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. 2009 Jul;66(1):63-74.
doi: 10.1002/ana.21673.

Activation of metabotropic glutamate receptor 5 improves recovery after spinal cord injury in rodents

Kimberly R Byrnes  1 , Bogdan Stoica, Angela Riccio, Ahdeah Pajoohesh-Ganji, David J Loane, Alan I Faden
Affiliations

Activation of metabotropic glutamate receptor 5 improves recovery after spinal cord injury in rodents

Kimberly R Byrnes et al. Ann Neurol. 2009 Jul.

Abstract

Objective: Activation of metabotropic glutamate receptor 5 (mGluR5) has neuroprotective properties in vitro and has been reported to limit postischemic lesion volume in vivo. Previously, mGluR5 has been identified on microglia in vitro, but the effects of mGluR5 activation on inflammation in vivo or on recovery after spinal cord injury is unknown.

Methods: Rats received intrathecal infusion of the selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) for 7 days after moderate impact spinal cord injury at T9. Complementary studies examined CHPG effects on activated spinal microglia cultures.

Results: Functional motor recovery was significantly increased by CHPG treatment up to 28 days after injury, with improvements in weight bearing, step taking, and coordination of stepping behavior. CHPG treatment significantly reduced lesion volume and increased white matter sparing at 28 days after injury. Administration of CHPG attenuated microglial-associated inflammatory responses in a dose-dependent fashion, including expression of ED1, Iba-1, Galectin-3, NADPH oxidase components, tumor necrosis factor-alpha, and inducible nitric oxide synthase. Because mGluR5 is expressed by microglial cells in the rat spinal cord, such effects may be mediated by direct action on microglial cells. mGluR5 stimulation also reduced microglial activation and decreased microglial-induced neurotoxicity in spinal cord microglia cultures; the latter effects were blocked by the selective mGluR5 antagonist MTEP.

Interpretation: These data demonstrate that mGluR5 activation can reduce microglial-associated inflammation, suggesting that the protective effects of mGluR5 agonists may reflect this action. Ann Neurol 2009;66:63-74.

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Conflict of interest statement

Potential conflict of interest: Drs. Faden and Byrnes are co-inventors on a patent application that has been filed by Georgetown University related to the technology that is described in this paper.

Figures

Fig 1
Fig 1
Functional effects of metabotropic glutamate receptor 5 (mGluR5) activation after traumatic spinal cord injury. Hind-limb locomotor function was assessed using the Basso–Beattie–Bresnahan (BBB) score at days 1, 7, 14, 21, and 28 after injury (A). CHPG treatment resulted in a significant improvement in BBB score by day 14 after injury, which continued through day 28. Squares = vehicle; triangles = CHPG. The slope of the line was also assessed (B). CHPG treatment resulted in a significantly greater rate of recovery than vehicle. Bars represent mean ± standard error of the mean. *p < 0.05.
Fig 2
Fig 2
Magnetic resonance imaging (MRI)–based lesion volume measurements after traumatic spinal cord injury. At 28 days after injury, rats underwent T2-weighted MRI. Hyperin-tense regions (arrows) indicate lesion sites in vehicle-treated (A) and CHPG-treated (B) animals. The MRI-based lesion volume of these hyperintense regions was assessed and demonstrated a significant reduction with CHPG treatment (C). Bars represent mean ± standard error of the mean. *p < 0.05.
Fig 3
Fig 3
Histological effects of metabotropic glutamate receptor 5 (mGluR5) activation after traumatic spinal cord injury. Using Eriochrome-stained tissue slides (A), we calculated cavity (B) and spared white matter volume (C). CHPG infusion resulted in a significant reduction in cavitation, supporting magnetic resonance imaging (MRI) findings. CHPG treatment also increased remaining white matter around the lesion area, which stains blue with the Eriochrome dye. Representative images obtained from the lesion epicenter. Bars represent mean ± standard error of the mean. *p < 0.05. Scale bar = 500μm.
Fig 4
Fig 4
Metabotropic glutamate receptor 5 (mGluR5) activation reduces Iba-1 staining after spinal cord injury in a dose-dependent manner. At 7 days after injury, spinal cord tissue at the lesion site was immunolabeled with Iba-1, a marker for activated microglia/macrophages. Immunolabeling was greatest in tissue that received infusion of vehicle (A). Infusion of 10mM CHPG reduced Iba-1 immunostaining (B). Immuno-staining was further reduced after treatment with 41mM CHPG (C). Quantitation of immunolabeling demonstrated a dose-dependent trend with CHPG treatment, and 41mM CHPG significantly reduced immunopositive pixel density in comparison with vehicle-treated tissue. Representative images obtained at 1mm caudal to lesion epicenter. Scale bar = 500μm. Bars represent mean ± standard error of the mean. *p < 0.05.
Fig 5
Fig 5
Metabotropic glutamate receptor 5 (mGluR5) activation reduces tumor necrosis factor-α (TNF-α) secretion at 24 hours after spinal cord injury. TNF-α was measured by enzyme-linked immunosorbent assay in the spinal cord tissue 24 hours after contusion injury. TNF-α was significantly increased in animals receiving contusion injury and vehicle treatment in comparison with sham-injured rats. Infusion of CHPG into the spinal cord at 30 minutes after injury resulted in a significant reduction in TNF-α protein detection. Bars represent mean ± standard error of the mean. *p < 0.05.
Fig 6
Fig 6
Metabotropic glutamate receptor 5 (mGluR5) activation alters the inflammatory response after spinal cord injury (SCI). Microglial-related inflammatory products p22 phox (A), ED1 (B), Galectin-3 (C), and inducible nitric oxide synthase (iNOS) (D) were found to be significantly suppressed 7 or 28 days after SCI in animals treated with CHPG in comparison with control, as measured by Western blotting. Representative Western blots for 7- or 28-day samples are shown. Bars represent mean ± standard error of the mean. *p < 0.05.
Fig 7
Fig 7
Metabotropic glutamate receptor 5 (mGluR5) activation reduces ED1 expression at 28 days after spinal cord injury (SCI). ED1 expression was measured in spinal cord tissue samples using fluorescence immunohistochemistry. Vehicle-treated tissue (A) demonstrated a large amount of staining for ED1 (green) around a large cavity in the center of the tissue, which was decreased in CHPG-treated tissue (B). TO-PRO-3–stained nuclei (blue) provide contrast. Images are taken from 2mm rostral to the lesion epicenter. Quantitation of ED1-positive pixel density showed that this reduction is statistically significant (C). Bars represent mean ± standard error of the mean. *p < 0.05. Scale bar = 500μm.
Fig 8
Fig 8
Metabotropic glutamate receptor 5 (mGluR5) activation reduces gp91phox expression at 28 days after spinal cord injury (SCI). Immunolabeling for gp91phox (green) was performed at 72 hours (A, B) and 28 days (C, D) after injury in vehicle- (A, C) and CHPG-treated (B, D) tissue. TO-PRO-3–stained nuclei (blue) provide contrast. Quantitation of gp91phox-positive pixel density demonstrated a significant reduction in CHPG-treated tissue at 28 days after injury (E). Images are taken from 2mm caudal to the lesion epicenter. Bars represent mean ± standard error of the mean. *p < 0.05. Scale bar = 500μm.
Fig 9
Fig 9
Microglia express metabotropic glutamate receptor 5 (mGluR5). Double labeling for mGluR5 and various markers of microglia was performed to determine whether microglia express mGluR5 in the spinal cord. mGluR5 (green) is expressed on Galectin-3–(red; A, B) or OX42 (red, C)-positive microglia (arrows) in injured spinal cord. Double labeling is shown magnified in (B). Areas of double labeling are indicated by yellow. Negative controls, in which the primary antibody for mGluR5 is OX42, are shown (D), and lack cellular labeling observed in (A–C).
Fig 10
Fig 10
CHPG inhibits lipopolysaccharide (LPS)-induced activation in spinal cord–derived microglia. Spinal cord microglial activation was measured by proliferation (MTS assay, A) and nitric oxide production (B) at 24 hours after stimulation. Both measurements were significantly inhibited by pretreatment with the metabotropic glutamate receptor 5 (mGluR5) agonist, CHPG (100μM). Bars represent mean ± standard error of the mean. **p < 0.01 vs control; #p < 0.05 vs LPS.
Fig 11
Fig 11
CHPG inhibits microglial-induced neurotoxicity. Neuronal number, as measured by counting neuronal nuclei–positive(NeuN+) cells 24 hours after microglia/neuron coculture. The number of NeuN+ cells was reduced by the coincubation of lipopolysaccharide (LPS)-stimulated microglia with neurons; this was reversed by the pretreatment of microglia with CHPG. Addition of the metabotropic glutamate receptor 5 (mGluR5) antagonist, MTEP (100μM), inhibited the effect of CHPG on microglial-induced neurotoxicity, demonstrating an mGluR5-mediated effect by CHPG. Bars represent mean ± standard error of the mean. *p < 0.05 vs control; #p < 0.05 vs LPS; +p < 0.05 vs CHPG + LPS.
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