Contrast-Enhanced MicroCT Assessment of Degeneration Following Partial and Full-Width Injuries of the Mouse Lumbar Intervertebral Disc
All animal procedures were performed with approval from the Washington University School of Medicine IACUC, with all methods approved by relevant regulatory agencies. C57BL/6J female mice aged 3 to 4 months were used (BW: 20 to 25 g). They were housed under standard rearing conditions (in a temperature chamber [21 ± 1 °C] room with normal 12-hour light/dark cycles).
The partial-width injury mimics a localized injury to the annulus fibrosus, aka annular tear injury, in humans19. Whether a partial-width lesion is associated with IVD degeneration in humans remains unresolved.20.21. We first assessed the feasibility of partial-width wounding on six animals, which were euthanized shortly after recovery from anesthesia. These IVDs were harvested, and then the thickness of the fibrous ring and the depth of the wound were measured using contrast-enhanced microCT (CEµCT) and histological analyses.
A second cohort of animals was randomly divided into three groups at the time of surgery: partial-width (PW) wound (n=18), full-width (FW) wound (n=18) and Sham (n= 15) with all animals undergoing retroperitoneal surgical exposure of lumbar IVD to compare progression of IVD response after injury. Sample sizes were determined from preliminary studies with a histological score as the primary outcome. These groups were allowed to recover after surgery and then assessed cross-sectionally at 2, 4, and 8 weeks after surgery (n = 5/sham and n = 6/injury). The wound was delivered to the L6/S1 IVD with the L5/6 IVD used as an internal control. Tissues were assessed using CEµCT and histological analyzes for degeneration. All experiences follow the ethical guidelines set by ARRIVE.
Retroperitoneal approach to intervertebral disc procedure
Mice were anesthetized with isoflurane gas in oxygen via a face mask (3–4% induction and 2–2.5% maintenance at a flow rate of 1 L/min; Highland Medical Equipment) and received a preoperative intradermal injection of lidocaine (7 mg/kg; Hospira, Inc.). The left flank was then shaved from the ventral to dorsal midline and the skin was sterilized. The skin was prepared for aseptic surgery by washing with 70% ethanol and povidone iodine. Under microscopic guidance, a retroperitoneal dissection was used to expose the lateral aspect of the spine with a Penfield dissector. The pelvis and hip can be rotated back to expose a wider working space while maintaining the pelvic bone and distal femur (Fig. S4). The Penfield dissector was used to pull the peritoneal wall to expose the psoas muscle and protect the abdominal organs (Fig. 1A). The psoas muscle was retracted back and forth by initially moving the tissues attached to the anterior surface of the pelvis with a cotton swab and then held in place with a metal spatula (Fig. 1A,B). The spine and intervertebral disc were exposed from the ventral midline to the posterior edge of the disc at L5/6 and L6/S1 levels (Fig. 1B). Since the upper margin of the pelvic bone indicates the L6 vertebral body (Fig. 1E), the location of the L5/6 and L6/S1 IVDs could be confirmed (Fig. 1B,C). Extension to the cranial space beyond L5/6 was also easily achieved by blunt dissection.
All mice were monitored every 8–12 h for 4 days after surgery to monitor activity levels and wound recovery, if any. Of 58 mice that underwent surgery, one mouse was euthanized 2 days after surgery due to wound dehiscence, and this animal was replaced in the next surgical cohort. For pain management, mice received an IP injection of carprofen (5 mg/kg; Zoetis, Inc) every 8–12 h supplemented with carprofen tablets (2 mg/tablet; Bio-Serv). Additional details and images of the surgical procedure can be found in Supplementary Methods, Fig. additional S1 and on microCT analysis in Fig. Additional S2.
Partial and full width wounds
For the partial-width wound, a distance of 0.3 mm from the tip end of scalpel blade #11 was measured and marked with a microcaliper under a microscope (Fig. 3A). The distance of 0.3 mm was determined from our preliminary studies. The edge of the slide was allowed to insert into the IVD until the 0.3 mm marking was no longer visible under the microscope. The injury site was closely observed under a microscope to confirm that there is no NP leakage.
For the full-width wound, a 33G needle was inserted through the lateral IVD shaft of the IVD and allowed to penetrate both sides of the annulus fibrosus (e.g., “bilateral”)6,8,10,14,15,16,22,29 (Fig. S3). Unlike the partial-width injury, NP hernias were observed after the full-width injury.
Contrast-enhanced microCT (CEμCT) CT
Three to four samples from each group were included for contrast-enhanced microCT analysis. Each functional spine unit was incubated in 175 mg/mL dilute solution from Ioversol stock (OptiRay 350; Guerbet, St. Louis) in PBS at 37°C. After 24 h of incubation, the samples were scanned using a µCT40 (Scanco Medical, CH) at a voxel size of 10 µm (45 kVp, 177 uA, high resolution, integration 300 ms). Ioversol is a hydrophilic contrast agent allowing visualization of FA and NP on microCT, and its measurements have been shown to correlate with water and glycosaminoglycan contents17.22.
CEµCT data was exported as a DICOM file for analysis in a custom MATLAB program (https://github.com/WashUMRC/ContouringGUI). After an initial median filter (sigma=0.8, support=5), functional units of the spine were isolated from surrounding soft tissues not part of the IVD by tracing an outline around the outer edge every 5 cross-sectional slices and transforming them by linear interpolation. The IVD was manually segmented from the vertebral bodies with the same methodology as above. The remaining voxels were referred to as the entire disc mask. The NP was thresholded from the FA followed by a morphological closure and then a morphological opening to first fill the interior holes and then smooth the boundaries. Volumes and average attenuations (intensity) were calculated from the regions inside the masks of the NP and the whole disk. The loudness was determined from the total number of voxels contained in the mask, and the attenuation was taken as the average 16-bit grayscale value of the voxels. Visualizations of the microCT were obtained using the image processing application OsiriX (Pixmeo, Geneva). FA thickness, depth of partial-width injury, and disc height index (DHI) were measured along the midsagittal plane. The DHI was calculated as the ratio of the height to the width of the DIV. The height of the IVD was taken as the average at 5 equidistant points along the midsagittal plane (Fig. S2A). The NP intensity to disc intensity (NI/DI) ratio, defined as the average attenuation of the voxels in the NP mask divided by the full disc mask (Fig. S2B), is a fully three-dimensional measure that quantifies the relative size and hydration to inform relative changes in degeneration23. The evaluation was randomized and the evaluator was blinded to the sample group when processing the data.
Histological preparation and evaluation
After microCT, samples were fixed for 24 h in 10% neutral buffered formalin followed by 3 days of decalcification in Immunocal (StatLab 1414-X). Samples were embedded in paraffin, sectioned at a thickness of 10 µm, and then stained with Safranin-O and Fast Green. The histological classification system recently developed by Melgoza et al. in 2021 was used to quantify degeneration at the lesioned level (L6/S1), allowing quantification of changes in several compartments of the IVD, nucleus pulposus, annulus fibrosus, endplates and interface boundaries24. Histological classification was done blind. The morphology and NI/DI determined from the CEµCT were used to further inform the level of degeneration of the injured level (L6/S1) compared to the internal control (L5/6).
Measurement of AF thickness and partial-width lesion depth
CEμCT of injured L6/S1 and histological analysis of uninjured L5/6 from the first cohort of mice were used to measure FA thickness. The CEμCT on the injured L6/S1 was used to measure the depth of the injury as defined by the shortest perpendicular distance between the outer edge of the annulus fibrosus and the visually observable outline of the injury site.
All statistics were examined for normality and nonparametric tests were used accordingly. A 2-way ANOVA was used to assess the effects of injury and post-surgical time on histological grade with adjusted post hoc comparisons between groups (Tukey). Paired you assays were used to compare the contrast-enhanced microCT parameters of the injured IVD with the adjacent uninjured IVD in the same animal. Comparisons and effects were considered significant when the p-value is less than or equal to 0.05. All statistics were run using GraphPad Prism 9.3.1 (GraphPad, San Diego, CA).