Elucidating the Potential Mechanisms Underlying Distraction Spinal Cord Injury-Associated Neuroinflammation and Apoptosis

Abstract

The incidence of distraction spinal cord injury (DSCI), which results from spinal cord ischemia due to vascular compromise and spinal cord tract disturbances, remains high. Furthermore, because no ideal animal model that mimics DSCI in clinical settings is available thus far, the related molecular mechanisms underlying DSCI remain unclear. Thus, this study aimed to establish a porcine model of DSCI and investigate the neuroinflammation and apoptosis mechanisms in these pigs. Before surgery, all pigs were randomly divided into three groups: sham group, osteotomy surgery only; the incomplete distraction spinal cord injury (IDSCI) and complete distraction spinal cord injury (CDSCI) group, osteotomy plus DSCI surgery with a motor-evoked potential (MEP) amplitude decreased by approximately 75% and 100%, respectively. After surgery, modified Tarlov scoring and MRC muscle strength scoring were used to evaluate neurologic function in each group. We observed the distracted spinal cord using MRI, and then all pigs were sacrificed. Inflammatory cytokine levels in the spinal cord and cerebrospinal fluid (CSF) were also analyzed. We used immunofluorescence staining to assess the neuronal and microglial structure and function and astrocyte hyperplasia in the central DSCI lesions (T15). Western blotting was used to determine the expression of apoptosis-related proteins. Results showed that the modified Tarlov scoring and muscle strength decreased significantly in the two DSCI groups. T2-MRI showed a relative enhancement at the center of the DSCI lesions. H&E and Lxol fast blue staining revealed that spinal cord distraction destroyed the normal structure of spinal cord tissues and nerve fiber tracts, exacerbating inflammatory cell infiltration, hyperemia, and edema. The IL-1β, IL-6, and TNF-α levels increased in the spinal cord and CSF following DSCI. Immunofluorescence staining results indicated the GFAP, Iba-1 expression increased following DSCI, whereas the NeuN expression reduced. Moreover, DSCI promoted the protein expression of P53, Bcl-2-associated X protein (Bax), and Caspase-3 in the spinal cord tissues, whereas it reduced the Bcl-2 expression. This study successfully established a porcine DSCI model that closely mimics DSCI in clinical settings, and clarified the mechanisms underlying DSCI-associated neuroinflammation and apoptosis; thus, our findings highlight potential DSCI-treatment strategies for further establishing suitable drug therapies.

The Role of Microglia/Macrophages Activation and TLR4/NF-κB/MAPK Pathway in Distraction Spinal Cord Injury-Induced Inflammation

“…The inflammatory environment which occurs after an SCI promotes the activation and proliferation of microglia/macrophages and the release of proinflammatory cytokines, as well as other toxic mediators, which is closely related to neuronal apoptosis and neurite injury ( Guadagno et al, 2013 ). The results of our previous study confirmed that TNF-α, IL-1β, and IL-6 levels increased significantly in damaged spinal cord tissue and cerebrospinal fluid after DSCI, while the survival rate of neurons decreased ( Han et al, 2022 ). After undergoing mechanical stimulation such as distraction, the expression of TLRs in activated microglia of the impaired spinal cord tissue was significantly increased ( Carpentier et al, 2008 ).…”

“…The surgical procedure for the DSCI model in Bama miniature pigs was consistent with that in our previous studies ( Han et al, 2022 ). After anesthetizing the animals, a midline longitudinal incision was made extending from thoracic vertebra 13 (T13) to lumbar vertebra 2 (L2; Figure 1D ).…”

“…The release of proinflammatory cytokines after the activation of microglia and macrophages is directly related to the level of inflammation, which is the key cause of neuronal apoptosis and spinal cord tissue destruction ( Papa et al, 2013 ). The results of our previous study ( Han et al, 2022 ) showed that IL-1β, IL-6, and TNF-α levels increased in the spinal cord tissue of pigs after being subjected to a DSCI, indicating a significant transformation of the injured spinal cord tissue to a pro-inflammatory environment. In the present study, neurological dysfunction did not heal in the Bama miniature pigs within the first 7 days after the DSCI.…”

“…The final distraction distance was found to be 74.3% (68–84%) of the segmental vertebral height. The technique used in our previous study ( Han et al, 2022 ), involving a T15 global column osteotomy plus pedicle screw fixation, was also utilized in the present study to successfully establish the DSCI model, for which the Tarlov and ILMS scores showed significant and stable neurological function defects after DSCI. The method of stable and repeatable large-animal model establishment used in the present study is a reliable guarantee for subsequent experiments.…”

“…In a previous study, we successfully established DSCI porcine models and found that the expression of the microglial marker Iba-1, as well as the proinflammatory cytokines TNF-α, IL-1β, and IL-6, increased after DSCIs ( Han et al, 2022 ). In the present study, we used a Bama miniature pig DSCI model as the research object, and further explored the activation of microglia and macrophages, as well as the changes in TLR4-mediated NF-κB and MAPK pathway activity after DSCI, to provide evidence for finding potential anti-inflammatory targets for these injuries.…”