This study focused on the interaction between neurophysiological and haemodynamic responses at dynamic neck positions after CCSCI. The present study clearly demonstrates that the behavioural assessment, DSSEPs and DMEPs deteriorated with compression time and different neck positions. At 1 and 2 wpi, DSSEPs were negatively affected at all neck positions and were significantly deteriorated at both extension and flexion, while the DMEPs were only affected at the flexion position. After 3 wpi, DSSEPs were all severely reduced and were identical at all neck positions. The DMEPs were always significantly deteriorated upon flexion after the compression, but were not damaged significantly upon neutrality and extension until 4 wpi. Dynamic MRI data at 4 wpi revealed that the sagittal diameter of the canal was compromised by more than a quarter (26.3%) and the spinal cord by more than half (53.7%) in CCSCI models and did not vary among different postures. The T2WI signal intensities of CCSCI models' cervical grey matter at flexion were significantly greater than those at neutral and extension, suggestive of severer ischemia at flexion. So we looked into the CCSCI models' perfusion at the compression site, and found it was significantly deteriorated at 4 wpi, especially upon cervical flexion. The dynamic change in SCBF was significantly correlated with the change in DMEP and DSSEP amplitudes upon flexion. These results could provide new insights for the development of drug therapy targeting spinal cord perfusion to prevent the progression and functional loss of CSM.
SSEPs and MEPs are distinct modalities that can be used to monitor the spinal cord. SSEPs reflect axonal conduction in the ascending sensory tracts in the posterior columns. MEPs monitor the descending motor system located in the anterior and lateral corticospinal tracts and the anterior horn motor neuron system, including the function of ischaemia-sensitive α motor neurons. The different conductive pathways of SSEPs and MEPs result in different characteristics of SSEPs and MEPs as indicators of spinal cord function, with respect to spinal cord ischaemia response time and false-positive and false-negative results.[33] Ueta et al.[34] investigated the effects of focal compression on physiologic integrity; electrophysiological manifestations including MEPs, SEPs and spine-to-spine potentials, spinal cord circulation and clinical status in four different directions of compression in pigs. Fehlings et al. reported the characteristics of MEP and SEP changes,[35] as well as their relationships with the severity of the injury and the SCBF[36] in spinal cord injury rat models. Lee et al.[37] and Hu et al.[38] reported SEP changes in two different CSM rat models using titanium screws and water-absorbable polyurethane polymers. Here, we used a well-established CCSCI rat model, in which the cervical cord was compressed by a gradually expanding water-absorbable polyurethane polymer.[39] The SCBF did not show deficits immediately after compression, and BBB scores and IPT tests were normal at 1 day after surgery, indicating that unexpanded compression material could be placed into the epidural space of the canal with no harm. The CCSCI models developed significant behavioural dysfunction, demonstrated by a significant drop in BBB scores and IPT angles after 1 wpi, which was in line with other studies.[32, 40] In addition, we detected significant DSSEP changes rather than DMEP changes upon a neutral neck position at 1 wpi, indicating early somatosensory tract disruption. DSSEP deterioration was more severe upon extension and milder upon a neutral position, suggesting that it is dynamically reversible. DMEPs showed significant deterioration upon flexion and remained relatively unchanged at neutral and extension at 1 wpi, indicating that dorsal compression disrupted the motor conduction pathway only in the flexed position. After 3 wpi, the DSSEP severely deteriorated at all neck positions, including neutral positions, indicating that the recuperation capability decreased. Until 4 wpi, the DMEP at neutral and extension positions also showed significant abnormalities compared with the sham group but was still significantly better than that at the flexion position. We assume this was because dorsal compression had much less influence on the anterior horn and motor conduction pathways. CSM patients suffering from hypertrophic ligamentum flavum generally present ataxia, which is mainly caused by dorsal funiculus disruption, but present full strength in all 4 extremities.[41] Ueta et al.[34] noted that SEPs were lost first during posterior, circumferential, and lateral acute transient compressions, while MEPs were lost first during anterior compression in pigs, which coordinated with our findings.
To better understand the mechanisms of varied DMEP and DSSEP performance at dynamic neck positions, we performed dynamic MRI of both sham and CCSCI rats. The relative T2WI signal intensities of the ROI in cervical grey matter in both the sham and CCSCI rats were analysed. The T2WI intensity was the greatest upon flexion compared with that at neutral or extension positions. This finding was in line with many clinical studies.[42, 43] While some authors assumed that cervical canal expansion at flexion permits better visualization of intramedullary hyperintensity on T2WI,[42, 43] others concluded that hyperintensities were more related to spinal cord ischaemia.[44] The significance of these lesions in CSM patients is also controversial in the literature.[45] Some studies have reported that the pattern of intramedullary hyperintensity lesions is an important criterion.[46, 47] In this study, the spinal canal remained constant at all three positions; also, we found a significant hyperintense pencil-like lesion pattern on sagittal T2WIs and the so-called “snake eyes” or “owl’s eyes pattern” with hyperintense signal conversion on axial T2WIs of CCSCI models upon flexion (see Fig. 5D, H). Previous studies reported that these typical “pencil-like” lesions on sagittal orientation and "snake eyes" lesions on axial orientation were important MRI characteristics of spinal cord infarction, indicating partially symmetrical small infarcts in the anterior horns.[44, 48] Intramedullary T2WI hyperintensity represents a variety of histologic changes, including oedema, ischaemia, demyelination, gliosis, microcavities, and cavities. Similarly, histological analysis in this study showed that the compressed spinal cord exhibited structural damage in its anterior horn, including fragmentation of neuronal nuclei, pyknosis, neuropil damage, degradation of the extracellular matrix, interstitial oedema, cytoplasmic reduction, and cavity formation. It has been reported that under chronic focal spinal cord compression, there was a decrease in blood flow in the compressed segment, which contributed to the pathogenesis of myelopathy.[18] Our previous study showed that 4 weeks of chronic compression led to a severe ischaemia-hypoxia environment, as demonstrated by increased HIF-1α expression,[25] and significantly decreased microvascular density, as demonstrated by micro-CT and immunohistochemistry data.[39, 49] Thus, the increased T2WI hyperintensity in our study was more likely to represent transient ischaemia upon flexion. We assume that the CCSCI models, whose spinal cord circulation was already compromised, suffered more perfusion decrease upon flexion than sham mice. The increased T2WI hyperintensity at flexion in CCSCI models is suggestive of a transient decrease in blood perfusion of the cord, which still needs to be further confirmed.
LDF is a non-invasive method for blood flow measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord at different neck positions. The technique uses tissue backscattered light to qualitatively assess the blood flow rate and has been a useful method for quantifying perfusion of the spinal cord in several animal and human studies.[50–52] Brain et al.[53] first reported ischemia as one of the crucial pathophysiological mechanisms in CSM. Pathological changes such as vessel wall thickening and hyalinization in the anterior spinal artery and parenchymal arterioles[53], and decreased number of vessels as indicated by the decreased laminin staining have been identified in chronic compressed cervical cords[54]. Kurokawa et al[18] previously reported an approximately 20% reduction in blood flow in the compressed segment (C5-C6) compared with that of the rostral (C3–C4) segment in CCSCI rat models using hydrogen clearance to measure blood flow. In our study, the reduction value was 33% in neutral position. The SCBF data in the two studies were not comparable because Kurokawa et al[18] did not quantify their compression degree. Moreover, we firstly report a 27% reduction of SCBF upon flexion compared with neutral position in CCSCI rat models at 4 wpi. Since the vasculature of the spinal cord is organized such that its ventral aspect and grey matter are supplied centrifugally by the central artery and the anterior part of the vasocorona, both of which arise from the ASA, ventral spinal cord compression caused by flexion position could probably exacerbate the ischaemic condition by compressing the ASA. We have previously reported that CSM patients with a "snake-eye" appearance of T2WI intramedullary hyperintensity suffered more severe flexion DSSEP deterioration in the clinic, probably caused by decreased SCBF at flexion.[12] Our LDF data directly indicated the perfusion loss in the compressed spinal cord during cervical flexion, as the studies mentioned above have suggested.
Finally, our data also indicated that motor functional loss was significantly related to decreased SCBF in CCSCI rat models at a flexed neck position. These findings are in general agreement with those of Bennett[55], who examined the effect of focal spinal cord ischaemia induced by segmental dorsal and ventral rhizotomy on MEPs and SSEPs in the cat spinal cord. He observed that MEPs more sensitively reflected spinal cord ischaemia than SSEPs but he did not measure SCBF. Fehlings et al.[36] reported that MEP amplitudes rather than SSEP amplitudes were significantly correlated with SCBF and injury severity (compression forces) in clip compression spinal cord injury rat models. Shine et al.[56] reported that MEPs disappeared prior to SSEPs after introducing ischaemia by aortic cross clamping; moreover, early MEP disappearance suggested a poor neurological outcome in patients undergoing thoracoabdominal aortic aneurysm repair. These animals and clinical studies indicated that compared with SSEPs, MEPs were more easily affected by spinal cord perfusion status. However, there are a number of other possible explanations for the high sensitivity of DMEPs to cervical flexion. For example, the corticospinal tract and the anterior horn motor neuronal system are mainly located at the ventral aspect of the spinal cord. Gooding et al. used dog models of cervical vascular insufficiency that were derived via selective vascular ligation to show that vascular disruption caused demyelination, glial fibrosis, and necrosis, especially in the corticospinal tracts.[57] Histological abnormalities in all dogs with combined compression and ischaemia were more pronounced than in dogs with either compression or ischaemia alone.[57] Thus, DMEP deterioration upon flexion in our CCSCI rat models was also more likely to be due to the combinatorial effects of ischaemia and direct neuronal compression.