1. objectives Methods Results Conclusions References
TIGAR protects neurons against ischemic injury through reducing intracellular ROS
Mei Li1, Jinhua Gu1, Jianbin Ge1, Rong Han1, Meiling Sun1, Yuqiang Ding2, Zhenghong Qin1*
objectives
1 Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, 2 Department of Anatomy and Neurobiology, Tongji University School of Medicine
Soochow University
Methods
To investigate if ischemia/reperfusion resulted in up-regulation of TIGAR and if TIGAR played a neuroprotective role in vivo and in vitro models of stroke.
Mouse model of ischemia/reperfusion was produced with occlusion and reopen of middle cerebral artery and neuron cell OGD-reoxygenation model was produced by deprivation of glucose/oxygen in culture medium and reoxygenation. The expression of TIGAR in neurons under normal and ischemic conditions was determined with immunofluorescence and immunoblotting. The up-regulation and down-regulation of TIGAR were achieved with lentivirus-mediated TIGAR overexpression and TIGAR knockdown, respectively. The effects of manipulation of TIGAR on neuronal injury and oxidative stress were assessed in vivo and in vitro ischemic models.
Figure 2. Expression of TIGAR was induced in ischemic brains. ICR mice were subjected to MCAO/reperfusion and were killed at indicated time. The whole ischemic cortical tissue was dissected for Western blot analysis. A: TIGAR was up-regulated in ischemic brain 3-6 hours after reperfusion. B: TIGAR was up-regulated in cultured neurons 3-12 hours after reoxygenation.
Figure 5. Knockdown of TIGAR decreased NADPH levels and increased intracellular ROS levels. Primary neurons were infected with LV-TIGAR or LV-sh_TIGAR and subjected to OGD/reoxygenation. GSH and NADPH were determined 3 hours after reoxygenation. A: GSH levels. B: NADPH levels. C: ROS production was analyzed using flow cytometry.
Results
Figure 6. Knockdown of TIGAR increased cell death by a ROS-mediated event. Primary cortical neurons were infected with LV-TIGAR or LV-sh_TIGAR and subjected to OGD/reoxygenation in the presence of NADPH or NAC. A: Cell viability. B: LDH release. Knockdown of TIGAR-induced cell death was reversed by supplementation with NADPH and NAC. C: TTC staining of mouse brain. ICR mice were administered LV-sh_scramble or LV-sh_TIGAR and subjected to MCAO/reperfusion and were killed at 24 hours after reperfusion. Mice were administered NADPH at the onset of MCAO.
Figure 3. TIGAR protected neurons against ischemic damage. ICR mice were administered LV-TIGAR or LV-sh_TIGAR and subjected to MCAO/reperfusion and were killed at indicated time. The whole ischemic cortical tissue was dissected for Western blot analysis. A: Efficiency of overexpression and knockdown of TIGAR by LV-TIGAR and LV-sh_TIGAR, respectively. B and C: TTC staining. TIGAR overexpression with LV-TIGAR reduced ischemic infarction and TIGAR knockdown with LV-sh_TIGAR increased ischemic infarction.
Conclusions
In summary, current studies demonstrated that up-regulation of TIGAR protected neurons against oxidative stress in ischemic stroke. TIGAR promoted neuronal survival through increasing production of NADPH and reducing intracellular levels of ROS. Our data provide a new insight into endogenous defensive mechanisms in ischemic stroke and suggest that TIGAR maybe a new therapeutic target for stroke prevention and treatment.
References
Figure 1. TIGAR expression in mouse brain and cultured neurons. A: Expression of TIGAR in brain, primary cortical neurons and astrocytes. Note: TIGAR was mainly expressed in neurons. B: Immunofluoresecence of TIAGR in mouse brain. Double immunofluorescence of TIGAR and GFAP (astrocytes marker) or NeuN (neuronal marker) was performed. C: Immunofluoresecence of TIGAR in mixed culture of primary neurons and astrocytes cells. Double immunofluorescence of TIGAR and MAP-2 (neuronal marker) or GFAP was performed. N = nucleus.
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Figure 4. TIGAR promoted neuronal survival under OGD/reoxygenation condition. Primary neurons were infected with LV-TIGAR or LV-sh_TIGAR and subjected to OGD/reoxygenation. A: Western blot analysis of efficiency of overexpression and knockdown of TIGAR by lentivirus in cultured neurons (MOI = 10). B: Cell viability assay. C: LDH release.