Douglas L. Feinstein

Lipid Emulsion Infusion Rescues Dogs From Bupivacaine-Induced Cardiac Toxicity

Background and Objectives We previously demonstrated in rats that intravenous infusion of a lipid emulsion increases survival in resuscitation from severe bupivacaine cardiac toxicity. The present studies were undertaken to determine if this method is similarly effective in a non-rodent model using a larger animal. Methods Bupivacaine, 10 mg/kg, was administered intravenously over 10 seconds to fasted dogs under isoflurane general anesthesia. Resuscitation included 10 minutes of internal cardiac massage followed with either saline or 20% lipid infusion, administered as a 4-mL/kg bolus followed by continuous infusion at 0.5 mL/kg/min for 10 minutes. Electrocardiogram (EKG), arterial blood pressure (BP), and myocardial pH (pHm) and pO2 (pmO2) were continuously measured. Results Survival after 10 minutes of unsuccessful cardiac massage was successful for all lipid-treated dogs (n = 6), but with no survivors in the saline controls (n = 6) (P < .01). Hemodynamics, PmO2, and pHm were improved during resuscitation with lipid compared with saline treatment in which dogs did not recover. Conclusions We found that infusing a lipid emulsion during resuscitation from bupivacaine-induced cardiac toxicity substantially improved hemodynamics, pmO2, and pHm and increased survival in dogs.

Peroxisome proliferator-activated receptor-γ agonists prevent experimental autoimmune encephalomyelitis

The development of clinical symptoms in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) involves T‐cell activation and migration into the central nervous system, production of glial‐derived inflammatory molecules, and demyelination and axonal damage. Ligands of the peroxisome proliferator‐activated receptor (PPAR) exert anti‐inflammatory effects on glial cells, reduce proliferation and activation of T cells, and induce myelin gene expression. We demonstrate in two models of EAE that orally administered PPARγ ligand pioglitazone reduced the incidence and severity of monophasic, chronic disease in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein peptide and of relapsing disease in B10.Pl mice immunized with myelin basic protein. Pioglitazone also reduced clinical signs when it was provided after disease onset. Clinical symptoms were reduced by two other PPARγ agonists, suggesting a role for PPARγ activation in protective effects. The suppression of clinical signs was paralleled by decreased lymphocyte infiltration, lessened demyelination, reduced chemokine and cytokine expression, and increased inhibitor of kappa B (IkB) expression in the brain. Pioglitazone also reduced the antigen‐dependent interferon‐γ production from EAE‐derived T cells. These results suggest that orally administered PPARγ agonists could provide therapeutic benefit in demyelinating disease.

Heat Shock Protein 70 Suppresses Astroglial-inducible Nitric-oxide Synthase Expression by Decreasing NFκB Activation

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20-60 min at 43°C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFκB p65 subunit, suggesting perturbation of NFκB activation. A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFκB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

Lipid infusion accelerates removal of bupivacaine and recovery from bupivacaine toxicity in the isolated rat heart.

Background and Objectives: Infusion of a lipid emulsion has been advocated for treatment of severe bupivacaine cardiac toxicity. The mechanism of lipid rescue is unknown. These studies address the possibility that lipid infusion reduces cardiac bupivacaine content in the context of cardiac toxicity. Methods: We compared the effects of a 1% lipid emulsion with standard Krebs buffer after inducing asystole in isolated rat heart with 500 μmol/L bupivacaine. We compared times to first heart beat and recovery of 90% of baseline rate pressure product (RPP = heart rate × [left ventricular systolic pressure − left ventricular diastolic pressure]) between controls and hearts receiving 1% lipid immediately after bupivacaine. We also used minibiopsies to compare control bupivacaine tissue content with hearts getting lipid immediately after an infusion of radiolabeled bupivacaine. We then compared bupivacaine efflux from hearts with and without lipid infusion started 75 seconds after radiolabeled bupivacaine was administered. Results: Infusion of lipid resulted in more rapid return of spontaneous contractions and full recovery of cardiac function. Average (± SEM) times to first beat and to 90% recovery of rate pressure product were 44.6 ± 3.5 versus 63.8 ± 4.3 seconds (P < .01) and 124.7 ± 12.4 versus 219.8 ± 25.6 seconds (P < .01) for lipid and controls, respectively. Lipid treatment resulted in more rapid loss of bupivacaine from heart tissue (P < .0016). Late lipid infusion, 75 seconds after bupivacaine infusion ended, increased the release of bupivacaine measured in effluent for the first 15-second interval compared with controls (183 vs. 121 nmol, n = 5 for both groups, P < .008). Conclusions: Lipid emulsion speeds loss of bupivacaine from cardiac tissue while accelerating recovery from bupivacaine-induced asystole. These findings are consistent with the hypothesis that bupivacaine partitions into the emulsion and supports the concept of a “lipid sink.” However, the data do not exclude other possible mechanisms of action.

Expression and function of inducible nitric oxide synthase in neurons.

Enzymatically derived nitric oxide (NO) has been implicated in numerous physiological and pathological processes in the brain. Whereas during development NO participates in developmental and maturation processes, excess NO production in the adult in response to inflammation, injury, or trauma participates in both cell death and repair. The expression and activity of the inducible isoform of NO synthase (iNOS) play a pivotal role in sustained and elevated NO release. Recent evidence suggests that neurons can respond to proinflammatory stimuli and take part in brain inflammation. Neuronal iNOS expression has been described in different experimental settings, including cytokine stimulation of neuronal cell lines and primary neurons in vitro as well as in animal models of stroke and neurodegeneration. This article outlines different conditions leading to iNOS gene transcription and expression in neurons and neuronal cells and highlights the potential impact on human brain inflammation and neurodegeneration.

Peroxisome proliferator-activated receptor-γ agonists induce neuroprotection following transient focal ischemia in normotensive, normoglycemic as well as hypertensive and type-2 diabetic rodents

Thiazolidinediones (TZDs) are synthetic agonists of the ligand‐activated transcription factor peroxisome proliferator‐activated receptor‐γ (PPARγ). TZDs are known to curtail inflammation associated with peripheral organ ischemia. As inflammation precipitates the neuronal death after stroke, we tested the efficacy of TZDs in preventing brain damage following transient middle cerebral artery occlusion (MCAO) in adult rodents. As hypertension and diabetes complicate the stroke outcome, we also evaluated the efficacy of TZDs in hypertensive rats and type‐2 diabetic mice subjected to transient MCAO. Pre‐treatment as well as post‐treatment with TZDs rosiglitazone and pioglitazone significantly decreased the infarct volume and neurological deficits in normotensive, normoglycemic, hypertensive and hyperglycemic rodents. Rosiglitazone neuroprotection was not enhanced by retinoic acid × receptor agonist 9‐cis‐retinoic acid, but was prevented by PPARγ antagonist GW9662. Rosiglitazone significantly decreased the post‐ischemic intercellular adhesion molecule‐1 expression and extravasation of macrophages and neutrophils into brain. Rosiglitazone treatment curtailed the post‐ischemic expression of the pro‐inflammatory genes interleukin‐1β, interleukin‐6, macrophage inflammatory protein‐1α, monocyte chemoattractant protein‐1, cyclooxygenase‐2, inducible nitric oxide synthase, early growth response‐1, CCAAT/enhancer binding protein‐β and nuclear factor‐kappa B, and increased the expression of the anti‐oxidant enzymes catalase and copper/zinc‐superoxide dismutase. Rosiglitazone also increased the expression of the anti‐inflammatory gene suppressor of cytokine signaling‐3 and prevented the phosphorylation of the transcription factor signal transducer and activator of transcription‐3 after focal ischemia. Thus, PPARγ activation with TZDs might be a potent therapeutic option for preventing inflammation and neuronal damage after stroke with promise in diabetic and hypertensive subjects.

Peroxisome proliferator-activated receptor gamma agonists protect cerebellar granule cells from cytokine-induced apoptotic cell death by inhibition of inducible nitric oxide synthase

Cerebellar granule cells (CGCs) can express the inducible isoform of nitric oxide synthase (iNOS) in response to inflammatory stimuli. We demonstrate that induction of iNOS in CGCs by bacterial lipopolysaccharide and pro-inflammatory cytokines results in cell death that was potentiated by excess L-arginine and inhibited by the selective iNOS inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. The NO-mediated cell death was accompanied by increased caspase-3-like activity, DNA fragmentation and positive terminal transferase dUTP nick end labeling (TUNEL), suggesting that apoptosis mediates CGC cell death. Incubation of CGCs with the non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen or indomethacin, or with 15-deoxy-delta12,14 prostaglandin J2 (PGJ2) downregulates iNOS expression and reduces subsequent cell death. Since in other cell types, both NSAIDs and PGJ2 can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma) and downregulate cytokine levels and iNOS expression, and since CGCs express PPARgamma in vivo and in vitro, our data suggest that activation of CGC PPARgamma mediates iNOS suppression and reduced cell death. Because PPARgamma is expressed in brains of Alzheimers Disease (AD) patients, in which neuronal iNOS expression and apoptotic cell death have been described, these results may help explain the basis for the beneficial effects of NSAIDs in AD.

Induction of nitric oxide synthase in rat C6 glioma cells

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte‐derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO2 production. However, addition of either tumor necrosis factor‐a (TNF‐α) or interferon‐γ (IFN‐γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose‐dependent manner (EC50 values of 39 ng/ml of TNF‐α and 9.4 U/ml of IFN‐γ), and the effect of TNF‐α could be further potentiated (twofold) by the presence of interleukin‐1β. The simultaneous presence of TNF‐α and IFN‐γ yielded a greater response than either cytokine alone; however, the respective EC50 values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca2+‐independent conversion of l‐arginine to l‐ citrulline, with an apparent Km of 51.2 nM, and this activity could be blocked by l‐arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.

Resuscitation with Lipid versus Epinephrine in a Rat Model of Bupivacaine Overdose

Background:Lipid emulsion infusion reverses cardiovascular compromise due to local anesthetic overdose in laboratory and clinical settings. The authors compared resuscitation with lipid, epinephrine, and saline control in a rat model of bupivacaine-induced cardiac toxicity to determine whether lipid provides a benefit over epinephrine. Methods:Bupivacaine, 20 mg/kg, was infused in rats anesthetized with isoflurane, producing asystole in all subjects. Ventilation with 100% oxygen and chest compressions were begun immediately, along with intravenous treatment with 30% lipid emulsion or saline (5-ml/kg bolus plus continuous infusion at 0.5 ml · kg−1 · min−1) or epinephrine (30 &mgr;g/kg). Chest compressions were continued and boluses were repeated at 2.5 and 5 min until the native rate–pressure product was greater than 20% baseline. Electrocardiogram and arterial pressure were monitored continuously and at 10 min, arterial blood gas, central venous oxygen saturation, and blood lactate were measured. Effect size (Cohen d) was determined for comparisons at 10 min. Results:Lipid infusion resulted in higher rate–pressure product (P < 0.001, d = 3.84), pH (P < 0.01, d = 3.78), arterial oxygen tension (P < 0.05, d = 2.8), and central venous oxygen saturation (P < 0.001, d = 4.9) at 10 min than did epinephrine. Epinephrine treatment caused higher lactate (P < 0.01, d = 1.48), persistent ventricular ectopy in all subjects, pulmonary edema in four of five rats, hypoxemia, and a mixed metabolic and respiratory acidosis by 10 min. Conclusions:Hemodynamic and metabolic metrics during resuscitation with lipid surpassed those with epinephrine, which were no better than those seen in the saline control group. Further studies are required to optimize the clinical management of systemic local anesthetic toxicity.

Noradrenergic regulation of inflammatory gene expression in brain.

It is now well accepted that inflammatory events contribute to the pathogenesis of numerous neurological disorders, including multiple sclerosis (MS), Alzheimers disease (AD), Parkinsons disease, and AIDs dementia. Whereas inflammation in the periphery is subject to rapid down regulation by increases in anti-inflammatory molecules and the presence of scavenging soluble cytokine receptors, the presence of an intact blood-brain barrier may limit a similar autoregulation from occurring in brain. Mechanisms intrinsic to the brain may provide additional immunomodulatory functions, and whose dysregulation could contribute to increased inflammation in disease. The findings that noradrenaline (NA) reduces cytokine expression in microglial, astroglial, and brain endothelial cells in vitro, and that modification of the noradrenergic signaling system occurs in some brain diseases having an inflammatory component, suggests that NA could act as an endogenous immunomodulator in brain. Furthermore, accumulating studies indicate that modification of the noradrenergic signaling system occurs in some neurodiseases. In this article, we will briefly review the evidence that NA can modulate inflammatory gene expression in vitro, summarize data supporting a similar immunomodulatory role in brain, and present recent data implicating a role for NA in attenuating the cortical inflammatory response to beta amyloid protein.