FTY720 reduces inflammation and promotes functional recovery after spinal cord injury

Abstract

A robust and complex inflammatory cascade is known to be a prominent component of secondary injury following spinal cord injury (SCI). Specifically, the concept of trauma-induced autoimmunity has linked the lymphocyte population with neural tissue injury and neurologic deficit. FTY720, a sphingosine receptor modulator that sequesters lymphocytes in secondary lymphoid organs, has been shown to be effective in the treatment of a variety of experimental autoimmune disorders. Accordingly, by reducing lymphocyte infiltration into the spinal cord following SCI, this novel immunomodulator may enhance tissue preservation and functional recovery. In the present study, a moderate to severe contusion SCI was simulated in adult Long-Evans hooded rats. Using flow cytometry we showed that daily FTY720 treatment dramatically reduced T-cell infiltration into the SCI lesion site at 4 and 7 days post-injury, while other inflammatory cell populations were relatively unaltered. To assess functional recovery, three groups of injured animals (treated, vehicle, and injury only) were evaluated weekly for hindlimb recovery. Animals in the treated group consistently exhibited higher functional scores than animals in the control groups after 2 weeks post-injury. This finding was associated with a greater degree of white matter sparing at the lesion epicenter when cords were later sectioned and stained. Furthermore, treated animals were found to exhibit improved bladder function and a reduced incidence of hemorrhagic cystitis compared to control counterparts. Collectively these results demonstrate the neuroprotective potential of FTY720 treatment after experimental SCI.

FIG. 1.

FTY720 treatment reduces T-cell infiltration into the spinal cord after injury. After SCI, animals were injected with either vehicle or FTY720 for 1, 4, or 7 days. A 1-cm segment of the cord centered on the impact site was then harvested from each animal and prepared for flow cytometry. (A) Representative dot plots of the injury site of a vehicle-injected animal showing the 7-AAD+ population used for analysis (gate P5). (B) Representative dot plots of the injury site of a vehicle-injected animal showing the percentages of CD3⁺/CD4⁺ double-positive cells (gate Q2) and of (C) CD3⁺/CD8⁺ double-positive cells (gate Q2) within the 7-AAD + gate. (D) A graphic summary of the mean percentages of 7-AAD+ events that were CD3⁺/CD4⁺ and CD3⁺/CD8⁺ for treated and vehicle animals at the various time points. As shown by this flow cytometric paradigm, FTY720 treatment reduces infiltration of both CD3⁺/CD4⁺ helper and CD3⁺/CD8⁺ cytotoxic T-cell populations into the cord after SCI (*p < 0.05 compared with vehicle-injected rats).

FIG. 2.

FTY720 promotes hindlimb recovery after SCI. After SCI, the animals were randomly placed in one of three groups: injury only, vehicle, and FTY720-treated. Functional assessments were performed weekly for 6 weeks. (A) Open-field assessment of hindlimb locomotor function using the BBB scale. Starting at week 3, treated rats as a group performed significantly better than the control groups. No significant differences were noted between the vehicle and injury only groups at any time point. (B) The inclined-plane test. Treated animals outperformed members of the control groups starting at week 2, and continued to do so for the duration of the study. (C) For all time points, linear regression analysis revealed a very strong correlation between the BBB and inclined-plane scores (r = 0.87; *p < 0.05 compared with injury-only and vehicle rats).

FIG. 3.

FTY720 improves bladder function after SCI. Manual bladder expression was performed three times daily for the first week after SCI, and two times daily thereafter until spontaneous micturition was re-established. The residual urine volumes of each animal were measured and recorded during each expression session to provide an average daily urine volume for the first 2 weeks post-SCI. Furthermore, during urine collection gross hematuria was noted when observed. (A) The mean residual urine volumes of treated rats were consistently and significantly lower than those of the control groups. (B) The number of days required until spontaneous micturition was re-established as defined by an average daily volume of <2 mL for 3 consecutive days. Treated animals did not require extended assistance in expelling urine and regained spontaneous bladder function by day 6, while control animals did not reach this milestone until day 10. (C) FTY720 treatment significantly reduced the incidence of gross hematuria compared to the control groups (*p < 0.05 compared with injury-only and vehicle-treated rats).

FIG. 4.

FTY720 treatment does not adversely affect body weight. Animal body weights were measured and recorded throughout the duration of the behavioral outcome study. Treatment with FTY720 did not affect body weight following SCI. The x-axis is presented on a logarithmic scale.

FIG. 5.

Myelin sparing correlated with locomotor recovery. At the conclusion of the behavioral outcome study (6 weeks post-SCI), the animals were sacrificed and cords were harvested and cryosectioned. Sections at the level of the SCI epicenter were stained with eriochrome cyanine (EC) to determine the extent of white matter sparing. (A) A representative EC-stained cross section at the level of the SCI epicenter from a vehicle animal. (B) A comparable section from a FTY720-treated animal. (C) At the level of the SCI epicenter, FTY720 treatment resulted in a significant increase in the percentage of spared myelin compared to vehicle treatment. (D) A linear regression analysis of the final BBB locomotor scores and spared myelin percentages at the epicenter exhibited a very strong correlation (r = 0.75). (E) In addition to the myelin-sparing analysis completed at the injury epicenter, adjacent cord sections at every 0.5 mm up to 5 mm away from the epicenter were similarly processed and analyzed. This analysis revealed that FTY720 treatment resulted in a significant increase in the percentage of spared myelin compared to vehicle treatment in adjacent areas of the injury epicenter, especially in regions caudal. Negative distances on the x-axis denote rostral direction, whereas positive distances denote caudal direction (scale bars 300 μm; *p < 0.05 compared with vehicle rats).