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  • Author or Editor: Andrew D. Parent x
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Eddie Perkins, Hitoshi Kimura, Andrew D. Parent and John H. Zhang

Object. Whether cerebral vasospasm occurs only in surface vessels or also in parenchymal arterioles is debatable. The present study was undertaken to evaluate comprehensively the microvasculature of the brainstem after experimental subarachnoid hemorrhage (SAH).

Methods. Nine mongrel dogs of either sex, each weighing between 18 and 24 kg, underwent double blood injections spaced 48 hours apart; the injections were infused into the cisterna magna immediately after angiography of the basilar arteries (BAs). Three additional dogs assigned to a control group received no blood injections. The dogs were killed on Day 7. Axial sections obtained from the midpontine region of both control dogs and animals subjected to SAH were evaluated with respect to the morphological characteristics of vessels and neurons, and for ultrastructural changes.

Severe vasospasm occurred in the BAs of all dogs subjected to SAH. Nevertheless, in these animals, the luminal areas and vessel perimeter in parenchymal arterioles, but not in parenchymal venules, were observed to have increased when compared with those of control dogs (p < 0.01, t-test). No corrugation of the internal elastic lamina was observed and smooth-muscle and endothelial cells remained normal at the ultrastructural level in the dogs with SAH.

Conclusions. In this model, vasospasm of the BAs did not extend into the region of the pons to affect the intraparenchymal arterioles. Dilation of the parenchymal arterioles might serve as compensation for reduced blood flow. Thus, no neuronal ischemia or infarction resulted in the pontine region of the brain.

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Yasushi Miyagi, Robin C. Carpenter, Toshinari Meguro, Andrew D. Parent and John H. Zhang

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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Toshinari Meguro, Christoph P. R. Klett, Betty Chen, Andrew D. Parent and John H. Zhang

Object. Oxyhemoglobin (OxyHb) released from hemolysed erythrocytes has been considered to be responsible for cerebral vasospasm after subarachnoid hemorrhage. The authors previously reported that OxyHb produced apoptosis in cultured vascular endothelial cells. The change in intracellular Ca++ homeostasis was expected to be one of the possible mechanisms of the cytotoxic effects of OxyHb. This study was undertaken to investigate the protective effects of Ca++-channel blockers on OxyHb-induced apoptosis.

Methods. Cultured bovine coronary artery and brain microvascular endothelial cells (passages 5–9) were used. A cell density study, immunohistochemical staining, and DNA fragmentation analysis were performed to confirm apoptosis. Various concentrations (1–50 µM) of OxyHb were used for 24- to 72-hour incubations with and without Ca++-channel blockers.

Oxyhemoglobin produced cytotoxicity leading to cell detachment from the culture dish in time- and concentration-dependent manners. The highest dose (50 µM) of OxyHb produced cell detachment after a 24-hour incubation, and the lower doses (1–10 µM) produced cell detachment after 48 to 72 hours. Immunohistochemical analysis showed that apoptosis occurred in cells that were still attached to the side of the culture dish after 48 to 72 hours of OxyHb treatment (5 µM). The OxyHb (10 µM) produced DNA ladders at 48 to 72 hours. Three Ca++-channel blockers were used to prevent the toxic effect of OxyHb. The voltage-dependent Ca++-channel blocker nicardipine (1 µM), the voltage-independent Ca++-channel blocker econazole (10 µM), and the inorganic Ca++-channel blocker lanthanum (100 µM) all failed to prevent cell detachment or DNA ladders produced by OxyHb. These results were similar in both cell lines.

Conclusions. Oxyhemoglobin produced apoptotic changes in cultured vascular endothelial cells, and Ca++-channel blockers did not prevent OxyHb-induced apoptosis.

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Kotaro Ogihara, Alexander Y. Zubkov, David H. Bernanke, Adam I. Lewis, Andrew D. Parent and John H. Zhang

Object. Oxyhemoglobin (OxyHb) is one of the most important spasmogens for cerebral vasospasm that follows aneurysmal subarachnoid hemorrhage. The cytotoxic effect of OxyHb has been documented in endothelial and smooth-muscle cells; however, the pattern of cell death—necrosis or apoptosis—as the final stage of cell damage has not been demonstrated. This study was undertaken to determine if OxyHb induces apoptotic changes in cultured bovine aortic endothelial cells.

Methods. Confluent bovine aortic endothelial cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to culture dishes after exposure to OxyHb. To identify apoptotic changes, the investigators used three specific methods: DNA fragmentation (electrophoreses), the apoptotic body (transmission electron microscopy), and cleavage of poly (adenosine diphosphate ribose) polymerase (PARP [Western blotting]).

Conclusions. Oxyhemoglobin decreased cell density in a concentration- and time-dependent manner. Analysis of DNA showed a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder). Transmission electron microscopy demonstrated condensation of nuclei and apoptotic bodies in OxyHb-treated endothelial cells. Western blotting with the PARP antibody revealed that the 116-kD PARP was cleaved to the 85-kD apoptosis-related fragment. These results for the first time demonstrated that the OxyHb induces apoptosis in cultured endothelial cells.