Exercise modulates chloride homeostasis after spinal cord injury

Abstract

Activity-based therapies are routinely integrated in spinal cord injury (SCI) rehabilitation programs because they result in a reduction of hyperreflexia and spasticity. However, the mechanisms by which exercise regulates activity in spinal pathways to reduce spasticity and improve functional recovery are poorly understood. Persisting alterations in the action of GABA on postsynaptic targets is a signature of CNS injuries, including SCI. The action of GABA depends on the intracellular chloride concentration, which is determined largely by the expression of two cation-chloride cotransporters (CCCs), KCC2 and NKCC1, which serve as chloride exporters and importers, respectively. We hypothesized that the reduction in hyperreflexia with exercise after SCI relies on a return to chloride homeostasis. Sprague Dawley rats received a spinal cord transection at T12 and were assigned to SCI-7d, SCI-14d, SCI-14d+exercise, SCI-28d, SCI-28d+exercise, or SCI-56d groups. During a terminal experiment, H-reflexes were recorded from interosseus muscles after stimulation of the tibial nerve and the low-frequency-dependent depression (FDD) was assessed. We provide evidence that exercise returns spinal excitability and levels of KCC2 and NKCC1 toward normal levels in the lumbar spinal cord. Acutely altering chloride extrusion using the KCC2 blocker DIOA masked the effect of exercise on FDD, whereas blocking NKCC1 with bumetanide returned FDD toward intact levels after SCI. Our results indicate that exercise contributes to reflex recovery and restoration of endogenous inhibition through a return to chloride homeostasis after SCI. This lends support for CCCs as part of a pathway that could be manipulated to improve functional recovery when combined with rehabilitation programs.

Keywords: H-reflex; KCC2; NKCC1; complete transection; exercise; spinal cord injury.

Figure 1.

Spinal excitability gradually increases after SCI. A, FDD gradually decreases over time, reaching a plateau ∼28 d after SCI. Shown are significant differences at 5 Hz (intact and SCI-7d vs SCI-14d, SCI-28d, and SCI-56d; SCI-14d vs SCI-28d and SCI-56d) and at 10 Hz (intact and SCI-7d vs SCI-28d and SCI-56d; SCI-14d vs SCI-28d and SCI-56d). The enlarged recordings depict representative averages of H-reflex recordings after a train of stimulation at 0.3 Hz (light gray), 5 Hz (dark gray) and 10 Hz (black) when the FDD is impaired (top) or not (bottom). B, H_max/M_max ratio gradually increases with time after SCI and also plateaus at ∼28 d.

Figure 2.

Blocking NKCC1 mimics recovery after SCI. A, Representative averages of H-reflex recordings after a train of stimulation at 0.3 Hz (black) and 10 Hz (gray) in the same animal before (top) and 1 h after (bottom) bumetanide. The amplitude of the H-reflex is smaller at 10 Hz compared with 0.3 Hz with or without drugs, but the depth of modulation is much larger after bumetanide. B, Bumetanide improves FDD in chronic SCI animals. *p < 0.05; **p = 0.001.

Figure 3.

Exercise prevents the modulation of the expression of the cation chloride cotransporters KCC2 and NKCC1. Western blot analysis showing KCC2 (A) and NKCC1 (B) protein levels in the lumbar spinal cord (L4–L6) of intact, SCI-7d, SCI-28d, and SCI-28d+exercise animals. KCC2 decreased significantly after a complete SCI transection, whereas NKCC1 levels increased. Exercise returned KCC2 and NKCC1 expression levels toward intact. KCC2 and NKCC1 proteins were detected and quantified by Western blots using actin as a standard control (*compared with intact; §compared with SCI-28d+exercise). C, KCC2/NKCC1 ratio is decreased after SCI, but not in exercised animals. D, Regression analysis showing a negative relationship between KCC2 and NKCC1 protein levels in the lumbar enlargement (p = 0.008).

Figure 4.

Exercise restores spinal excitability 14 and 28 d after SCI. A, Exercise increased the depth of modulation of the FDD of the H-reflex in SCI animals at 14 and 28 d post-SCI. B, The H_max/M_max ratio is significantly decreased by 28 d post-SCI.

Figure 5.

Exercise restores KCC2 expression level in the lumbar spinal cord after SCI. A, Digital images showing KCC2 (green) and ChAT (red) immunoreactivity in the lumbar spinal cord of intact, SCI-28d, and SCI-28d+exercise rats. B, Labeling intensity was measured in the ventral horn area delineated by the dotted circle and normalized to intact. KCC2 expression is significantly decreased in the ventral gray matter of the lumbar spinal cord after SCI, but returns toward intact values with exercise. C, Labeling intensity was measured by averaging the integrated density of three lines drawn across the motoneuron cell body to assess KCC2 immunoreactivity along the motoneuronal membrane. Overall, KCC2 was decreased in the motoneuronal membrane after SCI and recovered with exercise. Scale bars: left: 100 μm; middle and right: 25 μm.

Figure 6.

Exercise does not modify NKCC1 protein expression level in the lumbar spinal cord after SCI. A–F, Immunolocalization of NKCC1 protein in the ventral lumbar spinal cord in the lumbar spinal cord of intact, SCI-28d, and SCI-28d+exercise rats. G, Labeling intensity was measured in the ventral horn area delineated by the dotted circle and normalized to intact. NKCC1 expression level was not changed after SCI nor with exercise. Scale bars: A, C, E, 50 μm; B, D, F, 10 μm.

Figure 7.

Blocking KCC2 suppresses exercise-dependent reflex recovery after SCI. A, Representative averages of H-reflex recordings after a train of stimulation at 0.3 Hz (black) and 10 Hz (gray) in the same SCI-28d+exercise animal before (top) and after (bottom) DIOA. Before drugs, the H-reflex is almost totally depressed at 10 Hz compared with 0.3 Hz, whereas DIOA dramatically decreases the depth of modulation. B, Overall, DIOA “impaired” FDD in SCI-7d animals and masked FDD recovery in SCI-28d+exercise animals.

Figure 8.

Relationship between KCC2 and BDNF expression. Expression of BDNF (A) and TrkB (B) in the lumbar spinal cord (L4–L6) of intact, SCI-7d, SCI-28d, and SCI-28d+exercise animals. The expression of BDNF and TrkB decreased significantly after a complete spinal cord transection. Exercise returned BDNF expression levels toward intact. BDNF and TrkB proteins were detected and quantified by Western blots using actin as a standard control (*p < 0.05 vs intact; § p < 0.05 vs SCI-28d+exercise). There is a significant relationship between BDNF and KCC2 (C, p = 0.028) but not NKCC1 expression (D, p = 0.264).

Figure 9.

Schematic diagram of an immature CNS neuron (left, high [Cl⁻]_i) and an adult CNS neuron (right, low [Cl⁻]_i). Opening GABA_AR leads to Cl⁻ exit and depolarization in immature neurons, whereas it causes Cl⁻ entry and hyperpolarization in the adult neuron. The switch from excitatory effect of GABA to inhibition induced by modulation in KCC2 and NKCC1 expression during development is reversed after SCI. We suggest that this change in chloride cotransporter expression that is involved in the development of hyperreflexia after SCI is reverted by exercise.