Different mechanisms of L-arginine induced dilation of brain arterioles in normotensive and hypertensive rats.

We evaluated the response of pial arterioles to L-arginine in anesthetized normotensive rats and spontaneously hypertensive rats equipped with a closed cranial window. Topical application of 10(-6)-10(-4) mol/l L-arginine, which is known to be the endogenous substrate for the synthesis of nitric oxide, induced dose-dependent arteriolar vasodilation. The response was more pronounced in hypertensive than in normotensive rats (at the concentration of 10(-4) mol/l L-arginine: 18.3 +/- 3.3% vs. 6.7 +/- 1.7%, respectively, means +/- S.E.). The stereoisomer D-arginine had no effect in hypertensive rats. Topical application of the nitric oxide synthase inhibitor N-nitro-L-arginine converted L-arginine-induced dilation to constriction in normotensive and hypertensive rats. The cyclooxygenase inhibitor indomethacin (5 micrograms/ml cerebrospinal fluid) also blocked the dilation in both strains. Photochemical endothelial injury blocked L-arginine-induced dilation in normotensive rats, but only partly antagonized the response in hypertensive animals. Intravenous or topical pretreatment with the free radical scavenger superoxide dismutase significantly reduced the dilating response to 10(-4) mol/l L-arginine in hypertensive rats. Superoxide dismutase did not significantly change the response to L-arginine in normotensive animals. It is concluded that nitric oxid formation in the endothelium and liberation of cyclooxygenase products cause L-arginine-induced dilation in normotensive rats. While nitric oxide and cyclooxygenase products are also involved in L-arginine-induced dilation in spontaneously hypertensive rats, superoxide radicals contribute to the enhanced response in this strain. This mechanism appears to be specific for the hypertensive animals and is only partly dependent on an intact endothelium.

Is nitric oxide involved as a mediator of cerebrovascular changes in the early phase of experimental pneumococcal meningitis?

Nitric oxide (NO) is a mediator of haemodynamic changes and cytotoxicity in in vivo models of inflammation such as endotoxaemic shock. The purpose of this study was to investigate whether NO may be involved in the increase of cerebral blood flow (CBF), intracranial pressure (ICP) and brain water content, known to occur in the early phase of pneumococcal meningitis. Rats injected intracisternally with live pneumococci were either untreated or received 5 mg kg-1 i.v. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Pretreatment with L-NAME prevented the increase in CBF, ICP and brain water content, as seen in untreated animals. CBF tended to return towards baseline when rats were treated with L-NAME 2 h after pneumococcal injection. Whereas none of the untreated and L-NAME-pretreated animals died during the 6 h observation period, 3 out of 9 rats treated with L-NAME and 7 out of 9 rats with simultaneous i.v. injection of L-NAME and L-arginine died. Our results provide preliminary evidence that NO may be involved as a mediator of CBF changes and oedema formation in the early phase of pneumococcal meningitis in the rat. NO inhibition, however, may have detrimental effects of still unidentified cause, as indicated by the increased mortality in treated animals. Further studies with analysis of the causes of mortality, structurally different NO synthase inhibitors and direct evaluation of NO synthase induction are needed to further support this hypothesis.

Long term monitoring of immunoreactive endothelin-1 and endothelin-3 in ventricular cerebrospinal fluid, plasma, and 24-h urine of patients with subarachnoid hemorrhage.

Endothelins (ETs), peptides that were originally isolated from endothelial cells, have extremely potent and long-lasting vasoconstricting effects on cerebral vessels in vitro and in vivo. Observations that astrocytes produce these peptides and that their ET production can be stimulated, e.g. by thrombin, and potentiated via a self-enhancing autoregulatory mechanism may have shed new light upon the pathogenesis of cerebrovasospasm (CVS). ETs are present at low levels in normal human cerebrospinal fluid (CSF). Few and contradictory reports exist on ET levels in subarachnoid hemorrhage (SAH)-associated CVS. We monitored ventricular CSF, plasma, and 24-h urine levels of immunoreactive endothelin-1 (ET-1) and endothelin-3 (ET-3) in seven patients with SAH, who did (five) or did not (two) develop CVS in the course of their disease, as well as in two patients with different conditions (acoustic neuroma/postoperative meningitis; hydro-/hematocephalus) over 7-19 days. A distinct peak of both ET-1 and ET-3 in CSF of patients with SAH coincided with clinically documented signs of CVS and was absent in CSF of patients with SAH but no CVS. CSF levels of ET-1 and ET-3 displayed a striking parallelism in all subjects. Plasma ET-1 levels were essentially in the normal range. ET-3 was not detectable in plasma under our assay conditions. The excretion profiles of ET-1 and ET-3 in 24-h urine revealed again a predominantly parallel behavior of the two peptides. Interestingly, patients with high ET levels in CSF showed simultaneous peaks in urinary ET excretion, expressed as nanograms per gram of creatinine. Our findings support an association of ETs with the pathogenic events following SAH. The well-documented effects of these peptides on cerebral vessels suggest they are mediators rather than markers of disease.

Angiotensin II induces endothelium-dependent vasodilation of rat cerebral arterioles.

We evaluated the response of cerebral arterioles to angiotensin II (ANG II) in anesthetized rats equipped with a closed cranial window. Topical application of 10(-10)-10(-5) M ANG II induced dose-dependent arteriolar vasodilation. Maximum vasodilation of 24 +/- 2.2% (+/- SE) was attained at a concentration of 10(-6) M ANG II. The dilation in response to ANG II was blocked by 3 micrograms/ml indomethacin, a cyclooxygenase inhibitor, and was reversed to minimal vasoconstriction by 10(-5) M methylene blue, a substance that has been reported to eliminate endothelium-dependent vasodilation. Coapplication of indomethacin with methylene blue reduced the arteriolar response to ANG II to a similar extent as the application of indomethacin alone. Indomethacin or methylene blue did not inhibit the vasodilation induced by 10(-5) M adenosine, which is not endothelium and cyclooxygenase dependent. Mercury light illumination of the pial vessels after intravenous injection of fluorescein dye, a technique that has been used by others to functionally damage endothelial cells, reversed ANG II (10(-6) M)-induced vasodilation into a -14.2 +/- 2.3% constriction while not affecting the response to adenosine. Our data suggest that ANG II produces vasodilator responses of rat cerebral arterioles by the release of a factor that is derived from the endothelium and may be generated through a cyclooxygenase-dependent mechanism.

Confocal laser microscopy to study microcirculation on the rat brain surface in vivo.

We demonstrate the feasibility of using confocal laser microscopy (CLM) to study the microcirculation on the rat brain surface. Using a closed cranial window model in vivo, microvessels in the size range of capillaries (8 microns) and dynamic events were observed through an intact dura. This study is the first step in the development of a system which simultaneously monitors vascular and metabolic processes using CLM.