Effects of halothane, alpha-chloralose, and pCO2 on injury volume and CSF beta-endorphin levels in focal cerebral ischemia.

Anesthetic agent, arterial pCO2 level, and opioid peptides have all been implicated in the pathophysiology of experimental stroke models. The effects of halothane, alpha-chloralose, and differing concentrations of arterial pCO2 on injury volume and CSF beta-endorphin levels were studied in a feline model of experimental focal cerebral ischemia. The type of anesthetic agent used had no effect on injury volume following 6 h of focal cerebral ischemia. Over a 6-h period, beta-endorphin levels significantly increased from 10.1 +/- 5.0 fmol/mL at zero time to 14.4 +/- 7.2 fmol/mL at 6 h under halothane anesthesia (p < 0.05), whereas they did not significantly change (10.1 +/- 6.7 to 7.8 +/- 4.7 fmol/mL) under alpha-chloralose anesthesia. In contrast, hypercapnia had no effect on beta-endorphin levels, but significantly increased injury volume from 30.6 +/- 5.7% of the ipsilateral hemisphere under normocapnic conditions to 37.1 +/- 5.9% under hypercapnic conditions (p < 0.05). These results suggest that hypercapnia increases injury volume in a feline model of focal cerebral ischemia, and pCO2 should be controlled in experimental focal cerebral ischemia models.

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes.

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7alpha-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the "acidic" and "neutral" pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7alpha-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 microM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 microM), dibutyryl-cAMP (5O microM), nor squalestatin 1 (15O nM-1.0 microM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 microM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-beta-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7alpha-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [14C]cholesterol remained relatively high and consisted of beta-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.

Evaluation of delayed treatment of focal cerebral ischemia with three selective kappa-opioid agonists in cats.

BACKGROUND AND PURPOSE: The purpose of this study was to determine the therapeutic efficacy of three kappa-opioid agonists used for delayed treatment of experimental focal cerebral ischemia. METHODS: Forty halothane-anesthetized cats underwent permanent occlusion of the right intracranial internal carotid, middle cerebral, and anterior cerebral arteries via a transorbital, microsurgical approach. Six hours after occlusion, animals received a blinded bolus injection, and a subcutaneous osmotic pump was implanted to provide continuous release for 7 days. The injection and pump contained either saline or one of three kappa-agonists: dynorphin (1-13), U-50,488, or DuP E3800. Survival, neurological function, tissue damage, and brain weight were assessed. RESULTS: As a group, kappa-agonist-treated animals had higher survival (P < .02), less tissue damage (P < .02), and lower brain weight (P < .05) than saline controls. U-50,488 more effectively improved survival (P < .03) than dynorphin (P < .07) or E3800 (P < .07). Each of the three kappa compounds improved tissue damage (dynorphin, P < .02; U-50,488, P < .05; E3800, P < .05). Greater improvement in neurological function was seen after treatment with dynorphin (P < .05) than with U-50,488 (P < .6) or E3800 (P < .7). The only significant reduction in brain weight was seen after dynorphin treatment (P < .01). CONCLUSIONS: Compounds that act at the kappa subclass of opiate receptors are effective in increasing survival, improving neurological function, and decreasing tissue damage and edema in a cat model of focal cerebral ischemia. The current study provides support for the benefits of treatment of acute cerebrovascular ischemia with kappa-opioid agonists. The agents may prove to be of superior clinical utility because of efficacy even when administered 6 hours after the onset of stroke.

The effect of dietary n-3 fatty acids on in vivo platelet aggregation in the cerebral microcirculation.

Diets enriched in n-3 fish oil have been suggested to decrease coronary artery disease in part through their ability to decrease cyclooxygenase-dependent platelet aggregation. However little is known concerning the effect of n-3 fatty acids on in vivo platelet aggregation. The purpose of these experiments was to determine whether dietary n-3 fatty acids affect the rate at which platelet aggregation occurs in cerebral arterioles. Fish oil (200 mg eicosapentaenoic acid + 143 mg docosahexaenoic acid/kg), corn oil or water was given daily by gavage to mice (n = 30) for six weeks and then in vivo platelet aggregation was induced by the light plus dye method, which injuries the endothelium. Two additional groups of mice were acutely treated with saline or indomethacin (0.5 mg/kg, ip), with the latter serving as a positive control for therapeutic inhibition of platelet aggregation. Serum thromboxane B2 was analyzed by RIA. All fed groups gained weight equally. Serum thromboxane B2 was decreased by 40% in the fish oil group (p = 0.05 vs. corn oil, p = 0.07 vs. water). The mean (+/- SE) time to first aggregate in pial arterioles was 101 +/- 6, 91 +/- 6 and 101 +/- 9 seconds in the fish corn oil and water groups, respectively. Indomethacin significantly increased the time to first arteriolar aggregate by 35% (p < 0.002) and caused an 80% reduction in serum thromboxane. These studies show dietary fish oil produces a moderate reduction in serum TxB2 level and does not affect arteriolar platelet aggregation whereas indomethacin produces a drastic TxB2 reduction and significantly slows platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Variations in corticomotor and somatosensory evoked potentials: effects of temperature, halothane anesthesia, and arterial partial pressure of CO2.

The effects of temperature, halothane concentration, and arterial partial pressure of CO2 on corticomotor evoked potentials (CMEPs) and somatosensory evoked potentials (SSEPs) were studied. Hypothermia causes an increase in CMEP and SSEP latencies. Corticomotor evoked potential amplitude increases with hypothermia to reach a maximum at or below 28 degrees C. As the temperature decreases from 42 degrees C, SSEP amplitude initially increases to reach a maximum between 36 and 34 degrees C and then decreases with further reductions in temperature. Increased arterial partial pressure of CO2 decreases amplitude and increases latencies of the CMEPs and SSEPs. The concentration of halothane has no effect on CMEP amplitude or latency. However, SSEP amplitude is inversely related to halothane concentration, and SSEP latency is directly related to halothane concentration. These results suggest that physiologic variables must be carefully measured when evoked potentials are utilized in any animal or human study. Moreover, each type of evoked potential has a unique response to alterations of these variables.

14,15-Epoxyeicosatrienoic acid inhibits platelet aggregation in mouse cerebral arterioles.

BACKGROUND AND PURPOSE: Epoxygenase metabolites of arachidonic acid are produced by several tissues and have been shown to inhibit in vitro platelet aggregation. The purpose of the present investigation was to determine whether 14,15- or 8,9-epoxyeicosatrienoic acid, epoxygenase derivatives of arachidonic acid, affect the speed of platelet aggregation in in vivo mouse cerebral arterioles. METHODS: We performed a craniectomy in 116 anesthetized male mice and observed the pial arterioles by microscopy. We induced in situ platelet aggregation using a mercury light and intravascularly injected fluorescein dye. RESULTS: Indomethacin (0.5 mg/kg i.p.), a known cyclooxygenase inhibitor, and 14,15-epoxyeicosatrienoic acid (0.3 mg/kg i.v.) increased the time necessary for the light plus dye to induce the first arterial platelet aggregate by 35% and 26%, respectively, whereas 8,9-epoxyeicosatrienoic acid (0.3 mg/kg i.v.) had no effect. Analysis of mouse serum by radioimmunoassay showed that the degree of inhibition of platelet aggregation by indomethacin and epoxyeicosatrienoic acids correlated with the degree of inhibition of thromboxane production. CONCLUSIONS: We conclude that 14,15-epoxyeicosatrienoic acid is a potent inhibitor of in vivo platelet aggregation but cannot conclusively confirm that its effect on aggregation occurs via its reduction of platelet thromboxane A2. Because epoxyeicosatrienoic acids are produced by several tissues, including brain and vascular tissue, they may be important in vivo modulators of platelet aggregation and hemostasis.

Effect of fish oil n-3 fatty acids on cerebral microcirculation.

Dietary fish oil containing the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) is being consumed by many individuals in an effort to reduce thrombosis and heart disease. However, little is known about how these fatty acids can affect cerebrovascular function. The purpose of the present study was to begin to examine the effects of these fatty acids on cerebral arteriolar diameter and to compare their effects with that of arachidonic acid (AA). Pial arteriolar diameter responses to the topically applied fatty acids [0.2-200 micrograms/ml cerebrospinal fluid (CSF)] were measured in rabbits using in vivo microscopy and the acute cranial window technique. Prostaglandin E2 (PGE2) formed by the brain in response to AA, EPA, and DHA was measured in CSF using radioimmunoassay. EPA induced a dose-dependent dilation response of which the maximum was 29%, whereas the maximal dilation produced by AA was 100%. The arteriolar effect of EPA was reduced by indomethacin or superoxide dismutase plus catalase, indicating vasoactivity due to oxygen radicals formed by cyclooxygenase metabolism of EPA. DHA itself had no effect on diameter or adenosine-induced dilation but reduced dilation by AA when coapplied with AA. AA induced a 65-fold maximal increase in PGE2, whereas EPA and DHA had comparatively little effect. These results imply that substitution of n-3 fatty acids for AA in brain phospholipids may result in less cyclooxygenase-dependent cerebrovascular reactivity. This alteration in reactivity may produce important effects with respect to the brain's blood flow response to a number of physiological and pathological challenges.

Brain kininogen following experimental brain injury: evidence for a secondary event.

Previous studies have shown that following experimental brain injury cerebral arterioles dilate and display endothelial lesions and reduced responsiveness to hypocapnia. These abnormalities are caused by cyclo-oxygenase-dependent free radical generation. There is evidence that the kallikrein-kinin system may in part stimulate the cyclooxygenase-dependent damage since bradykinin is a powerful stimulator of prostaglandin formation and it has recently been shown that a specific kinin receptor blocker decreases the arteriolar abnormalities caused by injury. In order to further examine the hypothesis that the kallikrein-kinin system is important in inducing damage, rat brain tissue was examined for kininogen, the precursor of kinins, at 10 minutes and 1, 3, 6, 15, 24, 48, and 72 hours after injury. A fluid-percussion brain injury device was attached over the right cerebral cortex of rats and a 1.6-atmosphere pressure injury was administered. The kininogen content was determined by a radioimmunoassay procedure in tissues which were free of intravascular blood. After injury, bleeding was confined mainly to the right hemisphere. The kininogen content in the right hemisphere was significantly elevated by one hour after injury, continued to rise until 15 hours after injury, then was significantly decreased by 2 days after injury. In the left hemisphere, kininogen was significantly elevated at 1 hour postinjury, returned toward control levels over the 3- to 6-hour period after injury, then was again elevated at 15 hours after injury. These studies also show that brain water and cerebrovascular permeability were greater at 15 hours postinjury than at earlier time points. The data further support a role for the kallikrein-kinin system in brain injury and, when considered with the results of other studies, suggest that a secondary event is occurring in the 12- to 24-hour period after neural injury. The authors hypothesize that this secondary event is related to endothelial and vascular repair and may be important for the return of normal cerebrovascular function.

Laser-Doppler assessment of brain microcirculation: effect of systemic alterations.

There is a need for new technical approaches whereby the cerebral microcirculation can be easily and continuously assessed. The objective of this study was to determine whether laser-Doppler (LD) flowmetry can be utilized to assess changes in cerebral cortical blood flow and to determine whether changes in blood perfusion measured by LD flowmetry correlate with simultaneously measured changes in flow measured by H2 clearance in cats or with changes in pial arteriolar diameter measured with a microscope in rabbits equipped with a closed cranial window. In the rabbit experiments a 0.84-mm-diam LD probe was inserted through a cranial window port, and in the cat experiments the probe was fixed adjacent to the H2 probe. The probe was fixed at a distance of 1-2 mm from the cortical surface, where it and its associated electronics detect changes in blood cell velocity and blood volume within a tissue volume of approximately 1 mm3. Volume and velocity are multiplied to provide a flow signal. When cerebral blood flow in cats was decreased by hyperventilation-induced hypocapnia and increased by norepinephrine-induced hypertension, the percent changes in LD flow and H2 clearance flow changed linearly (r = 0.94, slope = 0.97). When arterial PCO2 was increased from 28 to 48 mmHg in the rabbit experiments, the pial arterioles dilated 19 +/- 4% (mean +/- SE) and LD flow increased by 74 +/- 9%, LD flow changes which would be predicted by a third power relationship of diameter to flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Laser-Doppler assessment of brain microcirculation: effect of local alterations.

The objective of this study was to determine whether changes in blood perfusion measured by laser-Doppler (LD) flowmetry correlate with simultaneously measured changes in pial arteriolar diameter after local application of vasoactive agents on the brain surface. A closed cranial window was implanted in anesthetized rabbits. A 0.84-mm-diam LD probe was inserted through one window port and fixed at a distance of 1-2 mm from the cortical surface. The probe detects changes in perfusion within a tissue volume of approximately 1 mm3. The diameter of pial arterioles in the area adjacent to the LD probe was simultaneously measured with a microscope and image-splitting device. Topical application of bradykinin (80 nM to 8 microM), which stimulates the formation of dilator prostaglandins and O2 radicals, induced a dose-dependent arteriolar dilation and increase in LD flow. Topical application of 33 microM 2-chloroadenosine, a stable analogue of adenosine, induced the same degree of pial arteriolar dilation as 8 microM bradykinin but produced a much larger increase in LD flow, probably due to its greater tissue penetration and stability. At 5 min after bradykinin washout the arterioles had nearly returned to their control diameter, whereas LD flow was still increased. Similarly, there was a discrepancy between LD flow and diameter changes after washout of 2-chloroadenosine. We conclude that LD flowmetry is a useful technique for continuous assessment of cortical blood flow in response to topically applied agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of bradykinin- and kallikrein-induced cerebral arteriolar dilation by a specific bradykinin antagonist.

We have previously shown that topical brain application of kallikrein, an enzyme which converts kininogen to bradykinin, induces rabbit pial arteriole dilation. The purpose of the present investigation was to utilize a newly developed competitive kinin receptor antagonist to test the hypothesis that kallikrein-induced dilation was due to the conversion of brain kininogen to vasoactive kinins. As in our previous study, we measured rabbit pial arteriole diameter with a microscope using the closed cranial window technique. The kinin antagonist (6 microM) reduced the dose-dependent dilation produced by bradykinin and blocked the dilation induced by kallikrein. In addition, the kinin antagonist was specific since it did not alter the cerebral arteriole dilation produced by adenosine, acetylcholine, or vasoactive intestinal polypeptide. These experiments provide further evidence for a possible role of the endogenous brain kallikrein-kinin system in the modulation of the cerebral circulation and provide the necessary pharmacologic foundation for future use of this antagonist in testing the role of kinins in the normal or altered cerebral circulation.

Effect of the thromboxane A2 mimetic U 46619 on pial arterioles of rabbits and rats.

Our previous experiments have shown that thromboxane A2 is a strong contractor of cerebral arterial smooth muscle strips. The objective of these experiments was to determine if U 46619, a stable thromboxane A2 mimetic, affects the cerebral microcirculation in vivo. Pial arteriole diameter in rabbits and rats was measured with a microscope using the closed cranial window technique. Topical application of 10(-11) to 10(-6) M U 46619 induced dose-dependent arteriole vasoconstriction in both species. In rabbits and rats the maximum vasoconstriction was 9.7 +/- 1.3% (mean +/- SEM) and 14.0 +/- 0.5%, respectively. In rats, 10(-7) and 10(-6) M U 46619 induced intravascular platelet aggregation accompanied by a further decrease in diameter and transient occlusion of the arterioles and venules. U 46619 had no significant effect on rabbit pial arterioles that were predilated by hypercapnia or hypercapnia plus hypoxia. Our data suggest that in animals with a normal vasculature, thromboxane A2 may be a moderate constrictor of cerebral arterioles and that this constrictor effect is prevented by hypercapnia and hypoxia.