Modulation of Matrix Metalloproteinases Activity in the Ventral Horn of the Spinal Cord Re-stores Neuroglial Synaptic Homeostasis and Neurotrophic Support following Peripheral Nerve Injury

Abstract

Modulation of extracellular matrix (ECM) remodeling after peripheral nerve injury (PNI) could represent a valid therapeutic strategy to prevent maladaptive synaptic plasticity in central nervous system (CNS). Inhibition of matrix metalloproteinases (MMPs) and maintaining a neurotrophic support could represent two approaches to prevent or reduce the maladaptive plastic changes in the ventral horn of spinal cord following PNI. The purpose of our study was to analyze changes in the ventral horn produced by gliopathy determined by the suffering of motor neurons following spared nerve injury (SNI) of the sciatic nerve and how the intrathecal (i.t.) administration of GM6001 (a MMPs inhibitor) or the NGF mimetic peptide BB14 modulate these events. Immunohistochemical analysis of spinal cord sections revealed that motor neuron disease following SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) response in the ventral horn of the spinal cord, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters (glutamate GLT1 and glycine GlyT1), increased levels of the neuronal glutamate transporter EAAC1, and a net increase of the Glutamate/GABA ratio, as measured by HPLC analysis. These molecular changes correlated to a significant reduction of mature NGF levels in the ventral horn. Continuous i.t. infusion of both GM6001 and BB14 reduced reactive astrogliosis, recovered the expression of neuronal and glial transporters, lowering the Glutamate/GABA ratio. Inhibition of MMPs by GM6001 significantly increased mature NGF levels, but it was absolutely ineffective in modifying the reactivity of microglia cells. Therefore, MMPs inhibition, although supplies neurotrophic support to ECM components and restores neuro-glial transporters expression, differently modulates astrocytic and microglial response after PNI.

Fig 1

Low (A) and high (B-C) magnification of spinal cord sections stained for glial markers. A) Low magnification (5x) sections show the entire spinal cord in SHAM and SNI rats treated with ACSF. Note, in SNI-ACSF sections, the significant asymmetry of the glial markers expression (indicative of the injured side) and the selective reaction in the dorsal and ventral horn. Scale bar: 200 μm. B) Magnifications (20x) of the ventral horn showing reactive gliosis. Scale bar: 50 μm. C) High magnification (40x) morphological details of reactive microglia and astrocytes. Scale bar: 50 μm.

Fig 2. Evaluation of glial markers in the ventral horn of spinal cord.

Sections (A and C) and densitometric quantitation (B and D) of ventral horn of lumbar spinal cords from SHAM and SNI-operated animals treated for 7 days with BB14 (0.9 mg/kg b.w.), GM6001 (100 mg/kg. b.w.) or ACSF (vehicle) and immunostained for GFAP (A–B) or Iba1 (C–D). Data are the mean±SEM (**p≤0.001, ACSF vs. SHAM/BB14/GM6001; ANOVA and Holm–Sidak test). Scale bar: 50 μm.

Fig 3. Endogenous NGF expression in the ventral horn.

(A) Sections of lumbar spinal cord prepared from SHAM and SNI rats treated for 7 days with ACSF (vehicle), BB14 (0.9 mg/kg b.w.), or GM6001 (100 mg/kg. b.w.) and immunostained with NGF antibody. Scale bar: 50 μm. (B) Densitometric quantitation of NGF levels. Data are the mean±SEM (**p≤0.001, ACSF vs. SHAM/BB14/GM6001; ANOVA and Holm–Sidak test). Scale bar: 50 μm.

Fig 4. Expression of spinal glial and neuronal aminoacid transporters.

Sections of ventral horn of lumbar spinal cord from SHAM and SNI animals treated for 7 days with ACSF (vehicle), BB14 (0.9 mg/kg b.w.), or GM6001 (100 mg/kg. b.w.) and immunostained for glial glutamate (A–B) or glycine (C–D) transporters, or the neuronal glutamate transporter EAAC1 (E–F). Data are the mean±SEM (**p≤0.001, ACSF vs. SHAM/BB14/GM6001; ANOVA and Holm–Sidak test). Scale bar: 50 μm.

Fig 5. HPLC analysis of Glutamate/GABA ratio.

Amino acid levels were measured by HPLC in the ventral horn of lumbar spinal cord dissected from SHAM and SNI animals treated for 7 days with BB14 (0.9 mg/kg b.w.), GM6001 (100 mg/kg. b.w.) or ACSF (vehicle). The Glutamate/GABA ratio was calculated as described in M&M. Data are the mean±SEM (**p≤0.001, ACSF vs. SHAM/BB14/GM6001; ANOVA and Holm–Sidak test).