Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury - PubMed (original) (raw)

Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury

Simone Di Giovanni et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.

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Figures

Fig. 1.

Fig. 1.

Cell cycle and DNA damage-related mRNA and protein changes after TBI. (a) Shown is a dendrogram (gene tree) obtained nucleating altered transcripts between sham and injured brains. Bar graph (Right) shows the color code for gene expression levels from low (blue) to high (red). These transcripts are up-regulated between 4 and 72 h after TBI (gene names in yellow indicate a Welch t test P value <0.05 between sham and injured at one corresponding time point). (b) Table shows gene name, accession number, and quantitation (fold changes) of mRNA changes by Affymetrix for the transcripts represented in the dendrogram. Genes with asterisks indicate a Welch t test P value <0.05 between sham and injured at the corresponding time point. (c) RT-PCR confirms expression of mRNAs for GADD45, PCNA, c-Myc, and p27 (ribosomal protein L-19 was used as control). (d) Immunoblots show increased GADD45 and PCNA protein levels in injured (I), versus sham (S) brains at 24 h in two parallel experiments. β-Actin is a loading control. (e) Immunoblotting at 72 h shows induction of cyclin D1 and inhibition of p27 protein expression after TBI in injured (I) versus sham (S) brains; flavopiridol (F) rescues p27 expression and inhibits cyclin D1. β-Actin is a loading control.

Fig. 2.

Fig. 2.

Flavopiridol promotes neuronal survival after DNA damage and inhibits astrocyte proliferation by blocking cell cycle proteins and cell cycle progression. (a and b) Flavopiridol treatment protects cells from death induced by etoposide (LDH release) (a) and number of Hoechst-positive cells (b) in primary cortical neurons at 24 h. (c) Immunoblotting shows expression of cell cycle proteins and caspase 3 after etoposide treatment, and etoposide plus flavopiridol treatment from rat cortical neuronal cells after treatment with etoposide (lane E) and after etoposide plus flavopiridol (lane E+F) at 24 h. (d) Flavopiridol blocks proliferation of cultured rat astrocytes. Shown are data from MTT cell viability assay in astrocytes after serum stimulation and treatment with flavopiridol. Values on y axis are expressed in OD, reflecting number of viable cells. (e) Flavopiridol completely suppresses cyclin D1 and PCNA protein expression in proliferating astrocytes. Immunoblotting at 48 h after serum stimulation (lane FBS) shows induction of cyclin D1 and PCNA compared with control resting astrocytes (lane C). Flavopiridol in duplicate experiments, administered 1 h before FBS completely suppresses cyclin D1 and PCNA protein expression (lanes FBS+F). (f) Flavopiridol reduces S phase in proliferating astrocytes to basal levels (FACS).

Fig. 3.

Fig. 3.

Immunofluorescence shows the expression of cyclin D1 and active caspase 3 in cortical neurons after TBI, and effects of flavopiridol treatment. (a) Double immunofluorescence in coronal sections of the brain cortex around the injury site shows staining for cyclin D1 in vehicle, and flavopiridol-treated animal after TBI, and for the neuronal marker NeuN. Cyclin D1 expression is induced in cortical neurons after TBI and strongly reduced after flavopiridol compared with vehicle (arrows). (Original magnification: ×125.) Bar graphs show quantitation of cortical neurons expressing cyclin D1 in ipsilateral and contralateral cortex in vehicle-treated rats, compared with flavopiridol (ipsilateral) (**, P < 0.01). (b) Double immunofluorescence in coronal sections of the brain cortex around the injury site shows staining for cyclin D1 in vehicle, and flavopiridol-treated animal after TBI, and for active caspase 3. Flavopiridol strongly reduces cyclin D1-positive and active caspase 3-positive neurons (arrows). (Original magnification: ×125.) Bar graphs show quantitation of cortical neurons coexpressing cyclin D1 and cleaved caspase 3 in ipsilateral and contralateral cortex in vehicle-treated rats, compared with flavopiridol (ipsilateral) (**, P < 0.01).

Fig. 4.

Fig. 4.

Immunofluorescence in glial cells after TBI and flavopiridol treatment. (a) Double immunofluorescence in coronal sections of brain cortex around the injury site shows staining for cyclin D1 and for astrocyte marker GFAP in vehicle, and flavopiridol-treated animals after TBI. Strong reduction of cyclin D1-positive and GFAP-positive cells is present after flavopiridol compared with vehicle (arrows). (Original magnification: ×125.) Bar graphs show quantitation of these data (**, P < 0.01). (b) Double immunofluorescence in coronal sections of hippocampus (CA1 and CA2) on the injury site shows staining for cyclin D1 and GFAP in vehicle, and flavopiridol-treated animal after TBI. Strong reduction of cyclin D1-positive and GFAP-positive cells is present after flavopiridol compared with vehicle (arrows). (Original magnification: ×125.) Bar graphs show quantitation of these data (*, P < 0.05; **, P < 0.01). (c) Double immunofluorescence in coronal sections of brain cortex around the injury site shows staining for cyclin D1 and for activated microglia marker OX-42 in vehicle, and flavopiridol-treated animals after TBI. Just sporadic GFAP-positive cells are present after flavopiridol (arrows). (Original magnification: ×125.) (d) Immunostaining for GFAP and nuclear marker (TOPRO) in ipsilateral cortex after TBI, comparing vehicle and flavopiridol-treated rats at 28 days posttrauma. Flavopiridol (250 μM) limits the injury-induced reactive gliosis and tissue loss. (Original magnification: ×125.)

Fig. 5.

Fig. 5.

Flavopiridol significantly reduces lesion volume 21 days after TBI in rats. (a) Bar graph shows significant reduction of lesion volume 21 days after TBI (30 min after the injury, at 250 μmol i.c.v.; n = 7; lesion volumes: mean ± SEM); (**, P < 0.01 vs. FP plus vehicle). (b) Representative T2-weighted MRI of vehicle and flavopiridol-treated rats shows marked reduction of lesion volume. (c) Flavopiridol improves cognitive performance as measured by the Morris water maze test (***, P < 0.001 compared with sham control animals). (d) Flavopiridol restores motor function close to control levels. Results are shown as daily mean ± SEM (**, P < 0.01 and ***, P < 0.001 vs. sham controls).

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References

    1. Raghupathi, R. (2004) Brain Pathol. 14, 215-222. - PMC - PubMed
    1. McGraw, J., Hiebert, G. W. & Steeves, J. D. (2001) J. Neurosci. Res. 63, 109-115. - PubMed
    1. Yakovlev, A. G. & Faden, A. I. (2001) Mol. Neurobiol. 24, 131-144. - PubMed
    1. Liou, A. K., Clark, R. S., Henshall, D. C., Yin, X. M. & Chen, J. T. (2003) Prog. Neurobiol. 69, 103-142. - PubMed
    1. Faden, A. I. (2002) Curr. Opin. Neurol. 15, 707-712. - PubMed

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