Background
Spinal cord injury (SCI) can lead to devastating complications, including permanent neurological damage [
1‐
3]. In acute traumatic spinal cord injury, ischemia and the following reperfusion play a critical role in primary mechanical and secondary pathophysiological mechanisms [
4‐
6]. After the initial rapid compression and trauma, spinal cord ischemia occurs via various mechanisms, including direct injury to the microvasculature, reduced spinal cord blood flow, and disrupted spinal cord autoregulation [
5]. In the following passages, restoration of vascular perfusion and surgical intervention are an essential part of the treatment approach to avoid persistent compression; however, reperfusion
per se can cause further damage [
7‐
9].
Spinal cord ischemia–reperfusion damage is also an important cause of postoperative neurological deficits following decompression surgery, which are rare but very serious. For example, 1.5–6.3% of patients with cervical spondylotic myelopathy suffer postoperative delayed paraplegia related to ischemia–reperfusion injury of the spinal cord [
7‐
9]. In such cases, neuroprotection against ischemia–reperfusion is crucial to prevent spinal cord injury.
Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been shown to minimize the severity of ischemia–reperfusion injury in many organs including the brain, heart, kidney, and lung [
10‐
14]. Statins attenuate neuronal injury and promote neurologic recovery after cerebral ischemia in experimental animal models and in vitro cellular models [
15‐
18]. Statins are frequently used as cholesterol-lowering agents, but their protective effect against ischemia depends on other actions as well [
19], including modification of oxidative stress [
16,
20‐
22], anti-inflammatory effects, and immunomodulation [
15,
18].
Statins have been repeatedly reported to be neuroprotective against spinal cord injury, demonstrating neurologic and histopathologic improvements [
23‐
27]. Especially simvastatin, since it readily crosses the blood–spinal cord barrier, could be widely used to treat spinal cord injuries in clinical practice [
24]. As yet, the underlying mechanism has not been fully studied. In models of cerebral ischemia, simvastatin attenuated neuronal death by reducing the production and toxicity of oxidative stress-related markers [
28,
29]. However, statins’ beneficial antioxidant properties in spinal cord neurons have not yet been investigated.
In this study, we sought the efficacy of simvastatin in attenuation of SCI-induced pathology. We first demonstrated that ischemia–reperfusion injury elicits motor neuron death and cytotoxicity in this model of SCI, and then investigated whether simvastatin treatment recovers those deteriorations of spinal cord neurons against oxidative stress as its neuroprotective mechanism of action.
Discussion
The present study demonstrated that motor neuron death, cytotoxicity, and oxidative stress induced by ischemia–reperfusion injury are significantly attenuated by simvastatin. To our knowledge, this is the first study to suggest the protective role of statins against ischemic injury of spinal cord neurons devising a primary motor neuron culture.
OGD and reoxygenation are well established and reliable neuronal cellular injury models that mimic changes that occur after ischemic insult in vivo [
38‐
40]. Neurons are the cell type most sensitive to ischemic injury. Previous studies of cerebral ischemia–reperfusion have demonstrated that ischemia–reperfusion injury leads to obvious morphological neuronal changes, decreased cell survival, and dramatic increases in LDH release [
38‐
40]. Our model of motor neuron ischemia–reperfusion yielded similar damages; motor neurons of primary cultured rat spinal cord resulted in a significant decrease in cell viability as evidenced by WST-8. After OGD, almost twice as much LDH had leaked out through the injured cell membrane as was leaked from healthy cells, implying increased neurotoxicity. Also OGD and reoxygenation dramatically increased DCFDA value, implying enhanced free radical generation in cells.
Statins reduce OGD and reoxygenation-induced neuronal injury [
28,
40]. Statins reduce cerebral infarct volume [
41,
42], improve perfusion deficits [
43], and facilitate cognitive improvement [
17] mostly using the experimental preclinical stroke models [
39,
43]. Protective effect of statins against ischemic injury has also been reported in other organs, such as myocardial ischemia–reperfusion injury [
11,
44], ischemic acute kidney injury [
14], and intestinal ischemia–reperfusion injury [
45]. We obtained similar results; simvastatin effectively attenuated ischemia–reperfusion-induced spinal cord motor neuron death. Quantitative analysis showed an increase in cell survival after simvastatin treatment at concentrations from 0.1 to 10 μM.
Similarly, ischemia–reperfusion-evoked LDH release was reduced by simvastatin in a dose-dependent manner, which supports the protective effect of simvastatin against ischemia–reperfusion-induced cytotoxicity. LDH leakage indicates cytotoxicity as a result of cell membrane disintegration [
20]. The elevated LDH following OGD in our study significantly decreased with simvastatin treatment at concentrations of 0.1, 1, and 10 μM, indicating that it attenuates the cellular injury/death induced by ischemia. Our basic research performed at the cellular level demonstrated the safety margin of simvastatin in terms of motor neuron protection.
Ischemic injury leads to production of massive amounts of ROS that directly damage the main cellular constituents [
18,
22]. In addition, reperfusion to an ischemic organ, a restoration of oxygen levels in hypoxic tissues, also stimulates ROS production [
46,
47]. However, the CNS is extremely sensitive to oxidative stress due to delicate lipid layers of its cell membranes and low levels of antioxidant enzymes [
48]. Oxidative stress responses in the CNS vary among different cell types. Neurons have relatively low antioxidant capacity and limited scope to upregulate it upon increased oxidative stress, so they are much more vulnerable to oxidative damage than other cells in CNS [
49,
50]. Previous studies investigated the correlation between increased production of ROS and neuronal death following ischemia/hypoxia [
51,
52]. Simvastatin has been reported to have pleiotropic effects, including reducing oxidative stress [
18,
19,
53].
In our study, simvastatin significantly decreased free radical production induced by ischemia–reperfusion, implying that the drug modifies ischemia–reperfusion-induced oxidative stress. Numerous studies have reported that statins decrease ROS production in tissue and in vivo models and in cultured neurons derived from embryonic rat brain tissue [
18,
20,
29] and following major vascular surgery [
54] including thoracoabdominal aneurysm repair [
55,
56] and decompression surgery of degenerative changes of the spine including cervical spondylotic myelopathy [
57‐
59]. Our study results agree with these findings, showing that simvastatin reduces the production of DCFDA in spinal cord cells exposed to OGD and reoxygenation. DCFDA is one of the most widely used techniques for directly measuring the redox state of a cell [
60].
Despite numerous reports of the neuroprotective effects of statins [
19,
28,
29,
61‐
63], some investigators have reported that they are toxic to neurons in vitro [
64,
65]. The toxicity versus protective effects of statins are dependent upon various factors, including the pharmacological characteristics of the individual statin agent, concentration of the drug, and cholesterol content of the neural cell used in the experiment [
66]. Therefore, before the main experiment, we evaluated simvastatin’s toxicity to healthy motor neurons using concentrations ranging from 0.1 to 50 μM, based on the previous studies [
28,
66‐
70]. Cellular viability, assessed by WST-8, was not affected at any experimental simvastatin dose from 0.1 to 10 μM. However, with a dose of 50 μM, the viability of the normal motor neuron was significantly reduced. This finding is consistent with that of previous reports and suggests possible cytotoxicity of high-dose statins [
71]; that high dose was not applied in the main experiments.
The limitation of this study is that we did not determine the precise mechanism of action of simvastatin. Our capabilities did not allow us to examine whether the protective effects were due to direct reduction of cholesterol or whether indirect/nonspecific mechanisms of the statins modify cellular signaling. Further studies will help to elucidate the exact mechanism and the long-term effects of statins for clinical application. Second, there were no additional experiments to show antagonizing the neuroprotective effect of simvastatin, by coapplication of other drugs, such as mevalonate, one of the downstream products of HMG-CoA reductase. Third, it is significant that our spinal cord cells were cultured from embryonic 14–15 fetus because only embryonic cells can be used for extended culture. However, younger cells are more sensitive to neurotoxins, and therefore, the results should generalize to adults with caution.