Subarachnoid Hemorrhage Induces Gliosis and Increased Expression of the Pro-inflammatory Cytokine High Mobility Group Box 1 Protein - PubMed (original) (raw)

Subarachnoid Hemorrhage Induces Gliosis and Increased Expression of the Pro-inflammatory Cytokine High Mobility Group Box 1 Protein

Kentaro Murakami et al. Transl Stroke Res. 2011.

Abstract

Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with high rates of morbidity and mortality. Surviving SAH patients often suffer from neurological impairment, yet little is currently known regarding the influence of subarachnoid blood on brain parenchyma. The objective of the present study was to examine the impact of subarachnoid blood on glial cells using a rabbit SAH model. The astrocyte-specific proteins, glial fibrillary acidic protein (GFAP) and S100B, were up-regulated in brainstem from SAH model rabbits, consistent with the development of reactive astrogliosis. In addition to reactive astrogliosis, cytosolic expression of the pro-inflammatory cytokine, high-mobility group box 1 protein (HMGB1) was increased in brain from SAH animals. We found that greater than 90% of cells expressing cytosolic HMGB1 immunostained positively for Iba1, a specific marker for microglia and macrophages. Further, the number of Iba1-positive cells was similar in brain from control and SAH animals, suggesting the majority of these cells were likely resident microglial cells rather than infiltrating macrophages. These observations demonstrate SAH impacts brain parenchyma by activating astrocytes and microglia, triggering up-regulation of the pro-inflammatory cytokine HMGB1.

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Conflict of interest statement

Disclosure The authors declare no conflicts of interest.

Figures

Fig. 1

Fig. 1

Distribution of blood following experimental SAH in rabbits. Ventral view of brains obtained from a healthy control (left panel) and a 5-day SAH animal (right panel). Brainstem tissue, used throughout this study, was obtained 1–2 mm below the brain surface from regions denoted by black rectangular boxes

Fig. 2

Fig. 2

Enhanced expression of glial fibrillary acidic protein (GFAP) in brainstem from SAH model animals. a GFAP immunostaining is increased in brainstem from SAH model animals. Images demonstrate enhanced GFAP immunostaining in brainstem obtained from a 5-day SAH animal. Bar graph expresses GFAP immunostaining, in arbitrary density units (ADU), in tissue obtained from un-operated control (_n_= 6), 5-day sham-operated (_n_=4), 2-day SAH (_n_=4), and 5-day SAH (n_=6) animals. Three images per animal were randomly selected for analysis. Scale bar: 50 μm. **p<0.01 versus control and §§_p<0.01 versus sham using Tukey–Kramer multiple comparison test. b GFAP protein is increased in brainstem following SAH. Western blot demonstrating immunoreactive bands corresponding to GFAP and β-tubulin using 5 μg of protein obtained from control (_n_=4), sham-operated (_n_=3), 2-day SAH (n_=4), and 5-day SAH (n_=3) brainstem. GFAP band intensity was quantified using NIH software (ImageJ) and expressed as a ratio to β-tubulin band intensity. **p<0.01, *p<0.05, versus control and §§_p<0.01, §_p<0.05 versus sham using Tukey–Kramer multiple comparison test

Fig. 3

Fig. 3

Enhanced expression of S100B and HMGB1 in brainstem from SAH rabbits. a Top panel representative image of an RT–PCR gel using S100B- and GAPDH-specific primers. Bottom panel summarized RT–PCR data obtained from RNA isolated from brainstem of control (_n_=5), sham-operated (_n_=5), 2-day SAH (_n_=6), and 5-day SAH (_n_=6) animals using quantitative densitometric analysis. S100B band intensity is expressed relative to GAPDH band intensity for each animal. b Top panel representative image of an RT–PCR gel using HMGB1-specific primers. Bottom panel quantification of HMGB1 mRNA in brainstem from control (_n_=5), sham-operated (_n_=5), 2-day (_n_=4), and 5-day SAH (_n_=5) animals. Data were obtained using quantitative densitometric analysis of RT–PCR gels. HMGB1 band intensity is expressed relative to GAPDH band intensity for each animal. c Top panel Western blot demonstrating immunoreactive bands corresponding to HMGB1 using 5 μg of protein obtained from control, sham-operated, 2-day SAH, and 5-day SAH brainstem. Bottom panel quantification of HMGB1 protein levels in brainstem from control (_n_=4), sham-operated (_n_=3), 2-day (_n_=4), and 5-day SAH (n_=3) animals obtained using Western blot. HMGB1 band intensity was quantified using NIH software (ImageJ) and expressed as a ratio to β-tubulin band intensity. *p<0.05 versus control and §_p<0.05 versus sham using Tukey–Kramer multiple comparison test

Fig. 4

Fig. 4

Cytosolic expression of HMGB1 in Iba1-positive cells of brainstem from SAH animals. a, b HMGB1 immunostaining images obtained from brainstem of a control (a) and a 5-day SAH (b) animal. c, d Merged images of HMGB1 immunos-taining (green) and DAPI nuclear staining (blue). e, f Merged images of HMGB1 immunostaining (green) and Iba1 immunostaining (red). Enlarged images on the right side of each panel highlight the increased number of Iba1-positive cells with cytosolic staining of HMGB1 in brainstem from SAH animals. Scale bars represent 5 μm. g Quantification of the density of cells staining positively for Iba1 and cytosolic HMGB1. Values were obtained from averaging three to four brainstem images per animal (_n_=4 animals per group). **p<0.01, Student t test

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