Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury

Abstract

Chronic neuropathic pain is a frequent consequence of spinal cord injury (SCI). Yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. Previous studies have demonstrated that SCI results in excessive ATP release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. We sought to assess the role of connexin 43 (Cx43) as a mediator of CNS inflammation and chronic pain. To determine the extent of Cx43 involvement in chronic pain, a weight-drop SCI was performed on transgenic mice with Cx43/Cx30 deletions. SCI induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. Notably, SCI-induced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with Cx43/Cx30 deletions, but fully developed in transgenic mice with only Cx30 deletion. SCI-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with Cx43/Cx30 deletions, when compared with littermate controls. In comparison, a standard regimen of post-SCI treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than Cx43 deletion. These findings suggest Cx43 is critically linked to the development of central neuropathic pain following acute SCI. Since Cx43/Cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain.

Fig. 1. Immunohistochemical staining of injured and noninjured spinal cord

(A) Immunohistochemical staining of GFAP (green), DAPI (blue), and Cx43 (red) in longitudinal spinal cord slices of Cx43/Cx30 KO mice and littermate controls. As expected, in Cx43/Cx30 KO, GFAP-expressing cells express no Cx43. Meanwhile, littermate controls express Cx43 in GFAP-expressing cells and exhibit an upregulation of CX43 at injury sites.

Fig. 2. Deletion of Cx43 does not significantly change lesion volume or locomotor recovery following mild spinal cord injury

(A) Schematic diagram of experimental design. Mild spinal cord injury was inflicted by a weight-drop impact (3-g weight-drop with tip diameter of 0.5-mm flat surface at T10 and T12) in adult female mice. Locomotor function was scored according to the Basso Mouse Scale (BMS) on a bi-weekly basis, whereas thermal hyperalgesia and mechanical allodynia were quantified daily. Animals were perfusion-fixed after 2 months and spinal cords were harvested for histology. (B) The graph depicts BMS scores for locomotor functions as a function of time after spinal cord injury. Deletion of Cx43/Cx30 has no significant effects on recovery of motor function (n = 6, P > 0.05, ANOVA, mean ± SEM). (C) The severity of spinal cord injury was quantified as the atrophy volume, since it is difficult to delineate the borders of the tissue lesion following mild spinal cord injury. Cx43/Cx30 KO and their littermate controls exhibit a comparable degree of atrophy close to the injury site.

Fig. 3. Cx30 KO plays no role in the development in chronic pain after spinal cord injury

Cx30KO and wild-type animals do not differ in development of mechanical allodynia (A) or heat hyperalgesia (B) after spinal cord injury. At all time points tested, no significant difference was noted (P > 0.1, one-way ANOVA, n = 10, mean ± SEM).

Fig. 4. Deletion of Cx43 reduced mechanical allodynia and heat hyperalgesia after mild spinal cord injury

(A) Graph comparing the development of mechanical allodynia over the course of 2 months after mild spinal cord injury. Four weeks after spinal cord injury, mechanical allodynia was significantly less pronounced in Cx43/Cx30 KO than in littermate controls (*P < 0.01, one-way ANOVA, n = 6, mean ± SEM). (B) Development of heat hyperalgesia over the course of 2 months after mild spinal cord injury. Starting from 4 weeks after spinal cord injury and lasting for the remaining observation period, thermal hyperalgesia was significantly reduced in Cx43/Cx30 KO compared with littermate controls (*P < 0.01, ANOVA, n = 6, mean ± SEM).

Fig. 5. Minocycline also reduces the development of neuropathic pain symptoms after mild spinal cord injury, but less efficiently than deletion of Cx43

(A) Bar histogram shows the effects of minocycline on mechanical allodynia. Significant attenuation of mechanical allodynia was observed 4–8 weeks after spinal cord injury in mice receiving minocycline compared with vehicle controls exposed to the same injury (**P < 0.05, ANOVA, n = 12) (B) Bar histogram shows the effects of minocycline on the development of heat hyperalgesia. Heat hyperalgesia was significantly reduced in mice receiving minocycline 4–8 weeks after spinal cord injury compared with vehicle controls (**P < 0.05, ANOVA, n = 12, mean ± SEM). (C and D) Graph comparing the analgesic effects of minocycline versus deletion of Cx43/Cx30. Both set of data was normalized to littermate controls exposed to the same injury. Deletion of Cx43/Cx30 leads to a significantly greater reduction of mechanical allodynia and heat thermal hyperalgesia after spinal cord injury (P < 0.01, ANOVA, n = 6).

Fig. 6. Deletion of Cx43 reduced astrogliosis after mild spinal cord injury

(A) Representative confocal images of longitudinal sections of spinal cord at site of injury 3 day, 1 week, and 1 month after the traumatic event. The sections were immunolabeled against GFAP (Top: littermate controls, Bottom: Cx43/Cx30 KO). Blue: DAPI; Green: GFAP. (B) Quantitative analysis of immunofluorescence intensity of GFAP in Cx43/Cx30 KO and littermate controls. Deletion of Cx43 reduced GFAP immunolabeling after traumatic injury in Cx43/Cx30 KO compared with littermate controls, reflecting reduced gliosis (P < 0.05, ANOVA, n = 6).