Enhanced expression of transforming growth factor beta 1 in the rat brain after a localized cerebral injury

Abstract

It is becoming clear that transforming growth factor beta (TGF beta) may be a key factor regulating inflammatory and tissue specific wound responses. Because the formation of a glial-collagen scar at CNS lesion sites is thought to contribute to the pathology associated with penetrating CNS injuries, and because in the periphery TGF beta 1 stimulates fibroblast deposition of scar tissue, we used in situ hybridization and immunohistochemistry to investigate the effect of a defined cerebral lesion on the local expression of TGF beta 1. Induction of TGF beta 1 mRNA and protein is relatively diffuse in the neuropile around the margins of the lesion at 1, 2 and 3 days, but becomes localized to the region of the glial scar at 7 and 14 days. The signal intensity for TGF beta 1 mRNA and protein is maximal between 2 and 3 days and decreases between 7 and 14 days after lesion. The predominant cell types in the neuropile localizing TGF beta 1 mRNA and protein have the morphological characteristics of astrocytes, although macrophages are also detected. An induction of TGF beta 1 mRNA was also observed in endothelial cells of the meninges, hippocampal fissure and choroid plexus, at 2 and 3 days. However, this is dramatically reduced by 7 days and has disappeared by 14 days. These results suggest a role for TGF beta 1, not only in inflammation, but also in the tissue-specific glial scar formation that occurs in the CNS. Furthermore, they suggest a potential therapeutic use of TGF beta 1 antagonists in the CNS to help limit the pathogenesis associated with matrix deposition in the wound.

Fig. 1

Bright field micrograph of the brain 1 day and 7 days after injury. The caudal (panels A and B) and rostral (panels C, D) portions of the lesion are shown. The curved open arrows show the region of the lesion in the cortex at 1 day (panels A, C) and 7 days (panels B and D) after the lesion. Bar = 250 μm. Note that the glial scar is well established by 7 days. The corpus callosum (large closed arrow), hippocampal fissure (closed curved arrow), the choroid plexus (straight open arrow with tail), dentate gyrus (DG) and CA1 region of the hippocampus are shown for orientation.

Fig. 2

Dark field micrographs showing TGFβ1 mRNA in the caudal region of the wound. TGFβ1 mRNA (T7) is observed at 1 day (panel A₁), 2 days (panel B₁), 3 days (panel C₁), 7 days (panel D₁) and 14 days (panel E₁) following the lesion. When sections are hybridized with the sense strand (T3) the signal is minimal (panels A₂–E₂). The curved open arrows show the margin of the lesion. TGFβ1 mRNA, but not the control signal, is also observed in the corpus callosum (panels D₁ and D₂) as shown by the closed straight arrows and in the meninges (panels A₁–D₁ and A₂–E₂) as shown by straight open arrows. The signal is most intense at 2 and 3 days after lesion. At 7 days and 14 days the signal is less diffuse and only present within the boundary of the well defined glial scar. Bar = 250 μm.

Fig. 3

Dark field micrographs showing TGFβ1 mRNA in the rostral region of the wound. TGFβ1 mRNA (T7) is observed at 1 day (panel A₁), 2 days (panel B₂). 3 days (panel C₁). 7 days (panel D₁)and 14 days (panel E₁) following the lesion. When sections are hybridized with the sense strand (T3) the signal is minimal (panels A₂–E₂). The curved open arrows show the margin of the lesion. TGFβ1 mRNA, but not the control signal, is also observed in the corpus callosum (panels B₁, D₁, E₁ and B₂, D₂, E₂) as shown by the closed straight arrows and in the choroid plexus (panels A₁–D₁ and A₂–E₂) as shown by straight open arrows with tails. The signal appears considerably diminished by 14 days. Bar = 250 μm.

Fig. 4

High magnification bright field micrographs of cells in the margin of the wound. TGFβ1 mRNA (T7) is observed associated with selective cells in the margin of the lesion at 1 day, (panel A) 2 days (panel B), 3 days (panel C), 7 days (panel D) and 14 days (panel E) after the lesion but not in cells hybridized with the sense strand (T3, row 3). Initial signs of the glial scar are seen at 3 days and are well defined by 7 days, at which time the mRNA appears less diffuse. High magnification reveals that these cells are typically glial cells (row 2). Bar = 10 μm.

Fig. 5

The presence of TGFβ1 mRNA in the corpus callosum 7 days following a defined lesion. There is an intense signal for TGFβ1 mRNA (Panels A and C) in the corpus callosum at the point of lesion penetration. Control sections show no signal (T3, panels B and D). Bar = 10 μm.

Fig. 6

TGFβ1 mRNA in the meninges, hippocampal fissure and choroid plexus after injury. Intense TGFβ1 mRNA (T7) is observed in the meninges (panel A) and hippocampal fissure (panel B) but not in the control sections hybridized with the sense strand (T3). In the choroid plexus, TGFβ1 mRNA is almost non-detectable in uninjured animals (0 day, C1) but very intense following injury (panels D₁, E₁). Control sections show no signal (T3). Bright field of low-power micrographs are shown in panels C₂ and D₂. Bar = 25 μm.

Fig. 7

Immunolocalization of TGFβ1 after injury. Immunoreactive TGFβ1 is seen diffusely in the neuropile along the borders of the lesion after 1 day and this increases at 3 days. Staining is residual by 14 days and mostly confined to the macrophages remaining in the center of the wound. Bar = 10 μm.