Sean R. Hall

Endothelial cysteinyl leukotriene 2 receptor expression mediates myocardial ischemia-reperfusion injury.

Cysteinyl leukotrienes (CysLTs) have been implicated as inflammatory mediators of cardiovascular disease. Three distinct CysLT receptor subtypes transduce the actions of CysLTs but the role of the endothelial CysLT2 receptor (CysLT2R) in cardiac function is unknown. Here, we investigated the role of CysLT2R in myocardial ischemia-reperfusion (I/R) injury using transgenic (tg) mice overexpressing human CysLT2R in vascular endothelium and nontransgenic (ntg) littermates. Infarction size in tg mice increased 114% compared with ntg mice 48 hours after I/R; this increase was blocked by the CysLT receptor antagonist BAY-u9773. Injection of 125 I-albumin into the systemic circulation revealed significantly enhanced extravasation of the label in tg mice, indicating increased leakage of the coronary endothelium, combined with increased incidence of hemorrhage and cardiomyocyte apoptosis. Expression of proinflammatory genes such as Egr-1, VCAM-1, and ICAM was significantly increased in tg mice relative to ntg controls. Echocardiographic assessment 2 weeks after I/R revealed decreased anterior wall thickness in tg mice. Furthermore, the postreperfusion time constant tau of isovolumic relaxation was significantly increased in tg animals, indicating diastolic dysfunction. These results reveal that endothelium-targeted overexpression of CysLT2R aggravates myocardial I/R injury by increasing endothelial permeability and exacerbating inflammatory gene expression, leading to accelerated left ventricular remodeling, induction of peri-infarct zone cellular apoptosis, and impaired cardiac performance.

Endothelial Progenitor Cell and Mesenchymal Stem Cell Isolation, Characterization, Viral Transduction

Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have emerged as potentially useful substrates for neovascularization and tissue repair and bioengineering. EPCs are a heterogeneous group of endothelial cell precursors originating in the hematopoietic compartment of the bone marrow. MSCs are a rare population of fibroblast-like cells derived from the bone marrow stroma, constituting approximately 0.001-0.01% of the nucleated cells in the marrow. Both cells types have been isolated from the bone marrow. In addition, EPC can be isolated from peripheral blood as well as the spleen, and MSC has also been isolated from peripheral adipose tissue. Several approaches have been used for the isolation of EPC and MSC, including density centrifugation and magnetic bead selection. Phenotypic characterization of both cell types is carried out using immunohistochemical detection and fluorescence-activated cell sorting analysis of cell-surface molecule expression. However, the lack of specific markers for each cell type renders their characterization difficult and ambiguous. In this chapter, we describe the methods that we use routinely for isolation, characterization, and genetic modification of EPC and MSC from human, rabbit, and mouse peripheral blood and bone marrow.

Intrathecal lidocaine prevents cardiovascular collapse and neurogenic pulmonary edema in a rat model of acute intracranial hypertension.

Sympathetic hyperactivity during sudden intracranial hypertension leads to cardiovascular instability, myocardial dysfunction, and neurogenic pulmonary edema. Because spinal anesthesia is associated with sympatholysis, we investigated the protective effects of intrathecal lidocaine in a rodent model. Halothane-anesthetized rats were given a 10-&mgr;L intrathecal injection of saline (n = 10) or lidocaine 1% (n = 6). A subdural balloon catheter was inflated for 60 s to produce intracranial hypertension. Hemodynamics were monitored, and hearts and lungs were harvested for histological examination. In Saline versus Lidocaine-Treated rats, peak mean arterial blood pressure during balloon inflation was 115 ± 4 mm Hg versus 78 ± 8 mm Hg (P < 0.05), mean arterial blood pressure 30 min after balloon deflation was 47 ± 2 mm Hg versus 67 ± 3 mm Hg (P < 0.05), and lung weight was 1.54 ± 0.03 g versus 1.41 ± 0.04 g (P < 0.05), respectively. Cardiac dysrhythmias and electrocardiographic changes were more frequent in the Saline-Treated group (P < 0.05). Saline-Treated rats had extensive, hemorrhagic pulmonary edema, whereas the Lidocaine-Treated rats had only patchy areas of lung abnormality. Histological changes in the myocardium were rare, and no difference was found between the two groups. We conclude that intrathecal lidocaine prevents cardiovascular collapse and neurogenic pulmonary edema in a rat model of acute intracranial hypertension.

Nitric oxide synthase inhibitors attenuate acute and chronic morphine withdrawal response in the rat locus coeruleus: an in vivo voltammetric study

Previous studies have demonstrated that activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) associated with opioid and non-opioid drug withdrawal syndromes. Using an in vivo voltammetric approach, we have examined the role of nitric oxide (NO), which mediates NMDA receptor function, in this withdrawal-induced LC hyperactivity. In the anaesthetized rat, acute morphine treatment (10 micrograms, i.c.v.) suppressed (55.7 +/- 4.4% of baseline) the catechol oxidation current (CA-OC) recorded from the LC using differential normal pulse voltammetry (DNPV). A subsequent intravenous injection of naloxone (2 mg/kg, i.v.) reversed the drug-induced inhibition of LC response and produced an increase (118.9 +/- 2.3% of baseline) in CA-OC above baseline, indicative of an acute withdrawal response. Systemic (100 mg/kg) and intracerebroventricular (i.c.v.) (100 micrograms) pretreatment of animals with the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) blocked the naloxone-induced LC withdrawal response without influencing the inhibitory effect of morphine on LC activity. In animals chronically infused with morphine (15 micrograms/h, i.c.v., 5 days) a naloxone challenge (2 mg/kg, i.v.) produced significant increase (253.7 +/- 19.3% of baseline) in CA-OC signal. This LC withdrawal response was significantly reduced by pretreatment with L-NAME (100 micrograms, i.c.v.) or N omega-nitro-L-arginine (L-NOARG 10 micrograms, i.c.v.). In unanaesthetized animals pretreated with chronic morphine, systemic (100 mg/kg) and central L-NAME (100 micrograms) pretreatment suppressed some of the behavioural signs of withdrawal precipitated by naloxone (10 mg/kg) injection. As doses of the NOS inhibitors used in this study have previously been reported to produce significant inhibition of brain NOS activity, their effect on opioid withdrawal response most likely is due to NOS inhibition. The results of this study indicate that NO plays an intermediary role in the LC neuronal hyperactivity associated with both acute and chronic morphine withdrawal.

Excitatory action of N-methyl-d-aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl-D-aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 microliters min-1) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1 +/- 32.2% above baseline) in the catechol oxidation current (CA.OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4 +/- 6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg-1 i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA.OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) (100 micrograms) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of L-NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CA.OC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.

The Release of Spinal Prostaglandin E2 and the Effect of Nitric Oxide Synthetase Inhibition During Strychnine-induced Allodynia

The removal of spinal glycinergic inhibition by intrathecal strychnine produces an allodynia-like state in rodents. Our objective was to measure spinal prostaglandin E2 (PGE2) release during strychnine-allodynia and examine the effects of N&ohgr;-nitro-l-arginine (l-NOARG), an inhibitor of nitric oxide synthetase. Under halothane, rats were fitted with intrathecal and spinal microdialysis catheters, and microelectrodes implanted into the locus coeruleus for measurement of catechol oxidation current (CAOC) using voltammetry. Animals were then administered urethane and treated as follows: 1) baseline control 10 min, intrathecal strychnine (40 &mgr;g) 10 min, 10 min of hair deflection, and 2) 10-min control followed by intrathecal strychnine (40 &mgr;g) with hair deflection for 60 min. Spinal dialysate samples were collected for PGE2 levels determined by using immunoassay. In separate experiments, the effect of intrathecal strychnine (40 &mgr;g) followed by hair deflection was studied in rats pretreated with intrathecal l-NOARG (50 nmol). After intrathecal strychnine, hair deflection significantly increased spinal PGE2 release (619% ± 143%), locus coeruleus CAOC (181% ± 6%), and mean arterial pressure (123% ± 2%) P < 0.05. Pretreatment with intrathecal l-NOARG significantly inhibited strychnine-allodynia. In this model, hair deflection evokes spinal PGE2 release, locus coeruleus activation, and an increase in mean arterial pressure. l-NOARG pretreatment attenuated the locus coeruleus CAOC, a biochemical index of strychnine-allodynia, suggesting a mediator role of nitric oxide. A mediator role of nitric oxide is also implicated, helping to explain the pathophysiology of this allodynic pain.

Central dexmedetomidine attenuates cardiac dysfunction in a rodent model of intracranial hypertension

PurposeTo determine if central sympathetic blockade by dexmedetomidine, a selective alpha2 adrenergic receptor agonist, prevents cardiac dysfunction associated with intracranial hypertension (ICH) in a rat model.MethodsFollowing intracisternal administration of dexmedetomidine (1 μg · μl−1, 10 μL volume) or the stereoisomer levomedetomidine (1 μg· μL−1, 10 μL volume) in halothane-anesthetized rats, a subdural balloon catheter was inflated for 60 sec to produce ICH. Intracranial pressure, hemodynamic, left ventricular (LV) pressures and electrocardiographic (ECG) changes were recorded. Plasma and myocardial catecholamines and malondialdehyde (MDA) levels were measured.ResultsAfter levomedetomidine administration, subdural balloon inflation precipitated an increase in mean arterial pressure (149 ± 33% of baseline), heart rate (122 ± 19% of baseline), LV systolic pressure (LVP), LV end-diastolic pressure (LVEDP), LV developed pressure (LVDP), LV dP/dtmax and rate pressure product (RPP) (132 ± 19%, 260 ± 142%, 119 ± 15%, 126 ± 24% and 146 ± 33% of baseline value, respectively). ICH decelerated LVP fall (τ), as t increased from 7.75 ± 1. 1 to 14.37 ± 4.5 msec. Moreover, plasma norepinephrine levels were elevated ( 169 ± 50% of baseline) and there was the appearance of cardiac dysrhythmias and other ECG abnormalities. This response was transient and cardiac function deteriorated in a temporal manner. Intracisternal dexmedetomidine prevented the rise in plasma norepinephrine, blocked the ECG abnormalities, and preserved cardiac function. Moreover, dexmedetomidine attenuated the rise in MDA levels.ConclusionsThe results demonstrate that dexmedetomidine attenuates cardiac dysfunction associated with ICH. Our results provide evidence for the role of central sympathetic hyperactivity in the development of cardiac dysfunction associated with ICH.RésuméObjectifDéterminer si !e biocage sympathique central par ia dexmédétomidine, un agoniste sélectif des récepteurs alpha2 adrénergiques, prévient ia dysfonction cardiaque associée à l’hypertension intracrânienne (HIC) chez un modèle rat.MéthodeAprès l’administration intradstemale de dexmédétomidine (1 μg · μL−1, volume de 10μL) ou du stéréoisomère lévomédétomidine (1 μg · μL−1, volume de 10 μL) chez des rats anesthésiés à l’halothane, un cathéter sous-dural à ballonnet a été gonflé pendant 60 s pour produire une HIC. La pression intracrânienne, l’hémodynamique, les pressions ventriculaires gauches (VG) et les changements électrocardiographiques (ECG) ont été enregistrés. Les niveaux plasmatiques et myocardiques de catécholamines et de malondialdéhyde (MDA) ont été mesurés.RésultatsAprès l’administration de lévomédétomidine, le gonflement du ballonnet sous-dural a précipité une hausse de la tension artérielle moyenne (149 ± 33 % des mesures de base), de la fréquence cardiaque (122 ± 19 % de la base), la tension systolique VG (TVG), la tension télédiastolique VG (TTDVG), la tension développée du VG (TDVG), dP/dtmax VG et le produit tension-fréquence cardiaque PJF (132 ± 19%, 260 ± 142 %, 119 ± 15 %, 126 ± 24% et 146 ± 33% des valeurs de base, respectivement). L’HIC a décéléré la chute de la TVG (τ), à mesure que t augmentait de 7,75 ± 1,1 à 14,37 ± 4,5 msec. De plus, les niveaux plasmatiques de norépinéphrine étaient élevés (169 ± 50 % de la base) et des dysrythmies cardiaques sont apparues avec d’autres anomalies ECG. La réaction a été une détérioration transitoire de la fonction cardiaque d’une manière temporelle. La dexmédétomidine intracisternale a empêché l’élévation de norépinéphrine plasmatique, bloqué les anomalies ECG et préservé la fonction cardiaque. La dexmédétomidine a diminué la hausse des niveaux de MDA.ConclusionLes résultats démontrent que la dexmédétomidine atténue la dysfonction cardiaque associée à HIC. Ils mettent en évidence le rôle de l’hyperactivité sympathique centrale dans le développement de dysfonction cardiaque associée à l’HIC.

Spinal Action of Ketorolac, S(+)- and R(-)-ibuprofen on Non-noxious Activation of the Cathechol Oxidation in the Rat Locus Coeruleus Evidence for a Central Role of Prostaglandins in the Strychnine Model of Allodynia

BACKGROUND Blockade of spinal glycine receptors with intrathecal strychnine produces an allodynia-like state in the anesthetized rat. Innocuous hair deflection in the presence of intrathecal strychnine induces a nociceptive-like activation of catechol oxidation in the locus coeruleus and enhances cardiovascular responses. Because prostaglandins play a central role in augmenting pain, this study evaluated the effect of intrathecal nonsteroidal antiinflammatory drugs in strychnine-induced allodynia. METHODS In urethane-anesthetized rats, changes in catechol oxidation in the locus coeruleus, measured using in vivo voltammetry, and cardiovascular parameters evoked by hair deflection of caudal dermatomes were determined after strychnine (40 microg) or saline were administered intrathecally. Subsequently, the effects of 30 microg ketorolac, 10 microg S(+)-ibuprofen, and 10 microg R(-)-ibuprofen administered intrathecally were evaluated. RESULTS After strychnine was administered intrathecally, hair deflection evoked an increase in the locus coeruleus catechol oxidation (peak, 149.7+/-7.2% of baseline) and mean arterial blood pressure (peak, 127.5+/-3.8% of baseline). These responses were not observed after saline was administered intrathecally. All hair deflection-evoked, strychnine-dependent peak responses were attenuated significantly with intrathecally administered ketorolac and S(+)-ibuprofen but not with R(-)-ibuprofen. CONCLUSIONS Locus coeruleus catechol oxidation is a sensitive biochemical index of strychnine-induced allodynia and is correlated temporally with the cardiovascular responses evoked by hair deflection during spinal glycinergic inhibition. The ability of intrathecally administered ketorolac and S(+)-ibuprofen, but not R(-)-ibuprofen, to suppress the locus coeruleus catechol oxidation and cardiovascular peak responses evoked during strychnine-induced allodynia provide evidence that central prostaglandins play an important role in the abnormal sensory processing of strychnine-induced allodynia.

Mannitol but not dantrolene prevents myocardial dysfunction following intra-cranial hypertension in rats.

Cardiac complications stemming from intra‐cranial hypertension may result from impaired intra‐cellular Ca2+ homeostasis. The aim of this study was to examine the effects of dantrolene, a blocker of sarcoplasmic reticulum (SR) Ca2+ release, on myocardial dysfunction associated with intra‐cranial hypertension in rats. Dantrolene (10 mg) with and without 15% mannitol was administered to halothane‐anesthetized rats prior to induction of intra‐cranial hypertension by subdural balloon inflation. Its effects were compared to 3% and 15% mannitol and 5% Pentaspan®. Dantrolene with mannitol or 15% mannitol alone prevented the transient intra‐cranial hypertension‐induced hyperdynamic response and ensuing circulatory collapse that was found in animals pre‐treated with 3% mannitol solution or pentaspan. Moreover, hemodynamic function was preserved irrespective of TnI cleavage. However, only animals treated with high dose 15% mannitol exhibited lower lipid peroxidation content in the heart. In contrast, pre‐treatment with dantrolene alone did not prevent the cardiac complications associated with intra‐cranial hypertension. In conclusion, 15% mannitol attenuated the cardiopulmonary complications associated with intra‐cranial hypertension. Dantrolene without mannitol was without effect. Since mannitol exhibits free radical scavenging properties, protection could be the result of a decrease in oxidative stress after intra‐cranial hypertension.

Activation of the Rostral Ventrolateral Medulla in an Acute Anesthetized Rodent Strychnine Model of Allodynia

UNLABELLED After the administration of intrathecal strychnine, allodynia is manifested as activation of supraspinal sites involved in pain processing and enhancement of cardiovascular responses evoked by normally innocuous stimuli. The objective of this study was to investigate the effect of strychnine-induced allodynia on adrenergic neuronal activity in the C1 area of the rostral ventrolateral medulla (RVLM), a major site involved in cardiovascular regulation. The effect of intrathecal strychnine (40 microg) or saline followed by repeated hair deflection to caudal lumbar dermatomes in the urethane-anesthetized rat was assessed by measuring voltammetric changes in the RVLM catechol oxidation current (CA x OC), mean arterial pressure (MAP), and heart rate (HR). After the administration of intrathecal strychnine, hair deflection evoked a significant and sustained increase in the RVLM CA x OC and MAP (peak 146.4%+/-5.6% and 159%+/-18.4% of baseline, respectively; P < 0.05). There was a nonsignificant increase in HR (peak 128%+/-8.2%). In the absence of hair deflection, there was no demonstrable change. Intrathecal saline-treated rats failed to demonstrate changes in RVLM CA x OC, MAP, or HR. In the present study, we demonstrated that, after the administration of intrathecal strychnine, innocuous hair deflection evokes temporally related neuronal activation in the rat RVLM and an increase in MAP. This suggests that the RVLM mediates, at least in part, the cardiovascular responses during strychnine allodynia. IMPLICATIONS Neural injury-associated pain, as manifested by allodynia, is resistant to conventional treatment. In a rat model of allodynia, we demonstrated activation of the brain region involved in sympathetic control. Innovative therapies that target this region may be successful in managing this debilitating condition.