Figure 1.
Nissl and TTC staining of the lesioned brain at different time points after focal ischemia.
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.
Figure 2.
The expression of TGF-β receptors at 24 h following MCAO.
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.
Figure 3.
The induction of TGF-β receptors at 72 h following MCAO.
Dark- and bright-field images demonstrate the induction of mRNA of the TGF-β receptors. The lesion sites are indicated by star symbols (*) and the borders of lesions are demarcated by white dots. A: TGF-β RI expression is induced in the penumbra immediately outside the lesion as well as within the infarct area. B: The mRNA of TGF-β RII is also induced in the penumbra and also within the lesion. C: TGF-β RIII mRNA expressing cells were present within the lesioned area with appearance suggesting expression in blood vessels. D: ALK1 was also seen to be induced around the vessels within the infarct area. The fields indicated by the white arrows in A–D is enlarged and shown in bright field in E–H. Cells expressing TGF-β RI (E), TGF-β RII (F), TGF-β RIII (G), and Alk1 (H) are indicated by black arrowheads. The mRNA of TGF-β RIII and Alk1 are around blood vessels. Abbreviations: cc - corpus callosum, LV - lateral ventricle. Scale bars: 1 mm for A-D, and 50 µm for E–H.
Figure 4.
Alterations in the mRNA levels of receptors of TGF-βs following MCAO.
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.
Figure 5.
The induction of TGF-β receptors at 1 month following MCAO.
The induction of mRNA of TGF-β receptors is demonstrated by dark-field images of sections labeled with in situ hybridization histochemistry. The lesion sites are indicated by star symbols (*) and the borders of lesions are demarcated by white dots. A: TGF-β RI mRNA is abundant within the infarct area. Outside the lesion, TGF-β RI is distributed in the cerebral cortex as in not operated control animals. B: The level of TGF-β RII mRNA is markedly elevated within the lesion. C: TGF-β RIII also appeared in the infarct area. The inlet shows a bright-field image demonstrating the presence of autoradiography grains above non-epithelial cells. D: ALK1 mRNA was slightly elevated within the lesion. Abbreviations: cc - corpus callosum, LV - lateral ventricle. Scale bars: 1 mm for all panels and 20 µm for the inlet in panel C.
Figure 6.
TGF-β RI mRNA expression in neurons of the intact cerebral cortex.
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.
Table 1.
Types of TGF-β receptor-expressing cells based on double labeling with cell type markers.
Figure 7.
TGF-β RI and RII are induced in microglial cells.
Double labeling of TGF-β RI and RII mRNA and immunoreactivity of the microglia marker Iba1 at 72 h following MCAO. A: A low magnification picture of a section double labeled with TGF-β RI and Iba1. The border of the lesion is indicated by black dots. A rectangle and a large black arrow show the position of high magnification pictures in Aa and Ab, respectively. Aa: Single labeled TGF-β RI mRNA expressing neurons are indicated by white, and Iba1-immunoreactive microglia by black arrowheads in the intact cortex away from the lesion, respectively. In the penumbra, double labeled cells are found, some examples are shown by small black arrows. Ab: A high magnification picture shows that the black in situ hybridization signal of TGF-β RI is located above Iba1-imunoreactive cell bodies (brown precipitate) within the lesion. B: TGF-β RII is also expressed in microglia as demonstrated by double labeling of TGF-β RII mRNA (black in situ hybridization signal) and immunoreactivity of Iba1 (brown precipitate). TGF-β RII mRNA expressing microglias are indicated by black arrows. Abbreviations: cc - corpus callosum, LV - lateral ventricle. Scale bars: 1 mm for A, 100 µm for Aa and 50 µm for Ab and B.
Figure 8.
TGF-β RIII and ALK1 are induced in endothelial cells within the lesion.
Double labeling of TGF-β RIII and ALK1 mRNA and immunoreactivity of the microglial marker Iba1, the endothelial marker vWF, and the marker of smooth muscle cells of vessels, □SMA at 72 h after MCAO. A: Double labeling of ALK1 mRNA and alpha-SMA immunoreactivity in a low magnification image. The lesion site is indicated by star symbol (*). The border of the lesion is shown by black dots. B: A higher magnification picture of a section double labeled with ALK1 mRNA and alpha-SMA immunoreactivity. Single labeled ALK1 mRNA expressing cells are indicated by white and alpha-SMA-immunoreactive smooth muscle cells by black arrowheads. C: Double labeling of ALK1 mRNA and vWF-immunoreactive endothels. The field indicated by the black arrow shows an example of double labeling. D: ALK1 mRNA (black grains, some cells indicated by white arrowheads) do not co-localize with Iba1 immunoreactivity (brown precipitate, some cells indicated by black arrowheads). E: TGF-β RII mRNA is present in vWF-immunoreactive endothels as well as in some non-endothelial cells in the representative figure. The last 3 panels demonstrate that TGF-β RIII mRNA is expressed in endothelial cells at 72 h after MCAO. F: A double labeling of TGF-β RIII and alpha-SMA shows no co-localization. A single labeled TGF-β RIII mRNA expressing cell is indicated by black, while an alpha-SMA immunoreactive smooth cell by a white arrowhead. G: TGF-β RIII-expressing cells contain vWF immunoreactivity. Double labeled cells are indicated by the black arrow. H: TGF-β RIII mRNA is not present in expressed in microglia. The white arrowhead points to a TGF-β RIII-expressing cell while the black arrowhead shows an Iba1-immunoreactive microglia. Abbreviations: cc - corpus callosum, LV - lateral ventricle. Scale bars: 1 mm for A, 50 µm for B, C, D and E, and 20 µm for F, G and H.
Figure 9.
Schematic figure on TGF-β signaling at 72 h after MCAO.
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.