Cell death.

<p>Activated caspase-3 immunoreactivity and TUNEL staining 3h after SAH. Panels A-B: 4-color fluorescence staining for TUNEL, NeuN, collagen-IV, and DAPI. Panel A: typical micrographs from male and female SAH animals. Each channel is shown as a separate image. Small arrows: TUNEL-positive neurons; large arrowheads: TUNEL-positive vascular cells. Note the greater frequency of TUNEL-positive neurons in male as compared to female. Panel B: average numbers of TUNEL-only, TUNEL+NeuN, and TUNEL+collagen-IV profiles. TUNEL-only and TUNEL-NeuN profiles are significantly greater in males than in females. Panel C: average numbers of profiles positive for activated caspase-3 (Cas) only, Cas+NeuN, and Cas+collagen-IV. All three indexes are significantly greater in male animals. Panel D: Fluoro-Jade B-positive cells (arrows) in representative SAH male and female brain sections. Panel E: Average numbers of Fluoro-Jade B-positive cells in SAH animals. Data are mean ± sem from 5 animals per gender. * significantly gender difference (p <0.05). </p

Cerebral inflammation.

<p>Luminal platelet aggregates and neutrophil accumulation in animals sacrificed 3 hours after SAH. Panel A: representative images of neutrophil staining. Note the greater number of neutrophils in male as compared to female brain. Scale bar = 500 μm. Panels B, C: average numbers of neutrophils and vascular platelet aggregates per whole brain section and per image field, respectively. Both parameters are greater in male as compared to female brains. Data are mean ± sem from 5 animals per gender. * significantly difference than females (p <0.05).</p

Attenuation of Cerebral Ischemic Injury in Smad1 Deficient Mice

<div><p>Stroke results in brain tissue damage from ischemia and oxidative stress. Molecular regulators of the protective versus deleterious cellular responses after cerebral ischemia remain to be identified. Here, we show that deletion of Smad1, a conserved transcription factor that mediates canonical bone morphogenetic protein (BMP) signaling, results in neuroprotection in an ischemia-reperfusion (I/R) stroke model. Uninjured mice with conditional deletion of <i>Smad1</i> in the CNS (<i>Smad1</i> cKO) displayed upregulation of the reactive astrocyte marker GFAP and hypertrophic morphological changes in astrocytes compared to littermate controls. Additionally, cultured <i>Smad1</i>^<i>-/-</i> astrocytes exhibited an enhanced antioxidant capacity. When subjected to I/R injury by transient middle cerebral artery occlusion (tMCAO), <i>Smad1</i> cKO mice showed enhanced neuronal survival and improved neurological recovery at 7 days post-stroke. This neuroprotective phenotype is associated with attenuated reactive astrocytosis and neuroinflammation, along with reductions in oxidative stress, p53 induction, and apoptosis. Our data suggest that Smad1-mediated signaling pathway is involved in stroke pathophysiology and may present a new potential target for stroke therapy.</p></div

<i>Smad1</i> deletion reduces oxidative stress and apoptosis following stroke.

<p>(A) Fluorescent images and quantification of <i>in vivo</i> HEt assay reflecting ROS levels on the ipsilateral hemisphere at 7 days post-stroke. The extent of oxidized nucleotides was measured by IHC for 8-oxo-dG levels at 24 h post-stroke. (B) IHC and quantification of activated Caspase 3 (aCasp3) and p53 on ipsilateral hemisphere at 7 days post-stroke. n = 3 for HEt, 8-oxo-dG, aCasp3, n = 4 for p53, unpaired Student’s t-test, *p<0.05; ***p<0.001. Scale, 50 μm.</p

Attenuated reactive astrocytosis after stroke in <i>Smad1</i> cKO mice.

<p>(A) Images of IHC for reactive astrocyte marker GFAP on ipsilateral hemisphere at 7 days (top two panels) or 3 months (bottom panels) post-stroke. The border between the stroke core and peri-infarct area is outlined by dotted lines. Arrows point to striatal infarct core. (B) Enlarged IHC images of boxed areas in (A) at the cortical peri-infarct area (blue box in D) with the indicated reactive astrocyte markers GFAP, Nestin, and Vimentin. Enlarged images of GFAP IHC highlight the hypertrophic morphology of GFAP⁺ astrocytes in mutants. (C) Quantification of the number of GFAP⁺ astrocytes and the intensity of GFAP immunoreactivity (IR) at the peri-infarct area shown in (B). n = 4, one-way ANOVA for the number of astrocytes, unpaired Student’s t-test for GFAP intensity, ***p<0.001, Ipsi, ipsilateral; Ctra, contralateral cortex. (D) Diagram of infarct territory affected by tMCAO (blue) and peri-infarct cortical area (blue box). (E) Reactive astrocytosis was similarly attenuated in the ipsilateral hippocampus of <i>Smad1</i> cKO mice. Scale, 500 μm (A), 100 μm (B), and 200 μm (E).</p