Simvastatin ameliorates cauda equina compression injury in a rat model of lumbar spinal stenosis

Abstract

Lumbar spinal stenosis (LSS) is the leading cause of morbidity and mortality worldwide. LSS pathology is associated with secondary injury caused by inflammation, oxidative damage and cell death. Apart from laminectomy, pharmacological therapy targeting secondary injury is limited. Statins are FDA-approved cholesterol-lowering drug. They also show pleiotropic anti-inflammatory, antioxidant and neuroprotective effects. To investigate the therapeutic efficacy of simvastatin in restoring normal locomotor function after cauda equina compression (CEC) in a rat model of LSS, CEC injury was induced in rats by implanting silicone gels into the epidural spaces of L4 and L6. Experimental group was treated with simvastatin (5 mg/kg body weight), while the injured (vehicle) and sham operated (sham) groups received vehicle solution. Locomotor function in terms of latency on rotarod was measured for 49 days and the threshold of pain was determined for 14 days. Rats were sacrificed on day 3 and 14 and the spinal cord and cauda equina fibers were extracted and studied by histology, immunofluorescence, electron microscopy (EM) and TUNEL assay. Simvastatin aided locomotor functional recovery and enhanced the threshold of pain after the CEC. Cellular Infiltration and demyelination decreased in the spinal cord from the simvastatin group. EM revealed enhanced myelination of cauda equina in the simvastatin group. TUNEL assay showed significantly decreased number of apoptotic neurons in spinal cord from the simvastatin group compared to the vehicle group. Simvastatin hastens the locomotor functional recovery and reduces pain after CEC. These outcomes are mediated through the neuroprotective and anti-inflammatory properties of simvastatin. The data indicate that simvastatin may be a promising drug candidate for LSS treatment in humans.

Fig. 1

Representative sagittal MRI showing the orientation of the surgically implanted silicone blocks. Silicone blocks were surgically implanted into the epidural spaces of L4-L5 and L5-L6. The animals were scanned with T Bruker 7T MRI scanner to determine the proper orientation and placement of the silicone blocks. The image shows the silicone blocks are precisely placed in the epidural space under L4-L5 and L5-L6 vertebrae

Fig. 2

Simvastatin reduces locomotor deficits in CEC rats. Simvastatin (5 mg/kg body wt.) improved motor function of CEC rats evaluated by rotarod test. Simvastatin group exhibited significantly higher locomotor recovery compared to the vehicle group from day 7 onward. Results are presented as latency in seconds and data are expressed as mean ± SD (n=24). Sham operated animal had no locomotor deficit on any day tested (data not shown). ** p<0.05 and *** p<0.001 vs. vehicle

Fig. 3

Simvastatin enhances the threshold of pain in CEC rats. Rats subjected to CEC surgery and treated either with vehicle or simvastatin after measuring the baseline pain threshold (Day 0) using analgesy meter (a) and dynamic plantar aesthesiometer (b). Pain threshold was measured from post CEC day 2 to 14. Vehicle group had significant hypersensitivity for 4 days measured with AM and 6 days measured with DPA when compared with the baseline values. After that, the animals spontaneously recovered normal sensitivity to pain threshold. Pain threshold of simvastatin group resembled to that of the baseline value on all the days tested. The results demonstrate the antinociceptive property of simvastatin. Data are represented as mean ± SD. Each group consisted of 6 animals. **p<0.05, ***p<0.001 vs. baseline and simvastatin group

Fig. 4

Simvastatin inhibits cellular infiltration in the spinal cord of rats on day 14 after CEC. Spinal cord from each group was extracted and analyzed by H&E staining (a). Spinal cord of sham animals showed normal tissue architecture without any cellular infiltration. However, spinal cord from vehicle group showed increased infiltration of cells and the cellular architecture was also altered with large vacuoles. Simvastatin group had normal cellular architecture in addition to reduced cellular infiltration. Images are representative of sections from 5 animals in each group (magnification 600X). The infiltrated cells were stained for activated microglia (CD68) and cytotoxic Tcells (b). Vehicle group showed increased infiltration of CD 68+ and CD 8+cells in the spinal cord. Simvastatin treatment significantly decreased in infiltration of activated microglia and cytotoxic T cells in the spinal cord of CEC rats. Immunoreactive cells were counted in 4 regions of single section (n=6). Summary of the quantification of immunolabeling of CD 68 and CD 8 is given (C, D). Data are given as mean ± SD. ** p<0.05 vs. sham and simvastatin

Fig. 5

Simvastatin inhibits CEC-induced demyelination. Simvastatin treatment preserved myelin levels in the spinal cord from CEC rats as determined by LFB staining (a). EM was also employed to determine the myelin degeneration (arrows b) and g-ratio of axons (d). Spinal cord of sham animals showed normal levels of myelin. While the vehicle group showed significant loss of myelin, measured in terms of LFB intensity, simvastatin group had myelin level comparable to that of sham group (a & c) Images are representative of sections from five animals from each group (magnification 600X). g-ratio, a measure of myelin level also showed simvastatin mediated myelin protection in the CEC rats (d). Images are representative of sections from three animals from each group (magnification 20000X)

Fig. 6

Simvastatin reduces inflammation mediated astroglial activation following spinal stenosis. Simvastatin treatment significantly decreased the activation of astroglial cells on day 3 and 14 after CEC measured in terms of GFAP positive area of the spinal tissue section examined (a: upper panel). In addition, simvastatin also decreased the number of TNF-α positive cells in the spinal cord of CEC rats on day 3 and 14 post spinal stenosis (a lower panel). Photomicrographs are representative of 3 animals in each group (magnification 400X). The respective histogram of GFAP positive area and number of TNF-α positive cell of spinal sections of different experimental groups are given in B and C. GFAP positive area and number of TNF-α positive cell were counted in 4 different locations on each spinal tissue section. Data are presented as mean ± SD of n=3 in each group

Fig. 7

Simvastatin reduces neuronal apoptosis in spinal cord from the CEC rats. Treatment with simvastatin significantly decreased the cellular apoptosis determined by TUNEL assay (a). Colocalization study of TUNEL and NeuN showed that most of the apoptotic cells are neurons (b). The graph (c) shows the number of cells double positive to TUNEL and NeuN observed in sham, vehicle and simvastatin groups on post CEC day 3 and 14. Photomicrographs are representative of 3 animals in each group. Fig C represents the number of cells counted over 5 different locations on each section in each group. ## p<0.001 vs. sham, ** p<0.001 vs. vehicle