerbates neuronal apoptosis after spinal cord injury (SCI). Inhibiting MGMT reduces this apoptosis, promoting functional recovery in SCI rats.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Spinal cord injury (SCI) triggers secondary degeneration, including neuronal apoptosis.
- Understanding the molecular mechanisms driving apoptosis post-SCI is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the role of MGMT (O6-methylguanine-DNA methyltransferase) in neuronal apoptosis following SCI.
- To elucidate the underlying molecular mechanisms of MGMT's action in SCI.
Main Methods:
- Western blot analysis to assess MGMT expression changes over time in injured spinal cord tissues.
- Immunohistochemistry to determine MGMT localization within neurons post-SCI.
- In vitro studies using H2O2 stimulation to confirm MGMT's role in apoptosis and p53 pathway activation.
- In vivo experiments using lentivirus-mediated inhibition of MGMT in a rat SCI model.
Main Results:
- MGMT expression significantly upregulated, peaking at 21 days post-SCI, and localized to neurons.
- SCI induced apoptosis markers (increased p53, Bax, cleaved caspase-3, cleaved caspase-9; decreased Bcl-2).
- Co-localization of cleaved caspase-3 with MGMT suggested MGMT's involvement in apoptosis.
- In vitro, MGMT activation of the p53 signaling pathway was confirmed.
- Inhibition of MGMT in vivo reduced neuronal apoptosis, increased GAP43 expression, and improved hindlimb locomotor function.
Conclusions:
- MGMT plays a significant role in mediating neuronal apoptosis after spinal cord injury.
- Targeting MGMT presents a potential therapeutic strategy for mitigating apoptosis and promoting functional recovery post-SCI.
