Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice

Abstract

Background: Glial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear.

Methods: We tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses.

Results: We found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI.

Conclusions: These studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.

Figure 1

Systemically administered anti-TNF therapy does not affect motosensory functions or lesion size after SCI. (A) Analysis of Basso Mouse Scale (BMS) scores in mice treated systemically every third day with either XPro1595, etanercept, or saline for 8 weeks (56 days) showed comparable locomotor performance in the open field arena (P = 0.64), though all groups significantly improved their BMS score over time (****P <0.0001, two-way repeated masures ANOVA). (B) Luxol fast blue stained thoracic spinal cord sections from mice treated with either saline, XPro1595, or etanercept and allowed 8 weeks survival after SCI. Scale bar: 100 μm. (C) Catwalk analysis showing changes in front and hind limb stride length and front- and hind limb base of support (BOS) over time after SCI. No difference was observed between saline-, XPro1595-, and etanercept-treated mice; however, all mice displayed significant changes in stride length (**P <0.01 for hind limbs and *****P <0.0001 for front limbs, respectively) and BOS on the front limbs (**P <0.01) over time. (D) Gridwalk analysis showing changes in the average number of foot falls errors and average number of foot slips errors over time after SCI. No difference was observed between saline-, XPro1595-, and etanercept-treated mice; however, all mice displayed significant changes over time both in foot falls errors (***P <0.001) and foot fall slips (***P <0.001). (E) Analysis of thermal hyperalgesia after SCI showed no differences in latencies to remove left (P = 0.27) or right (P = 0.16) hind limbs after stimulation between saline-, XPro1595-, or etanercept-treated mice. All mice did, however, display significant changes over time in the latency to remove both the left and right hind limbs after stimulation (****P <0.0001).

Figure 2

Centrally administered XPro1595 improves motor functions and decreases lesion size after SCI. (A) Analysis of BMS scores in mice treated centrally for three consecutive days with either saline, XPro1595, or etanercept showed that XPro1595-treated mice significantly improved their BMS score from 3 to 35 days after SCI compared to both saline- and etanercept-treated mice (*P <0.05 and ***P <0.001, two-way repeated measures ANOVA). All groups of mice significantly improved their BMS score over time (***P <0.001). (B) Rung walk analysis showed that XPro1595-treated mice significantly decreased their number of mistakes compared to saline- and etanercept-treated mice (*P <0.05 and **P <0.01). (C) Thermal stimulation using the Hargreave’s test showed no differences in latency time to withdraw paws between saline-, XPro1595-, and etanercept-treated mice. All mice decreased their latency to remove their paws over time after SCI (****P <0.0001). (D) Luxol fast blue stained thoracic spinal cord sections from mice treated with either saline, XPro1595, or etanercept and allowed 35 days survival after SCI. Scale bar: 100 μm. (E) Analysis of lesion volumes 35 days after SCI showed that the lesion size was significantly smaller in XPro1595-treated mice compared to both saline- and etanercept-treated mice (one-way ANOVA, followed by Tukey’s test). (F) Representative thoracic spinal cord sections from saline-, XPro1595-, and etanercept-treated mice stained for anti-GFAP allowed 35 days survival. Scale bar: 100 μm. (G) Quantification of GFAP protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. Data are normalized to β-actin protein expression. Results, expressed as percent of control, are the mean ± SEM of three animals per group. *P <0.05, **P <0.01, and ***P <0.001 versus control by one-way ANOVA with Tukey’s test.

Figure 3

Open field test analysis of SCI mice treated centrally with anti-TNF therapy for three consecutive days and allowed 35 days of survival after SCI. (A,B) Analysis of locomotor activity in mice treated centrally with either saline, XPro1595, or etanercept and allowed 35 days survival after SCI showed that all mice travelled a similar distance (A) at comparable speeds (B) in the open field test. (C,D) Analysis of anxiety-related behavior in the open field test showed that XPro1595-treated mice displayed decreased anxiety-related behavior represented by increased center/perimeter ratio (C) and increased number of zone changes into the perimeter and center area (D). (E,F) The number of droppings (E) was comparable in all groups of mice, whereas the number of groomings was increased both in XPro1595- and etanercept-treated mice compared to saline-treated mice. *P <0.05 and **P <0.01, one-way ANOVA followed by Tukey’s test.

Figure 4

Changes in Iba1 protein expression following central anti-TNF treatment after SCI. (A) Quantification of Iba1 protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. Data are normalized to β-actin protein expression. Representative experiments are shown. Results, expressed as percent of control, are the mean ± SEM of three animals per group. #P <0.05 versus XPro1595 and etanercept; *P <0.05, **P <0.01, and ***P <0.001 versus control by one-way ANOVA with Tukey’s test. (B) Representative low magnification photomicrographs of thoracic spinal cord sections from saline-, XPro1595-, and etanercept-treated mice allowed 7 and 35 days survival after SCI and representative high magnification photomicrographs from saline-treated mice allowed 7 and 35 days survival and stained for anti-Iba1. Estimation of the total number of Iba1⁺ cells 7 days after SCI (upper graph) and densitometric analysis of Iba1⁺ cells 35 days after SCI (lower graph) displayed no difference in microglial/macrophage numbers between the different treatment groups. Scale bars: low magnification: 100 μm and high magnification: 10 μm.

Figure 5

Changes in MBP, TNFR2, TLR4, and GAP43 protein expression following central anti-TNF treatment after SCI. (A) Quantification of MBP protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. (B) Quantification of TNFR2 protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. (C) Quantification of TLR2 protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. (D) Quantification of GAP43 protein expression in spinal cord tissue of saline-, XPro1595-, and etanercept-treated mice at 7 and 28 days after SCI. Data are normalized to β-actin protein expression. Representative experiments are shown. Results, expressed as percent of control, are the mean ± SEM of three animals per group. *P <0.05, **P <0.01, ***P <0.001, and ****P <0.0001 by one-way ANOVA with Tukey’s test.