Asefa Gebrewold

Effects of ethanol on terminal arterioles and muscular venules: direct observations on the microcirculation.

The present experiments, using an image-splitting television microscope recording system, show for the first time the quantitative effects of topical, intra-arterial, and intravenously administered ethanol on terminal arterioles (15–23 μm i.d.) and muscular venules (28–39 μm i.d.), in situ, in a splanchnic terminal vascular bed. Irrespective of the route of administration, ethanol (0.05–10%) dose-dependently dilated both terminal arterioles and muscular venules. These ethanol-induced dilator responses were not modified by cholinergic, histaminergic (neither H1- nor H2-blockers), serotonergic, or prostaglandin synthetase antagonists. β-Adrenergic blockade significantly enhanced the vasodilator effects of the alcohol. In addition, locally as well as systemically administered ethanol dose-dependently inhibited the constrictor effects of catecholamines (epinephrine and norepinephrine), angiotensin, vasopressin, and barium chloride on the microscopic resistance and capacitance vessels. Catecholamine-, angiotensin-, and vasopressin-induced constrictor responses on terminal arterioles were more sensitive to inhibition by ethanol than were the agonist-induced contractile responses on muscular venules. The sensitivity of barium chloride responses to ethanol inhibition, in the two types of muscular microvessels, was the reverse of that observed with the other agonists. The duration of the arteriolar constrictor responses to catecholamines was reduced significantly upon application of ethanol, whereas the duration of Ba2--induced venular constrictions was increased after application of the alcohol. Low doses of ethanol (i.e., 0.01–0.05%) did not potentiate constrictor responses to norepinephrine, epinephrine, angiotensin, vasopressin, or Ba2+ These findings indicate that ethanol can (i) exert direct effects on microvascular smooth muscle: (ii) attenuate microvascular constrictor responses induced by vasoactive substances; and (iii) possibly interfere with microvascular smooth muscle cell inactivation or disposition of catecholamines and Ba2+. It is suggested that the direct vasodepressant actions on microscopic resistance and capacitance vessels may play a significant role in alcohol-induced peripheral vasodilatation seen in man and animals.

Role of brain [Mg2+]i in alcohol-induced hemorrhagic stroke in a rat model: A 31P-NMR in vivo study

One hundred percent of anesthetized rats administered 6.6 gm/kg of ethanol IP died within 10-35 min of alcohol injection; upon autopsy of the brain all demonstrated profound subarachnoid and intracranial bleeding, clear signs of hemorrhagic stroke. Pretreatment of rats with 4 mumol/min MgCl2, but not saline, via IV administration (for 30-45 min), prevented hemorrhagic stroke in all animals so treated with 6.6 gm/kg ethanol. Administration of the stroke dose of alcohol resulted in rapid (within 3-5 min) and marked deficits in whole brain intracellular free Mg ([Mg2++]i) as observed by in vivo 31P-NMR spectroscopy. Intracellular pH (pHi) and the phosphocreatine [PCr]/[ATP] ratio also fell following a significant fall in brain [Mg2+]i). Brains of rats that exhibited strokelike events, upon death and autopsy, demonstrated continued and marked intracellular acidosis with progressive fall in the [PCr]/[ATP] ratio and elevation of inorganic phosphate (Pi) and [H+]i; these events were not accompanied by any rises in systemic arterial blood pressure. Rats pretreated with MgCl2 exhibited relatively stable brain [Mg2+]i, and essentially unchanged pHi, [PCr], [ATP], or [Pi] following alcohol administration, although such animals exhibited threefold alterations in plasma Mg2+, as measured by ion selective electrodes. These observations suggest that high alcohol ingestion can result in severe vasospasm, ischemia, and rupture of blood vessels probably as a consequence of depletion of brain [Mg2+]i, events that can be prevented by Mg2+ pretreatment.

Cocaine-induced cerebral vascular damage can be ameliorated by Mg2+ in rat brain

Cocaine HCl (10 micrograms/ml) delivered perivascularly to the surface of the rat brain resulted in rapid contraction of pial arterioles, which reduced the diameters by 26% compared to controls. This was followed by venular vasospasm and rupture of postcapillary venules and micro-hemorrhages at postcapillary sites. Administration of Mg aspartate HCl, by intraarterial or intravenous infusion (1, 10 and 20 mumol/min), before or after the cocaine, produced dose-dependent inhibition (20-85%) of the cocaine-induced arteriolar spasms and prevention and attenuation of the venular vasculotoxicity and hemorrhaging. These data suggest that magnesium salts might be useful agents in the treatment of cocaine-induced intoxication and prevention of brain damage.

Pyrrolidine Dithiocarbamate Attenuates Alcohol-Induced Leukocyte–Endothelial Cell Interaction and Cerebral Vascular Damage in Rats

Effects of chronic (14-day) pretreatment of orally administered pyrrolidine dithiocarbamate (PDTC) (100 or 200 mg/kg/day) on alcohol-induced venular cerebrovasospasm, microvessel rupture, leukocyte-endothelial chemoattraction, and microhemorrhaging was studied by direct, quantitative in vivo high-resolution TV microscopy of the intact rat brain. Sham animals chronically treated with placebo exhibited concentration-dependent venular cerebrovasospasm, endothelial-leukocyte rolling and attraction, microvessel rupture. and focal hemorrhages, irrespective of route (i.e., perivascular, systemic) of ethanol administration. PDTC pretreatment either prevented or ameliorated greatly the cerebrovasospasm, leukocyte-endothelial chemoattraction, and brain vascular damage induced by ethanol. These new data suggest that alcohol induces cerebral vascular and brain damage by reperfusion injury events, which trigger induction of proinflammatory factors, and transcription factor NF-kappaB and lipid peroxidation of vascular smooth muscle and endothelial cell membranes; these proinflammatory, pro-oxidant, and redox events could play a crucial role in the pathogenesis of alcohol-induced cerebral ischemia and stroke.

Magnesium depletion impairs myocardial carbohydrate and lipid metabolism and cardiac bioenergetics and raises myocardial calcium content in-vivo: Relationship to etiology of cardiac diseases

This study examines the effects of Mg depletion on myocardial bioenergetic, carbohydrate, lipid and phospholipid metabolism. Rats were studied after long‐term (12 week) selective dietary restriction of Mg (20% normal dietary intake). Myocardial biopsy samples were examined for glucose 6‐phosphate and glycogen to evaluate carbohydrate pathways and for glycerol phosphate and mitochondrial fatty acid oxidation and phospholipid contents to evaluate lipid and phospholipid turnover. Dietary Mg deficiency resulted in falls in myocardial glycogen, glucose‐6‐phosphate, glycerol phosphate, as well as the contents of phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphatidyl glycerol (DPG), phosphatidyl inositol (PI) and total phospholipid phosphorus. These observations demonstrate impaired phospholipid metabolism, probably at the biosynthetic level. The mitochondrial oxidation of long‐chain fatty acids was also impaired after Mg depletion. Mg depletion (serum Mg fell 60%) also resulted in significant falls in myocardial [ATP], phosphocreatine (PCr), and Mg with a concomitant rise in myocardial Ca content. These observations are consistent with the tenet that prolonged low [Mg2+]. can result in marked reduction in oxygen and substrate delivery to the cardiac myocytes, with concomitant changes in membrane phospholipids (potentially resulting in a pro‐oxidant state) probably as a result of coronary vasoconstriction.

Alcohol-Induced Vascular Damage of Brain Is Ameliorated by Administration of Magnesium

Ethanol ingestion can cause irreversible neuronal and vascular damage in the brain and stroke-like events. Using an intact in vivo rat brain (pial) model, TV image-intensification, cultured cerebral vascular muscle cells, digital-image analysis, and a novel Mg2+ ion-selective electrode to measure extracellular ionized Mg2+, studies were designed to determine whether: 1) perivascular or systemic administration (i.v. or intra-arterial) of magnesium aspartate HCI (MgA) exert vasodilator effects on arterioles (65-130 microm o.d.) and venules (60-135 microm); 2) nonvasodilator doses of MgA could modify vascular spasms induced by BaCl2 and ethanol; 3) nonvasodilator doses of MgA could ameliorate or prevent the cerebral vascular damage induced by high doses of ethanol; and 4) ethanol depletes cerebral vascular muscle of intracellular Mg ions ([Mg2+]i). Perivascular application of MgA (0.01-100 micromol) produced dose-dependent vasodilatation of cerebral arterioles and venules; arterioles yielded greater vasodilator responses compared to venules. Nonvasodilator doses of Mg (1.0, 4.0 micromol/min), administered i.v. or intra-arterially, into a branch of the internal carotid artery, prevented: 1) the spasmogenic actions of ethanol and Ba2+; and 2) the vasculotoxic actions (rupture of postcapillary venules and focal hemorrhages) of ethanol. In addition, ethanol depleted cerebral vascular muscle cells of [Mg2+]i; blood levels of ionized Mg2+ rose after IP ethanol. Despite the fact that systemic infusion of low nonvasodilator doses did not result in dilatation of the pial arterioles and venules, plasma total and ionized Mg rose 18-230%, depending upon dose of MgA and time of plasma sampling. These data support the idea that Mg2+ can act as a local vasodilator on brain microvessels and possess antispasmodic properties on brain arterioles and venules. In addition, our results indicate that Mg may possess some unique cerebral vascular protective properties against the vasculotoxic effects of ethanol. Lastly, these findings suggest ethanol-induced cerebrovasospasm and vascular damage appear to be associated with a rapid loss of [Mg2+]i from cerebral vascular muscle cells.

Magnesium Deficiency Exacerbates Brain Injury and Stroke Mortality Induced by Alcohol: A 31P-NMR In Vivo Study

Mimicking in rats the reduced level of dietary magnesium (Mg) intake, seen in present-day Western World populations, short-term (4 weeks) restriction of Mg intake (30-35% normal) resulted in a 40% loss in brain intracellular free Mg2+ ions ([Mg2+]i) and significant rises in brain intracellular pH (pHi) and phosphocreatine ([PCr]) but no change in [ATP] or [Pi] as measured by 31P-NMR spectroscopy. Such Mg-deficient animals (serum Mg fell 65%), when given ED40 stroke doses of ethanol, demonstrated a 100% stroke mortality. These findings indicate that: 1) moderate, short-term Mg deficiency makes the brain vulnerable to hypoxic-lethal stroke insults induced by alcohol administration, and 2) brain [Mg2+]i appears to play an important role in finely regulating brain pHi and [PCr].

Short-term reduction in dietary intake of magnesium causes deficits in brain intracellular free Mg2+ and [H+]i but not high-energy phosphates as observed by in vivo 31P-NMR

31P-NMR spectroscopic studies were performed in vivo on brains of rats fed 30-35% normal dietary Mg intake for 6 weeks. Within 2 weeks of the moderately restricted Mg diet serum Mg fell 50%, and brain intracellular free [Mg2+]i fell 15%; within 3 weeks of restricted diet, brain [Mg2+]i fell 40% and remained at this level for the additional 3 weeks. Intracellular pH (pH[i]) progressively rose in a reciprocal manner for 4 weeks. At no interval of time did brain phosphocreatine (PCr), [ATP], or inorganic phosphate change despite the fall in brain [Mg2+]i, brain pH(i) and serum Mg. The Mg-deficiency-induced cytosolic loss of protons (resulting in an alkaline cytosol) could be a compensatory mechanism to stabilize [PCr], [ATP] and [ADP] levels via creatine kinase, thus maintaining cytosolic phosphorylation potential. The rise in pH(i) associated with Mg-deficiency would also account for increased cerebral vascular muscle contractility under these conditions. Lastly, these studies indicate that brain [Mg2+]i may change without a concomitant change in cell [ATP], and that brain [Mg2+]i may be a useful marker for total body Mg2+ status.

Continuous osmotic minipump infusion of alcohol into brain decreases brain [Mg2+] and brain bioenergetics and enhances susceptibility to hemorrhagic stroke: An in vivo 31P-NMR study

31P-NMR spectroscopic studies were performed in vivo on brains of rats chronically infused for 7 and 14 days with 30% ethanol (in the third cerebral ventricle). Peripheral blood alcohol concentration (BAC) rose to between 16.5-30.5 mg/dl. Brain intracellular free Mg2+ ([Mg2+]i) fell 33-39%, brain mitochondrial cytosolic phosphorylation potential (CPP) fell 31-48%, and brain phosphocreatine (PCr) fell approximately 15%; however, neither brain intracellular free hydrogen ion concentration (pHi) nor brain intracellular inorganic phosphate (Pi) were affected significantly by the chronic release of ethanol from the brain implants. Correlations were found between [Mg2+]i and [PCr] and between [Mg2+]i and CPP. Although brain free [MgADP] was not affected, [MgATP] fell by almost 20% accompanied by a 35-40% rise in free [ADP]. Interestingly, 14-day surgical implantation of 0.9% sterile saline into the third cerebral ventricle was associated with a 20% fall in brain [Mg2+]i and a 35% fall in CPP; however, PCr, ATP, or pHi was not significantly altered. Systemic administration of 4 g/kg ethanol into the 7- and 14-day chronic ethanol animals resulted in a 9- and 12-fold increase in hemorrhagic stroke mortality compared to naive, control rats. Eating habits, grooming, gait and arterial blood pressure were not affected by the chronic brain implantation of ethanol. These data lend support to the notion, primarily based on epidemiologic evidence, that chronic exposure to alcohol can pose a high risk for hemorrhagic stroke. Our alcohol pump-implanted rats also might provide a new model of slow, moderate alcohol intoxication.

HDFx: A Recently Discovered Biologic and its Potential Use in Prevention andTreatment of Hemorrhagic Fever Viruses and Antibiotic-Resistant Superbugs

Recently, we have reported on the discovery of a new, conserved protein (35-40 kDa) that protects rats, mice, guinea-pigs, and rabbits against lethal hemorrhage, endotoxins, live lethal bacterial and fungal microorganisms, and traumatic injuries when given prophylactally and therapeutically. HDFx was found to stimulate several arms of the innate immune system (e.g. macrophages, NK cells). HDFx was also found to stabilize the microcirculation, prevent rupture and leakage of postcapillary venules, prevent adhesion of platelets to endothelium and loss of platelets, stabilize falls in arterial blood pressure, and prevent stasis and pooling of blood in the postcapillary vessels, as observed by intra-vital high-resolution TV microscopy. HDFx also stimulates phagocytic uptake of foreign particulate matter and bacteria by liver Kupffer cells, splenic macrophages, and circulating macrophages. It also prevents explosive release of cytokines and chemokines from macrophages and lymphocytes in animals subjected to live bacteria, endotoxins, trauma and combined injuries. Surprisingly, HDFx was found to accelerate wound healing and aid the regeneration of tissues. Repeated administration of HDFx, over many months, does not result in either diminished protective activity or detectable organ or tissue pathologies. One of the major consequences of infections and wars/conflicts is loss of the ability to regenerate normal physiologic functions of numerous organs and tissues. A major characteristic of invasion of the body by septic-endotoxic microorganisms and hemorrhagic fever viruses (HFVs) is that these entities eventuate in rupture of the microvessels in the capillary circulation of numerous organs and tissues leading to massive blood and fluid loss, making the body susceptible to superimposed infections and loss of immuno-competence. About 100 million people are infected worldwide, annually, with about 60,000 to 75,000 deaths per year from HFVs. Added to these numbers are the numerous hospital-borne and food-borne infections along with infections resulting from major disasters (hurricanes, tornados, earthquakes, etc.) that cause 75,000 to 100,000 deaths per year in the U.S.A. alone. The ability and uniqueness of HDFx to minimize infections, accelerate wound healing, and promote tissue regeneration should greatly aid treatment and recovery of these victims and be of great value in infections from HFVs and on battlefields.