Ischemia-reperfusion injury after spinal cord decompressive surgery-An in vivo rat model

Abstract

Background: Although decompression surgery is the optimal treatment for patients with severe degenerative cervical myelopathy (DCM), some individuals experience no improvement or even a decline in neurological function after surgery, with spinal cord ischemia-reperfusion injury (SCII) identified as the primary cause. Spinal cord compression results in local ischemia and blood perfusion following decompression is fundamental to SCII. However, owing to inadequate perioperative blood flow monitoring, direct evidence regarding the occurrence of SCII after decompression is lacking. The objective of this study was to establish a suitable animal model for investigating the underlying mechanism of spinal cord ischemia-reperfusion injury following decompression surgery for degenerative cervical myelopathy (DCM) and to elucidate alterations in neurological function and local blood flow within the spinal cord before and after decompression.

Methods: Twenty-four Sprague-Dawley rats were allocated to three groups: the DCM group (cervical compression group, with implanted compression material in the spinal canal, n = 8), the DCM-D group (cervical decompression group, with removal of compression material from the spinal canal 4 weeks after implantation, n = 8), and the SHAM group (sham operation, n = 8). Von Frey test, forepaw grip strength, and gait were assessed within 4 weeks post-implantation. Spinal cord compression was evaluated using magnetic resonance imaging. Local blood flow in the spinal cord was monitored during the perioperative decompression. The rats were sacrificed 1 week after decompression to observe morphological changes in the compressed or decompressed segments of the spinal cord. Additionally, NeuN expression and the oxidative damage marker 8-oxoG DNA were analyzed.

Results: Following spinal cord compression, abnormal mechanical pain worsened, and a decrease in forepaw grip strength was observed within 1-4 weeks. Upon decompression, the abnormal mechanical pain subsided, and forepaw grip strength was restored; however, neither reached the level of the sham operation group. Decompression leads to an increase in the local blood flow, indicating improved perfusion of the spinal cord. The number of NeuN-positive cells in the spinal cord of rats in the DCM-D group exceeded that in the DCM group but remained lower than that in the SHAM group. Notably, a higher level of 8-oxoG DNA expression was observed, suggesting oxidative stress following spinal cord decompression.

Conclusion: This model is deemed suitable for analyzing the underlying mechanism of SCII following decompressive cervical laminectomy, as we posit that the obtained results are comparable to the clinical progression of degenerative cervical myelopathy (DCM) post-decompression and exhibit analogous neurological alterations. Notably, this model revealed ischemic reperfusion in the spinal cord after decompression, concomitant with oxidative damage, which plausibly underlies the neurological deterioration observed after decompression.

Keywords: 8‐oxoG DNA; degenerative cervical myelopathy; spinal cord ischemia–reperfusion injury; surgical decompression.

FIGURE 1

(A) Physical drawing of the material. (B) Image of material removed during decompression surgery.

FIGURE 2

(A) Intraoperative images of compression and decompression procedures; (B) experimental cycle and behavior testing regime.

FIGURE 3

Pre‐experimental rat weight and weight changes during the experiment in DCM‐D group rats. (A) There was no significant change in the weight of the rats before compression across all the three groups. (B) There was no statistical difference in body weight between the DCM‐D group of rats after 4 weeks of compression surgery(4w) and 1week of decompression surgery(D‐1w).

FIGURE 4

(A) There were no significant differences in baseline data among the three groups. (B) Following 4 weeks of compression, there were statistically significant variations in pain thresholds observed among the three groups (DCM group, DCM‐D group, and sham group), with a p < 0.0001 obtained from a two‐tailed t test. No significant difference was found between the DCM and DCM‐D groups. (C) One week after completion of decompression surgery, notable disparities in pain thresholds emerged between the sham group and the DCM‐D group, as well as between the DCM‐D group and the DCM group (p < 0.05; two‐tailed t test; n = 4). (D) Within DCM‐D group, assessments before compression and after 4 weeks of compression have significant difference (p < 0.001), after 4 weeks of compression and after 1 week of decompression revealed that decompression significantly alleviated pain (p < 0.001). These findings were derived using a linear mixed model approach with n = 4 participants.

FIGURE 5

(A) There were no significant differences in standardized grip strength among the three groups before compression. (B, C) A comparison of standardized grip strength was conducted between rats at 1 week and 4 weeks after compression. Significant differences were observed between the sham group and the DCM group at both time points (*p < 0.05). Specifically, at 1 week, there was a highly significant difference (***p < 0.001), while at 4 weeks, the difference remained statistically significant (**p < 0.01). No significant difference was found between the DCM group and the DCM‐D group. The analysis employed a two‐tailed t test with a sample size of n = 7. (D, E) One week after compression, there were significant differences in peak standardized grip strength (***p < 0.001) and mean standardized grip strength (****p < 0.0001) between the Sham group and DCM‐D group. A significant difference was also observed between the Sham group and the DCM group (****p < 0.0001). No significant difference in peak standardized grip strength was found between the DCM‐D group and the DCM group. Notably, a significant difference in mean standardized grip strength (*p < 0.05) was observed. The statistical analysis employed a two‐tailed t test with sample sizes of n = 3 for the Sham groups and n = 4 for the other groups. (F) In the DCM‐D group, peak standardized grip strength of 4 weeks after decompression decreased compared to 1 week after compression (ANOVA for repeated measures; n = 4). (G) Rat grip test diagram.

FIGURE 6

The gait of rats did not improve after decompression. (A) Representative gait images of three groups of rats, blue and purple for right forelimb and right hindlimb, and yellow and green for left forelimb and left hindlimb. The upper and lower panels of the DCM‐D group images show the gait of rats before and after decompression, respectively. (B) After 4 weeks of compression, the swing speed of the forelimbs of the sham group was significantly higher than that of the compression group (*p < 0.05). There was no significant difference in the standing time and stride length of the forelimbs and hindlimbs. In the decompression group, the forelimb stride length after decompression was significantly shorter than that before decompression (*p < 0.05), and the hindlimb swing speed and stride length after decompression were significantly lower than those before decompression (*p < 0.05). There was no significant difference in other gait parameters. Two‐tailed t test, n = 4.

FIGURE 7

Spinal cord blood perfusion gradually increased within 10 min of decompression. (A) Blood perfusion images of rats were captured at four time points: Before decompression, immediately after decompression, 5 min after decompression, and 10 min after decompression. The region of interest (ROI) highlighted in the figure corresponds to the dorsal spinal cord beneath the exposed C6 lamina. Brighter colors indicate higher blood perfusion. (B) Statistical analysis revealed that the average blood perfusion in this region was significantly greater 5 min after decompression than that before decompression (one‐way ANOVA, n = 4). (C) Furthermore, there was a significant increase in perfusion at 10 min after decompression compared to that observed at 5 min post‐decompression. Additionally, the rate of perfusion during the initial 0–5 min period was higher than that during the subsequent 5–10 min interval (two‐tailed t test, n = 4). (D) Anatomy of the cervical spine and spinal cord of rats during blood flow testing.

FIGURE 8

HE staining of compressed segments in three groups of rats. Yellow arrow, neuron nuclear condensation; green arrow, glial cell; black arrow, vacuolated cell; red arrow, vascular congestion.

FIGURE 9

(A) Representative confocal images of rat cervical spinal cord sections stained with 8‐oxoG DNA and NeuN are shown (scale bar, 100 μm). (B) At 1 week post‐decompression (5 weeks after compression surgery), the proportion of neurons exhibiting 8‐oxoG DNA was significantly increased in the decompression group compared to the Sham group. Each group consisted of n = 3,4 animals. Statistical analysis revealed a highly significant difference (**p < 0.001) using one‐way ANOVA followed by Tukey's post hoc test. (C) The number of NeuN‐positive cells in the dorsal horn of the spinal cord at 1 week post‐decompression did not show any significant differences between the DCM‐D, DCM, and Sham groups.

FIGURE 10

The sagittal and transverse MRI images of DCM rats depict the presence of compression material, indicated by the arrow within a white square in the spinal canal. This material is located beneath the C6‐7 lamina, situated on the posterior aspect of the spinal cord. The three line segments a, b, and c in the figure represent the diameter of the spinal canal, which is employed in the calculation of the spinal cord compression rate. Compression rate is quantified to be 53.8%.