Continuous Hypodynamic Change of Cerebrospinal Fluid Flow as A Potential Factor Working for Experimental Scoliotic Formation

Abstract

Scoliosis is often associated with syringomyelia (SM). As an important role in SM formation, the influence from abnormal cerebrospinal fluid (CSF) flow is still unclear to scoliosis. The aim of this experimental work is to explore the connection between CSF flow and scoliosis through imaging and histological analysis on the basis of a kaolin-induced scoliotic rabbit model. For imaging observation, in 40 kaolin-induced rabbits by C7 spinal cord injection, through pre- and postoperative MRI and radiography, CSF flow and scoliosis formation were detected at consecutive phases. According to the final formation of scoliosis until postoperative week 12, the kaolin-induced rabbits were divided into 2 groups. Through comparing the 2 groups, the relationship between the changes of CSF flow velocity and scoliosis formation were reviewed and analyzed. For histological observation, another 20 kaolin-induced rabbits were used for consecutive histological observations of spinal cord at postoperative 3-day, 2-week, 4-week and 6-week. After kaolin-induction, abnormal spinal coronal curve was observed from postoperative week 6 in the 37 survived rabbits. At postoperative week 12, scoliosis formation was detected in 73.0% kaolin-induced rabbits and the mean Cobb angle was 27.4°. From the comparison between scoliotic and non-scoliotic groups, the difference of the velocities of CSF flow was more obviously from postoperative week 4 to 12, especially after week 6. In the scoliotic group, the peak velocity of CSF flow was diseased gradually following scoliosis formation after induction. Moreover, the decrease of the peak velocities of CSF flow from preoperation to postoperative 12 weeks (ΔVmax), including up-flow (ΔVUmax) and down-flow (ΔVDmax), were positively correlated to the final scoliotic Cobb angle (P < 0.01). Through histological observation at different phases, the distinctive pathological changes of the spinal cord included early inflammatory reaction, adhesion and blockage in the subarachnoid space and the central canal, perivascular space enlargement, central canal expansion, which suggested the CSF flow being blocked by multiple ways after kaolin-induction. In conclusion, experimental scoliosis can be successfully induced by intraspinal kaolin injection. In this model, continuous hypodynamic change of CSF flow was correlated to the formation of scoliosis, which could be an important factor of scoliotic pathogenesis being explored furtherly.

Figure 1

Posterior-anterior radiographs of the spine and scoliosis development (Rabbit 21#). Preoperative coronal imaging of spine (A). Postoperative 6-week, a tiny thoracic curve appearance and the Cobb angle was 12° (B). Postoperative 12-week, the thoracic scoliosis developed to 54° (C). Post-anatomic observation of the thoracic scoliosis and the apical vertebra was marked by the white arrow (D).

Figure 2

Pre- and postoperative 12-week MRI scanning of the cervical spine cord with kaolin-induction (Rabbit 21#). Preoperative sagittal and transverse MRI imaging of the cervical spine cord (A,C). Postoperative 12-week, the syrinx was observed in the sagittal and transverse MRI imaging of cervical spine cord (B,D).

Figure 3

The changes of CSF flow at consecutive phases after kaolin-induction (group S vs group Non-S). At C0 level, after kaolin-induction, the peak velocities of CSF flow (VDmax and Vumax) were decreased from week 4 to 12 (A,B). At C7 level with a similar trend as at C0, after kaolin-induction, the peak velocities of CSF flow (VDmax and Vumax) were also decreased from week 4 to 12 (C,D). ^*P < 0.05, ^**P < 0.01.

Figure 4

In group S, from preoperation to postoperative 4, 6, 8, 10, 12 weeks, the peak velocities (VDmax and VUmax) of CSF flow at C0 and C7 were decreased gradually, which was combined with the occurrence and progression of scoliosis from postoperative 6 weeks.

Figure 5

Relationship of CSF flow change and scoliosis formation at postoperative 12 weeks. ∆VDmax and Cobb angle at level of C0. Pearson correlation analysis shows a significant positive correlation of ∆VDmax versus Cobb angle (Pearson r = 0.595; P ≤ 0.001, n = 27). Line represents linear regression of data (y = 7.265x+ 4.808; r² = 0.3542) (A). ∆VUmax and Cobb angle at level of C0. Pearson correlation analysis shows a significant positive correlation of ∆VUmax versus Cobb angle (Pearson r = 0.673; P ≤ 0.001, n = 27). Line represents linear regression of data (y = 7.240x+ 4.140; r² = 0.4523) (B). ∆VDmax and Cobb angle at level of C7. Pearson correlation analysis shows a significant positive correlation of ∆VDmax versus Cobb angle (Pearson r = 0.668; P ≤ 0.001, n = 27). Line represents linear regression of data (y = 7.636x+ 3.103; r² = 0.4460) (C). ∆VUmax and Cobb angle at level of C7. Pearson correlation analysis shows a significant positive correlation of ∆VUmax versus Cobb angle (Pearson r = 0.683; P ≤ 0.001, n = 27). Line represents linear regression of data (y = 8.588x+2.716; r² = 0.4662) (D).

Figure 6

The extramedullary and intramedullary histological changes at postoperative 6-week with kaolin-induction, HE staining, 40× magnification (A). Focus of inflammatory infiltration in the thickening pia mater (arrow), 200× magnification (B). Focus of obstruction by the kaolin granulation and the inflammatory adhesion in the subarachnoid space (arrow), 200× magnification (C). Focus of the expanded central canal (arrow), 200× magnification (D).

Figure 7

The changes of the central canal after kaolin-induction. To compare with the normal spinal cord tissue with regular and complete layer of ependymal cells (A), at postoperative 3-day with kaolin-induction, the central canal was tightly blocked by kaolin crystals, neutrophil cell and inflammatory exudation; the spinal cord parenchymal change was characterized by congestion, edema with neutrophil infiltration (B); at postoperative 2-week, the ependymal cells layer was interrupted and the central canal began to be expanded, and neutrophils infiltrated and macrophages engulfed the kaolin crystals in the spinal cord parenchyma with perivascular space (Virchow-Robin space) expansion (C); at postoperative 4-week, the central canal was further expanded with the ependymal cells proliferation, and lymphocytes infiltrated in the spinal cord parenchyma and accumulated in the expanded perivascular space (Virchow-Robin space) (D); at postoperative 6-week, the expansion of central canal became more obvious and asymmetric with disordered arrangement of surrounding structures (E). To compare with the control, the areas of the central canal gradually increased from postoperative 3-day to 6-week (F). *P < 0.05, **P < 0.01. (HE staining, 200× magnification).

Figure 8

The process of building a rabbit scoliotic model by kaolin-induction. The experimental rabbit was placed prone on a self-made frame to raise the cervicothoracic junction (A). A 2 cm midline incision was made from the seventh cervical vertebra (C7) to the first thoracic vertebra (T1), and the basis of C7 spinous process was exposed (B). After cutting the C7 spinous process, a tiny part of the lamina and ligamentumflavum were resected for exposing the dura (arrow) (C). Kaolin was injected from the subarachnoid space to the center of the spinal cord layer by layer through the dura being navigated by a stereotaxic apparatus (D).

Figure 9

The measurement of CSF flow by PC-cine MRI observation. CSF flow was measured at C0 (line a) and C7 (line b) levels (A). CSF flow was analyzed by Argus postprocessing program, the peak velocities of CSF flow were measured on the basis of the CSF velocity-time curve, including up-flow (marked by VUmax) and down-flow (marked by VDmax) (B).