Time-Course Alterations of Monoamine Levels and Cerebral Blood Flow in Brain Regions after Subarachnoid Hemorrhage in Rats

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Abstract

To investigate the possible correlation between changes in monoaminergic neuronal activity and cerebral blood flow (CBF) in the same brain regions after subarachnoid hemorrhage (SAH), monoamine levels were analyzed by both HPLC-ECD and fluorohistochemistry techniques, and CBF was measured by using colored microspheres. At the second day of SAH, significant and nonsignificant reductions in blood flow were seen in the examined brain regions with a marked increase in CBF appearing in the telencephalon and hypothalamus on the third day. Significant reductions of monoamine levels in most brain regions were also observed on the second day after SAH, whereas norepinephrine (NE) levels in midbrain increased to 1.5 times compared to the normal level. These reductions were sustained until the fourth day of SAH, although at the third day, serotonin (5-HT) and dopamine levels in the hippocampus and 5-HT levels in the cerebellum were significantly elevated. In fluorohistochemical studies, the fluoro-intensities of monoamines, particularly catecholamines, in the midbrain dorsal NE bundle were enhanced at the second day after SAH. These NE neurons originated from the A6 cell group close to the area where homologous blood was applied through the cisterna magna. The results obtained after SAH show an apparent correlation between changes in monoamine levels and CBF in norepinephrine (NE)-rich areas. These results suggest that SAH-induced neuronal dysfunctions, particularly with NE neurons, are caused not only by reductions of blood flow but also by hemorrhage.

Introduction

Clinical studies on subarachnoid hemorrhage (SAH) have reported marked fluctuations in monoamine levels and blood circulation [2, 3, 13, 17, 21, 22]. For instance, in studies on the fluctuation of adrenergic neuronal activity, significant increases in norepinephrine (NE), epinephrine and 3-methoxy-4-hydroxyphenylglycol (MHPG) levels have been detected in the serum and cerebrospinal fluid (CSF) of patients in whom vasospasm and other severe conditions have been caused by SAH [2, 4, 16, 19, 22, 36]. Such extraordinary increases were more pronounced in unconscious patients than under ordinary conditions occurring after SAH and might be caused by central adrenergic neuronal system dysfunction induced by damage to hypothalamic functions [2, 6, 8, 29, 30, 35]. Furthermore, in examining dopaminergic and serotonergic neuronal activity in SAH patients, alterations in metabolites of dopamine (DA) and serotonin (5-HT) in serum and CSF have been demonstrated in SAH patients. Thus, changes of monoamine levels in SAH patients have been clearly observed, although it is difficult to say whether these changes are caused by brain dysfunction and/or the extraordinary changes in cerebral blood flow (CBF) caused by SAH. In addition, reductions of CBF have been measured in most brain regions at the initial stage of SAH.

In related animal studies, the Svendgaard group [6, 7, 29, 30] prepared a reproducible experimental animal model for SAH. According to their report, homologous blood was injected into the cisterna magna of rats inducing SAH, and the functional mechanism of noradrenergic neurons was examined. They suggested that changes in CBF and glucose metabolism after SAH might be regulated via the ascending noradrenergic neuronal pathway, which originates from the A1 and A2 nuclei in the medulla oblongata [[7]]. Specifically, changes in blood flow and metabolism seen after SAH were dominated by hypothalamic functions. However, to explain all symptoms seen after SAH by means of hypothalamic functions alone might prove difficult, because time-dependent changes in CBF and monoaminergic neuronal activities in the same brain regions after SAH have never been examined.

In this study, our specific aim was to quantitatively analyze time-dependent changes in blood flow and monoaminergic neuronal activity after SAH. In order to carry out main goal, the fluctuations in monoamine levels and CBF in eight discrete homogenized brain regions of SAH rats were simultaneously examined at 10 min, 1, 2, 3, and 4 days after SAH using an HPLC-ECD method and a colored microsphere method. To examine the correlative changes in CBF and monoaminergic neuronal activities in the brain regions after SAH, we applied half of each brain homogenate to measure changes in CBF and half for changes in monoamine levels. Therefore, neither in vivo microdialysis nor electrophysiological measurements were considered suitable methods for our specific investigation, because both methods can detect changes in CBF and monoamine levels only in small or specific brain regions. Furthermore, we have focused on changes in hypothalamic noradrenergic neuronal activity after SAH by fluorohistochemical detection, because this neuronal activity has been suggested to play an important role in regulating the homeostasis of brain functions.

These results may provide important clues by which we may examine correlations between CBF and monoaminergic neuronal activities.

Section snippets

Preparation of SAH Model Rats

Normally fed male Wistar rats (350–400 g; CLEA Animal Lab., Tokyo, Japan) were anesthetized by nitrate and 4% halothane. Blood-gas and arterial pressure were measured through the femoral artery, and these levels (MABP: 100–120 mmHg, PO2: 100–110 mmHg, PCO2: 35–40 mmHg) were regulated by respirator using a servoventilator (SN-480-7, Shinano Co, Ltd, Tokyo, Japan). Anesthesia was maintained with 70% nitrous-oxide and 30% oxygen.

The SAH rat model was prepared in accordance with Svendgaard's method

Blood Flow and Monoamine Levels in the Normal Rat Brain (Sham-Operation) Regions

Blood flow in each brain region is tabulated as the mean value ± SD in Table 1. Values were obtained from the brain regions of normal (sham operation) rats by means of the microsphere method. In Table 2, the monoamine levels in each brain region are shown as the mean ± SD. The monoamine contents varied in each brain region, although no notable variations in blood flow appeared. In the following experiments, the values obtained from normal rats (sham operation) were calculated as 100% when

CBF after SAH

The aim of our present investigation was to evaluate the mechanism of brain dysfunction due to severe contracture of microblood vessels at the acute phase of SAH. Therefore, we focused on the time relationship between variations of this vasocontraction, but not vasospasm, and changes in brain monoamine levels after SAH, although reductions in CBF after SAH have been discussed in previously published work [3, 13, 17, 21, 26].

According to other reports, reduced CBF can be detected at both acute

Acknowledgements

We thank Ms. Seiko Yajima for secretarial work and Dr. George Lawlor for his editorial assistance. Partial financial supports from The Science Research Promotion Fund of Japan Private School Promotion Foundation and The Japan Health Sciences Foundation (Y.W. and T.S.) are gratefully acknowledged.

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