GLT1 overexpression reverses established neuropathic pain-related behavior and attenuates chronic dorsal horn neuron activation following cervical spinal cord injury

Abstract

Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. Following SCI, chronic dysregulation of extracellular glutamate homeostasis has been shown to play a key role in persistent central hyperexcitability of superficial dorsal horn neurons that mediate pain neurotransmission, leading to various forms of neuropathic pain. Astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake, particularly in the spinal cord. In our unilateral cervical contusion model of mouse SCI that is associated with ipsilateral forepaw heat hypersensitivity (a form of chronic at-level neuropathic pain-related behavior), we previously reported significant and long-lasting reductions in GLT1 expression and functional GLT1-mediated glutamate uptake in cervical spinal cord dorsal horn. To therapeutically address GLT1 dysfunction following cervical contusion SCI, we injected an adeno-associated virus type 8 (AAV8)-Gfa2 vector into the superficial dorsal horn to increase GLT1 expression selectively in astrocytes. Compared to both contusion-only animals and injured mice that received AAV8-eGFP control injection, AAV8-GLT1 delivery increased GLT1 protein expression in astrocytes of the injured cervical spinal cord dorsal horn, resulting in a significant and persistent reversal of already-established heat hypersensitivity. Furthermore, AAV8-GLT1 injection significantly reduced expression of the transcription factor and marker of persistently increased neuronal activation, ΔFosB, in superficial dorsal horn neurons. These results demonstrate that focal restoration of GLT1 expression in the superficial dorsal horn is a promising target for treating chronic neuropathic pain following SCI.

Keywords: adeno-associated virus; astrocyte; contusion; glutamate transporter; neuropathic pain.

Figure 1. Intraspinal injections of AAV8-eGFP transduced superficial regions of the dorsal horn

(A) Experimental paradigm showing unilateral C5 contusion and AAV8 injection into superficial dorsal horn at C6 and C7. (B) eGFP reporter expressing cells in dorsal horn. (C) Higher magnification image showing eGFP reporter expressing cells in dorsal horn. (D) Astrocyte-like morphology of eGFP+ cells.

Figure 2. AAV8-GLT1 selectively transduced astrocytes of superficial dorsal horn

(A–C) eGFP expression in GFAP+ astrocytes. (D–F) Lack of co-localization of Olig2 and eGFP. (G–I) Lack of co-localization of Tuj1 and eGFP. (J) Percentage of eGFP+ cells expressing GFAP, Olig2 or Tuj1. (K) Percentage of GFAP, Olig2 or Tuj1 expressing cells that were eGFP labeled. (L) Percentage of eGFP transduced area in dorsal horn. n = 3 mice/group.

Figure 3. AAV8-GLT1 increased GLT1 expression in superficial dorsal horn

(A–C) Lack of His-tag expression in AAV8-eGFP injected dorsal horn. (D–F) Co-localization of His-tag with GLT1 protein in dorsal horn. (G–I) Expression of exogenous His-tag+ GLT1 in GFAP+ astrocytes. Increased GLT1 expression in laminae I-II of AAV8-GLT1 (K) compared to AAV8-eGFP (J) mice. Quantification of GLT1 immunostaining intensity in: superficial dorsal horn (L), n = 6/group; in ventral portion of the dorsal horn (M) n = 3/group; in ventral dorsal horn (N), n =3/group; in white matter (O), n = 3/group

Figure 4. GLT1 overexpression attenuated already-established neuropathic pain-related behavior following cervical contusion

(A) Contused mice receiving AAV8-GLT1 showed an attenuated decrease in ipsilateral forepaw withdrawal latency compared to injured animals that received AAV8-eGFP or injury only. Mice receiving laminectomy-only or laminectomy-only followed by AAV8-eGFP injection showed no change in withdrawal latency compared to pre-surgery baseline. * SCI+AAV8-GLT1 vs. SCI+AAV8-eGFP. # SCI+AAV8-GLT1 vs. Laminectomy-only. % SCI+AAV8-GLT1 vs. Laminectomy+AAV8-GLT1. (B–D) There were no significant differences in grip strength for ipsilateral forepaw, contralateral forepaw or both hindpaws between injured AAV8-eGFP and AAV8-GLT1 mice. SCI+AAV8-eGFP, n = 9; SCI+AAV8-GLT1, n = 9; SCI-only, n = 4; Laminectomy-only, n = 4; Laminectomy+AAV8-GLT1, n = 4.

Figure 5. GLT1 overexpression reduced chronic dorsal horn neuron activation following cervical contusion

(A–C) Reduced numbers of ∆FosB+ neurons (A, arrowheads) in ipsilateral laminae I-II in AAV8-GLT1 mice compared to AAV8-eGFP (p = 0.02). Cresyl violet staining illustrates epicenter in AAV8-eGFP (D, top) and AAV8-GLT1 (D, bottom) groups. Lesion size (E), lesion volume (F) and motor neuron counts (G) were not different between groups. n = 6–8/group.