Anat Cell Biol.  2017 Sep;50(3):207-213. 10.5115/acb.2017.50.3.207.

Lithium ameliorates rat spinal cord injury by suppressing glycogen synthase kinase-3β and activating heme oxygenase-1

Affiliations
  • 1Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju, Korea. shint@jejunu.ac.kr, healthy@jejunu.ac.kr
  • 2HAEMALGEUN Veterinary Medical Clinic, Jeju, Korea.

Abstract

Glycogen synthase kinase (GSK)-3β and related enzymes are associated with various forms of neuroinflammation, including spinal cord injury (SCI). Our aim was to evaluate whether lithium, a non-selective inhibitor of GSK-3β, ameliorated SCI progression, and also to analyze whether lithium affected the expression levels of two representative GSK-3β-associated molecules, nuclear factor erythroid 2-related factor-2 (Nrf-2) and heme oxygenase-1 (HO-1) (a target gene of Nrf-2). Intraperitoneal lithium chloride (80 mg/kg/day for 3 days) significantly improved locomotor function at 8 days post-injury (DPI); this was maintained until 14 DPI (P<0.05). Western blotting showed significantly increased phosphorylation of GSK-3β (Ser9), Nrf-2, and the Nrf-2 target HO-1 in the spinal cords of lithium-treated animals. Fewer neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) were observed in the spinal cords of the lithium-treated group compared with the vehicle-treated group. Microglial activation (evaluated by measuring the immunoreactivity of ionized calcium-binding protein-1) was also significantly reduced in the lithium-treated group. These findings suggest that GSK-3β becomes activated after SCI, and that a non-specific enzyme inhibitor, lithium, ameliorates rat SCI by increasing phosphorylation of GSK-3β and the associated molecules Nrf-2 and HO-1.

Keyword

Spinal cord injuries; Lithium; Glycogen synthase kinase-3β; NF-E2-related factor-2; Heme oxygenase-1

MeSH Terms

Animals
Blotting, Western
Glycogen Synthase Kinases
Glycogen Synthase*
Glycogen*
Heme Oxygenase-1*
Heme*
Hemorrhage
Lithium Chloride
Lithium*
Phosphorylation
Rats*
Spinal Cord Injuries*
Spinal Cord*
Glycogen
Glycogen Synthase
Glycogen Synthase Kinases
Heme
Heme Oxygenase-1
Lithium
Lithium Chloride

Figure

  • Fig. 1 Locomotor outcomes as evaluated by Basso, Beattie, and Bresnahan (BBB) scoring (n=5/daily). The BBB scores were very low in both groups to 3 days post-injury; on day 8 post-injury, the BBB score in the lithium-treated group was significantly higher than that in the vehicle-treated group. This difference persisted until 14 days post-injury. **P<0.01 vs. vehicle-treated group.

  • Fig. 2 Histological profiles of the spinal cords of vehicle-treated (A, C) and lithium-treated groups (B, D) 4 days post-injury. (A, B) Low-magnification images of sagittal sections. (C, D) High-magnification images of the squares in panels (A) and (B), respectively. A–D, hematoxylin and eosin staining. Scale bars=100 µm (A, B), 20 µm (C, D).

  • Fig. 3 Ionized calcium-binding protein 1 (Iba-1) immunostaining of the core cord regions of vehicle-treated (A) and lithium-treated groups (B) on day 4 post-injury. Iba-1–positive microglial cells were evident in all regions, including the white and gray matter. Ramified microglial cells and many inflammatory cells immunostained for Iba-1 in the core cord regions of both groups. Scale bars=100 µm (A, B). (C) Bar graphs: semi-quantitative analysis of Iba-1 levels (~17 kDa protein) using a simple Western blotting system. Normalization was achieved by reprobing the membranes with an anti–β-actin antibody. Means±SE (n=5 per group) are shown. *P<0.05 vs. the vehicle-treated group. SCI, spinal cord injury; DPI, days post-injury.

  • Fig. 4 Western blotting to detect glycogen synthase kinase (GSK)-3β in rats with spinal cord injury (SCI). (A) Representative immunoblots of phosphorylated GSK (p-GSK)-3β (Ser9), total GSK-3β (~46 kDa), and β-actin (~45 kDa). (B) The p-GSK-3β level increased significantly in the spinal cords of lithium-treated rats. To quantify GSK-3β phosphorylation, the levels of the phosphorylated form were normalized to those of total GSK-3β. Normalization was achieved by reprobing the membranes with an anti–β-actin antibody. Means±SEM (n=5 per group) are shown. *P<0.05 vs. the lithium-treated group. DPI, days post-injury.

  • Fig. 5 Western blotting to detect nuclear factor erythroid 2-related factor-2 (Nrf-2) and heme oxygenase-1 (HO-1) in the spinal cords of rats with spinal cord injury (SCI). (A , B) Representative immunoblots of Nrf-2 (~57 kDa), HO-1 (~32 kDa), and β-actin (~45 kDa). Bar graphs: Both the Nrf-2 and HO-1 levels increased significantly and Nrf-2 was translocated from the cytoplasm to the nucleus in the spinal cords of lithium-treated rats. Normalization was achieved by reprobing the membranes with an anti–β-actin antibody. Means±SEM (n=5 per group) are shown. *P<0.05, **P<0.01 vs. the lithium-treated group. Cyto, cytoplasm; Nu, nucleus; OD, optical density; DPI, days post-injury.


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