Endothelium-dependent responses in the microcirculation observed in vivo.

Endothelium-dependent responses were first demonstrated 40 years ago in the aorta. Since then, extensive research has been conducted in vitro using conductance vessels and materials derived from them. However, the microcirculation controls blood flow to vital organs and has been the focus of in vivo studies of endothelium-dependent dilation beginning immediately after the first in vitro report. Initial in vivo studies employed a light/dye technique for selectively damaging the endothelium to unequivocally prove, in vivo, the existence of endothelium-dependent dilation and in the microvasculature. Endothelium-dependent constriction was similarly proven. Endothelium-dependent agonists include acetylcholine (ACh), bradykinin, arachidonic acid, calcium ionophore A-23187, calcitonin gene-related peptide (CGRP), serotonin, histamine and endothelin-1. Normal and disease states have been studied. Endothelial nitric oxide synthase, cyclooxygenase and cytochrome P450 have been shown to generate the mediators of the responses. Some of the key enzyme systems generate reactive oxygen species (ROS) like superoxide which may prevent EDR. However, one ROS, namely H2 O2 , is one of a number of hyperpolarizing factors that cause dilation initiated by endothelium. Depending upon microvascular bed, a single agonist may use different pathways to elicit an endothelium-dependent response. Interpretation of studies using inhibitors of eNOS is complicated by the fact that these inhibitors may also inhibit ATP-sensitive potassium channels. Other in vivo observations of brain arterioles failed to establish nitric oxide as the mediator of responses elicited by CGRP or by ACh and suggest that a nitrosothiol may be a better fit for the latter.

Dimethylsulfoxide and ethanol, commonly used diluents, prevent dilation of pial arterioles by openers of K(ATP) ion channels.

Ethanol and dimethylsulfoxide are commonly used as diluents for water-insoluble drugs. Both are antioxidants. An earlier study of cats presented pharmacological evidence indicating that oxidants could open the K(ATP) ion channel in cerebral surface arterioles [pial arterioles] and that antioxidants including dimethylsulfoxide and L-cysteine prevented opening of these channels. Ethanol was not tested. The present study extends the older observations to a second species, the rat, and examines ethanol as well as dimethylsulfoxide and L-cysteine. A microscope and image splitter were used to measure arteriolar diameters under a closed cranial window in pentobarbital-anesthetized, paralyzed rats. Drugs were topically applied. Dose-dependent dilations produced by two well-established openers of the K(ATP) ion channel were inhibited in dose-dependent manner by ethanol at doses from 0.01% to 0.075%. Above this dose, the effect disappeared. Dilation by sodium nitroprusside was not affected. Dimethylsulfoxide and L-cysteine inhibited dilation produced by pinacidil. Dimethylsulfoxide inhibited pinacidil in a dose-dependent manner at doses from 0.01% to 0.2%. L-Cysteine inhibited pinacidil. Since all the inhibitory drugs have antioxidant properties, their effect may be a reflection of that property as suggested in an earlier paper. Ethanol and dimethylsulfoxide inhibited in doses frequently present when these agents are used as solvents. When investigators use these solvents to dissolve water-insoluble, topically applied drugs, we suggest that they first test the possibility that their observations are being made under conditions in which opening of the K(ATP) ion channel is inhibited.

Evidence for a K(ATP) ion channel link in the inhibition of hypercapnic dilation of pial arterioles by 7-nitroindazole and tetrodotoxin.

7-Nitroindazole, an inhibitor of neuronal nitric oxide synthase, reportedly inhibits hypercapnic dilation, but tetrodotoxin, an inhibitor of neuronal transmission, reportedly does not. Thus, evidence does not uniformly support the hypothesis of a neurogenic link to the hypercapnic response. Others suggest the hypercapnic response is mediated by a K(ATP) ion channel. In the following studies, we observed that topically administered tetrodotoxin inhibited dilations produced by hypercapnia. In addition, topical tetrodotoxin and either topical or intraperitoneal 7-nitroindazole, inhibited dilations produced by the K(ATP) channel openers, cromakalim and pinacidil. Inhibition of hypercapnic dilation and inhibition of dilation by the openers of the K(ATP) channel was immediately reversed by either L-lysine or L-arginine, amino acids previously shown to facilitate opening of the channel. The data strongly supports the previous conclusion that there is a K(ATP) ion channel link in the response of pial arterioles to hypercapnia. The location of the channel is not established by these data, nor is it known whether the action of tetrodotoxin on the channel was direct or indirect.

Leukocyte accumulation and hemodynamic changes in the cerebral microcirculation during early reperfusion after stroke.

BACKGROUND AND PURPOSE: Leukocytes contribute to cerebral ischemia-reperfusion injury. However, few experimental models examine both in vivo behavior of leukocytes and microvascular rheology after stroke. The purpose of the present study was to characterize patterns of leukocyte accumulation in the cerebral microcirculation and to examine the relationship between leukocyte accumulation and microcirculatory hemodynamics after middle cerebral artery occlusion and reperfusion (MCAO-R). METHODS: Male rats (250 to 350 g) were anesthetized and ventilated. Tail catheters were inserted for measurement of arterial blood gases and administration of drugs. Body temperature was maintained at 37 degrees C. Animals were subjected to 2 hours of MCAO by the filament method. A cranial-window preparation was performed, and the brain was superfused with warm, aerated artificial cerebrospinal fluid. Reperfusion was initiated by withdrawing the filament, and the pial microcirculation was observed by use of intravital fluorescence microscopy. Leukocyte accumulation in venules, arterioles, and capillaries; leukocyte rolling in venules; and leukocyte venular shear rate were assessed during 1 hour of reperfusion. RESULTS: We found significant leukocyte adhesion in cerebral venules during 1 hour of reperfusion after 2 hours of MCAO. Leukocyte trapping in capillaries and adhesion to arterioles after MCAO-R tended to increase compared with controls, but the increase was not significant. We also found that shear rate was significantly reduced in venules during early reperfusion after MCAO. CONCLUSIONS: A model using the filament method of stroke and fluorescence microscopy was used to examine white-cell behavior and hemodynamics in the cerebral microcirculation after MCAO-R. We observed a significant increase in leukocyte rolling and adhesion in venules and a significant decrease in blood shear rate in the microcirculation of the brain during early reperfusion. Leukocytes may activate and damage the blood vessels and surrounding brain cells, which contributes to an exaggerated inflammatory component to reperfusion. The model described can be used to examine precisely blood cell-endothelium interactions and hemodynamic changes in the microcirculation during postischemic reperfusion. Information from these and similar experiments may contribute to our understanding of the early inflammatory response in the brain during reperfusion after stroke.

Cyclin D1 and Cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits.

BACKGROUND AND PURPOSE: The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We hypothesized that spinal motor neurons might be lost by programmed cell death and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for cyclin D1 and cyclin-dependent kinases (Cdk) 4. METHODS: We used a rabbit spinal cord ischemia model with a balloon catheter. Spinal cord was removed at 8 hours and 1, 2, and 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. In situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), DNA fragment with gel electrophoresis, Western blot analysis for cyclin D1 and Cdk4, and temporal profiles of cyclin D1 and Cdk4 immunoreactivity were investigated. RESULTS: Most motor neurons were preserved until 2 days but were selectively lost at 7 days of reperfusion. Immunocytochemistry showed positive TUNEL selectively at 2 days of reperfusion in spinal motor neuron nuclei. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunoreactivity of cyclin D1 and Cdk4 proteins was induced selectively at 8 hours in motor neuron nuclei, which eventually died. CONCLUSIONS: These results indicate that induction of cyclin D1 and Cdk4 may be implicated in programmed cell death change after transient spinal cord ischemia in rabbits.

The presence, origin, and significance of A beta peptide in the cell bodies of neurons.

Interest in the A beta amyloid in Alzheimer disease (AD) has largely focused on the A beta in the neuropil, an extracellular site. Here much attention has been given to the possibility that A beta acts as a neurotoxin. However, increasing emphasis is now being given to the relationship between neurofibrillary tangles (NFT) and the degree of cognitive decline, as opposed to the relationship between decline and senile plaques, the sites of extracellular A beta deposition. This review focuses attention on the existence and significance of A beta in the cell body of the neuron. The review brings together diverse strands of literature indicating: (1) the tau-positive, paired helical filaments that are the main component of NFT are not themselves the source of the amyloid-like staining (Congo red birefringence) of PHF, and are not, in fact, an "amyloid"; (2) there is A beta within the cytoplasm of neurons affected by AD and in other conditions characterized by tau-positive neurofibrillary tangles; (3) peptides derived from portions of the A beta precursor can bind to PHF; (4) the affected neurons are the source of extracellular A beta in their vicinity and are also unable to maintain the synaptic structures that depend upon the integrity of the neuronal cell body; and (5) debates about whether the intracellular A beta is an amyloid depend upon beliefs about its tertiary structure and assumptions concerning the relationship between the size of self-aggregating A beta molecules, their tertiary structure, and their ability to generate Congo red birefringence without necessarily being detected as ultrastructural filaments 5-10 nm wide. Based upon this literature, it is suggested that the Congo red birefringence generated by NFT is caused by A beta, intimately bound to the NFT. Moreover, whether defined as an amyloid or not, the A beta in the neuronal cell body indicates an abnormal processing of the precursor molecule on the way to its ultimate transmembrane domain. Deranged neuronal functioning, which leads to this abnormal processing and/or the intracellular A beta itself, may be the cause of subsequent functional and morphologic abnormalities in the brain.

Is the EDRF in the cerebral circulation NO? Its release by shear and the dangers in interpreting the effects of NOS inhibitors.

Evidence from investigations of brain microcirculation (pial arterioles) reveals at least 3 different endothelium (EC) dependent mechanisms for dilation. Only one of the three can be triggered by acetylcholine (ACh) and in this vascular bed it is only this path that is dependent upon endothelial nitric oxide synthase (NOS) which produces nitric oxide (NO) from arginine. In this vascular bed the ACh sensitive path cannot be triggered by bradykinin (BK). This state of affairs appears to differ from that found in other beds or in endothelium cultured from conductance vessels. In the cerebral microcirculation there is considerable pharmacological evidence that the endothelium derived relaxing factor (EDRF) for ACh is not NO itself but may contain NO. In many experimental vascular settings the release of the NOS dependent EDRF is shear dependent. In the cerebral microcirculation there are several studies suggesting, in vivo, that this is correct. Among these are the following: (1) vessels narrow when shear is reduced after carotid ligation, and remain so along with unresponsiveness to ACh for at least ten minutes following resumption of flow. This may be important in developing stroke. The collapse is not passive due to low pressure. We know this because the narrowed vessels with their low intraluminal shear and pressure are still capable of large dilation by the NO donor, sodium nitropruside; (2) the antiplatelet effects of EC which are mediated, in part, by the EDRF for ACh are enhanced for 10 to 20 minutes following the transient increase and return of shear within these vessels. If the reverse is also true, reductions of shear may have important harmful proaggregant effects on platelets (and leukocytes) in the microvascular bed of developing infarcts. However most of the cited work depends upon pharmacological inhibitors of NOS to "prove" that NOS and an EDRF/NO are involved. In the last three years evidence in cats and rats shows that many of the NOS inhibitors also block K channels in cerebrovascular smooth muscle and that arginine, the "antidote" to the NOS inhibitors keeps the channels open. This latter work must force a reexamination of the conclusions reached in many studies.

A review of vasomotor responses of arterioles on the surface of the mouse brain: the necessary prelude to studies using genetically manipulated mice.

OBJECTIVE: To review the published data concerning the vasomotor responses of arterioles on the surface of the mouse brain. This information is essential to the planning of studies using genetically manipulated mice to investigate the control of cerebral vascular resistance. RESULTS: Cerebral vasomotor responses of mice have been reported using a wide variety of vasoactive agents. The responses are usually like those of other laboratory animals. Some agents are capable of eliciting opposing dilating and constricting responses. The initial tone of the arteriole is one of several factors that can determine the direction of the response elicited by such agents. Endothelium-dependent dilators and constrictors have been described. There are a variety of endothelium-derived relaxing factors (EDRFs). Only one of these is synthesized by nitric oxide synthase (NOS). Antisense data suggests that both the endothelial and the neuronal isoforms of NOS may exist in the endothelial cells of these vessels. Several diseases can be modeled in mice and cerebrovascular responses studied. Studies of genetically modified mice suggest that the endothelial form of NOS contributes to microvascular events, which limit ischemic damage to brain parenchyma. However, during and following ischemia there may be loss of this NOS or inability to mobilize a storage form of the EDRF, which it produces. CONCLUSIONS: Data available from studies of mice provide a good basis for planning further studies, examining cerebrovascular control mechanisms in health and disease, by workers now using genetically modified mice. The latter represent a powerful and increasingly popular tool for this purpose.

Do no harm versus the greatest good for the greatest number: health care and the clash of ethical imperatives.

For two millennia, more or less, doctors all over the world have taken the Hippocratic oath to "do no harm." However, these words often have conflicted with social policy. Particularly in this century, some governments and other institutions have encouraged--or insisted on--placing the good of the state ahead of the duty to individual patients. Sterilizing "handicapped" and "feebleminded" persons in Germany long before the creation of the concentration camps is a prominent example of a program initiated under the guise of sacrificing few to improve the welfare of all. America likes to think it is fundamentally different from the Germany that carried out the sterilization program. However, the social and economic forces in Germany that placed national health above the principle of do no harm and encouraged doing the greatest good for the greatest number are similar to what is developing in America--a concordance between economic forces and a social philosophy that is willing to curtail access of some individuals to treatments that they need, want, and can pay for to achieve a hypothetical goal of better national health.

Histopathologic clues to the pathways of neuronal death following ischemia/hypoxia.

This review describes histopathologic observations made with both light and electron microscopy using both conventional staining techniques and histochemistry. Several conditions are analyzed: Ischemic cell change; delayed neuronal death; selective vulnerability. The histopathologic support for the calcium hypothesis and for the excitotoxic hypothesis explaining neuronal death is also reviewed. The findings lead to several suggestions relevant to attempts at developing interventional therapies administered after the onset of ischemia/hypoxia. (1) Except in gerbils, delayed neuronal death and more rapid neuronal death appear to be on the same continuum of cellular events. The lag between ischemia and either onset or termination of these shared events depends upon the severity and/or duration of ischemia/hypoxia. We still do not know whether the "delay," when it occurs, is a delay between ischemia and initiation of the lethal sequence or is, instead, a delay between an immediate initiation of the sequence and its lethal termination. (2) Selective vulnerability (e.g., of CA1 sector in hippocampus) is only relative. The changes are again those of ischemic cell change and are identical to the changes seen elsewhere in more severe ischemia. (3) There is histopathologic support for both the calcium hypothesis and for the cytotoxic hypothesis. Indeed, there is histopathologic support linking the two hypotheses and linking these mechanisms to the appearance of ischemic cell change. However, the histopathologic data are surprisingly sparse. The role of either hypothesis in explaining neuronal death in all areas of brain, in all types of ischemic insult, and at all times following such an insult remains to be established. (3) Apoptosis may be an important mode of neuronal death following ischemia. It differs from acute ischemic cell change; nevertheless, both calcium overload and/or excitotoxic neurotransmitters may trigger apoptosis. (4) Third cell change has been described: Eosinophilic neurons that are not shrunken and whose nuclei are not pyknotic but contain clumped chromatin. The pathogenesis and fate of these neurons remains uncertain. It is possible that they represent early apoptotic neurons. Adequate assessment of apoptosis and its relationship (to both these neurons and to neurons displaying classical ischemic cell change) may depend upon dual staining with conventional aniline dyes and with histochemical techniques designed to detect intranuclear fragments of DNA.