Nitric oxide contributes to opioid release from glia during hypoxia

Activation of neuronal nitric oxide (NO) synthase contributes to increased CSF concentrations of the opioids methionine enkephalin and leucine enkephalin during hypoxia in the newborn pig. NO and these opioids, in turn, contribute to hypoxic pial artery dilation. However, the cellular site of origin for opioids detected in CSF cannot be determined using this in vivo model. The present study, therefore, was designed to determine if NO contributes to opioid release from piglet glia grown in primary culture. Glial cell cultures produced more methionine enkephalin than leucine enkephalin under basal conditions. Administration of SNP and 8-Br cGMP to glial cells increased release of both opioids (471±58 vs. 1181±148 pg/mg protein methionine enkephalin before and after SNP 10⁻⁶ M). SNP also increased release of cGMP. Exposure of piglet glial cells to lower than normal O₂ increased the release of both opioids (503±61 vs. 1488±186 pg/mg protein methionine enkephalin before and after hypoxia, (P_O2≈15 mmHg). Hypoxia also increased the release of cGMP from glia while the NO synthase inhibitor N-nitro- -arginine blocked that release. These data show that NO/cGMP and hypoxia release opioids from glia. Additionally, hypoxia releases NO/cGMP from glia. These data therefore suggest that NO contributes to opioid release from glia during hypoxia.     

Stimulus duration-dependent contribution of Kca channel activation and cAMP to hypoxic cerebrovasodilation

Activation of calcium sensitive (K_ca) K channels and cAMP contribute to pial artery dilation observed during a 10-min exposure to hypoxia. Recent studies show that pial dilation during a 20- or 40-min hypoxic exposure was less than that observed during a 5- or 10-min exposure indicating that stimulus duration determines the nature of the vascular response to hypoxia. The present study was designed to determine if the stimulus duration modulates the contribution of K_ca channel activation and cAMP-dependent mechanisms to hypoxic pial artery dilation in piglets equipped with a closed cranial window. The K_ca channel antagonist iberiotoxin had no influence on pial dilation during 5 min of hypoxia (pO₂≈25 mmHg), decremented the dilation during 10- and 20-min exposure, but had no effect on the dilation during a 40-min exposure (33±1% vs. 32±3%, 33±1% vs. 25±1%, 23±1% vs. 19±1%, and 21±2% vs. 17±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after iberiotoxin). NS1619, a K_ca channel agonist, induced pial dilation during hypoxia that was attenuated by 20- and 40-min but not by 5- and 10-min exposure durations. Similarly, the cAMP antagonist Rp 8-Bromo cAMPs had no influence on pial dilation during 5 min of hypoxia, decremented the dilation during a 10-min exposure, but had no effect on the dilation during a 20- or 40-min exposure (36±1% vs. 34±2%, 34±1% vs. 22±1%, 24±2% vs. 21±2%, and 21±2% vs. 19±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after Rp 8-Bromo cAMPs). Additionally, CSF cAMP was unchanged during 5 min, elevated during 10 min, but such elevations were attenuated during 20- and 40-min hypoxic exposure. Pial vasodilation to a cAMP analogue during hypoxia was attenuated by 20- and 40-min but not by 5- and 10-min hypoxic exposure durations. These data show that K_ca channel activation and cAMP contribute to hypoxic pial artery dilation in a stimulus duration-dependent manner. These data suggest that diminished pial artery dilation during longer hypoxic exposure results from attenuated K_ca channel and cAMP-dependent mechanisms.     

Lesions of the rostral ventrolateral medulla reduce the cerebrovascular response to hypoxia

Sympathoexcitatory neurons of the rostral ventrolateral medulla are tonically active and required for maintenance of resting levels of arterial pressure. They are also selectively excited by hypoxia and responsible for the associated sympathoexcitation. Since electrical or chemical stimulation of RVL will increase regional cerebral blood flow (rCBF) independently of changes in regional cerebral glucose utilization (rCGU) we investigated whether the RVL was also required to maintain resting levels of rCBF and also participated in the cerebrovascular vasodilation elicited by hypoxia. Rats were anesthetized (chloralose; 40 mg/kg, s.c.), paralyzed (tubocurarine) and ventilated (100% O₂). rCBF was measured in 10 dissected brain regions using [¹⁴C]iodoantipyrine; rCGU was measured by 2-deoxy-d-[¹⁴C]glucose. in controls (n = 6) rCBF ranged56 ± 5 in corpus callosum to101 ± 6ml/min× 100g in inferior colliculus. Hypoxic-hypoxia (PaO₂ - 36 ± 1mmHg, n = 6) increased rCBF in all structures maximally, at 204% of control, in occipital cortex. Hypercapnia (PaCO₂ = 63.5 ± 0.9, n =5) also increased rCBFP < 0.01) maximally to 199% of control in superior colliculus, Spinal cord transection with maintenance of arterial pressure did not affect resting rCBF and increased the vasodilation to hypoxia (PaO₂ = 39 ± 1mmHg, n = 5) from 2- to 3-fold in all structuresP < 0.01). Bilateral lesions within the RVL had no effect on resting rCBF or rCGU. However, they significantly reduced, in all areas by 50–69% (P < 0.01, n = 5), the cerebrovascular dilation elicited by hypoxia but not hypercapnia, Bilateral lesions in the spinal trigeminal nucleus (PaO₂ = 35 ± 1; n = 6), or transection of the IXth and Xth cranical nerves did not affect the rCBF response to hypoxia(PaO₂ = 41 ± 2; n = 6) (P > 0.05) indicating that the effect of RVL Lesions was not attributable to interference with arterial baro- or chemoreceptor reflexes. We conclude that neurons within RVL are not responsible for maintaining tonic levels of rCBF. However they contribute to the cerebrovascular vasodilation elicited by hypoxia but not hypercapnia. The cerebrovascular response to hypoxia appears reflexive and, in part, due to stimulation of oxygen-sensing neurons in RVL. In contrast, the vasodilation elicited by hypercapnia reflects local chemical signals in the cerebral microcirculation     

Extracellular pH changes in the superfused cat carotid body during hypoxia and hypercapnia

Extracellular pH changes were measured in the superfused cat carotid body with double barreled pH glass microelectrodes, under constant pH (7.45 ± 0.02), temperature (35°C) and flow (3.6 ml/min) of the superfusion medium. Changes of pO₂ in the medium from about 188 Torr (30% O₂) to 35 or 12 Torr (5% and 2% respectively) called hypoxia, induced a change of the pH signal of about 0.1 units indicating acidification of the tissue. Medium pH monitored with pH macroelectrode did not change during hypoxic stimulation. An increase of pCO₂ in the medium from about 20 Torr (3%CO₂, pH7.45 ± 0.02) to 70 Torr (12%CO₂, pH6.98 ± 0.01) called hypercapnia, under constant pO₂ (188 ± 2Torr), temperature (35°C) and flow (3.6 ml/min) resulted in acidification of the tissue of about 0.3 pH units. Extracellular pH changes during hypoxia did not occur when the superfusion medium had no glucose; however, pH changes during hypercapnia persisted under these conditions. The hypoxic and hypercapnic chemosensory response of the sinus nerve were decreased or abolished during glucose deprivation in a time-dependent manner. Replacement of glucose with 2-deoxyglucose in the medium led to a similar pattern, i.e. inhibition of the hypoxic and hypercapnic chemosensory nerve response and of the extracellular hypoxic pH changes. These results indicate that glycolysis takes place and contributes to O₂ and CO₂-chemoreception in the carotid body.     

Modification in swallowing and ventilation co‐ordination during hypercapnia,hypoxia, and tachypnea in unrestrained animals

Background It has been demonstrated that aspirations could occur during respiratory failure, explained by a lack of co‐ordination between swallowing and ventilation. To test this hypothesis, we examined the co‐ordination of ventilation and swallowing in a completely unrestrained rat model during different level of hypercapnia, during hypoxia, and during tachypnea. Methods A total of 50 male Wistar rats (250–350 g) were studied in a barometric plethysmograph to analyze swallowing and ventilation during swallowing, at different gas concentration [room air (G1), 10% of O₂ and 0% of CO₂ (G2), 21% of O₂ and 5% of CO₂ (G3), 21% of O₂ and 10% of CO₂ (G4), tachypnea (G5)]. Key Results During hypoxia, there was no difference between G2 and G1 regarding the swallowing parameters and ventilatory parameters. During hypercapnia, there was an increase in swallowing during inspiration in G4 (16 ± 20%P < 0.01) compared with G1. The analysis of ventilatory parameters during swallowing showed an increase in tidal volume (VT) and mean inspiratory time (VT/TI) (P < 0.001) with no change in respiratory cycle duration (TTOT), inspiratory time (TI), and expiratory time (TE) when compared with G1. During tachypnea (G5), the VT decreased (P < 0.05) without any change in VT/TI. Conclusions & Inferences Our results on animal demonstrated that hypercapnia increased swallowing during inspiration, which was not the case for tachypnea or hypoxia, and could explain some aspirations during respiratory failure.     

Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg²⁺-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 5–7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg²⁺-treated group received MgSO₄ 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg²⁺ concentrations increased from1.6 ± 0.1mg/dl to17.7 ± 3.3mg/dl.³H-MK-801 binding was used as an index of NMDA receptor modification. TheB_max in control, hypoxic and Mg²⁺-treated hypoxic piglets was1.09 ± 0.17, 0.70 ± 0.25and0.96 ± 0.14pmoles/mg protein, respectively. TheK_d for the same groups were10.02 ± 2.04, 4.88 ± 1.43and8.71 ± 2.23nM, respectively. TheB_max andK_d in the hypoxic group were significantly lower compared to the control and Mg²⁺-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg²⁺. The activity of Na⁺, K⁺ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg²⁺-treated hypoxic groups. These findings show that MgSO₄ prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction. We suggest that the administration of Mg²⁺ prior to and during hypoxia may be neuroprotective in vivo, possibly by reducing the NMDA receptor-mediated influx of calcium.     

Role of cAMP and K channel-dependent mechanisms in piglet hypoxic/ischemic impaired nociceptin/orphanin FQ-induced cerebrovasodilation

This study was designed to determine the role of altered cAMP and K⁺ channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ) following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC/oFQ elicits pial dilation via release of cAMP, which, in turn, activates the calcium sensitive (K_ca) and the ATP-dependent K⁺ (K_ATP) channel. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased pO₂ to 35±3 mmHg with unchanged pCO₂. Topical NOC/oFQ (10⁻⁸, 10⁻⁶ M) induced vasodilation was attenuated by ischemia/reperfusion (I+R) and reversed to vasoconstriction by hypoxia/ischemia/reperfusion (H+I+R) at 1 h of reperfusion (control, 9±1 and 16±1%; I+R, 3±1 and 6±1%; H+I+R, −7±1 and −12±1%). Such altered dilation returned to control values within 4 h in I+R animals and within 12 h in H+I+R animals. NOC/oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I+R animals and reversed to decreases in cAMP concentration in H+I+R animals (control, 1037±58 and 1919±209 fmol/ml; I+R, 1068±33 and 1289±30 fmol/ml; H+I+R, 976±36 and 772±27 fmol/ml for absence and presence of NOC/oFQ 10⁻⁶ M, respectively). Topical 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) pial dilation was unchanged by I+R but blunted by H+I+R (control, 10±1 and 20±1%; I+R, 11±1 and 20±2%; H+I+R, 0±1 and 0±2%). Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K_ATP channel activators, respectively, elicited dilation that was blunted by both I+R and H+I+R while NS1619, a K_ca channel activator, elicited dilation that was unchanged by I+R but blunted by H+I+R. These data indicate that impaired NOC/oFQ dilation following I+R results form altered adenylate cyclase and K_ATP channel-dependent mechanisms. These data further indicate that impaired NOC/oFQ dilation following H+I+R results not only from altered adenylate cyclase and K_ATP channel but also from altered cAMP and K_ca channel-dependent mechanisms.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

Acetylcholine release from cat carotid bodies

Hypoxia, hypercapnia and acidosis stimulate the carotid body (CB) sending increased neural activity via a branch of the glossopharyngeal nerve to nucleus tractus solitarius; this precipitates an impressive array of cardiopulmonary, endocrine and renal reflex responses. However, the cellular mechanisms by which these stimuli generate the increased CB neural output are only poorly understood. Central to the understanding of these mechanisms is the determination of which agents are released within the CB in response to hypoxia, and serve as the stimulating transmitter(s) for chemosensory nerve endings. Acetylcholine (ACh) has been proposed as such an agent from the outset, but this proposal has been, and remains, controversial. The present study tests two hypotheses: (1) The CB releases ACh under normoxic/normocapnic conditions; and (2) The amount released increases during hypoxia and other conditions known to increase neural output from the CB. These hypotheses were tested in 12 experiments in which both CBs were removed from the anesthetized cat and incubated at 37°C in a physiological salt solution while the solution was bubbled with four different concentrations of oxygen and carbon dioxide. The incubation medium was exchanged at 10 min intervals for 30 min (three periods of incubation). The medium was analyzed with high performance liquid chromatography-electrochemical detection for ACh content. Normoxic/normocapnic conditions (21% O₂/6% CO₂) produced a total of 0.639±0.106 pmol/150 μl (mean±S.E.M.; n=12). All stimulating conditions produced larger total outputs: 4% O₂/2% CO₂ produced 1.773±0.46 pmol/150 μl; 0% O₂/5% CO₂, 0.868±0.13 pmol/150 μl; 4% O₂/10% CO₂, 1.077±0.21 pmol/150 μl. These three amounts were significantly greater than the normoxic/normocapnic condition, but indistinguishable among themselves. Further, the amount of ACh released did not diminish over the 30 min of stimulation.These data support the concept that during hypoxia ACh functions as a stimulating transmitter in the CB, and are consistent with the earlier reports of cholinergic enzymes and receptors found in the CB.     

Contribution of Kca channel activation to hypoxic cerebrovasodilation does not involve NO

Although nitric oxide (NO) and calcium sensitive K⁺ channel (K_ca) activation contribute to hypoxic pial artery dilation in the piglet, responses to the NO releasers SNP and SNAP are unchanged by the K_ca channel antagonist iberiotoxin. These data suggest that NO does not elicit dilation via K_ca channel activation. The present study was designed to determine if dilation by K_ca channel activation is mediated by NO in newborn pigs equipped with a closed cranial window. NS1619 (10⁻⁸, 10⁻⁶ M), a K_ca agonist, produced dilation that was unchanged by the NO synthase inhibitor, -NNA (10⁻⁶ or 10⁻³ M) (11±1 and 20±1 vs. 11±1 and 18±1% before and after -NNA 10⁻³ M). NS1619 dilation also was not associated with increased CSF cGMP and was unchanged by Rp 8-Bromo cGMPs, a cGMP antagonist (9±1 and 17±1 vs. 9±1 and 16±2% before and after Rp 8-Bromo cGMPs 10⁻⁵ M). Iberiotoxin (10⁻⁷ M) attenuated hypoxic dilation but hypoxia associated CSF cGMP release was unchanged (418±11 and 897±31 vs. 419±10 and 896±25 fmol/ml for control and moderate hypoxia before and after iberiotoxin). Coadministration of -NNA with iberiotoxin further decremented hypoxic pial dilation and blocked the hypoxia-associated rise in CSF cGMP. These data show that pial artery dilation by K_ca channel activation is not mediated by NO/cGMP. Further, these data suggest that NO and the K_ca channel act at different sites in their contributions to hypoxic pial artery dilation.     

Neuroprotection of mild hypothermia: differential effects

To estimate whether mild hypothermia during repetitive hypoxia provides a neuroprotective effect on brain tissue, hippocampal slice preparations were subjected to repetitive hypoxic episodes under different temperature conditions. Slices of guinea pig hippocampus (n=40) were placed at the interface of artificial cerebrospinal fluid (aCSF) and gas (normoxia: 95% O₂, 5% CO₂; hypoxia: 95% N₂, 5% CO₂). Evoked potentials (EP) and direct current (DC) potentials were recorded from hippocampal CA1 region. Slices were subjected to two repetitive hypoxic episodes under the following temperature conditions: (A) 34°C/34°C, (B) 30°C/30°C and (C) 34°C/30°C. Hypoxic phases lasted until an anoxic terminal negativity (ATN) occurred. The recovery after first hypoxia lasted 30 min. Tissue function was assessed regarding the latency of ATN and the recovery of evoked potentials. The ATN latencies with protocol A (n=25) for the first and second hypoxia were 5.9±1.3 min (mean±S.E.M., 1st hypoxia) and 2.4±0.9 min (2nd hypoxia), with protocol B the latencies (n=7) were significantly longer: 25.2±7.1 min and 15.6±7.7 min. With protocol C (n=8), the latencies were 5.6±1.8 and 3.3±0.5 min. No differences were seen in the recovery of the EPs with protocols A–C. Our results suggest that a mild hypothermia is only neuroprotective if applied from an initial hypoxia onwards.     

Cerebral extracellular lactate concentration and blood flow during chemical stimulation of the nucleus tractus solitarii in anesthetized rats

The extracellular lactate concentration and blood flow in the cerebral cortex of urethane-anesthetized, paralyzed and artificially ventilated rats were monitored continuously and simultaneously using an enzyme electrode and a laser Doppler flowmeter (LDF), respectively, during chemical stimulation of the nucleus tractus solitarii (NTS) by microinjection of -glutamate (1.7 nmol 50 nl). Chemical stimulation of the NTS significantly decreased the arterial blood pressure (ABP) from 85 ± 17 to 68 ± 14 mmHg, heart rate from 418 ± 13 to 402 ± 19 beats · min⁻¹ and cerebral blood flow (CBF) by 17.9 ± 6.2% (P < 0.001). However, chemical stimulation of the NTS significantly increased the lactate concentration by 58.9 ± 17.3 μM (P < 0.001). Barostat maneuver, which held systemic ABP constant during chemical stimulation of the NTS attenuated the responses in CBF and lactate concentration by 30 and 27%, respectively. The onset of the increase in lactate concentration was delayed about 19 s after that of the CBF decrease. Circulatory lactate produced no significant change in the cerebral extracellular lactate concentration. These results indicate that chemical stimulation of the NTS induces an increase in extracellular lactate concentration in the cerebral cortex through a decrease in CBF via cerebral vasoconstriction.     

The effects of graded hypercapnia on the activation flow coupling response due to forepaw stimulation in α-chloralose anesthetized rats

Activation flow coupling (AFC), changes in cerebral blood flow (CBF) due to changes in neural activity with functional stimulation, provides the physiological basis of many neuroimaging techniques. Hypercapnia leads to an increase in CBF while neural activity remains unaffected. Laser Doppler (LD) flowmetry was used to measure CBF changes (LD_CBF) in the somatosensory cortex due to periodic electrical forepaw stimulation (4 s in duration) before and during graded hypercapnia (3% CO₂, 5% CO₂ and 10% CO₂). With increasing CO₂ concentrations, the baseline LD_CBF progressively increased. The peak height (PH) of the LD_CBF response, expressed as a percent change from the observed baseline for each hypercapnic state, significantly decreased (P<0.05) with increasing CO₂ concentrations. However, the absolute magnitude of the LD_CBF change was independent of CO₂ concentration. The temporal dynamics of the LD_CBF response during hypercapnia were significantly prolonged compared to baseline conditions (P<0.05).     

Interaction between central-peripheral chemoreflexes and cerebro-cardiovascular control

Abstract We investigated the interaction between hypoxia and hypercapnia on ventilation and on cerebro-cardio-vascular control. A group of 12 healthy subjects performed rebreathing tests to determine the ventilatory response to hypoxia, at different levels of carbon dioxide (CO₂), and to normoxic hypercapnia.Oxygen saturation (SaO₂), end-tidal CO₂ (et-CO₂), minute ventilation, blood pressure, R-R interval and mid-cerebral artery flow velocity (MCFV) were continuously recorded. The hypoxic ventilatory response significantly increased under hypercapnia and decreased under hypocapnia (slopes L/min/% Sa O₂: –0.33±0.05, –0.74±0.02 and –1.59±0.3, p<0.0001, in hypocapnia, normocapnia and hypercapnia, respectively). At similar degrees of ventilation, MCFV increased more markedly during normocapnic hypoxia than normoxic hypercapnia; the slopes linking MCFV to hypoxia remained unchanged at increasing levels of et-CO₂, whereas the regression lines were shifted upward. The R-R interval decreased more markedly during normocapnic hypoxia than normoxic hypercapnia and the arterial baroreflex sensitivity was decreased only by hypoxia. Cardiovascular responses to hypoxia were not affected by different levels of et-CO₂. We conclude that concomitant hypoxia and hypercapnia, while increasing ventilation synergistically, exert an additive effect on cerebral blood flow. Increased sympathetic activity (and reduced baroreflex sensitivity) is one of the mechanisms by which hypoxia stimulates cardiac sympathetic activity.     

Effect of nimodipine on the autoregulation of cerebral blood flow studied by laser-Doppler flowmetry

The present work examines whether nimodipine impairs autoregulation of CBF during hypotension. The CBF of 16 anesthetized rabbits was measured with a laser-Doppler flowmetry probe placed on the external surface of a plexiglas window, chronically inserted in the skull. Autoregulation was triggered by aortic bleeding. First, the effects of three doses of nimodipine (1, 3 and 10 μg/kg) and the solvent were studied in 10 rabbits in which MABP was maintained at 50 mmHg for one minute. Second, 10 μg/kg i.v. nimodipine was administered to 6 rabbits in which MABP was kept at 30 mmHg for one minute. Before bleeding, the 10 μg/kg dose significantly decreased MABP (from 96 ± 11mmHg to 81 ± 11mmHg, P < 0.01) and increased CBF (from 104 ± 20%to147 ± 25%, P < 0.01) as compared to the solvent. In the first set of experiments, only the 10 μg/kg dose suppressed the autoregulatory vasodilation, but CBF was not different from control (84 ± 17%versus87 ± 12%), probably because of the previous induced vasodilation. In the second set of experiments, active vasodilation occurred and the CBF during hypotension was not different from control (72 ± 26%versus65 ± 11%). We conclude that under nimodipine the triggering of the active autoregulatory vasodilation is dependent on both the severity of hypotension and the previous nimodipine-induced vasodilation.     

Hypotension dilates pial arteries by KATP and Kca channel activation

Hypotension induced pial artery dilation is prostaglandin-dependent in the newborn pig. Prostaglandins, in turn, elicit vasodilation through cGMP and cAMP dependent mechanisms and K⁺ channel activation contributes to cyclic nucleotide induced vasodilation. The present study was designed to characterize the role of ATP sensitive (K_ATP) and calcium sensitive (K_ca) channel activation in hypotension induced pial artery dilation in newborn pigs equipped with a closed cranial window. Glibenclamide and iberiotoxin, K_ATP and K_ca channel antagonists, attenuated hypotension induced dilation (36±1 vs. 14±2% before and after iberiotoxin). Combined administration of these K⁺ channel antagonists eliminated the vascular response. Hypotension induced dilation was associated with elevated cerebrospinal fluid (CSF) cAMP but not cGMP concentration (1023±29 vs. 1566±39 fmol/ml for cAMP). L-NNA, a nitric oxide (NO) synthase inhibitor, and Rp 8-Br cGMPs, a protein kinase G inhibitor, had no effect but Rp 8-Br cAMPs, a protein kinase A inhibitor, attenuated hypotensive dilation (35±1 vs. 16±2% before and after Rp 8-Br cAMPs). Dilation by the cAMP analogue 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) was attenuated by glibenclamide and iberiotoxin (8±1 and 17±1 vs. 4±1 and 9±1% before and after glibenclamide). These data show that both K_ATP and K_ca channel activation contribute to hypotension induced dilation. These data suggest that dilation during hypotension results from the sequential release of prostaglandins and cAMP, which, in turn, activates both the K_ATP and K_ca channel.     

Brain blood flow during hypercapnia in fish: no role of nitric oxide

Very little is known about the regulation of cerebral blood flow (CBF) in lower vertebrates, especially fish. In mammals, hypercapnia causes cerebral vasodilation and increased CBF through mechanisms that involve the production of nitric oxide (NO). We have used epi-illumination microscopy in vivo to observe effects of hypercapnia on venular erythrocyte velocity, used as an index of CBF velocity, in rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius). Rainbow trout exposed to a pCO₂ of 7.5 mmHg displayed a small increase of CBF velocity in two out of five fishes, while dorsal aortic blood pressure (P_DA) did not change. Exposing trout to a pCO₂ of 22.5 mmHg, resulted in an 80% increase in CBF velocity and a 21% increase in P_DA. Trout exposed to a pCO₂ of 75 mmHg showed an additional increase in blood pressure, while no further increase was seen in CBF velocity compared to a pCO₂ of 22.5 mmHg. By contrast, no change in CBF velocity was seen in crucian carp, even at a pCO₂ of 75 mmHg. None of the circulatory changes seen in the trout could be blocked by superfusing the brain surface with the NO synthase blocker N^G-nitro- -arginine. The results point at striking species differences in the responses of CBF and P_DA to hypercapnia in fish, and that the hypercapnia induced increase in CBF velocity seen in rainbow trout is independent of NO production.     

Effects of birth asphyxia on neonatal hippocampal structure and function in the spiny mouse

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term = 39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5 min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP₃R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.     

l-Arginine ameliorates recirculation and metabolic derangement in brain ischemia in hypertensive rats

The effects of

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-arginine (a precursor of nitric oxide, NO) on cerebral blood flow (CBF), cerebrovascular resistance (CVR) and metabolites in the ischemic brain were examined in spontaneously hypertensive rats with bilateral carotid artery occlusion for 30 min followed by 60 min-recirculation. The administration of

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-arginine (300 mg/kg, i.v.) increased the CBF by an average of 11 ml·100 g⁻¹·min⁻¹ (P<0.05 vs. at rest), and N^ω-nitro-

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-arginine (

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-NNA, an inhibitor of NO synthase, 5 mg/kg, i.v.) reduced the CBF by 5–6 ml·100 g⁻¹·min⁻¹ with increase in the mean arterial pressure by 26 mmHg. During ischemia the CBF significantly decreased to below 8% of the resting values in all rats. The largest blood flow in postischemic hyperemia was 171±9% of the resting CBF in the rats with

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-arginine (P<0.05 vs.

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-NNA and saline), followed by 126±5 with saline and 109±3 with

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-NNA. The CVR at 60 min of recirculation was 3.291±0.144 mmHg·ml⁻¹·100 g⁻¹·min⁻¹ in the rats with saline, remained low level of 2.711±0.124 with

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-arginine (P<0.01 vs.

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-NNA and P<0.05 vs. saline) and in contrast, significantly increased to 5.732±0.184 with

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-NNA (P<0.01 vs.

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-arginine and saline, respectively). Tissue lactate with saline increased 2.3-fold at 60 min of recirculation, whereas the increase was inhibited to 1.4-fold after

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-arginine treatment (P<0.01 vs.

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-NNA) and in contrast, significantly increased 5.7-fold with

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-NNA. The ATP and glucose levels were better preserved in the rats with

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-arginine than in those with

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-NNA or saline. These findings support that the enhanced postischemic hyperemia is beneficial to the ischemic brain and the administration of

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-arginine may be potentially useful for the treatment of acute stroke.     

Controlled Hypercapnia Enhances Cerebral Blood Flow and Brain Tissue Oxygenation After Aneurysmal Subarachnoid Hemorrhage: Results of a Phase 1 Study

Background

This study investigated if cerebral blood flow (CBF) regulation by changes of the arterial partial pressure of carbon dioxide (PaCO₂) can be used therapeutically to increase CBF and improve neurological outcome after subarachnoid hemorrhage (SAH).

Methods

In 12 mechanically ventilated poor-grade SAH-patients, a daily trial intervention was performed between day 4 and 14. During this intervention, PaCO₂ was decreased to 30 mmHg and then gradually increased to 40, 50, and 60 mmHg in 15-min intervals by modifications of the respiratory minute volume. CBF and brain tissue oxygen saturation (StiO₂) were the primary and secondary endpoints. Intracranial pressure was controlled by an external ventricular drainage.

Results

CBF reproducibly decreased during hyperventilation and increased to a maximum of 141 ± 53 % of baseline during hypercapnia (PaCO₂ 60 mmHg) on all days between day 4 and 14 after SAH. Similarly, StiO₂ increased during hypercapnia. CBF remained elevated within the first hour after resetting ventilation to baseline parameters and no rebound effect was observed within this time-span. PaCO₂-reactivities of CBF and StiO₂ were highest between 30 and 50 mmHg and slightly decreased at higher levels.

Conclusion

CBF and StiO₂ reproducibly increased by controlled hypercapnia of up to 60 mmHg even during the period of the maximum expected vasospasm. The absence of a rebound effect within the first hour after hypercapnia indicates that an improvement of the protocol is possible. The intervention may yield a therapeutic potential to prevent ischemic deficits after aneurysmal SAH.