Representative light photomicrographs of the ventral gray matter of Nissl-stained transverse taken from the L4 spinal cord segment are shown in Figure 2A. There were more normal motor neurons at 48 hours after reperfusion in the simvastatin group (43 ± 14) than in the vehicle group (19 ± 18; P = 0.037) (Fig. 2B). There were significantly more normal motor neurons in the sham group (72 ± 12) than in the simvastatin group (P = 0.010).
Representative light photomicrographs of HE-stained sections in the ventrolateral white matter are shown in Figure 3A. Vacuolation in the white matter was widespread and prominent in the vehicle group, whereas only small and scattered vacuolation was observed in the simvastatin group. The percentage area of vacuolation in the simvastatin group (10% ± 4%) was significantly smaller than in the vehicle group (19% ± 5%; P = 0.030) (Fig. 3B). The sham group (1% ± 1%) had a significantly smaller percentage area of vacuolation than the simvastatin group (P = 0.003).
The relationships among MDI scores, the number of normal neurons, and percentage areas of vacuolation are shown in Figure 4. There was a significant negative correlation between MDI scores and the number of normal neurons (with sham group: Spearman correlation coefficient −0.89, P < 0.001; without sham group: Spearman correlation coefficient −0.73, P = 0.008). There was a significant positive correlation between MDI scores and the percentage areas of vacuolation (with sham group: Spearman correlation coefficient 0.84, P < 0.001; without sham group: Spearman correlation coefficient 0.60, P = 0.043). There was a significant negative correlation between the number of normal neurons and the percentage areas of vacuolation (with sham group: Spearman correlation coefficient −0.93, P < 0.001; without sham group: Spearman correlation coefficient −0.91, P < 0.001).
In this study, we demonstrated that simvastatin treatment significantly attenuated hindlimb motor dysfunction and histological damage in the gray and white matter of the spinal cord induced by ischemia/reperfusion in rats. The importance of evaluating both white matter and gray matter injury in spinal cord ischemia has been reported.18–20 Follis et al.19 also demonstrated that gray and white matter of the spinal cord exhibit different sensitivities to ischemic injury because of tissue-structural differences.19 Moreover, Kanellopoulos et al.20 reported that (2,3-dihydroxy-)6-nitro-7-sulfamoyl-(f)-quinoxaline-2,3-dione (NBQX), an AMPA/kainate glutamate receptor antagonist, preserved hindlimb motor function in a rat spinal cord ischemia/reperfusion model20; white matter damage was decreased in the NBQX group at 48 hours after reperfusion, whereas there was no improvement in gray matter damage.
In the central nervous system, white matter tissue is highly susceptible to ischemic damage. The myelin sheath accounts for 50% of white matter volume and is susceptible to severe damage by lipid peroxidation because it is composed of 70% to 80% lipid.21 Furthermore, oligodendrocytes are vulnerable to superoxide produced during ischemia/reperfusion because they contain only low levels of superoxide antagonists such as Mu-superoxide and catalase.22 Because simvastatin can inhibit lipid peroxidation and superoxide production after cerebral ischemia/reperfusion,7 the preservation of hindlimb motor function observed in the present study may have been associated with a protective effect of simvastatin against both white and gray matter injury. Although the complete mechanism by which simvastatin protects the spinal cord against ischemia/reperfusion injury is unknown, several mechanisms are suggested for neurons.7–10,12,13,15 Simvastatin reduces oxygen and glucose deprivation/reoxygenation- or N-methyl-D-aspartate-induced neuronal injury in vitro,9,15 and in rat cerebral ischemia models, cerebral infarction volume and production of 4-hydroxynonenal–conjugated protein in the cerebral cortex and caudate are reduced with simvastatin administration.7 In transient cerebral ischemia rat models, mRNA expression of interleukin-1β and tumor necrosis factor-α are suppressed in the cortex and hippocampus after simvastatin, with improvements in T-maze and circular water maze memory tests.10 These data also suggest that simvastatin should protect spinal cord neurons from ischemia/reperfusion injury. However, this requires further investigation.
There are several reports on the effects of varying timing of statin administration on ischemic neuronal injury.7–15 For example, simvastatin administration from 1 week to 48 hours before ischemia induces ischemic tolerance in rat cerebral ischemia models.8,10 However, evidence from animal and clinical studies suggests that spinal cord neuronal injury may continuously worsen even after ischemia/reperfusion,23–25 and in the present study, the spinal cord continuously deteriorated and near-complete paraplegia developed at 48 hours after reperfusion in the vehicle group. A potential cause of delayed neuronal damage is an overinflammatory response that continues after reperfusion.23 Furthermore, administration of pravastatin after reperfusion was reported to reduce the overinflammatory reaction and was neuroprotective at 5 days' recovery from transient cerebral ischemia in rats.11 Based on these data, we administered simvastatin from 1 week before ischemia and continued for the first 24 hours of postischemic reperfusion. It is possible that a selection of different time points for simvastatin administration may have resulted in different outcomes. Further studies are required to determine the optimal window for treatment.
The concentration of simvastatin used in our study was based on that previously reported,13 although it was higher than that used clinically. However, statin sensitivity differs among species, and the rat is less sensitive to statin than the human.7,8 As such, it may be unnecessary to administer higher doses of statin to induce ischemic tolerance in humans. Rhabdomyolysis is a serious side effect of high-dose statin. However, in the present study, we did not observe abnormal findings in urine that were suggestive of rhabdomyolysis in simvastatin-treated rats. Westwood et al.26 also reported that creatine kinase and muscle necrosis were not observed histologically in rats administered 60 mg/kg/d simvastatin for 43 days.26
A potential limitation of our study is that we only examined the efficacy of simvastatin up to 48 hours after reperfusion because the most severe spinal cord injury occurs at this time.24 In the clinical setting, however, paraplegia may develop at 1 to 5 days after spinal cord ischemia.27 In addition, some patients with cerebral infarction who discontinued chronic statin therapy in its acute phase had a higher mortality rate28,29 and infarct volume28 at long-term follow-up, suggesting that withdrawal from statin administration influences long-term prognosis. However, prophylactic treatment with statins in various animal models of transient cerebral ischemia was reported to reduce postischemic delayed neuronal death in the hippocampus at 3 and 5 days' recovery.30–32 Furthermore, a study of permanent cerebral ischemia reported that the rats treated with atorvastatin had reduced infarction volumes at 21 days after ischemia.33 Further studies are required to determine these long-term results including the influence of discontinuation of simvastatin administration.
In summary, simvastatin treatment attenuated hindlimb motor dysfunction at 24 and 48 hours after reperfusion and reduced both white and gray matter injury at 48 hours after reperfusion in our rat spinal cord ischemia model. These data suggest a potential application for simvastatin for neuroprotection in the perioperative management of descending and thoracoabdominal aortic surgery. Further studies on the timing of administration and long-term effects are required.
We are grateful to Prof. Shin-ichi Toyabe (Niigata University Crisis Management Office, Niigata University Medical and Dental Hospital) for his valuable help.
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