Three-Dimensional Design and Implementation of a Dual Compartment Imaging Chamber to Assess Effects of Hypertonic Saline on Periaxonal Swelling and Axonal Spheroid Formation Following Cervical Contusive Spinal Cord Injury in Real Time.

Abstract

Contusive and compressive spinal cord injury (SCI) induces pathological changes to spinal cord white matter (WM) including periaxonal swelling and resultant disruption of the axomyelinic interface, axonal swelling/spheroid formation, and secondary axonal transection. To further our knowledge of the role of vascular edema in these pathological changes to WM, we designed, and three-dimensional (3D) printed a dual-compartment imaging chamber separated by a semipermeable membrane to mimic and manipulate interstitial and vascular fluid compartments in real time. We hypothesized that hypertonic saline (HTS) applied to the "vascular" chamber would osmotically shift fluid out of the periaxonal space and preserve myelinated fibers after SCI. Adult male and female 6- to 8-week-old Thy1^YFP+ transgenic mice underwent a C5, mild contusive SCI (30 kilodyne, IH Impactor) in vivo, and their spinal cords were harvested for ex vivo imaging. Utilizing longitudinal two-photon excitation microscopy (2PE), we imaged both myelin (Nile red) and axons (YFP+) simultaneously up to 4 h after SCI. C5 contusive SCI conditions induced significant increases in periaxonal swelling and axonal spheroid formation within the dorsal column fibers over time. In contrast, perfusion of 3% and 5% HTS in the "vascular" compartment beginning 30 min after SCI was highly protective and significantly reduced periaxonal swelling and axonal spheroid formation from 1 h 30 min to the last hour recorded (4 h post-SCI) compared to normal saline (NS) controls. At 2 post-SCI, treatment with 3% and 5% HTS significantly (Kruskal-Wallis ANOVA on Ranks, H(3) = 3, p = 0.05, n = 5-6/group) reduced periaxonal swelling compared to NS (median, 25th percentile; 11.00, 4.00 versus 9.00, 7.00 versus 48.00, 29.50, respectively; Dunn's method, both p < 0.05). By 4 h post-SCI, treatment with 3% and 5% HTS significantly (H(3) = 15.74, p = 0.001, n = 5-6/group) decreased axonal spheroids compared to NS (5.00, 3.00 versus 4.00, 3.00 versus 95.00, 38.75, p = 0.001, p < 0.001, respectively). In contrast, 7.5% HTS had no beneficial effect. Collectively, these data provide insight into the dynamic interplay between interstitial fluid exchange within the periaxonal space and pathological changes in myelinated fibers following SCI. Delayed in vivo administration of 3% HTS significantly increased axonal survival and reduced periaxonal swellings 24 h post SCI compared to NS control, validating the translatability of our dual compartment imaging chamber (mean, standard deviation; 58.09, 3.34 versus 32.08, 5.98, p = 0.003; 595.19, 326.10 versus 1525.25, 259.82, p = 0.018, respectively). Our findings suggest that low-dose hypertonic solutions may have a protective effect in part by mitigating periaxonal swelling and thereby potentially reducing the occurrence of axonal spheroids within these denuded regions. These results enhance understanding of secondary axonal degeneration mechanisms and hold promise for targeted therapeutic interventions to improve outcomes in SCI.