Amiloride improves locomotor recovery after spinal cord injury

Abstract

Amiloride is a drug approved by the United States Food and Drug Administration, which has shown neuroprotective effects in different neuropathological conditions, including brain injury or brain ischemia, but has not been tested in spinal cord injury (SCI). We tested amiloride's therapeutic potential in a clinically relevant rat model of contusion SCI inflicted at the thoracic segment T10. Rats receiving daily administration of amiloride from 24 h to 35 days after SCI exhibited a significant improvement in hindlimb locomotor ability at 21, 28, and 35 days after injury, when compared to vehicle-treated SCI rats. Rats receiving amiloride treatment also exhibited a significant increase in myelin oligodendrocyte glycoprotein (MOG) levels 35 days after SCI at the site of injury (T10) when compared to vehicle-treated controls, which indicated a partial reverse in the decrease of MOG observed with injury. Our data indicate that higher levels of MOG correlate with improved locomotor recovery after SCI, and that this may explain the beneficial effects of amiloride after SCI. Given that amiloride treatment after SCI caused a significant preservation of myelin levels, and improved locomotor recovery, it should be considered as a possible therapeutic intervention after SCI.

FIG. 1.

(A) All rats (male Sprague-Dawley) were contused at T10 using an Infinite Horizon device and a force of 150 kDynes, 1 sec dwell time. All surgical procedures are described in Nesic et al., (2008). Naïve animals (n=6) were not subjected to any part of the surgical or postsurgical care protocols. All procedures complied with the recommendations in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas Medical Branch Animal Care and Use Committee. Hindlimb movement was assessed using the Basso, Beattie, and Bresnahan Scale, also known as the BBB scale (Basso et al., 1995). BBB scores were collected daily the first 14 days after injury, and once weekly thereafter. The BBB scale assigns values to the left and right hindlimbs corresponding to their locomotor ability. Combined left and right hindlimb BBB locomotor scores of SCI+amiloride (AML) (n=6) and SCI+vehicle (V) (n=6) animals are shown (mean±SE; * p<0.05; repeated measure two-way ANOVA without replication; SPSS statistical package, version 11.0). Animals were weighed weekly to adjust the administration of amiloride to account for weight increases over time for each individual animal. (B) Recovery of bladder function in vehicle- and amiloride-treated SCI rats. SCI rats cannot spontaneously empty their bladders during the first days after injury, and therefore the care of SCI rats includes manual emptying of their bladders twice a day. However, SCI rats over time recover their ability to spontaneously empty their bladders, and that was presented in this graph. Therefore, in our experiment, spontaneous emptying of bladders in both experimental groups was restored at ∼7 days after SCI. Mean±SE.

FIG. 2.

(A) A representative Western blot showing myelin oligodendrocyte glycoprotein (MOG) monomer band at ∼23kD from the site of injury (T10), 3 days and 7 days after SCI. Western blot methods are described in detail in Nesic et al., , , , 2010. As a loading control in all our Western blots we use β-actin (presented here as a band of ∼ 40kD). MOG antibody was purchased from Abcam (ab28766, dilution: 1ug/mL). Bar graph: We also analyzed MOG protein levels at different time points after SCI, 1 (n=5), 3 (n=5), 7 (n=3), 21 (n=5), and 35 days (n=6) after SCI (mean±SE). The 23 kD band was quantified and normalized, first to β-actin levels, and then to uninjured values (uninjured signal intensity=100%). β-actin antibody was purchased from Sigma (A5441, dilution 1:80,000). (B) We analyzed MOG levels in SCI rats with distinctly different levels of motor recovery 21 days or 56 days post-SCI as indicated by the BBB scores. We have chosen to analyze MOG levels in SCI rats whose BBB scores are different by ≥4 BBB units, given that we did not expect that smaller differences in BBB scores would have been translated into measurable alterations in MOG levels, considering inter-individual variations in MOG levels (see MOG bands for SCI rats with the same BBB scores). Higher MOG levels at the lesion site (T10) were associated with better hindlimb locomotor recovery at both time points after SCI: a positive association between MOG levels and motor recovery was nearly unchanged from 21 to 56 days after SCI. Bar graph: MOG monomer band intensities were quantified and normalized, first to β-actin levels, and then to uninjured values (uninjured signal intensity=100%). The corresponding BBB scores are written above each bar (mean±SE). (C) We analyzed MOG protein levels in three groups of rats: naive (n=5), SCI rats treated with vehicle (n=6), and SCI rats treated with amiloride (AML) (n=6). Their BBB scores are shown in Figure 1. MOG levels were markedly decreased at the lesion site 35 days after moderated SCI, whereas amiloride treatment significantly increased the MOG monomer and dimer levels (dimer 46kD band, believed to be the active MOG form; Clements et al., 2003). MOG dimer levels paralleled changes in monomer bands in all our experiments. (D) Quantitative analyses of the monomer band showed that MOG levels at T10 were significantly higher in amiloride-treated SCI rats (mean±SE *p=0.03 t-test). Although MOG levels at L4/5 decreased ∼40% after SCI, they were not significantly different from uninjured levels. This was primarily because of inter-individual variations, which also obscured a modest increase in MOG in amiloride-treated SCI rats. Therefore, it is possible that amiloride also affected myelin loss in lumbar segments that directly control hindlimb locomotion, but this remains to be analyzed further.