Adenosine triphosphate release inhibitors targeting pannexin1 improve recovery after spinal cord injury

Abstract

Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.

Keywords: adenosine triphosphate; purinergic P2X; receptors; spinal cord injuries.

Fig. 1

Schema of Brilliant Blue G and Brilliant Blue FCF inhibiting adenosine triphosphate release Brilliant Blue G inhibits P2X7R, ATP, and inflammation. Brilliant Blue FCF inhibits Panx1, inhibits ATP release, and inhibits inflammation. ATP: adenosine triphosphate BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G Panx1: P2X purinoceptor 7/pannexin1

Fig. 2

Functional recovery after spinal cord injury Fig. 2A: The Basso, Beattie, and Bresnahan score after SCI. The results are presented as the mean ± SEM. Fifteen rats were used and analyzed in each group. Significant differences were observed in Basso, Beattie, and Bresnahan scores between the BBG and vehicle groups at 4 days post-SCI, and the BBFCF group showed significant differences when compared with the vehicle group after 21 days. Significant differences were observed between the BBG and the BBFCF vehicle groups at 4 days post-SCI. ^* p<0.05 in the saline-treated and BBG groups. ^# p<0.05 in the saline-treated and BBFCF groups. ^† p<0.05 in BBG and BBFCF groups using Kruskal–Wallis and post hoc Dunn’s multiple comparison tests. Fig. 2B: Touch test results after SCI. The results are presented as the mean ± SEM. Only the touch test results for the BBG group were significantly different from those in the vehicle group 14 days after SCI. At 28 days post-SCI, the BBFCF group’s results were significantly different from those in the vehicle group. At 42 days after SCI, the results for the BBG and CBX groups were significantly different from those in the vehicle group. ^* p<0.05 and ^**p<0.01 in the saline-treated and BBG groups. ^# p<0.01 in the saline-treated and BBFCF groups. ^§ p<0.05 in the saline-treated and CBX groups using Kruskal–Wallis and post hoc Dunn’s multiple comparison tests. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone SCI: spinal cord injury SEM: standard error of the mean

Fig. 3

Immunohistochemical analysis of the injured spinal region 7 days after spinal cord injury Fig. 3A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3B:Quantification of Fig. 3A. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3D:Quantification of Fig. 3C. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3E:Immunohistochemical analysis of the CD68-positive myeloperoxidase-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3F:Quantification of Fig. 3E. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3G:Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3H:Quantification of Fig. 3G. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3J:Quantification of Fig. 3I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 SCI: spinal cord injury SEM: standard error of the mean

Fig. 3

Immunohistochemical analysis of the injured spinal region 7 days after spinal cord injury Fig. 3A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3B:Quantification of Fig. 3A. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3D:Quantification of Fig. 3C. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3E:Immunohistochemical analysis of the CD68-positive myeloperoxidase-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3F:Quantification of Fig. 3E. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3G:Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3H:Quantification of Fig. 3G. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3J:Quantification of Fig. 3I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 SCI: spinal cord injury SEM: standard error of the mean

Fig. 3

Immunohistochemical analysis of the injured spinal region 7 days after spinal cord injury Fig. 3A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3B:Quantification of Fig. 3A. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3D:Quantification of Fig. 3C. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3E:Immunohistochemical analysis of the CD68-positive myeloperoxidase-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3F:Quantification of Fig. 3E. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3G:Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3H:Quantification of Fig. 3G. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3J:Quantification of Fig. 3I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 SCI: spinal cord injury SEM: standard error of the mean

Fig. 3

Immunohistochemical analysis of the injured spinal region 7 days after spinal cord injury Fig. 3A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3B:Quantification of Fig. 3A. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3D:Quantification of Fig. 3C. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3E:Immunohistochemical analysis of the CD68-positive myeloperoxidase-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3F:Quantification of Fig. 3E. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3G:Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3H:Quantification of Fig. 3G. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3J:Quantification of Fig. 3I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 SCI: spinal cord injury SEM: standard error of the mean

Fig. 3

Immunohistochemical analysis of the injured spinal region 7 days after spinal cord injury Fig. 3A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3B:Quantification of Fig. 3A. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3D:Quantification of Fig. 3C. The results are presented as the mean ± SEM. ^**p<0.01 by Student’s t-test. Five rats were used and analyzed in each group. Fig. 3E:Immunohistochemical analysis of the CD68-positive myeloperoxidase-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3F:Quantification of Fig. 3E. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3G:Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3H:Quantification of Fig. 3G. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. Fig. 3I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 7 days after SCI. Bar: 40 µm. Fig. 3J:Quantification of Fig. 3I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Five rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 SCI: spinal cord injury SEM: standard error of the mean

Fig. 4

Immunohistochemical analysis of the injured spinal region 42 days after spinal cord injury Fig. 4A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4B: Quantification of Fig. 4A. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4D: Quantification of Fig. 4C. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4E:Immunohistochemical analysis of the CD68-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4F: Quantification of Fig. 4E. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4G: Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4H: Quantification of Fig. 4G. The results are presented as the mean ± SEM. ^***p<0.001 by Student’s t-test. Twelve rats were used and analyzed in each group. Fig. 4I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4J: Quantification of Fig. 4I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Twelve rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 N.S.: not significant according to Student’s t-test SCI: spinal cord injury SEM: standard error of the mean

Fig. 4

Immunohistochemical analysis of the injured spinal region 42 days after spinal cord injury Fig. 4A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4B: Quantification of Fig. 4A. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4D: Quantification of Fig. 4C. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4E:Immunohistochemical analysis of the CD68-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4F: Quantification of Fig. 4E. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4G: Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4H: Quantification of Fig. 4G. The results are presented as the mean ± SEM. ^***p<0.001 by Student’s t-test. Twelve rats were used and analyzed in each group. Fig. 4I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4J: Quantification of Fig. 4I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Twelve rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 N.S.: not significant according to Student’s t-test SCI: spinal cord injury SEM: standard error of the mean

Fig. 4

Immunohistochemical analysis of the injured spinal region 42 days after spinal cord injury Fig. 4A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4B: Quantification of Fig. 4A. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4D: Quantification of Fig. 4C. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4E:Immunohistochemical analysis of the CD68-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4F: Quantification of Fig. 4E. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4G: Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4H: Quantification of Fig. 4G. The results are presented as the mean ± SEM. ^***p<0.001 by Student’s t-test. Twelve rats were used and analyzed in each group. Fig. 4I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4J: Quantification of Fig. 4I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Twelve rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 N.S.: not significant according to Student’s t-test SCI: spinal cord injury SEM: standard error of the mean

Fig. 4

Immunohistochemical analysis of the injured spinal region 42 days after spinal cord injury Fig. 4A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4B: Quantification of Fig. 4A. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4D: Quantification of Fig. 4C. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4E:Immunohistochemical analysis of the CD68-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4F: Quantification of Fig. 4E. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4G: Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4H: Quantification of Fig. 4G. The results are presented as the mean ± SEM. ^***p<0.001 by Student’s t-test. Twelve rats were used and analyzed in each group. Fig. 4I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4J: Quantification of Fig. 4I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Twelve rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 N.S.: not significant according to Student’s t-test SCI: spinal cord injury SEM: standard error of the mean

Fig. 4

Immunohistochemical analysis of the injured spinal region 42 days after spinal cord injury Fig. 4A:Immunohistochemical analysis of the myeloperoxidase (MPO)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4B: Quantification of Fig. 4A. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4C:Immunohistochemical analysis of the CD8a-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4D: Quantification of Fig. 4C. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4E:Immunohistochemical analysis of the CD68-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4F: Quantification of Fig. 4E. The results are presented as the mean ± SEM. Twelve rats were used and analyzed in each group. Fig. 4G: Immunohistochemical analysis of the ionized calcium-binding adaptor molecule 1 (Iba1)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4H: Quantification of Fig. 4G. The results are presented as the mean ± SEM. ^***p<0.001 by Student’s t-test. Twelve rats were used and analyzed in each group. Fig. 4I:Immunohistochemical analysis of the glial fibrillary acidic protein (GFAP)-positive area in the injured spinal region 42 days after SCI. Bar: 40 µm. Fig. 4J: Quantification of Fig. 4I. The results are presented as the mean ± SEM. ^***p<0.001 using Student’s t-test. Twelve rats were used and analyzed in each group. BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1 N.S.: not significant according to Student’s t-test SCI: spinal cord injury SEM: standard error of the mean

Fig. 5

Hematoxylin and eosin staining analysis of the injured spinal region 42 days after spinal cord injury Fig. 5A: Hematoxylin and eosin staining of the injured area 42 days after SCI. Bar: 500 µm. Fig. 5B: Quantification of Fig. 5A. The results are presented as the mean ± standard error of the mean. ^***p<0.001 (n=15 rats in each group) using Student’s t-test. Fig. 5C: Schema of a longitudinal section of the spinal cord showing tissue damage, atrophy, and vacuolation after treatment with Brilliant Blue G and Brilliant Blue FCF. SCI: spinal cord injury BBFCF: Brilliant Blue FCF BBG: Brilliant Blue G CBX: carbenoxolone Panx1: P2X purinoceptor 7/pannexin1