In Vitro Culture And Identification Of Vascular Smooth Muscle Cells From Rat Intra- And Extracranial Arteries

Background and Objective: Atherosclerosis is one of the major causes of the ischemic stroke, but the risk factors and the mechanisms seem to be different between intra- and extracranial atherosclerosis. Vascular smooth muscle cells (VSMCs) are the major cellular component of the tunica media. After vascular injury, VSMCs characteristically exhibit phenotypic modulation, change from the quiescent "contractile" phenotype to the active "synthetic" phenotype, that can migrate and proliferate from media to the intima, thereby contribute to the progression of many vascular diseases such as atherosclerosis and restenosis. Therefore, primary culture of VSMCs in vitro can provide an important way to study the biological behavior of VSMCs and the pathogenesis and prevention strategy of the related vascular diseases. At present, the culture of extracranial arterial VSMCs has been widely used. In contrast, the culture of intracranial arterial VSMCs has rarely been reported, most of which are obtained from the bovines, pigs, dogs, rabbits with the relatively large diameter cerebral arteries. Moreover, the use of traditional enzymatic dispersion methods is limited because of their complex operations. The aim of the present study was to culture VSMCs derived from rat intracranial and extracranial arteries by using a modified enzymatic dispersion method of, providing a good in vivo model system for studying the molecular mechanisms and treatment of cerebrovascular diseases including intracranial and extracranial atherosclerosis.Methods:1. Primary culture of VSMCs: (1) Rat basilar artery VSMCs:Two healthy male Sprague-Dawley (SD) rats (250~300g) were sacrificed by cervical dislocation and decontaminated with 75% ethanol thoroughly. The basilar arteries were surgically isolated from the rat brain under sterile conditions. After removal of the adventitia, they were cut into approximately 0.2 mm rings and then digested with 0.1% typeⅠcollagenase for 5 hours and with 0.125% trypsin for another 10 minutes at 37℃, respectively. After digestion, cells were cultured in DMEM/F12 supplemented with 20% fetal calf serum. (2) Rat carotid artery VSMCs:A SD rat was placed in dorsal recumbency and a 2-cm longitudinal midline incision was made in the neck. The left common carotid artery was exposed and then excised (about 1.5 cm long) under sterile conditions quickly. After removal of both the adventitia and the intima, it was cut into approximately 0.2 mm rings and then digested and cultured according to the methods as mentioned above. 2. Subculture of VSMCs:When cultures reached 80-90% confluence, cells were subcultured using 0.25% trypsin for dissociation. 3. Purification of cells:The cells were purified by using a combination of manual scraping and differential attachment techniques. 4. Identification of VSMCs:VSMCs were identified by the morphological feature and growth pattern. In addition, since smooth muscleα-actin is considered as a specific and well-known differentiation marker of VSMCs, immunocytochemical staining of smooth muscleα-actin was also performed. A mouse monoclonal antibody against smooth muscleα-actin (1:300) was applied as the primary antibody to identify VSMCs in the culture. A goat anti-mouse biotinylated immunoglobulin conjugated with avidin-biotinylated horseradish peroxidase was used as the secondary antibody, followed by streptavidin biotin peroxidase complex (SABC) staining according to the manufacturer's instructions. 5. Cell viability:At passage 5, the number and viability of cultured cells were determined using a hemocytometer and the trypan blue (0.4%) dye exclusion assay.Results:After 3 days of incubation, primary cultures of cells isolated from the basilar artery or from the common carotid artery began to attach to the wall of the incubation dishes. After 2 weeks, cells were exhibited a spindle-shaped morphology with a classic"hill-and-valley"growth pattern at confluence. These features remained unchanged through five passages. The purity of fifth passaged VSMCs from the basilar artery or from the common carotid artery was greater than 97% to 98% as confirmed by their expression of smooth muscleα-actin. The viability of fifth passaged VSMCs from the basilar artery or the common carotid artery, measured by trypan blue dye exclusion, was more than 95% to 96%.Conclusion:The method described here is a relatively simple, reliable and inexpensive for establishing an in vitro cell culture model, which is suitable for studying the mechanism and treatment of cerebrovascular diseases such as intra- and extracranial atherosclerosis, and restenosis.