Intrapericardial injections using a novel technique.

Intrapericardial injections have been proposed as a means to specifically treat diseases of the myocardium, pericardium, and coronary vasculature. The pericardial space is potential drug reservoir, allowing sustained delivery of drug to the target tissue. In this study we have demonstrated a novel method for pericardial delivery in the mouse.

Contributors to cognitive impairment in congestive heart failure: a pilot case-control study.

BACKGROUND: Cognitive impairment and heart failure are both serious health problems related to population ageing. Impaired cognitive function is an important but underrecognized complication of congestive heart failure (CHF). The aim of the study was to examine the sociodemographic, clinical, neuroimaging and biochemical parameters affecting cognition in CHF. METHODS: Thirty-one patients with CHF (left-ventricular ejection fraction < 40%) and 24 controls without CHF, all free of clinically significant cognitive impairment, participating in a case-control study were assessed using a cognitive battery (CAMCOG), a depression scale, 6-min-walk test, left-ventricular ejection fraction, semi-quantitative magnetic resonance imaging, and cortisol, aldosterone and renin concentrations. RESULTS: The CHF patients had lower CAMCOG scores than controls (93.5 +/- 6.1 vs 99.9 +/- 2.4, P < 0.001) and had significantly lower scores on visuospatial, executive function, visual memory and verbal learning tasks. Concentrations of renin and aldosterone were higher in patients with CHF (5.4 +/- 6.0 vs 0.8 +/- 0.7 mU/L, P < 0.001 and 598.2 +/- 306.2 vs 346.0 +/- 201.5, P= 0.003). Right medial temporal lobe atrophy was more prominent in CHF (P= 0.030). Left medial temporal lobe atrophy and deep white matter hyperintensities showed moderate association with cognitive scores in CHF, whereas functional capacity and biochemical parameters were fairly correlated to cognition. CONCLUSION: Congestive heart failure is associated with a pattern of generalized cognitive decline. Structural brain changes, functional capacity and biochemical parameters are associated with the cognitive performance of patients with CHF, but their contribution appears modest. The design of a definitive case-control study is described.

Coronary heart disease is associated with regional grey matter volume loss: implications for cognitive function and behaviour.

Coronary heart disease (CHD) has been associated with impaired cognition, but the mechanisms underlying these changes remain unclear. We designed this study to determine whether adults with CHD show regional brain losses of grey matter volume relative to controls. We used statistical parametric mapping (SPM5) to determine regional changes in grey matter volume of T(1)-weighted magnetic resonance images of 11 adults with prior history of myocardial infarction relative to seven healthy controls. All analyses were adjusted for total grey and white matter volume, age, sex and handedness. CHD participants showed a loss of grey matter volume in the left medial frontal lobe (including the cingulate), precentral and postcentral cortex, right temporal lobe and left middle temporal gyrus, and left precuneus and posterior cingulate. CHD is associated with loss of grey matter in various brain regions, including some that play a significant role in cognitive function and behaviour. The underlying causes of these regional brain changes remain to be determined.

Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats.

The intracellular second messenger, cyclic guanosine monophosphate (cGMP), a soluble guanylate cyclase (GC) product, is a primary mechanism for the transduction of a nitric oxide (NO)-initiated signal in the central nervous system. NO is produced from L-arginine by neuronal nitric oxide synthase (NOS), which is found in sympathetic preganglionic neurons of the intermediolateral cell column. This suggests the possibility that NO is a modulator of sympathetic nervous activity (SNA) through a cGMP-mediated mechanism. The aim of this study was to determine the effects of intrathecally injected membrane-permeant 8-bromo-cGMP and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble form of GC, on arterial pressure in urethane anaesthetized (1.4 g kg(-1) I.P.) rats. The effects of intrathecal cGMP and ODQ on haemodynamic responses to haemorrhage were also investigated. Finally, L-arginine, the NO precursor, was also injected intrathecally, alone and in the presence of ODQ. Baseline mean arterial pressure (MAP) increased significantly after intrathecal 8-Br-cGMP injection (10 microl, 1, 3, 10, 30, 100 microm). A dose-effect relationship (1 microm to 100 microm) was also established (EC(50)=6.03 microm). During continuous haemorrhage, MAP was maintained in animals injected with 8-Br-cGMP, relative to the control group. Although no change in baseline MAP was observed as a result of intrathecal ODQ injection (10 microl, 100 mM), a greater rate of fall in MAP was observed during haemorrhage. Injecting L-arginine (10, 100, 1000 microm, 10 microl) showed a pressor effect that was consistent with the effect of the downstream messenger, cGMP. Furthermore, its pressor effect was blocked by ODQ pre-administration. The results indicate that cGMP increases blood pressure, and thus suggest that cGMP increases SNA. This supports the hypothesis that the sympathoexcitatory effects of spinal delivery of NO are mediated by a cGMP-dependent mechanism.

Hypotension is associated with diuretic resistance in severe chronic heart failure, independent of renal function.

BACKGROUND: Diuretic resistance and systemic hypotension are common in chronic heart failure (CHF), however, the two have not been associated. AIMS: Since blood pressure (BP) might be an important determinant of sodium excretion, we searched for an association between BP and diuretic dosage in severe CHF. METHODS: Our heart failure database was retrospectively reviewed for patients with severe left ventricular systolic dysfunction. The 54-patient cohort was divided on the basis of frusemide dosage (high-dose > or = 250 mg daily, n=26). RESULTS: Patients taking high-dose frusemide had higher serum creatinine, and lower systolic and diastolic BP. On logistic regression analysis, increased serum creatinine and reduced diastolic BP were independent predictors of the use of high-dose frusemide. Grouping these variables into tertiles, the odds ratio for the use of high-dose frusemide was 4.0 as diastolic BP decreased (p<0.01), and 6.8 as serum creatinine increased (p<0.001). CONCLUSIONS: We have found an association between hypotension and the use of high-dose frusemide in severe CHF, which is independent of renal function, and which may be an important physiologic mechanism of diuretic resistance in severe CHF.

Effect of anaesthetic and rat strain on heart rate responses to simulated haemorrhage.

AIM AND METHODS: Haemorrhage is characterized by two distinct responses, sympathoexcitation that evokes tachycardia and supports blood pressure, followed by sympathoinhibition contributing to bradycardia and hypotension. It has been shown that anaesthetics alter the response to haemorrhage and we hypothesized that rat strain may also influence the response. We investigated the effect of simulated haemorrhage on heart rate (HR) responses in three strains of conscious rats, and the effect of three common anaesthetics, by comparing HR responses under anaesthesia to the conscious response. Haemorrhage was simulated by constricting the inferior vena cava. We demonstrate differential effects of anaesthetics, including both maintenance and elimination of HR responses to haemorrhage depending on anaesthetic. RESULTS: We also show that both phases of the HR response differ in different conscious rat strains, and we have demonstrated a transient increase in HR during the decompensatory phase of haemorrhage, a novel 'second HR peak' with advanced hypotension. CONCLUSION: Both rat strain and anaesthetic influence HR responses to haemorrhage, and some anaesthetics appear less suitable than others for studies of haemodynamic responses in rats. There was evidence of an additional compensatory mechanism that operates at advanced levels of hypotension in the rat.

Neuropeptide Y mRNA expression in interneurons in rat spinal cord.

Neuropeptide Y (NPY)-immunoreactive axons are present within the spinal cord. Some of these axons originate from neurons in the brainstem. Other axons arise from within the spinal cord since NPY-immunoreactivity can be detected after complete spinal cord transection. To identify spinal neurons that might express NPY, we localized NPY mRNA in rat spinal cord using in situ hybridization histochemistry. NPY mRNA-containing neurons were localized in the dorsal horn, in medial laminae of the grey matter and in the lateral spinal nucleus in thoracic, lumbar and sacral cord. The location of some of these neurons, and their proximity to sympathetic preganglionic neurons, suggest some NPY-containing interneurons are likely to be involved in spinal as well as supraspinal autonomic reflex pathways.

Neurokinin-1 receptor immunoreactivity in hypotension sensitive sympathetic preganglionic neurons.

Substance P activation of neurokinin-1 (NK1) receptors on spinal sympathetic preganglionic neurons (SPN) influences blood pressure. We identified SPN likely to subserve the baroreceptor reflex and established if these neurons showed NK1 receptor-immunoreactivity. Nitroprusside (NP) infusion or inferior vena cava (IVC) constriction activated similar numbers of SPN. Of these, about 40% were NK1 receptor-immunoreactive after NP infusion, but only about 20% were NK1 receptor-immunoreactive after IVC constriction. The distribution of Fos/NK1 receptor SPN suggested that substance P may preferentially target sympathoadrenal SPN.

Time of day and access to food alter water intake in rats after water deprivation.

1. Drinking behaviour after water deprivation is one of the standard tests used to study thirst in humans and animals. Diurnal cycle and food availability are known to influence water intake, but have not been considered in previous studies of thirst after water deprivation. In the present study, we examined the effects of diurnal variation and food availability on water intake after 24 h water deprivation in rats. 2. All rats cycled through four treatments in varying order. These treatments were: (i) 24 h water deprivation with free access to food from 1900 h one day to 1900 h the next day, followed by free access to both food and water (Night-with-Food); (ii) 24 h water deprivation with free access to food from from 1900 h one day to 1900 h the next day, followed by free access to water but not food (Night-without-Food); (iii) 24 h water deprivation with free access to food from 0700 h one day to 0700 h the next day, followed by free access to both food and water (Day-with-Food); or (iv) 24 h water deprivation with free access to food from 0700 h one day to 0700 h the next day, followed by free access to water but not food (Day-without-Food). The amount of water consumed during the first 6 h, post-24 h water deprivation, was examined under each condition. 3. There was a significant diurnal effect (P < 0.001) and a significant food availability effect (P = 0.007) on the water consumed in the 6 h period after water deprivation. Most water was consumed by the Night-with-Food group and the least amount of water was consumed by the Day-without-Food group. These effects persisted after correction for water intake during 6 h periods from 0700 and 1900 h with and without food but without previous water deprivation. The diurnal and food availability effects on water consumption were independent (P = 0.5). 4. The coefficient of variability for each group suggests that the most sensitive measurements of water intake are obtained during the day in the absence of food. 5. We conclude that both the time of day and access to food independently alter water intake in rats subjected to a previous 24 h water deprivation. Our study also supports the validity of performing water intake measurements in thirst studies in rats during the day.

Coronary artery baroreceptor-mediated changes in arterial pressure: a pilot study in conscious and anaesthetized sheep.

1. Evidence suggesting the presence of coronary artery baroreceptors on coronary arteries has existed for over 30 years. 2. Evidence that activation of ventricular mechanoreceptors can elicit cardiovascular changes has been challenged, with those changes now thought to be due to coronary artery mechanoreceptors. 3. Studies have suggested that coronary artery mechanoreceptors act as coronary baroreceptors with a role in cardiovascular regulation. However, all evidence to date has been obtained in anaesthetized animal models in physiologically compromised intra-operative states. 4. The purpose of the present study was to design an ovine model that would allow the discrete stretch of coronary arteries without causing ischaemia or changing flow or intra-arterial pressure and that would confirm results seen in previous studies. In addition, the possibility that the technique could be used for studies of coronary artery baroreflexes in conscious sheep was investigated. 5. Controlled stretch of the proximal left anterior descending coronary artery elicited decreases in arterial pressure without changes in heart rate or electrocardiographic activity in halothane-anaesthetized sheep. Similar results were demonstrated in conscious sheep after surgical recovery of up to 2 weeks. 6. The present study supports the possibility that coronary artery baroreceptors exist and likely have a role in cardiovascular regulation. The results of the present study in anaesthetized sheep are in agreement with previous results in anaesthetized animals, but also provide the first demonstration of coronary baroreceptor activity in a conscious animal model, underscoring the potential use of the model in the study of coronary artery baroreceptors in the intact animal.

Tracer-toxins: cholera toxin B-saporin as a model.

We have shown previously that retrogradely-transported cholera toxin B (CTB)-saporin has eliminated sympathetic preganglionic neurons by 7 days after injection (Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F., Minson, J.B., 1999. NeuroReport 10, 307). To ascertain whether this tracer-toxin can kill other types of neurons that transport CTB retrogradely with a similar time course, we injected CTB-saporin into the facial nerves of rats and allowed them to survive for 7 days. Facial motoneurons were counted ipsilateral and contralateral to the injected nerves in sections of perfused medulla processed to reveal immunoreactivity for choline acetyltransferase (ChAT). There was a statistically significant decrease in the number of ChAT-immunoreactive neurons ipsilateral to the injected nerve in three out of nine rats. Inadequate injections were probably the reason that most rats showed no decrease in motoneurons numbers after treatment with CTB-saporin, since the staining intensity and numbers of facial motoneurons that showed CTB-immunoreactivity varied markedly between rats after retrograde tracing with unconjugated CTB. These results show that CTB-saporin can eliminate motoneurons as well as sympathetic preganglionic neurons, indicate that protocols for the injection of tracer-toxins should be optimized to ensure maximum neuronal death and support our contention that CTB-saporin should kill any central neuron that expresses GM1 ganglioside, the membrane component to which CTB binds.

Nitric oxide limits pressor responses to sympathetic activation in rat spinal cord.

N-methyl D-aspartate (NMDA) receptor stimulation is known to activate nitric oxide (NO) synthase, an enzyme present in a high proportion of sympathetic preganglionic neurons. In this study, we have examined the possibility that NO modulates the pressor responses elicited by NMDA receptor stimulation in the spinal cord. In experiments on anesthetized rats, we determined whether intrathecal administration of either 3-morpholinylsydnoneimine chloride (SIN-1), an NO donor, or N:(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, affected the response to stimulation of spinal NMDA receptors by NMDA (1 pmol to 1 micromol in 10-microL intrathecal administration). Intrathecal NMDA resulted in dose-dependent increases in blood pressure. SIN-1 (100 nmol) attenuated the pressor responses to NMDA (F(1,70)=12, P=0.001). Conversely, L-NAME (1 nmol to 1 micromol) augmented the pressor response to NMDA in a dose-dependent manner (F(3,161)=28.3, P<0.001). The effect of L-NAME to amplify the pressor response to NMDA was reversed by L-arginine but not by D-arginine. These results indicate that endogenous synthesis of NO in the spinal cord limits the pressor response to stimulation of spinal NMDA receptors.

The time course of haemodynamic, autonomic and skeletal muscle metabolic abnormalities following first extensive myocardial infarction in man.

We investigated the time course of genesis of skeletal muscle dysfunction and sympatho-vagal imbalance after myocardial infarction. We studied 22 normal controls, 22 patients with >6 months stable chronic heart failure and 10 patients after a first massive myocardial infarction at 1-3 weeks (the "early" period), 6-8 weeks ("mid") and 6-9 months ("late") following their infarct. Four patients developed overt heart failure. Forearm muscle metabolism was studied using (31)P magnetic resonance spectroscopy (MRS). Sympatho-vagal balance was assessed by heart rate variability and radiolabelled norepinephrine kinetics. Increased norepinephrine spillover (0.55+/-0.02 v 0.27+/-0.04 mg/min/m(2); P<0.01) and decreased heart rate variability were confined to those post-myocardial infarction patients who subsequently developed heart failure. Resting cardiac output was normal in all the post-myocardial infarction patients, although the response of cardiac output to supine bicycle exercise at the "mid" study point was less in the group who subsequently developed heart failure (9+/-1 v 41+/-8 %; P<0.005). In the MRS studies, there were no detectable differences between those who did or did not develop heart failure. The initial rate of ATP turnover, calculated from initial-exercise changes in pH and phosphocreatine (PCr), was increased in established chronic heart failure, but in the post-myocardial infarction patients a numerically similar increase reached statistical significance only in the early group (19+/-3 v 11+/-1 mM/min; P<0.005). The apparent maximum rate of oxidative ATP synthesis, calculated from post-exercise PCr recovery kinetics, was lower than control in the late post-myocardial infarction and established chronic heart failure groups 34+/-5 v 55+/-4 mM/min; P<0.03 and 38+/-3 v 55+/-4 mM/min; P<0.003, respectively). Skeletal muscle metabolism and autonomic function become abnormal after an extensive myocardial infarction. While skeletal muscle abnormalities are relatively slow to develop and unrelated to the degree of failure, excessive neurohormonal activation and impaired cardiac output response to exercise seem from an early stage to characterize patients who subsequently develop chronic heart failure.

Activation of spinal opioid receptors contributes to hypotension after hemorrhage in conscious rats.

Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a profound fall in blood pressure. This study examined the location and subtypes of opioid receptors that might contribute to hypotension after hemorrhage. Intrathecal naloxone methiodide (100 nmol) abolished the fall in blood pressure after hemorrhage (1.5% of body wt; mean arterial pressure 122 +/- 8 mmHg after naloxone methiodide vs. 46 +/- 5 mmHg in controls, P < 0. 001). Intracisternal naloxone methiodide was less effective than intrathecal naloxone methiodide, whereas intravenous naloxone methiodide, which does not cross the blood-brain barrier, did not alter the fall in blood pressure after hemorrhage. These results demonstrate that spinal opioid receptors contribute to hypotension after hemorrhage but do not exclude supraspinal effects. In separate experiments, the subtype-specific opioid antagonists ICI-174864 (delta-antagonist), norbinaltorphimine (nor-BNI; kappa-antagonist), and H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; mu-antagonist) were each administered intrathecally to determine the minimum dose that would attenuate hypotension during severe hemorrhage. These antagonists were effective at similar doses (3 nmol for CTOP, 6 nmol for ICI-174864, and 10 nmol for nor-BNI), although the binding affinities of these three different agents for their target receptors varied >1600-fold. Comparisons of the minimum effective doses of these antagonists in relation to their binding affinities provides strong evidence for the participation of delta-receptors in mediating hypotension after hemorrhage. In contrast, the dose at which nor-BNI was effective suggests an effect at delta-receptors but not kappa-receptors. The efficacy of CTOP, albeit at a high dose, also suggests an effect at mu-receptors.

Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons.

Aiming to ablate sympathetic preganglionic neurons (SPN) innervating a defined target, we injected saporin conjugated to cholera toxin B subunit (CTB) unilaterally into the superior cervical ganglion of rats. In spinal cord segments T1-T3, the numbers of cholinergic neurons in the intermediolateral cell column ipsilateral and contralateral to the injected ganglion were significantly different by 3 days post-injection. By day 14, 77% of ipsilateral cholinergic neurons had disappeared. A higher percentage of neurons were killed in T1-T2 than in T3. Comparing SPN counts from CTB-saporin injected rats and counts from rats receiving unconjugated CTB into the superior cervical ganglion indicated that 84% of SPN supplying the ganglion had died by 14 days. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons and may also eliminate other types of neurons that transport CTB.

Neurokinin-1 receptors and spinal cord control of blood pressure in spontaneously hypertensive rats.

In this study we examined blood pressure and heart rate responses to intrathecal administration of a synthetic NK1-receptor agonist, H2N-(CH2)4-CO-Phe-Phe-Pro-NmeLeu-Met-NH2 (GR 73,632), in spontaneously hypertensive rats (SHR) and their progenitor strain, the Wistar-Kyoto rat (WKY). Sodium pentobarbitone anaesthetised rats with implanted intrathecal catheters were paralysed (pancuronium dibromide) and artificially ventilated. Injection of GR 73,632 at the T9 spinal level evoked dose-dependent increases in mean arterial pressure (MAP) in WKY and SHR. SHR had a lower MAP response threshold than WKY but increase in response with increasing dose was less in SHR than WKY. Biphasic blood pressure responses at high doses were observed in both strains. Prior administration of the NK1-receptor antagonist (3 aR,7aR)-7,7-diphenyl-2-[1-imino-2(methoxyphenyl)ethyl] perhydroisoindol-4-one (RP 67,580) significantly reduced the pressor response in WKY but not SHR. The depressor response was not attenuated in either strain.

Animal models of heart failure.

Animal models of heart failure present homogenous groups of animals all with heart failure produced by a well defined lesion at a particular stage of evolution, in contrast to humans, who present with heart failure of uncertain duration from a wide variety of causes and with marked variation in age and pre-morbid health and fitness. Animal models of heart failure provide diseased groups of animals in which experimental procedures, not possible in humans, can be evaluated and in which new treatments can be tested before their safety is established in humans. An ideal model should have a common human counterpart and should closely mimic heart failure in humans. Thus the haemodynamic changes should include increased cardiac filling pressures and low cardiac output. There should be evidence of activation of the sympathetic nervous system and increased secretion of hormones such as renin, angiotensin, aldosterone, vasopressin, atrial natriuretic factor and endothelin. The clinical features of the human syndrome such as cardiomegaly, lung and peripheral oedema and decreased exercise tolerance should be present. Lastly, the model should be inexpensive and technically simple to produce and study. This paper reviews some commonly used models of heart failure in relation to the criteria listed above. There is no perfect animal model of heart failure and in practice one should match the model to the purpose of the study.

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion.

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.