Chromosomal substitution-dependent differences in cardiovascular responses to sodium pentobarbital.

In this study we addressed initial laboratory observations of enhanced cardiovascular sensitivity to sodium pentobarbital (PTB) in normotensive Dahl Salt Sensitive rats (SS) compared to Brown Norway (BN) rats. We also used unique consomic (chromosomal substitution) strains to confirm preliminary observations that such differences were related to chromosome 13. Increasing concentrations of PTB were administered sequentially to SS, BN, and SS strains with BN chromosomal substitutions until the point of cardiovascular collapse. Both spontaneous and controlled ventilation were studied. The effect of large (450 microg/mL) and small (35 microg/mL) concentrations of PTB on in situ transmembrane potential of mesenteric arterial vascular smooth muscle (VSM) cells was also measured in these animals with local sympathetic innervation both intact and eliminated. An analysis of variance was used to identify significant differences among groups. Despite virtually identical plasma clearance of PTB, cardiovascular collapse occurred at approximately 35%-45% smaller cumulative doses of administered PTB in SS and other strains compared with BN and SS.13BN (introgression of BN chromosome 13 into an SS) in both spontaneous and controlled ventilation. In neurally intact preparations, large dose PTB-induced VSM hyperpolarization was 4-5 times greater than the small dose in SS and SS.16BN but not in BN and SS.13BN strains. Denervation eliminated this strain difference. These results suggest that enhanced cardiovascular sensitivity to PTB in SS rats is related to greater hyperpolarization of VSM transmembrane potential in resistance vessels and this effect is associated with chromosome 13.

Sevoflurane depresses glutamatergic neurotransmission to brainstem inspiratory premotor neurons but not postsynaptic receptor function in a decerebrate dog model.

BACKGROUND: Inspiratory bulbospinal neurons in the caudal ventral medulla are premotor neurons that drive motoneurons, which innervate pump muscles such as the diaphragm and external intercostals. Excitatory drive to these neurons is mediated by N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors and is modulated by an inhibitory gamma-aminobutyric acid type A (GABAA)ergic input. The authors investigated the effect of sevoflurane on these synaptic mechanisms in decerebrate dogs. METHODS: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 minimum alveolar concentration sevoflurane on extracellularly recorded activity of single neurons was measured during localized picoejection of the GABAA receptor blocker bicuculline and the glutamate agonists AMPA and NMDA. Complete blockade of the GABAAergic mechanism by bicuculline allowed differentiation between the effects of sevoflurane on overall GABAAergic inhibition and on overall glutamatergic excitation. The neuronal responses to exogenous AMPA and NMDA were used to estimate the anesthetic effect on postsynaptic glutamatergic neurotransmission. RESULTS: One minimum alveolar concentration sevoflurane depressed the spontaneous activity of 23 inspiratory premotor neurons by (mean +/- SD) 30.0 +/- 21.0% (P < 0.001). Overall glutamatergic excitation was depressed 19.2 +/- 18.5% (P < 0.001), whereas overall GABAAergic inhibition was enhanced by 11.9 +/- 25.1% (P < 0.05). The postsynaptic responses to exogenous AMPA and NMDA did not change. CONCLUSION: One minimum alveolar concentration depressed the activity of inspiratory premotor neurons by a reduction of glutamatergic excitation and an increase in overall inhibition. The postsynaptic AMPA and NMDA receptor response was unchanged. These findings contrast with studies in inspiratory premotor neurons where halothane did not change overall inhibition but significantly reduced the postsynaptic glutamate receptor response.

Sevoflurane enhances gamma-aminobutyric acid type A receptor function and overall inhibition of inspiratory premotor neurons in a decerebrate dog model.

BACKGROUND: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic gamma-aminobutyric acid type A (GABAA)ergic inhibition. In a previous study, 1 minimum alveolar concentration (MAC) sevoflurane depressed overall glutamatergic excitatory drive and enhanced overall GABAAergic inhibitory drive to the neurons. This study investigated in further detail the effects of sevoflurane on GABAAergic inhibition by examining postsynaptic GABAA receptor activity in these neurons. METHODS: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC sevoflurane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor antagonist bicuculline and the GABAA agonist muscimol. Complete blockade of GABAAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABAA receptor function. RESULTS: One MAC sevoflurane depressed the spontaneous activity of 21 inspiratory premotor neurons by (mean +/- SD) 32.6 +/- 20.5% (P < 0.001). Overall excitatory drive was depressed 17.9 +/- 19.8% (P < 0.01). Overall GABAAergic inhibition was enhanced by 18.5 +/- 18.2% (P < 0.001), and the postsynaptic GABAA receptor function was increased by 184.4 +/- 121.8% (n = 20; P < 0.001). CONCLUSION: One MAC sevoflurane greatly enhanced GABAA receptor function on inspiratory premotor neurons and increased overall synaptic inhibition but to a smaller extent, indicating that the presynaptic inhibitory input was also reduced. Therefore, the anesthetic depression of spontaneous inspiratory premotor neuronal activity by 1 MAC sevoflurane in vivo is due to a combined effect on the two major ionotropic synaptic neurotransmitter systems with a decrease in overall glutamatergic excitation and a strong enhancement of postsynaptic GABAA receptor function.

Halothane enhances gamma-aminobutyric acid receptor type A function but does not change overall inhibition in inspiratory premotor neurons in a decerebrate dog model.

BACKGROUND: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic gamma-aminobutyric acid type A (GABA(A))-mediated (GABA(A)ergic) inhibition. In a previous study, 1 minimum alveolar concentration (MAC) halothane depressed overall glutamatergic excitatory drive but did not change overall inhibitory drive to the neurons. This study investigated in further detail the effects of halothane on GABA(A)ergic inhibition by examining postsynaptic GABA(A) receptor activity in these neurons. METHODS: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC halothane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABA(A) receptor antagonist bicuculline and the GABA(A) agonist muscimol. Complete blockade of GABAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABA(A) receptor function. RESULTS: One minimum alveolar concentration halothane depressed the spontaneous activity of 19 inspiratory premotor neurons by 22.9 +/- 29.1% (mean +/- SD; P < 0.01). Overall excitatory drive was depressed 23.6 +/- 16.9% (P < 0.001). Overall GABAergic inhibition was not changed (+8.7 +/- 27.5%; P = 0.295), but the postsynaptic GABA(A) receptor function was increased by 110.3 +/- 97.5% (P < 0.001). CONCLUSION: One minimum alveolar concentration halothane greatly enhanced GABA(A) receptor function on inspiratory premotor neurons but did not change overall synaptic inhibition, indicating that the presynaptic inhibitory input was reduced. Therefore, the anesthetic depression of spontaneous inspiratory premotor neuronal activity in the intact brainstem respiratory network is mainly due to a decrease in overall glutamatergic excitation.

Dynorphinergic mechanism mediating endomorphin-2-induced antianalgesia in the mouse spinal cord.

We have previously demonstrated that both endomorphin-1 (EM-1) and endomorphin-2 (EM-2) at high doses (1.75-35 nmol) given intrathecally (i.t.) or intracerebroventricularly produce antinociception by stimulation of mu-opioid receptors. Now, we report that EM-2 at small doses (0.05-1.75 nmol), which injected alone did not produce antinociception, produces anti-analgesia against opioid agonist-induced antinociception. The tail-flick (TF) response was used to test the antinociception in male CD-1 mice. Intrathecal pretreatment with EM-2 (0.02-1.75 nmol) 45 min before i.t. morphine (3.0 nmol) injection dose dependently attenuated morphine-induced TF inhibition. On the other hand, a similar dose of EM-1 (1.64 nmol) failed to produce any antianalgesic effect. The EM-2 (1.75 nmol)-produced anti-analgesia against morphine-induced TF inhibition was blocked by i.t. pretreatment with the mu-opioid antagonist naloxone or 3-methoxynaltrexone, but not delta-opioid receptor antagonist naltrindole, kappa-opioid receptor antagonist nor-binaltorphimine, or N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The EM-2-induced antianalgesic effect against morphine-induced TF inhibition was blocked by i.t. pretreatment with antiserum against dynorphin A(1-17), but not beta-endorphin, [Met]-enkephalin, [Leu]-enkephalin, or cholecystokinin antiserum (200 microg each). The i.t. EM-2 pretreatment also attenuated the TF inhibition induced by other mu-opioid agonists, [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin, EM-1 and EM-2, delta-opioid agonist deltorphin II, and kappa-opioid agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H). It is concluded that EM-2 at subanalgesic doses presumably stimulates a subtype of mu-opioid receptor and subsequently induces the release of dynorphin A(1-17) to produce antianalgesic effects against mu-, delta-, or kappa-agonists-induced antinociception. The EM-2-induced antianalgesia is not mediated by the release of [Met]-enkephalin, [Leu]-enkephalin, beta-endorphin, or cholecystokinin, nor does it involve kappa- or delta-opioid or NMDA receptors in the spinal cord.

Cholinergic reversal of isoflurane anesthesia in rats as measured by cross-approximate entropy of the electroencephalogram.

BACKGROUND: Pharmacologic modulation of the state of consciousness is of interest for clinical practice and for a better understanding of anesthetic mechanisms. The cholinergic activating system is an important regulator of the state of consciousness during general anesthesia. Entropy of the electroencephalogram has been proposed as a promising measure of anesthetic depth. The authors have shown that volatile anesthetics decrease cross-approximate entropy (C-ApEn) of the bihemispheric frontal electroencephalogram in rats. The effect of cholinergic agents on C-ApEn has not been examined. Here, the authors test the hypothesis that cholinergic activation reverses the effect of isoflurane anesthesia on C-ApEn. METHODS: An electroencephalogram in the 1- to 100-Hz range was recorded bipolarly, with epidural leads from the frontal cortex of both hemispheres, and used to calculate C-ApEn, which reflects statistical independence of bihemispheric electroencephalographic activity. Cholinesterase inhibitor, neostigmine (25 mug), or the muscarinic agonist oxotremorine (25 mug) were infused intracerebroventricularly while the rats were inhaling 1.0% (0.7 minimum alveolar concentration) isoflurane. In other animals, isoflurane was lowered to 0.4% (0.3 minimum alveolar concentration) to assess the electroencephalogram in a sedated, waking state. RESULTS: At 1.0% isoflurane, C-ApEn decreased by 54% compared with that at 0.4%, but the motor reflex response to tail pinch was still present. Cholinergic agents reversed the electroencephalogram-depressant effect of isoflurane, i.e., C-ApEn rose to the level measured at 0.4% isoflurane. The rise in C-ApEn was paralleled by the appearance of spontaneous limb and orofacial explorative movements, suggesting a return of consciousness. In contrast, cholinergic agents fully blocked the motor reflex to tail pinch. CONCLUSIONS: C-ApEn of the bihemispheric electroencephalogram correlates with the return of spontaneous motor signs but not with the nociceptive reflex. Cerebral cholinergic activation dissociates central and peripheral anesthetic effects. C-ApEn, a novel measure of interhemispheric electroencephalogram independence, is a promising correlate of depth of sedation and state of consciousness.

Reactive oxygen species precede the epsilon isoform of protein kinase C in the anesthetic preconditioning signaling cascade.

BACKGROUND: Protein kinase C (PKC) and reactive oxygen species (ROS) are known to have a role in anesthetic preconditioning (APC). Cardiac preconditioning by triggers other than volatile anesthetics, such as opioids or brief ischemia, is known to be isoform selective, but the isoform required for APC is not known. The authors aimed to identify the PKC isoform that is involved in APC and to elucidate the relative positions of PKC activation and ROS formation in the APC signaling cascade. METHODS: Isolated guinea pig hearts were subjected to 30 min of ischemia and 120 min of reperfusion. Before ischemia, hearts were either untreated or treated with sevoflurane (APC) in the absence or presence of the nonspecific PKC inhibitor chelerythrine, the PKC-delta inhibitor PP101, or the PKC-epsilon inhibitor PP149. Spectrofluorometry and the fluorescent probes dihydroethidium were used to measure intracellular ROS, and effluent dityrosine as used to measure extracellular ROS release. RESULTS: Previous sevoflurane exposure protected the heart against ischemia-reperfusion injury, as previously described. Chelerythrine or PP149 abolished protection, but PP101 did not. ROS formation was observed during sevoflurane exposure and was not altered by any of the PKC inhibitors. CONCLUSIONS: APC is mediated by PKC-epsilon but not by PKC-delta. Furthermore, PKC activation probably occurs downstream of ROS generation in the APC signaling cascade.

Halothane depresses glutamatergic neurotransmission to brain stem inspiratory premotor neurons in a decerebrate dog model.

BACKGROUND: Inspiratory bulbospinal neurons in the caudal ventral medulla are premotor neurons that drive phrenic motoneurons and ultimately the diaphragm. Excitatory drive to these neurons is mediated by N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors and modulated by an inhibitory gamma-aminobutyric acid(A) (GABA(A))ergic input. The authors investigated the effect of halothane on these synaptic mechanisms in decerebrate dogs. METHODS: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 minimum alveolar concentration (MAC) halothane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABA(A) receptor blocker bicuculline and the glutamate agonists AMPA and NMDA. Complete blockade of the GABA(A)ergic mechanism by bicuculline allowed differentiation between the effects of halothane on overall GABA(A)ergic inhibition and on overall glutamatergic excitation. The neuronal responses to exogenous AMPA and NMDA were used to estimate the anesthetic effect on postsynaptic glutamatergic neurotransmission. RESULTS: Halothane, 1 MAC, depressed the spontaneous activity of 21 inspiratory neurons by 20.6 +/- 18.0% (mean +/- SD; P = 0.012). Overall glutamatergic excitation was depressed 15.4 +/- 20.2% (P = 0.001), while overall GABA(A)ergic inhibition did not change. The postsynaptic responses to exogenous AMPA and NMDA were also depressed by 18.6 +/- 35.7% (P = 0.03) and 22.2 +/- 26.2% (P = 0.004), respectively. CONCLUSION: Halothane, 1 MAC, depressed the activity of inspiratory premotor neurons by a reduction of glutamatergic excitation. Overall inhibitory drive did not change. The postsynaptic AMPA and NMDA receptor response was significantly reduced. These findings contrast with studies in expiratory premotor neurons in which overall inhibition was significantly increased by halothane and there was no reduction in the postsynaptic glutamate receptor response.

Isoflurane-induced cerebral hyperemia is partially mediated by nitric oxide and epoxyeicosatrienoic acids in mice in vivo.

BACKGROUND: Despite intense investigation, the mechanism of isoflurane-induced cerebral hyperemia is unclear. The current study was designed to determine the contributions of neuronal nitric oxide synthase, prostaglandins, and epoxyeicosatrienoic acids to isoflurane-induced cerebral hyperemia. METHODS: Regional cerebral cortical blood flow was measured with laser Doppler flowmetry during stepwise increases of isoflurane from 0.0 to 1.2, 1.8, and 2.4 vol% end-tidal concentration in alpha-chloralose-urethane-anesthetized, C57BL/6 mice before and 45 min after administration of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI, 40 mg/kg, intraperitoneal), the cyclooxygenase inhibitor indomethacin (INDO, 10 mg/kg, intravenous), and the cytochrome P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 20 mg/kg, intravenous). RESULTS: Isoflurane increased regional cerebral cortical blood flow by 9 +/- 3, 46 +/- 21, and 101 +/- 26% (SD) at 1.2, 1.8, and 2.4 vol%, respectively. The increases in regional cerebral cortical blood flow were significantly (*P < 0.05) smaller after 7-NI (5 +/- 6, 29 +/- 19*, 68 +/- 15%*) or PPOH (4 +/- 8, 27 +/- 17*, 67 +/- 30%*), but not after administration of INDO (4 +/- 4, 33 +/- 18 [NS], 107 +/- 35% [NS]). The effect of combined treatment with 7-NI, PPOH, and INDO was not additive and was equal to that of either 7-NI or PPOH alone (5 +/- 5, 30 +/- 12*, 76 +/- 24%*). Chronic treatment of mice for 5 days with 7-NI (2 x 40 mg/kg, intraperitoneal) produced similar decreases in regional cerebral cortical blood flow as those seen with acute administration. Neither PPOH nor INDO conferred a significant additional block of the hyperemia in these animals. CONCLUSIONS: Nitric oxide and epoxyeicosatrienoic acids contribute to isoflurane-induced hyperemia. However, only approximately one third of the cerebral hyperemic response to isoflurane is mediated by autacoids. The remaining part of this response appears to be mediated by a direct action of isoflurane on smooth muscle by some yet-unknown mechanism.

Effects of halothane and sevoflurane on inhibitory neurotransmission to medullary expiratory neurons in a decerebrate dog model.

BACKGROUND: In canine expiratory bulbospinal neurons, 1 minimum alveolar concentration (MAC) halothane and sevoflurane reduced the glutamatergic excitatory drive at a presynaptic site and enhanced the overall gamma-aminobutyric acid (GABA)-mediated inhibitory input. The authors investigated if this inhibitory enhancement was mainly caused by postsynaptic effects. METHODS: Two separate anesthetic studies were performed in two sets of decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC halothane or sevoflurane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor agonist muscimol and the GABAA receptor antagonist bicuculline. Complete blockade of GABAA-mediated inhibition with bicuculline was used to assess the prevailing overall inhibitory input to the neuron. The neuronal response to muscimol was used to estimate the anesthetic effect on postsynaptic GABAA receptor function. RESULTS: Halothane at 1 MAC depressed the spontaneous activity of 12 expiratory neurons 22.2 +/- 14.8% (mean +/- SD) and overall glutamatergic excitation 14.5 +/- 17.9%. Overall GABA-mediated inhibition was enhanced 14.1 +/- 17.9% and postsynaptic GABAA receptor function 74.2 +/- 69.2%. Sevoflurane at 1 MAC depressed the spontaneous activity of 23 neurons 20.6 +/- 19.3% and overall excitation 10.6 +/- 21.7%. Overall inhibition was enhanced 15.4 +/- 34.0% and postsynaptic GABAA receptor function 65.0 +/- 70.9%. The effects of halothane and sevoflurane were not statistically different. CONCLUSION: Halothane and sevoflurane at 1 MAC produced a small increase in overall inhibition of expiratory premotor neuronal activity. The increase in inhibition results from a marked enhancement of postsynaptic GABAA receptor function that is partially offset by a reduction in presynaptic inhibitory input by the anesthetics.

Focal cerebral ischemia in rats produced by intracarotid embolization with viscous silicone.

Many factors contribute to the severity of neuronal cell death and the functional outcome in stroke. We describe an embolic model of focal cerebral ischemia in the rat that does not require craniotomy and is compatible with continuous measurement of regional CBF using multichannel laser Doppler flow (LDF) technique. Either a 22 microliters (large lesion) or 11 microliters (small lesion) bolus of viscous silicone was injected cephalad into the internal carotid artery. Upon injection, LDF decreased abruptly, most severely in the parietal cortex (-74% +/- 5%) in the large lesion and in the occipital cortex (-69% +/- 10%) in the small lesion model. Over the first hour, post-embolization LDF improved in most areas (e.g. -48% +/- 9% parietal, large lesion) but declined in the small lesion group in the occipital region (-81% +/- 8%). CBF measured by [C]14-IAP autoradiography 1 h post-embolization in the large lesion model demonstrated near-hemispheric ischemia (70% of hemisphere) with sparing of cingulate cortex. Autoradiography demonstrated that ischemia in the small lesion was largely cortical. Light microscopy of brains embolized with 11 microliters of dyed silicone showed filling of pial vessels with no silicone in the Circle of Willis or parenchyma. No animals in the large lesion group survived 24 h. Thirteen of 15 animals in the small lesion group survived for two weeks with resolution of initial hemiplegia, ocular asymmetry and weight loss. Hematoxylin-eosin staining two weeks post-embolization showed signs of severe hypoxia and infarction. In conclusion, the intracarotid silicone embolization technique produces a titrable, reproducible permanent ischemic injury by blocking perfusion in the pial circulation, and is amenable to multisite monitoring with laser Doppler flowmetry. The smaller embolus produces cortical infarction with high rate of survival and neurological recovery.

Alteration of vascular capacitance and blood flow distribution during halothane anesthesia.

We examined the effect of halothane on systemic vascular capacitance as well as on systemic vascular resistance using cardiopulmonary bypass in dogs. Venous outflows from two different vascular beds, the splanchnic and extrasplanchnic beds, were also measured. Under constant perfusion flow and constant central venous pressure, a change in reservoir blood volume inversely represented a change in systemic blood volume and then in systemic vascular capacitance, and a change in mean arterial pressure directly reflected a change in systemic vascular resistance. Administration of 1% and 2% halothane produced the blood concentrations of 0.58±0.14 mM and 1.34±0.06 mM, respectively. Systemic vascular resistance decreased by 12±6% and 40±4% during 1% and 2% halothane, respecitively. Systemic blood volume increased by 7±2 ml·kg-1 and 15±4 ml·kg-1 during 1% and 2% halothane, respectively. Halothane did not cause significant blood flow redistribution between the splanchnic and extrasplanchnic vascular beds. These results suggest that halothane causes an increase in systemic vascular capacitance as well as a decrease in systemic vascular resistance. This increase in vascular capacitance may contribute in part to a decrease in cardiac output during halothane anesthesia.