Upregulation of rho A and rho kinase messenger RNAs in the basilar artery of a rat model of subarachnoid hemorrhage

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Object. Rho A, a small guanosine triphosphate—binding protein, and rho kinases have been suggested to play an important role in the agonist-induced myofilament Ca++ sensitization and cytoskeletal organization of smooth-muscle cells. To discover their possible roles in the prolonged contraction seen in cerebral vasospasm, the authors investigated the messenger (m)RNA expressions of rho A and rho-associated kinases α and β in the basilar artery (BA) of a rat double cisternal blood—injection model.

Methods. An experimental subarachnoid hemorrhage (SAH) was achieved in rats by twice injecting autologous arterial blood into the cisterna magna of each animal. The mRNAs for rho A and rho-associated kinases α and β of the rat BA were analyzed using reverse transcription—polymerase chain reaction (RT-PCR). The cisternal blood injection induced a marked corrugation of elastic lamina and contraction of smooth-muscle cells observed with the aid of light and transmission electron microscopy in the rat BA on Days 3, 5, and 7. Results of the RT-PCR revealed that mRNAs for rho A and rho kinases α and β were expressed in the rat BA and that they were significantly upregulated and reached their peaks on Day 5.

Conclusions. The mRNA upregulation of these proteins indicates that activation of rho A/rho kinase—related signal transduction pathways is involved in the development of long-lasting contraction of cerebral arteries after SAH.

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Address reprint requests to: John H. Zhang, M.D., Ph.D., Department of Neurosurgery, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505. jzhang@neurosurgery.umsmed.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    A–D: Transmission electron micrographs showing the extent of corrugation of elastic lamina of the spastic BA after a staged cisternal blood injection. The micrographs were focused on the corrugation of the elastic lamina of the spastic arteries on Day 3 (B), Day 5 (C), and Day 7 (D) post-SAH. The micrograph in (A) is a sample from a control rat (no blood injection). There is no corrugation in the BA of the control rat. There are slight corrugations in a sample from a rat sacrificed on Day 3 and severe corrugations in samples from rats killed on Day 5 or 7. Bars = 4 µm. E: Graph showing the percentage change in the lumen diameter of the BA. The lumen in the BA from a control animal was designated 100% in imaging analysis (dashed line). The lumen values of rats with SAH on Days 3, 5, and 7 were calculated as the percentage of the control. Each value represents the mean ± SEM (vertical bar) from three animals. * p < 0.05.

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    Blot showing detection of rho A, rho kinases α and β mRNAs by RT-PCR in the rat BA. The predicted size of each PCR product is shown on the right side of the photograph. The DNA size marker (Lane 1) is ΦX174RF DNA/Hae III fragments, showing the bands at 872, 603, 310, 271/281, 234, and 194 bp. Lanes 2 through 5 represent GAPDH, 452 bp; rho A, 410 bp; rho kinase α, 221 bp; and rho kinase β, 303 bp, respectively.

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    Blots showing the relative extent of expression of rho A and rho kinases α and β mRNAs in the time course of experimental SAH. The PCR amplification was repeated for 28 cycles for rho A, 32 cycles for rho kinases α and β, and 24 cycles for GAPDH. Each lane shows the RT-PCR product of mRNA from three rats. The target indicates the expression of rho A and rho kinases in the BAs. The GAPDH is from the same samples as shown in the target and was amplified together with the target; GAPDH serves as an internal control.

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    Bar graphs showing the temporal changes in mRNA expression for rho A and rho kinases α and β in the BA of the rat SAH model. Each band volume in the agarose gel was scanned, and the extent of each mRNA expression was calculated based on the ratios of the rho A (A) and rho kinases α (B) and β (C) band volumes to the GAPDH band volumes. Data from different PCRs were normalized by the result from untreated BA (control), described as fold increases from control, and expressed as the mean ± SEM, based on the three different PCRs taken from nine to 10 animals in each group. The symbols on the left of the bars show the significance (* p < 0.05, ** p < 0.01 according to ANOVA, followed by Dunn's procedure as a multiple comparison) compared with control. n.s. = not significantly different from control.

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