Class I histone deacetylase (HDAC) inhibitor CI-994 promotes functional recovery following spinal cord injury

Abstract

Spinal cord injury (SCI) induces severe and long-lasting neurological disability. Accumulating evidence has suggested that histone deacetylase (HDAC) inhibitors exert neuroprotective effects against various insults and deficits in the central nervous system. In the present study, we assessed the effect of the class I HDAC inhibitor CI-994 in a mouse model of SCI. Following SCI, mice were treated with either dimethyl sulfoxide (control vehicle) or 1, 10, or 30 mg/kg CI-994. Level of acetylated histone H3 expression was increased in the motor cortex and spinal cord of 10 mg/kg CCI-994-treated mice after SCI. CI-994 increased histone H3 acetylation in the myeloperoxidase-positive neutrophils and CD68-positive microglia/macrophages in the spinal cord. Although it did not appear to contribute to corticospinal tract axonal reorganization, intraperitoneal injection of CI-994 promoted behavioral recovery following SCI. Furthermore, administration of CI-994 suppressed neutrophil accumulation, inflammatory cytokine expressions, and neuronal loss as early as 3 days following injury. Thus, our findings indicate that HDAC inhibitors may improve functional recovery following SCI, especially during the early stages of the disease.

Fig. 1. Changes in HDAC expression following SCI.

a–f Profiles of HDAC expression in the motor cortex a–d and spinal cord e, f following SCI. Levels of class I HDAC expression relative to those of GAPDH were measured via real-time PCR. Results represent the mean ± SE from three independent experiments. Relative levels of HDAC expression are presented as fold changes relative to the level in sham mice. n = 3. *p < 0.05; ANOVA with Tukey–Kramer test. g HDAC expression was not altered in the spinal cord of CI-994-treated mice compared with DMSO-treated control mice. HDAC1 and three expressions were examined 3 days after SCI. n = 3. N.S., not significant; Student’s t-test. HDAC: histone deacetylase; SCI: spinal cord injury; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

Fig. 2. CI-994 increases HDAC activity after SCI.

a, b Histone H3 acetylation was upregulated in the motor cortex a and spinal cord b of CI-994-treated mice. Western blot analysis revealed that 10 or 30 mg/kg CI-994 treatment once a day for 14 days tended to increase acetylated histone-H3 levels. n = 4. **p < 0.01, *p < 0.05; ANOVA with Tukey–Kramer test. c Time-course of histone H3 acetylation in the motor cortex. Mice were administrated with DMSO or 10 mg/kg CI-994 after SCI. n = 3. *p < 0.05; ANOVA with Tukey–Kramer test. d, e Class 1 HDAC activities were reduced in the motor cortex d and spinal cord e of CI-994-treated mice. n = 6. **p < 0.01, *p < 0.05; Student’s t-test

Fig. 3. CI-994 increases acetylated histone H3 level in immune cells.

a HDAC1 and HDAC3 expression was observed in MPO-positive cells from 3 day after SCI. Scale bar 20 μm. b–e Double-staining images of acetylated histone H3 and indicated cell-type specific markers in the spinal cord b–d and cortex e. Increased acetylated histone H3 in MPO-positive cells (Green in b) and CD68-positive cells (Red in c) in the spinal cord of CI-994-treated mice. Arrowheads: co-labeling of anti-acetylated histone H3 and cell-type specific markers. Scale bars 20 μm in the spinal cord images b–d; 50 μm in the cortex images e. n = 6. *p < 0.05; Student’s t-test. HDAC: histone deacetylase; SCI: spinal cord injury

Fig. 4. CI-994 treatment improves recovery of motor function following SCI.

a BMS scores after injury were significantly increased in CI-994-treated mice relative to those in vehicle (DMSO)-treated mice. b CI-994 treatment attenuated increases in the number of foot-slips in the beam walk test after SCI. c CI-994 treatment attenuated increases in the number of foot-falls in the grid walk test after SCI. d CI-994 treatment reduced the percentage and number of errors in the ladder walk test. e There was no significant difference among the groups in the inclined plane test. Results are presented as the mean ± SE. DMSO n = 21, CI-994 n = 22 in a; DMSO n = 15, CI-994 n = 16 in b–e. *p < 0.05, **p < 0.01. Two-way repeated-measures ANOVA with Tukey–Kramer test. SCI: spinal cord injury; BMS: Basso Mouse Scale

Fig. 5. CI-994 does not promote sprouting of corticospinal tract fibers following SCI.

a, b Representative images from the transverse sections of the cervical spinal cord a and sagittal sections of the lesion site, which show biotinylated dextran amine (BDA)-labeled corticospinal tract fibers (green) extending into the gray matter at 28 days following injury. Scale bar 100 μm (a, high magnification images in b), 200 μm (low-magnification images in b). c, d The ratio of the total number of sprouting corticospinal tract fibers c, or the number of the fibers at the indicated cervical or thoracic spinal cord levels d at 28 days after injury. No significant differences were observed between control (DMSO) and CI-994-treated mice. Student’s t-test. Sham: n = 7; DMSO, CI-994: n = 14. e Regions of reactive gliosis were visualized as GFAP-positive areas near the site of injury in control or CI-994-treated mice. dpi: days post injury. Scale bar 200 μm. SCI: spinal cord injury; GFAP: glial fibrillary acidic protein

Fig. 6. CI-994 reduces neutrophil infiltration following SCI.

a Schematic representation of injured spinal cord. Transverse sections were prepared from 6 mm rostral b, or adjacent to the lesion site (F). b The number of MPO-positive neutrophils was decreased in the injured spinal cord of CI-994-treated mice at 1 day and 3 days after SCI. dpi: days post injury. Scale bar: 200 μm. c Dot plots of isolated immune cells in the spinal cord, gated for live cell analysis. d Representative cytometry data for neutrophils (Ly6C⁺/Ly6G⁺ cells) in the injured spinal cord 3 days after SCI. e Quantitative data for accumulated neutrophils in the injured spinal cord up to 7 days after SCI. f Numbers of CD68 (ED-1)-positive activated microglia/macrophages were not altered in the injured spinal cord of CI-994-treated mice. Scale bar: 200 μm in low-magnification images, 100 μm in high magnification images. g Representative cytometry data for macrophages (Ly6C⁺/CD11b⁺ cells) in the injured spinal cord at the indicated days after SCI. hQuantitative data for accumulated macrophages in the injured spinal cord up to 7 days after SCI. Results are presented as the mean ± SE (n = 3–6). *p < 0.05, Two-way repeated-measures ANOVA. SCI: spinal cord injury; MPO: myeloperoxidase

Fig. 7. CI-994 ameliorates the loss of NeuN-positive neurons following SCI.

a Decreased cytokine/chemokine expression in the spinal cord of CI-994-treated mice. Cytokine/chemokine levels in CI-994-treated mice were normalized to the levels in DMSO (control)-treated mice. n = 4. b Representative images of immunohistochemical staining of neurons in the gray mater following SCI. Neurons were immunostained with anti-NeuN antibody. Scale bar: 200 μm. c The numbers of neurons in the gray matter from 2 mm rostral to lesioned sites to 2 mm caudal to lesioned sites were calculated at the indicated days after SCI. Results are presented as the mean ± SE of five mice per group. *p < 0.05, **p < 0.01; ANOVA with Tukey–Kramer test. SCI: spinal cord injury