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Interventions for preventing ischemia-reperfusion induced neuroapoptosis and injury

Updated: Apr 9, 2023

Introduction

Ischemia/Reperfusion (I/R) is the blockage and restoration of oxygen in cells that can cause cell injury and lead to cell death. Spinal cord ischemia, after open and endovascular thoracoabdominal aortic aneurysm repair, tends to occur as a permanent or transient complication. The spinal cord I/R is intended to restore blood to the spinal cord region that sustained an injury after surgery. Typically, a specific period of ischemia can cause permanent damage due to blood perfusion and can lead to paralysis in severe cases. Even given the advances in surgical techniques, spinal cord ischemia is difficult to prevent. Not much is known about strategies to mitigate this injury; however, research is analyzing different approaches.

The literature review is focused on the influence of various cellular and genetic apoptotic pathways on activating neuroapoptosis and what methods can prevent apoptotic genes or apoptosis promoters from expressing themselves. The emphasis of this literature review is on I/R conditions since such conditions are likely to induce neuroapoptosis. The research question for this review is as follows: what are viable methods that can be used to regulate the p53 gene or other pro-apoptotic pathways to prevent neuroapoptosis caused by spinal cord ischemia? This review will compare the various interventions to prevent or treat I/R-induced injury or neuroapoptosis that have been experimented with.


Role of miRNA-22 in p53 gene regulation

Research conducted at the Linyi Cancer Hospital in Shandong, China, experimented with rat cells to assess the influence of microRNA-22’s impact on inhibiting the p53 gene and preventing subsequent enzyme-mediated neuroapoptosis [1]. The research aimed to find whether a certain microRNA sequence (miR-22) can inhibit the P53 Upregulated Modulator of Apoptosis (PUMA) in our DNA that facilitates neuroapoptosis, or neuron cell death when activated by ischemic conditions. Since miR-22 is present in high amounts in the brain, the researchers assumed that miR-22 plays a strong role in preventing neuroapoptosis. After assessing the experiment’s results, they found that miR-22 directly regulates PUMA in tumor cells that were put in ischemic conditions and prevent it from initiating a cascade of formation of enzymes such as caspase-3 and -9.

The caspase enzymes are part of the pathway that leads to cell death. In cells where miR-22 was prevented from functioning, there was an increase in neuroapoptosis, a decreased presence of cleaved-caspase-3, and increased p53 expression. The rate of apoptosis was assessed using the PI and Annexin V assays and the TUNEL staining assay to analyze approximately 10,000 cells per sample. This indicates that miR-22 plays an important role in neurons to prevent cell death and could be used as an intervention to apoptosis in I/R injury in the brain. Since the overexpression of miR-22 showed a reduction in the rate of apoptosis of rat cells, the miR-22/PUMA may provide new insights to preventing cerebral injuries caused by I/R.


Inhibition of caspase-mediated apoptosis

Research conducted at China Medical University in Liaoning, China, implemented neurological assessments using the Tarlov criteria to assess the neurological deficits of lab rats after being affected by I/R [2]. The I/R in the rats caused the Tarlov scores to decrease significantly. Double immunofluorescence staining revealed that cells in the experimental group simulated with I/R conditions showed that p53 expression was significantly greater than that of cells in the control group. The staining concluded that PUMA was expressed nearly 5 times more and p53 nearly 2 times more in the experimental group compared to the control. This suggested that the p53 gene is actively involved in activating the caspase-3 enzyme-mediated apoptotic pathway when prompted by I/R conditions. PUMA was shown to promote mitochondria in cells to activate the caspase-3 apoptotic pathways. To inhibit this neuroapoptosis, the researchers performed an intrathecal injection of small interfering-PUMA (si-PUMA) and pifithrin-alpha (PFT-a) on the cells with elevated p53 gene expression. Subsequent western blot assay showed that both si-PUMA and PFT-a prevented the p53 gene’s activation of the caspase-3 pathway through mitochondria. In doing that, si-PUMA and PFT-a also improved Tarlov scores for neurological assessments of these treated rats, indicating improved behavioral function.


miR-130a and the PTEN/ PI3K/AKT pathway

A study published in the journal of Biomedicine & Pharmacotherapy analyzes the effect of the miR-130a sequence in promoting the PTEN/ PI3K/AKT pathway, a cellular pathway that uses the PTEN gene to suppress tumor proliferation and prevent cell death [3]. The researchers identified the disordered expression of the miR-130a sequence to be associated with the increased risk of ischemic stroke. They experimented with the miR-130a sequence to determine if it could be used as possible neuroprotection to I/R induced neuroapoptosis. After simulating ischemic conditions in an experimental group of pheochromocytoma-12 (PC12) cells derived from rats, the researchers determined that miR-130a expression was nearly 2 times lesser in these cells, thereby reducing miR-130a’s neuroprotective effect; however, ectopic expression of the miR-130a sequence in another group of affected cells showed an increased survival rate and decreased apoptosis rate of the cells according to the results of the Annexin V-FITC apoptosis detection kit. They also found that PTEN could nullify the impact of the miR-130a sequence; nevertheless, upregulating the miR-130a sequence activated the PI3K/AKT pathway that suppressed PTEN and helped prevent neuroapoptosis. The experiment uses a cancer-suppression pathway and a different microRNA sequence to prevent neuroapoptosis.


miR-199a-5p inhibition of the ECE1 gene

MicroRNA-199a-5p (miR-199a-5p) is known to be associated with heart and brain I/R injury [4]. Researchers in Liaoning, China, sought out to assess the impact of the miR-199a-5p sequence on the spinal cord I/R injury. They experimented with the impact of miR-199a-5p on the endothelin-converting enzyme 1 (ECE1), an enzyme that supports the neuroapoptosis pathway.

In simulating an I/R condition in rat spinal cord cells, the researchers found that the I/R had caused predicted damage to the lower limb motor of the rats. In cells administered with treatment, there were 18 differentially expressed miR-199a-5p sequences compared to the control group. These cells also demonstrated a dramatic reduction in apoptosis rate, indicating that miR-199a-5p sequences can be used to prevent I/R-induced neuroapoptosis. Their results demonstrated that this negative regulation of ECE1 protected the lab rats from spinal ischemic injury.

Mitophagy

Autophagosomes in cells are organelles that conduct the process of autophagy, the catabolism of components, and organelles inside the cell, to maintain a consistent number of organelles or simply eliminate organelles due to misconstructions or faultiness [5]. Mitophagy is the selective removal of a damaged or faulty mitochondrion in cells.

Research published in the Journal of Thoracic and Cardiovascular Surgery demonstrates a viable method of neuroprotection through the inhibition of miR-124 against spinal cord I/R injury [6]. The research indicated that mitophagy induction may be possibly involved with neuroprotection against I/R injury. The experiment involved the inhibition of miR-124 using antagomir-124. Antagomir-124 showed significant downregulation of miR-124 and p53 apoptosis stimulators in lab-simulated I/R cells in rats. This inhibition subsequently promoted the expression of becline-1 and LC3-II, components that inhibit the pro-apoptotic pathways.

Inhibition of miR-124 in ischemic spinal cord rat cells was correlated with mitophagy, reduced apoptosis rates, and improved neurological outcomes. The miR-124 inhibition approach introduces another method that is defocused from upregulating miR sequences and downregulating a unique sequence to achieve the same goal.


Conclusion

Research indicates that miRNA has a significant role in either promoting or suppressing PUMA or other pro-apoptotic pathways. While most approaches involve the upregulation of specific miR sequences to suppress pro-apoptotic pathways, some avenues approach neuroprotection through approaches such as downregulation of pro-apoptotic miR sequences or interfering with autophagy mechanisms.

Nevertheless, not much is known about definitive and promising approaches to prevent or protect from spinal cord ischemic injury caused by post-surgical I/R. The various approaches discussed in this literature review are viable methods that have demonstrated significant success.


References

  1. Jiao H, Chen R, Jiang Z, Zhang L, Wang H. miR-22 protect PC12 from ischemia/reperfusion-induced injury by targeting p53 upregulated modulator of apoptosis (PUMA). Bioengineered. 2020 [accessed 2020 Aug 24];11(1):209–218. https://www-tandfonline-com.libproxy.lib.unc.edu/doi/pdf/10.1080/21655979.2020.1729321?needAccess=true. DOI:10.1080/21655979.2020.1729321

  2. Li X-Q, Yu Q, Chen F-S, Tan W-F, Zhang Z-L, Ma H. Inhibiting aberrant p53-PUMA feedback loop activation attenuates ischaemia reperfusion-induced neuroapoptosis and neuroinflammation in rats by downregulating caspase 3 and the NF-κB cytokine pathway. J Neuroinflamm. 2018 [accessed 2020 Sep 1];15(1). http://libproxy.lib.unc.edu/login?url=https://www-proquest-com.libproxy.lib.unc.edu/docview/2108904573?accountid=14244. DOI:10.1186/s12974-018-1271-9

  3. Zheng T, Shi Y, Zhang J, Peng J, Zhang X, Chen K, Chen Y, Liu L. MiR-130a exerts neuroprotective effects against ischemic stroke through PTEN/PI3K/AKT pathway. Biomed Pharmacothera. 2019 Sep [accessed 2020 Sep 1]. https://reader.elsevier.com/reader/sd/pii/S0753332219315057?token=31E54742DE0427EB91A4E068975AE3BB8951102A3F4C5D68E5A4C1D57ABEDEFA573530AE4C5835BC57AD0D065F9EE492. DOI:10.1016/j.biopha.2019.109117

  4. Bao N, Fang B, Lv H, Jiang Y, Chen F, Wang Z, Ma H. Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat. Cell Mol Neurobiol. 2018 [accessed 2020 Sep 2];38(6):1293–1303. https://link-springer-com.libproxy.lib.unc.edu/article/10.1007/s10571-018-0597-2. DOI:10.1007/s10571-018-0597-2

  5. Liu K, Yan L, Jiang X, Yu Y, Liu H, Gu T, Shi E. Mechanisms of mitophagy. Nature. 2010 [accessed 2020 Sep 12];12:9–14. https://www.nature.com/articles/nrm3028#citeas. doi:10.1038/nrm3028

  6. Liu K, Yan L, Jiang X, Yu Y, Liu H, Gu T, Shi E. Acquired inhibition of microRNA-124 protects against spinal cord ischemia-reperfusion injury partially through a mitophagy-dependent pathway. Thorac Cardiovasc Surg. 2017 [accessed 2020 Sep 3];154(5):1498–1508. https://www-sciencedirect-com.libproxy.lib.unc.edu/science/article/pii/S0022522317310875?via%3Dihub. DOI:10.1016/j.jtcvs.2017.05.046

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