Alterations in the mRNA levels of receptors of TGF-βs following MCAO.

<p>The density of autoradiography grains (Y axis) was calculated for the TGF-β RI (A), the TGF-β RII (B), the TGF-β RIII (C), and Alk1 (D). Analysis was performed in the cortex and the striatum in 4 cases: 24 h after transient (1 h) and permanent MCAO, 72 h, and 1 month after transient MCAO. The penumbra was not examined 1 month after MCAO as it was not identifiable any more. The star symbol (*) indicates significantly (p<0.05) different values.</p

Nissl and TTC staining of the lesioned brain at different time points after focal ischemia.

<p>Coronal sections demonstrate the damage caused by the experimental manipulations. The lesion can be seen as the area with lighter appearance in the Nissl and TTC sections. The Nissl and the TTC stained sections are at different rostrocaudal levels because TTC labeling was applied to the frontal part of the brains while Nissl staining was performed on sections intermingled with those used for in situ hybridization histochemistry at a level where the size of the lesion was maximal. A: Sham operated; there is no sign of lesion. B: 24 hours after transient MCAO; the infract area is visible in the striatum and the cerebral cortex. C: 24 hours after permanent MCAO; the lesion is more pronounced than following transient MCAO. D: 72 hours following transient MCAO; the mass of invading cells are visible. E: 1 month after MCAO; some tissue disappears, while other parts of the infarct area are completely invaded by non-neuronal cells. Scale bars  = 1 mm.</p

The expression of TGF-β receptors at 24 h following MCAO.

<p>Dark-field photomicrographs of sections labeled by in situ hybridization histochemistry show the mRNA of TGF-β receptors. The lesion sites are indicated by star symbols (*) and the borders of lesions are demarcated by white dots. Away from the lesion, a low level of TGF-β RI expression is seen in the caudate putamen and a higher level in the cortex (A). The basal expression level of TGF-β RII (B), TGF-β RIII (C), and ALK1 mRNA (D) is very low. Abbreviations: cc - corpus callosum, LV - lateral ventricle. Scale bars  = 1 mm.</p

Schematic figure on TGF-β signaling at 72 h after MCAO.

<p>In the infarct area, TGF-β1 released from microglial cells acts on microglial and endothelial cells via TGF-β RI and Alk1, respectively. In the penumbra, TGF-β1 released from astrocytes and microglial cells act on these glial cells through TGF-β RI. Meanwhile, in the intact tissue away from the lesion, TGF-β2 and -β3 are released from neurons act on neurons and astrocytes by means of TGF-β RI.</p

TGF-β RI mRNA expression in neurons of the intact cerebral cortex.

<p>TGF-β RI predominantly expressed in neurons and not in glial cell types in the normal cerebral cortex. A: Double labeling of TGF-β RI mRNA and NeuN immunoreactivity. The black in situ hybridization signal co-localizes with brown NeuN immunoreactivity in a large number of cells. The back arrows point at some double labeled cells. The rectangle indicates the position of the high magnification picture in the inlet. It shows the presence of autoradiography grains above NeuN-immunopositive neuronal cells. B: Single labeled TGF-β RI mRNA expressing cells are indicated by black, and S-100 immunoreactive astrocytes by white arrowheads. C: TGF-β RI mRNA expressing neurons are indicated by black, and Iba1-immunoreactive microglias by white arrowheads. There are no double labeled cells present for TGF-β RI and the astrocyte marker S-100 or the microglia marker Iba1. Scale bars  = 200 µm for A, 50 µm for B and C, and 10 µm for the inlet in panel A.</p

Types of TGF-β receptor-expressing cells based on double labeling with cell type markers.

<p>Double labeling was performed by in situ hybridization histochemistry for the TGF-β receptors and immunohistochemistry for markers of different cell types at 72 h after MCAO. To identify the cell types the following markers were used: NeuN for neurons, S-100 for astroglial cells, Iba1 for microglial cells, and vWF for endothels. Data on TGF-β receptors and double labeled cells are presented in 3 different locations. The cell counts were performed in 400 µm×400 µm rectangular-shaped areas of the intact (away from the lesion), penumbral and lesioned cerebral cortex from 4 brains.</p><p>Types of TGF-β receptor-expressing cells based on double labeling with cell type markers.</p

The induction of TGF-β1, - β2, and -β3 mRNA 72 h and 1 mo after MCAO.

<p> <i>In situ</i> hybridization sections show the expression of TGF-β mRNA. The infarct areas are indicated by the star symbol (*) and the borders of the infarcts are demarcated with white dots. A: TGF-β1 expression is induced at the perimeter of the lesion as well as within the infarct area 72 h after MCAO. High magnification bright field photomicrographs demonstrate that TGF-β1 mRNA accumulates above cell bodies within the infarct area (Aa), as well as in the corpus callosum (Ab). The locations of these pictures are indicated by the arrowheads in the left panel. B: TGF-β2 is present in layers II, III, and V of the cerebral cortex. Only a few cells demonstrate increased expression level of TGF-β2 in the vicinity of the border of the lesion at 72 h after MCAO. C: TGF-β3 is present in some neurons in layer IV of the cerebral cortex, but the level of expression is not higher than that in normal animals D: The induction of TGF-β1 mRNA is demonstrated 1 month after MCAO. TGF-β1 mRNA is abundant within the infarct area except for in a relatively small core region. The field indicated by the large black arrowhead in A is enlarged and shown in bright-field in the inlet to demonstrate that even the densest TGF-β1 signal represents labeling of individual cells as autoradiography grains accumulated above cell bodies. E: TGF-β2 mRNA is present in a few cells at the perimeter of the infarct area. Outside the lesion, TGF-β2 mRNA is distributed in the cerebral cortex as in intact animals but is not induced above normal levels. Abbreviations: ac – anterior commissure, cc – corpus callosum, CP - caudate putamen, LV - lateral ventricle. Scale bar = 2 mm for A and E, 1 mm for C, and 50 µm for B.</p

The expression of TGF-β proteins in different brain regions around an ischemic lesion at 24 h following a 1 h transient MCAO.

<p>The number of cells was counted in 200×400 µm areas (0.08 mm²) of coronal brain sections in cortical layers I-VI and in the caudate putamen (CP). The number of autoradiography grains was counted above TGF-β-positive cell nuclei indicated by the accumulation of autoradiography grains. Measurements were performed around the ischemic lesion (dark gray) and in the corresponding brain area on the contralateral side of the brain (light gray). Sections from 5 animals were involved in the analysis. A–C: The number of cells expressing TGF-β1, -β2, and -β3 in 0.08 mm². D-F: The number of autoradiography grains proportional to the mRNA level of TGF- β1, -β2, and -β3 in single cells. The values around the ischemic lesion and in the corresponding brain area on the contralateral side of the brain were compared using paired Students t-tests, where data obtained from the same section formed the pairs to eliminate variation from the labeling intensity of an individual staining. The star symbol (*) indicates brain regions, in which the number of TGF-β-expressing cells or the mRNA level of the particular subtype of TGF-β in single cells was significantly (p<0.05) elevated.</p