Stable laser-Doppler flow-motion patterns in the human cutaneous microcirculation: Implications for prospective geroscience studies.

Microvascular function has been assessed by determining the rhythmic oscillations in blood flow induced by the vasomotion of resistance vessels. Although laser-Doppler flowmetry (LDF) allows simple, non-invasive evaluation of this flow-motion in the cutaneous microcirculation, the temporal and spatial reproducibility of such assessments remains unclear.In the present study, we investigated cutaneous flow-motion in three consecutive years in eight skin regions using LDF in six healthy young volunteers. The characteristic flow-motion frequency was determined using fast-Fourier transformation. Additionally, in two years a more traditional measure of microvascular reactivity, postocclusive reactive hyperemia (PORH) was evoked in the forearm after transient brachial artery occlusion (1-2-3 min) induced by cuff inflation.Well-defined flow-motion was found in six regions showing significant differences in frequency: the highest flow-motion frequency was found in the frontal and temporal regions (8.0 ± 1.1 and 8.5 ± 1.0 cycles/min, cpm, respectively, mean ± SD) followed by the scapular, infraclavicular and coxal regions (7.5 ± 1.3; 6.7 ± 1.1 and 6.5 ± 1.2 cpm, respectively). The lowest, stable flow-motion was found in the posterior femoral region (5.5 ± 1.0 cpm), whereas flow-motion was detectable only sporadically in the limbs. The region-dependent flow-motion frequencies were very stable within individuals either between the body sides, or among the three measurements, only the infraclavicular region showed a small difference (114 ± 17%∗, % of value in 1st year; ∗P < 0.05). However, PORH indices differed after 2-3 min occlusions significantly in consecutive years.We report that flow-motion frequencies determined from LDF signals show both region-specificity and excellent intra-individual temporal and spatial reproducibility suggesting their usefulness for non-invasive follow-up of microvascular reactivity.

Molecular hydrogen affords neuroprotection in a translational piglet model of hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is the major consequence of perinatal asphyxia (PA) in term neonates. Although the newborn piglet is an accepted large animal PA/HIE model, there is no consensus on PA-induction methodology to produce clinically relevant HIE. We aimed to create and to characterize a novel PA model faithfully reproducing all features of asphyxiation including severe hypercapnia resulting in HIE, and to test whether H2 is neuroprotective in this model. Piglets were anaesthetised, artificially ventilated, and intensively monitored (electroencephalography, core temperature, O2 saturation, arterial blood pressure and blood gases). Asphyxia (20 min) was induced by ventilation with a hypoxic-hypercapnic (6%O2 - 20%CO2) gas mixture. Asphyxia-induced changes in the cortical microcirculation were assessed with laser-speckle contrast imaging and analysis. Asphyxia was followed by reventilation with air or air containing hydrogen (2.1%H2, 4 hours). After 24 hours survival, the brains were harvested for neuropathology. Our PA model was characterized by the development of severe hypoxia (pO2 = 27 ± 4 mmHg), and combined acidosis (pH = 6.76 ± 0.04; pCO2 = 114 ± 11 mmHg; lactate = 12.12 ± 0.83 mmol/L), however, cortical ischemia did not develop during the stress. Severely depressed electroencephalography (EEG), and marked neuronal injury indicated the development of HIE. H2 was neuroprotective shown both by the enhanced recovery of EEG and by the significant preservation of neurons in the cerebral cortex, hippocampus, basal ganglia, and the thalamus. H2 appeared to reduce oxidative stress shown by attenuation of 8-hydroxy-2'-deoxyguanosine immunostaining. In summary, this new PA piglet model is able to induce moderate/severe HIE, and the efficacy of hydrogen post-treatment to preserve neuronal activity/function in this PA/HIE model suggests the feasibility of this safe and inexpensive approach in the treatment of asphyxiated babies.

Comparison of cerebrocortical microvascular effects of different hypoxic-ischemic insults in piglets: a laser-speckle imaging study.

The newborn pig is a widely accepted large animal model of hypoxic/ischemic (H/I) encephalopathy (HIE) of the term neonate appropriate for translational research. The methodology of the induction of H/I stress shows extensive variability of the literature, and little is known how these affect study outcome. The purpose of the present study was to determine the cerebrocortical microvascular effects of different H/I insults used in current HIE piglet models. For the semiquantitative study of cerebrocortical blood flow, we developed a methodological innovation: an operating microscope was converted into a custom-designed laser-speckle imager. Anesthetized, air-ventilated newborn pigs (n=7) were fitted with a closed cranial window. Speckle image series (2 ms, 1 Hz) were collected during baseline conditions, during transient bilateral carotid artery occlusion (BCAO), hypoxic (FiO(2)=0.1) hypoxia, hypoxia + BCAO, and asphyxia induced by suspending ventilation. Laser-speckle contrast analysis was performed off-line over parenchymal and arteriolar regions of interests, and pial arteriolar diameters were also determined for detailed analysis of cortical perfusion changes. Under normoxic conditions, transient BCAO did not affect parenchymal perfusion or pial arteriolar diameters. Hypoxia induced marked cortical hyperemia in 5 out of 7 piglets, with simultaneous increases in pial arteriolar diameters and arteriolar flow velocity, however, BCAO could not even affect these hypoxia-induced perfusion changes. In contrast to hypoxia or hypoxia + BCAO, asphyxia inevitably led also to severe cerebrocortical ischemia. In summary, acute reversible BCAO does not reduce cerebrocortical blood flow in the piglet, and thus it likely does not exacerbate the effect of hypoxic ventilation. Asphyxia elicits not only severe hypoxia, but also severe brain ischemia. These microcirculatory effects must be taken into consideration when assessing results obtained in the various HIE piglet models.

Diazoxide is protective in the rat retina against ischemic injury induced by bilateral carotid occlusion and glutamate-induced degeneration.

Diazoxide (DIAZ) has been shown to be neuroprotective in animal models of different brain pathologies. However, the direct protective effect of DIAZ in different in vivo models of retinal degeneration has not yet been shown. Therefore, the aim of the present study was to investigate the neuroprotective role of this compound in two rodent model systems: monosodium-glutamate (MSG)- and chronic bilateral carotid artery occlusion (BCAO)-induced retinal degeneration. Rats were subjected either to s.c. MSG treatment on postnatal days 1, 5 and 9, or to BCAO at 2 months of age, followed by intravitreal DIAZ treatment. Histological examination was carried out 14 or 21 days after treatments, respectively. MSG treatment destroyed almost the entire inner retina, with the inner nuclear and ganglion cell layers being fused. DIAZ treatment significantly ameliorated the MSG-induced retinal degeneration. BCAO led to a severe degeneration of all retinal layers, and DIAZ proved to be protective also in this model. Our results may have clinical implications in reducing glutamate-induced excitotoxicity or ischemic retinal degeneration in ophthalmic diseases.

Novel calretinin and reelin expressing neuronal population includes Cajal-Retzius-type cells in the neocortex of adult pigs.

Cajal-Retzius cells and their secreted product reelin are essential for the lamination of the cerebral cortex. In all species studied to date Cajal-Retzius cells form a transient neuronal population that almost completely disappears from the neocortex postnatally. Recently, in the hippocampal formation of adult domestic pig, we have found a large calretinin- and reelin-immunoreactive cell population that morphologically corresponded to Cajal-Retzius cells. In the present study, we examined calretinin- and reelin-immunoreactive neurons in layer I of the prefrontal, temporal, parietal and occipital neocortical areas of newborn, young adult and adult domestic pigs. Large numbers of bipolar or fusiform calretinin-positive cells were found in the upper half of layer I in all examined age groups. The morphology of these neurons resembled that of the Cajal-Retzius cells. Layer I was occupied by a dense calretinin-positive axonal plexus that was similar to the previously described axons of Cajal-Retzius cells in other species. In a similar location, where calretinin-positive cells occurred in layer I, large numbers of reelin-immunoreactive cells were found in all examined age groups. In addition, reelin colocalized with calretinin in layer I neurons. The number of calretinin and reelin-positive neurons decreased from 1 day to one year, but calretinin-positive Cajal-Retzius-type cells still comprised a remarkable large population in 12-month-old animals. Correlated light and electron microscopic examination of calretinin-labeled Cajal-Retzius-type cells indicated that these cells are integrated in the synaptic circuitry of the neocortex. Our results suggest that Cajal-Retzius cells do not disappear inevitably from the mature neocortex in all mammalian species. The function of this cell type is not known, but late persisting Cajal-Retzius-type cells in the domestic pig provide an opportunity to study their neuronal connections and the possible role of reelin in plasticity and regeneration of neocortex.

Unaltered cerebral Na+,K+-ATPase activity after hypoxic/ischemic injury in piglets.

We examined whether hypoxic/ischemic (H/I) stress decreased the cerebral Na(+),K(+)-ATPase enzyme activity (NEA) of newborn pigs. The effects of global ischemia (10 min), asphyxia (10 min), and incomplete forebrain ischemia (45 min) were analyzed in ten different brain regions. The lengths of the reperfusion periods varied between 15 min and 3 h. NEA was determined as the ouabain-sensitive fraction of the total ATPase activity of the sample. Marked regional differences in NEA were observed in all experimental groups, whereas NEA was not significantly affected in any of the brain structures investigated. The present results suggest that damaged brain Na(+),K(+)-ATPase may not be the cause of the neuronal-vascular impairment following H/I stress.

Cyclooxygenase-2 inhibitor NS398 preserves neuronal function after hypoxia/ischemia in piglets.

Anoxic stress attenuates NMDA-induced pial arteriolar dilation via a mechanism involving actions of cyclooxygenase (COX)-derived reactive oxygen species (ROS). We examined whether the selective COX-2 inhibitor NS398 would protect neuronal function after global hypoxia/ischemia (H/I) in piglets. Pial arteriolar responses to NMDA (10-100 micromol/l) were determined using intravital microscopy in anesthetized piglets before and 1 h after H/I. Study groups received vehicle, 0.3, 1, or 5 mg/kg NS398, or 0.3 mg/kg indomethacin (n = 7, 6, 6, 5 and 8, respectively) i.v. 20 min prior to H/I. H/I reduced NMDA- induced dilation to 44 +/- 6% (100 micromol/l NMDA, mean +/- s.e.m.) of the pre-ischemic response in vehicle animals (p < 0.05). However, NS398 dose-dependently protected arteriolar dilation to NMDA (77 +/- 8, 81 +/- 16, and 102 +/- 10% preservation at 0.3, 1 and 5 mg/kg, respectively). Indomethacin caused similar preservation. However, indomethacin but not NS398 reduced serum thromboxane B(2) levels to undetectable values. In conclusion, COX-2 appears to be a major source of ROS in the piglet cerebral cortex after H/I.

Impaired early neurologic outcome in newborn piglets reoxygenated with 100% oxygen compared with room air after pneumothorax-induced asphyxia.

Birth asphyxia is a serious problem worldwide, resulting in 1 million deaths and an equal number of neurologic sequelae annually. It is therefore important to develop new and better ways to treat asphyxia. In the present study we tested the effects of reoxygenation with room air or with 100% oxygen (O2) after experimental pneumothorax-induced asphyxia on the blood oxidative stress indicators, early neurologic outcome, and cerebral histopathology of newborn piglets. Twenty-six animals were studied in three experimental groups: 1) sham-operated animals (SHAM, n = 6), 2) animals reoxygenated with room air after pneumothorax (R21, n = 10), and 3) animals reoxygenated with 100% O2 after pneumothorax (R100, n = 10). In groups R21 and R100, asphyxia was induced under anesthesia with bilateral intrapleural room air insufflation. Gasping, bradyarrhythmia, arterial hypotension, hypoxemia, hypercarbia, and combined acidosis occurred 62 +/- 6 min (R21) or 65 +/- 7 min (R100; mean +/- SD) after the start of the experiments; then pneumothorax was relieved, and a 10-min reoxygenation period was started with mechanical ventilation with room air (R21) or with 100% O2 (R100). The newborn piglets then breathed room air spontaneously during the next 3 h. Blood oxidative stress indicators (oxidized and reduced glutathione, plasma Hb, and malondialdehyde concentrations) were measured at different stages of the experiments. Early neurologic outcome examinations (neurologic score of 20 indicates normal, 5 indicates brain-dead) were performed at the end of the study. The brains were next fixed, and various regions were stained for cerebral histopathology. In the SHAM group, the blood gas and acid-base status differed significantly from those measured in groups R21 and R100. In group R100, arterial PO2 was significantly higher after 5 (13.8 +/- 5.6 kPa) and 10 min (13.2 +/- 6.3 kPa) of reoxygenation than in group R21 (8.7 +/- 2.8 kPa and 9.2 +/- 3.1 kPa). The levels of all oxidative stress indicators remained unchanged in the study groups (SHAM, R21, and R100). The neurologic examination score in the SHAM group was 18 +/- 0, in group R21 it was 13.5 +/- 3.1, and in group R100 it was 9.5 +/- 4.1 (significant differences between SHAM and R21 or R100, and between R21 and R100). Cerebral histopathology revealed marked damage of similar severity in both asphyxiated groups. We conclude that the blood oxidative stress indicators and cerebral histopathology did not differ significantly after a 10-min period of reoxygenation with room air or with 100% O2 after pneumothorax-induced asphyxia, but reoxygenation with 100% O2 might impair the early neurologic outcome of newborn piglets.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

BACKGROUND: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. METHODS: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. RESULTS: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. CONCLUSIONS: Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

Kainic acid rapidly induces cyclooxygenase (COX)-2 in piglet cerebral cortex.

Ischemia/reperfusion (I/R) results in a robust induction of cyclooxygenase (COX)-2 in the newborn brain via unknown mechanisms, but glutamate release and activation of KA receptors may be involved. We examined effects of local KA (3-300 micromol/l for 10 min) treatment on cortical COX-2 expression in anesthetized piglets using a closed cranial window. Treated and corresponding control tissue samples were collected 0.5-10 h after treatment. COX-2 mRNA and protein levels were assessed using RNase protection assay and immunohistochemistry, respectively. KA elicited reproducible dose-dependent increases in cortical COX-2 mRNA unaffected by indomethacin or N(G)-nitro-L-arginine methyl ester pretreatment. COX-2 mRNA levels were elevated at 30 min, peaked at 2 h, but remained enhanced for up to 10 h after KA. Neuronal COX-2 immunoreactivity was also enhanced compared with the control side in all cortical layers 8h after KA. In summary, activation of KA receptors may be involved in the neuronal induction of COX-2 after I/R in the newborn.

Capsaicin-sensitive mechanisms are involved in cortical spreading depression-associated cerebral blood flow changes in rats.

We tested the hypothesis that capsaicin-sensitive mechanisms play a role in the cortical spreading depression (CSD)-related changes in cortical blood flow (CBF). CBF was measured with laser Doppler flowmetry in anesthetized rats. The animals were treated with capsaicin before (48 h-2 weeks) or during the experiments. This agent is thought to stimulate small-diameter sensory nerve fibers selectively and to deplete stored peptides. In the vehicle-treated group (n=8), the peak value of the CSD-associated hyperperfusion was 257+/-12% above the baseline (mean+/-SEM, P<0.05). In the groups treated with 20 and 40 microg/kg or 20 mg/kg capsaicin, there were only small decreases in CBF. In the groups treated with 100 mg/kg capsaicin, the CSD-associated hyperemia was reduced at 48 h (158+/-7%, P<0.05). However, at 96 h a transient hyperresponsiveness (390+/-38%, P<0.05) was observed, which had disappeared by 2 weeks. These results indicate that the manipulation of sensory neuropeptide stores results in a biphasic effect on CSD-induced CBF responses.

Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats.

Cerebral hypoxia is a major component of immediate and secondary cell damage caused by ischemia. Hyperbaric oxygen (HBO) is a potent means to increase the amount of oxygen dissolved in blood plasma. The effectiveness of HBO in clinical and experimental cerebral ischemia, however, is controversial. We sought to determine whether treatment with HBO initiated early after focal cerebral ischemia-onset protects the brain when experimental conditions such as brain temperature are controlled. Male Wistar rats (n=57) underwent reversible filament occlusion of the right middle cerebral artery (MCA) for 75 min. Animals were awakened after filament introduction and assessed for presence of forelimb paresis. Rats then underwent a 60-min course of either 100% O(2) at 1.0 atmosphere absolute (ata; control group), HBO 1.5 ata, or HBO 2.5 ata in a customized HBO chamber allowing physiological monitoring and pericranial temperature control. The filament was then removed. Seven days after ischemia, rat behavior was scored from 3-18 (18=normal) and brains were removed for histological analysis of infarct volume. Rats treated with HBO 2.5 ata had better mean+/-standard deviation (S.D.) behavioral scores (14+/-2; p<0.05) than control (10+/-3) or HBO 1.5-ata-treated animals (11+/-3). Similarly, total infarct volumes (mean+/-S.D.) were smaller in animals receiving HBO at 2.5 ata (76+/-65 mm(3); p<0.05) compared to control (129+/-83 mm(3)) and HBO 1.5-ata (119+/-68 mm(3))-treated groups. Cortical infarction occurred less frequently in HBO 2. 5-ata-treated than in control animals (44% vs. 71%; p<0.05). We conclude that HBO can improve outcome after temporary focal ischemia when treatment is started early after ischemia-onset but HBO dose appears important. Potential mechanisms include enhanced oxygen supply to marginally perfused cells.

[Reoxigenation after neonatal asphyxia with 21% or 100% oxygen in piglets]. / Ujszülöttkori asphyxiát követö reoxigenizáció sertéseken 21% vagy 100% oxigénnel.

Birth asphyxia represents a serious problem worldwide, resulting in 1 million deaths and an equal number of neurologic sequelae annually. It is therefore important to develop new and better ways to treat asphyxia. In the present study we tested the effect of reoxygenation with room air or 100% oxygen following experimental pneumothorax induced asphyxia on blood oxidative stress indicators, early neurologic outcome and cerebral histopathology of newborn piglets. 26 animals were studied in three experimental groups: sham-operated (SHAM, n = 6), reoxygenation with room air after pneumothorax (RORA, n = 10) and reoxygenation with 100% oxygen after pneumothorax (RO100, n = 10). In RORA and RO100 asphyxia was induced under anesthesia with bilateral intrapleural room air insufflation. Gasping, bradyarrhythmia, arterial hypotension, hypoxemia, hypercarbia and severe combined acidosis occurred 62 +/- 6 (RORA) and 65 +/- 7 min (RO100) after the start of the experiments, when the pneumothorax was relieved and ten min of reoxygenation period was started with mechanical ventilation with room air (RORA) or 100% oxygen (RO100). Then the spontaneously breathing animals were followed on room air during the next three hours. Blood oxidative stress indicators--as oxidized and reduced glutathione, plasma hemoglobin and malondialdehyde concentrations--were also measured at different stages of the experiments and early neurologic examinations (neurological score: 20 = normal, 5 = brain dead) were performed at the end of the study. Then the brains were fixed and stained. In SHAM blood gases and acid/base status differed significantly from values measured in RORA and RO100. In RO100 PaO2 was significantly higher at 5 (13.8 +/- 1.8 kPa) and 10 min (13.2 +/- 2.0 kPa) than in RORA (8.7 +/- 0.9, 9.2 +/- 1.0 kPa), respectively. All the measures of oxidative stress indicators remained unchanged in the study groups (SHAM, RORA, RO100). Neurologic examination scores from SHAM were 18 +/- 0, from RORA 13.5 +/- 1.0 and from RO100 9.5 +/- 1.3 (significant differences between SHAM and RORA and RO100, significant difference between RORA and RO100). Cerebral histopathology showed marked damage with similar severity in both asphyxiated groups. We conclude that blood oxidative stress indicators and cerebral histopathology did not differ significantly after 10 min reoxygenation either with room air or with 100% oxygen following pneumothorax induced asphyxia, but reoxygenation with 100% oxygen might impair the early neurologic outcome of newborn pigs.

Mitochondrial potassium channel opener diazoxide preserves neuronal-vascular function after cerebral ischemia in newborn pigs.

BACKGROUND AND PURPOSE: N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K(ATP)) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K(ATP) channels (mitoK(ATP)) are involved in the ischemic preconditioning-like effect of K(+) channel openers. We determined whether the selective mitoK(ATP) channel opener diazoxide preserves the vascular dilation to NMDA after I/R. METHODS: Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 micromol/L diazoxide; and coapplication of 100 micromol/L 5-hydroxydecanoic acid (5-HD), a K(ATP) antagonist with diazoxide. RESULTS: NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 micromol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 micromol/L NMDA-induced vasodilation after I/R was 53+/-19% (mean+/-SEM, n=8) in vehicle-treated controls versus 55+/-10%, 85+/-5%, and 99+/-15% in animals pretreated with 1, 5, and 10 micromol/L diazoxide (n=8, n=8, and n=12, respectively) and 60+/-15% in the group treated with 5-HD+diazoxide (n=5). CONCLUSIONS: The mitoK(ATP) channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK(ATP) channels may play a role in mediating the protective effect of other K(+) channel openers.

Cycloheximide rapidly inhibits cortical COX activity and COX-dependent pial arteriolar dilation in piglets.

We have previously shown that cycloheximide (CHX) preserved neuronal function after global cerebral ischemia in piglets, in a manner similar to indomethacin. To elucidate the mechanism of this protection, we tested the hypothesis that CHX would inhibit cyclooxygenase (COX) activity in the piglet cerebral cortex and vasculature. Pial arteriolar responses to hypercapnia, arterial hypotension, and sodium nitroprusside (SNP) were determined before and 20 min after treatment with CHX (0.3-1 mg/kg iv) using a closed cranial window and intravital microscopy. We also determined baseline and arachidonic acid (AA)-stimulated cortical PGF(2alpha) and 6-keto-PGF(1alpha) production before and 20-60 min after CHX (1 mg/kg iv) treatment, using ELISA kits. CHX did not affect baseline diameters (approximately 100 microm) but significantly decreased arteriolar dilation to COX-dependent stimuli, such as hypercapnia and hypotension, but not to COX-independent SNP. In the 1 mg/kg CHX-treated group, increases in vascular diameters were reduced from 22 +/- 2 to 10 +/- 2%, from 49 +/- 5 to 31 +/- 3% (means +/- SE, 5 and 10% CO2, respectively, n = 8), from 12 +/- 3 to 3 +/- 1%, and from 26 +/- 5 to 6 +/- 2% ( approximately 25 and 40% decreases in blood pressure, respectively, n = 6). CHX also inhibited conversion of exogenous AA to both PGF(2alpha) and 6-keto-PGF(1alpha); for example, 20 min after CHX treatment 10 microg/ml AA-stimulated PGF(2alpha) concentrations in the artificial cerebrospinal fluid decreased from 14.28 +/- 3.04 to 5.90 +/- 1.26 ng/ml (n = 9). Thus CHX rapidly decreases COX activity in the piglet cerebral cortex. This result may explain in part the preservation of neuronal function of CHX in cerebral ischemia.

Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries.

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5-12 h after global ischemia (2.5-10 min, n = 50), hypoxia (n = 3), or hypercapnia (n = 2) and from time-control (n = 19) or untreated animals (n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-L-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.

Cerebrovascular reactivity remains intact after cortical depolarization in newborn piglets.

Cerebrovascular reactivity is severely affected by ischemia, and changes in vascular responses have been reported after cortical spreading depression and head trauma as well. Cortical depolarization (CD) occurs during ischemia, cortical spreading depression, and head trauma, but its effects on cerebrovascular reactivity are unclear. We tested the hypothesis that CD induced by KCl diminishes the vascular responsiveness to various vasodilatory stimuli in piglets. Responses of pial arterioles were determined by changes in vascular diameter by use of a closed cranial window and intravital microscopy. Baseline arteriolar diameters were 105 +/- 3 microm (mean +/- SEM, n = 27). CD was elicited by topical administration of 1 mol/L KCl for 3 min. Vascular responses were measured before and 1 h after CD. KCl elicited CD and constricted arterioles by 54 +/- 4% (n = 27). N-methyl-D-aspartate induced dose-dependent vasodilation that was unaffected by CD; the percent changes were 9 +/- 1 versus 8 +/- 1 (before and after CD) at 10(-5) mol/L, 19 +/- 2 versus 18 +/- 3 at 5 x 10(-5) mol/L, and 29 +/- 2 versus 26 +/- 3 at 10(-4) mol/L (n = 9). Hypercapnic vasodilation was not diminished by CD; the percent changes were 15 +/- 2 versus 16 +/- 4 at 5%, and 27 +/- 5 versus 27 +/- 6 at 10% inspired CO2 (n = 8). Aprikalim and forskolin caused dilation that was also resistant to prior CD; the percent change values were 21 +/- 4 versus 18 +/- 3 and 16 +/- 2 versus 16 +/- 4 at 10(-6) mol/L, 36 +/- 5 versus 34 +/- 5 and 34 +/- 7 versus 37 +/- 7 at 10(-5) mol/L (n = 8), respectively. Finally, calcitonin gene-related peptide-induced vasodilation was unaffected by CD; percent changes were 15 +/- 3 versus 16 +/- 2 at 10(-7) mol/L and 26 +/- 4 versus 22 +/- 3 at 10(-6) mol/L (n = 8). The intact vascular responses after CD suggest that this component is not responsible for decreased cerebrovascular reactivity after ischemia, head trauma, or cortical spreading depression.

Inhibitors of protein synthesis preserve the N-methyl-D-aspartate-induced cerebral arteriolar dilation after ischemia in piglets.

BACKGROUND AND PURPOSE--Cerebral arteriolar dilation to N-methyl-D-aspartate (NMDA) is a neuronally mediated process that is sensitive to cerebral ischemia. We tested the hypothesis that pretreatment with transcription or translation inhibitors preserves the vascular response to NMDA after global cerebral ischemia. METHODS--Pial arteriolar diameters were measured in anesthetized piglets by use of a closed cranial window and intravital microscopy. Arteriolar responses to NMDA (10(-5) and 10(-4) mol/L) were measured before and 1, 2, and 4 hours after 10 minutes of ischemia. Ischemia was induced by increasing intracranial pressure. Subgroups were pretreated with vehicle, topical actinomycin D (Act-D) 10(-5) or 10(-6) mol/L, or intravenous cycloheximide (CHX) 1.0 or 0.3 mg/kg 15 minutes before ischemia. The effects of Act-D and CHX on vascular responses to NMDA without preceding ischemia were also examined. RESULTS--In the vehicle group, arteriolar responses to NMDA were clearly attenuated 1 hour after ischemia but returned to baseline at 2 to 4 hours. Preischemic compared with 1 hour postischemic arteriolar dilation to NMDA was 10+/-2% versus 1+/-0% at 10(-5) mol/L and 40+/-4% versus 20+/-4% at 10(-4) mol/L NMDA (mean+/-SEM; both P<0.05, n=7). In contrast, pretreatment with Act-D resulted in preservation of the arteriolar responses to NMDA 1 hour after ischemia. For 10(-6) mol/L (n=5) of Act-D, dilations were 6+/-2% versus 6+/-2% at 10(-5) mol/L and 51+/-9% versus 39+/-10% at 10(-4) mol/L of NMDA. For 10(-5) mol/L (n=5) of Act-D, arterioles dilated by 7+/-2% versus 7+/-2% at 10(-5) mol/L and 38+/-4% versus 35+/-4% at 10(-4) mol/L NMDA. Similarly, CHX preserved NMDA-induced vasodilation. For 0.3 mg/kg of CHX (n=5), dilations were 8+/-2% versus 8+/-1% at 10(-5) mol/L and 39+/-4% versus 28+/-6% at 10(-4) mol/L NMDA. For 1.0 mg/kg of CHX (n=5), arterioles dilated by 10+/-2% versus 6+/-2% at 10(-5) mol/L and 37+/-7% versus 35+/-6% at 10(-4) mol/L NMDA. In experiments without ischemia, NMDA-induced vasodilation before and 85 minutes after administration of Act-D or CHX was not significantly different. CONCLUSIONS--Vascular responses of cerebral arterioles to NMDA after ischemia are preserved by pretreatment with either Act-D or CHX. Without preceding ischemia, Act-D and CHX do not potentiate neuronal-vascular responses to NMDA. Our results suggest that continued or augmented protein synthesis is involved in the transient attenuation of NMDA-induced dilation during the early reperfusion phase and that inhibitors of protein synthesis may protect neurons against ischemic stress.

Beta-amyloid (1-42) peptide impairs blood-brain barrier function after intracarotid infusion in rats.

The effects of intracarotid infusions of beta-amyloid (1-42) peptide was studied on the permeability of brain vessels. Using a quantitative Evans blue method a dose-dependent increase of brain tissue albumin content was established following intracarotid injections of the peptide. Cerebral vessels of increased permeability were also demonstrated with a vital 'staining' technique. Lectin histochemistry revealed an almost complete abolition of specific lectin binding sites of affected endothelial cells. The findings indicate a significant deterioration by beta-amyloid (1-42) peptide of blood-brain barrier function and suggest that this may result from endothelial damage. It is assumed that altered permeability of cerebral vessels may be involved in the development of brain pathologies associated with Alzheimer's disease.