Neuroprotective Effects of the Glutamate Transporter Activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) following Traumatic Brain Injury in the Adult Rat

Abstract

Traumatic brain injury (TBI) in humans and in animals leads to an acute and sustained increase in tissue glutamate concentrations within the brain, triggering glutamate-mediated excitotoxicity. Excitatory amino acid transporters (EAATs) are responsible for maintaining extracellular central nervous system glutamate concentrations below neurotoxic levels. Our results demonstrate that as early as 5 min and up to 2 h following brain trauma in brain-injured rats, the activity (Vmax) of EAAT2 in the cortex and the hippocampus was significantly decreased, compared with sham-injured animals. The affinity for glutamate (KM) and the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) were not altered by the injury. Administration of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a GLT-1 activator, beginning immediately after injury and continuing for 24 h, significantly decreased neurodegeneration, loss of microtubule-associated protein 2 and NeuN (+) immunoreactivities, and attenuated calpain activation in both the cortex and the hippocampus at 24 h after the injury; the reduction in neurodegeneration remained evident up to 14 days post-injury. In synaptosomal uptake assays, MS-153 up-regulated GLT-1 activity in the naïve rat brain but did not reverse the reduced activity of GLT-1 in traumatically-injured brains. This study demonstrates that administration of MS-153 in the acute post-traumatic period provides acute and long-term neuroprotection for TBI and suggests that the neuroprotective effects of MS-153 are related to mechanisms other than GLT-1 activation, such as the inhibition of voltage-gated calcium channels.

Keywords: EAAT2/GLT-1; MS-153; glutamate uptake; neuroprotection; traumatic brain injury.

FIG. 1.

Effect of lateral fluid-percussion brain injury on glutamate uptake and GLT-1 expression. V_max (nmol.mg⁻¹.min⁻¹) of ³H-L-glutamate uptake was measured in synaptosomes prepared from the injured cortex (A) and hippocampus (B) of brain-injured or sham-injured rats as described in the Methods. (C) Representative immunoblots illustrating GLT-1, glutamate aspartate transporter (GLAST) and actin expression from brain-injured or sham-injured rat synaptosomes. (D) Quantitative densitometric analysis of immunoblots. Bars represent means and standard deviation, *p < 0.05.

FIG. 2.

Effect of (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) on Fluoro-Jade B (FJ-B) reactivity in the cortex and hippocampus following lateral fluid-percussion brain injury. Representative photomicrographs of FJ-B–stained sections from the cortex (A-C) and area CA3 of the hippocampus (D-F) from sham-injured (A, D), vehicle-treated brain-injured (B, D) and MS-153-treated brain-injured rats (C, F). Note the absence of FJ-B (+) profiles in sham-injured animals (A, D). Scale bar in panel D represents 100 μm for all panels. Quantitative analyses are illustrated in graphs (G) and (H) 1 day post-injury, (I) and (J) 7 days post-injury, and (K) and (L) 14 days post-injury. The dose and dosing of MS-153 are described in the Methods. All values are presented as means and standard deviation. ##p < 0.01 and ###p < 0.001, compared with sham values; *p < 0.05 and **p < 0.01, compared with saline-treated values.

FIG. 3.

Effect of (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) on NeuN immunoreactivity following lateral fluid-percussion brain injury. Representative photomicrographs illustrating NeuN immunoreactivity in the cortex from the left (injured) hemisphere (A, C) and the right hemisphere (B, D) at 24 h after injury in vehicle-treated (A, B) and MS-153-treated (C, D) brain-injured rats. Scale bar in panel C represents 100 μm for all panels. Analyses of NeuN (+) counts in the cortex at 1 (E), 7 (F) and 14 days (G) after injury. Bars represent mean and standard deviation. #p < 0.01; ##p < 0.001.

FIG. 4.

Effect of (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) on microtubule-associated protein 2 (MAP-2) immunoreactivity and calpain activation following lateral fluid-percussion brain injury. (A) Representative photomicrographs illustrating showing MAP-2 immunoreactivity in sham-injured rats (upper panel) and in saline-treated, brain-injured rats after traumatic brain injury (middle panel) and MS-153-treated, brain-injured rats (lower panel). Arrow represents the site of maximal cortical injury as denoted by a loss of MAP-2 immunoreactivity. (B) Quantitative analysis of MAP-2 loss. *p < 0.05. (C) Representative immunoblots depicting spectrin breakdown products (SBDPs) in lysates of cortex (upper panel) and hippocampus (lower panel) from sham- and brain-injured animals. Note the presence of the 150-145 kDa doublet in brain-injured samples. (D) Quantification of SBDPs in cortex (upper panel) and hippocampus (lower panel). #p < 0.05 and ##p < 0.01, compared with sham values; *p < 0.05, compared with saline-treated values.

FIG. 5.

Effect of (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) on glutamate uptake in synaptosomes ex vivo. (A) Glutamate uptake in synaptosomes from naïve rat brains following incubation with increasing concentrations of the active (R)- or inactive (S)-enantiomer of MS-153. (B) Graph illustrates kinetic analysis of L-glutamate uptake in synaptosomes from either naïve rat brains or injured-brains pre-incubated with 100 μM MS-153. Values for V_max and K_M are presented in the table; K_M was not statistically different between the groups. ***p < 0.001 drug, compared with phosphate-buffered saline buffer; ###p < 0.001 injured, compared with naïve values.