The Altered Expression Of Gap Junctional Proteins In Cerebral Vasospasm

The Altered Expression of Gap Junctional Proteins in Cerebral VasospasmObjective: Gap junctions formed from intercellular channels are the essential structures for contacted cells communications. Ions and small molecular substance (<1kDa) can be diffused between adjacent cells via intercellular channels, which are composed of connexins. It has been identified to date that three major connexins, namely Cx43, Cx40 and Cx37, express in walls of mammalian vasculature. However, the expression and distribution of connexins in the cells of cerebrovascular wall are still uncertain. Cerebral vasospasm after subarachnoid hemorrhage (SAH), the mechanism of which is still unknown, is a severe disease clinically. Because gap junctions play an important role in coordinating the vasomotion, we hypothesize that gap junctions may attend the process of cerebral vasospasm. The main objective of this study was to investigate the connexin type and its localization in the walls of basilar arteries of rabbits. The model of SAH was established on rabbits and then the alteration of mRNA for connexins from the tissue of basilar arteries was determined after cerebral vasospasm. It is expected to provide basis for further investigation on the role of gap junctions in cerebral vasospasm.Methods: 1) Detection of mRNA for connexins in the tissue of rabbits' basilar arteries New Zealand rabbits (n=8, as normal group), half female and half male, weigh 2.5~3.0kg. The brains from these rabbits were removed under anesthesia (Nembutal 65mg/kg weight). The basilar artery and its larger branches were dissected out from the brain stem. Arachnoid membraneadhered on those vessels was dissected away in Kerbs' solution. Vascular samples were kept in fluid nitrogen. The RT-PCR was performed after the total RNA isolated from the vascular tissues. Primers for Cx40, Cx45, Cx37, Cx43, and β-actin were used in the PCR. RT-PCR products electrophoresed on the agarose gels containing ethidium bromide (EB) were observed and photographed. 2) Localization of Cx45 and Cx43 proteins in the walls of rabbits' basilar arteries Same specie rabbits (n=8, as immunohistochemical group) were anatomized after anesthesia. Basilar arteries with part brain stem tissue were cut down and then kept in the fluid nitrogen. The immunohistochemical technique was applied to display the localizations of Cx45 and Cx43 proteins in the walls of those vessels. The results of immunohistology were observed with light microscopy. 3) The establishment of subarachnoid two hemorrhage model on rabbit The same type rabbits (n=6, as SAH model group) were penetrated at the cisternal magna under anesthesia. Arterial blood, 0.5ml/kg, was slowly injected into the space of cistern at 0h and 24h later respectively. The arteriography of all cerebral vessels was carried out through catheterization of the femoral artery prior to blood injection and 2 days after two hemorrhage respectively. The diameters prior to blood injection and 2 days after two hemorrhage were compared. 4) The semiquantitation of mRNA for connexins in those spasmodic vessels Eight rabbits were dealt with in the same ways as described above but with no arteriography carried out. Two days after twice blood injections, the animals were killed and then the total RNA in the tissue of basilar arteries was extracted. Semiquantitative RT-PCR was performed to measure the relative contain of mRNA for connexin40 in spasmodic vascular tissue. The average relative contain of mRNA for connexin40 in normal group was regarded as normal control. β-actin primers were used as internalcontrol for semiquantitative RT-PCR. The products of PCR were electrophoresed in agarose gel stained with EB and then photographed. Semiquantitation of connexin was done by densitometer scanning of the ethidium bromide-stained agarose gels. The density ratio of connexin40 RT-PCR band to theβ-actin RT-PCR band was used as a measure of connexin40 mRNA level in the tissue. The results of RT-PCR were taken down as means ±SE and analyzed wit...