Cerebrovascular connexin expression: effects of traumatic brain injury

Abstract

Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24 h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.

FIG. 1.

Comparison of connexin (Cx) mRNA expression in endothelial cells versus smooth muscle cells from cerebral arteries. mRNA for each of the four Cxs in the cerebral arteries of rats is shown. Cells were isolated from uninjured male Sprague-Dawley rats (n=6) using laser capture microdissection to select specific cells from each layer (endothelial layer: 2–10 cells per rat; smooth muscle layer: 10 cells per rat). Data are presented as mean±standard error (*p<0.05 versus corresponding endothelial cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DLU, digital light units).

FIG. 2.

Connexin 43 (Cx43) mRNA expression in smooth muscle cells. Cells were harvested from the cerebral arteries of male Sprague-Dawley rats at 2, 6, and 24 h after fluid-percussion traumatic brain injury (TBI; n=6/time point) or sham injury (sham; n=6/time point) using laser capture microdissection to select specific cells from the smooth muscle layer. Data are presented as mean±standard error (*p<0.05 versus corresponding sham animals; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DLU, digital light units).

FIG. 3.

Connexin 45 (Cx45) mRNA expression in endothelial cells of cerebral arteries. Cells were isolated at 2, 6, and 24 h after fluid-percussion traumatic brain injury (TBI; n=6/time point) or sham injury (sham; n=6/time point) using laser capture microdissection to select specific cells from the endothelial layer. Data are presented as mean±standard error (*p<0.05 versus corresponding sham animals; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DLU, digital light units).

FIG. 4.

Connexin (Cx) protein expression. Cerebral arteries of rats 24 h following fluid-percussion traumatic brain injury (TBI; T1–T6; n=6) or sham injury (sham; S1–S6; n=6) were evaluated using Western blot (A, upper panel), and densitometrically quantified to compare changes between injury groups (B, lower panel). Data are presented as median (solid line), first and third quartiles (boundaries of box), and range (error bars). The mean is also shown by the dashed lines (*p<0.05 versus corresponding sham animals; GAPDH, glyceraldehyde-3-phosphate dehydrogenase).