Sergio Palazzesi

Video-assisted orotracheal intubation in mice

Orotracheal intubation in mice is a complicated technique because of the peculiar oropharyngeal anatomy and the difficulty in visualizing the laryngis aditus. Here we report a new and simple method for rapid endotracheal intubation by using a small bore, straight fibre-optic arthroscope. Under endoscope-assisted visualization of the laryngis aditus, a polyethylene cannula, inserted on a guide-wire in order to facilitate the introduction of the tip across the vocal cords, was advanced in the trachea. The success rate of intubation was 100%. We were also able to re-intubate the mice 4 and 8 weeks later without any major complications. We conclude that this method can be easily and safely used for studies where controlled pulmonary ventilation is necessary.

Attenuation of aortic banding-induced cardiac hypertrophy by propranolol is independent of beta-adrenoceptor blockade.

Objective Racemic propranolol attenuates cardiac hypertrophy secondary to abdominal aortic banding-induced pressure overload by a mechanism independent of its effect on cardiac work load. This was only observed, however, using doses of propranolol that were much higher than those needed to induce β-adrenoceptor blockade. Thus, the question remains as to whether the antihypertrophic effect of propranolol depends on its ability to antagonize cardiac β-adrenoceptor-mediated action (positive chronotropic effect, trophic effect) or on β-adrenoceptor-independent action. Methods In a rat model of chronic pressure overload induced by abdominal aortic banding, we evaluated the effects on left ventricular hypertrophy (LVH) of the propranolol isomers, l-propranolol and d-propranolol, which compared to l-isomer is approximately 50-fold less potent as a β-adrenoceptor antagonist, but is similarly potent as a membrane-stabilizer, as well as of timolol, a non-selective β-adrenergic antagonist devoid of membrane stabilizing activity, and disopyramide, which is a membrane stabilizer, but not a β-adrenoceptor blocker. Results Compared to sham-operated rats, banded rats had 30% greater left ventricular to body weight (LVW/BW) ratio (P < 0.01). The increase in LVW/BW ratio was significantly attenuated by treatment with 40 and 80 (but not 10) mg/kg per day of l-propranolol. Left ventricular hypertrophy was also prevented by d-propranolol, 40 and 80 mg/kg per day, and disopyramide, 50 mg/kg per day, whereas timolol, 30 and 60 mg/kg per day, showed no antihypertrophic effect. In separate groups of banded rats in which the reduction in heart rate induced by propranolol (80 mg/kg per day) was prevented by chronic cardiac pacing at 375 b.p.m., hypertrophy was again prevented, indicating that the effects of l-propranolol on LVH are not related to a reduction in cardiac work load. Conclusions In the aortic banding-induced model of LVH: (i) the antihypertrophic effect of propranolol is independent of its β-adrenergic blocking activity; and Iii) since disopyramide and D-propranolol also proved to be able to antagonize banding-induced LVH, the hypothesis is proposed that membrane-stabilizing activity, among the ancillary properties of propranolol, most likely accounts for the antihypertrophic effect of this drug.

Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure‐overloaded hearts

Cardiac hypertrophy is a homeostatic response to elevated afterload. Na+/H+ exchanger (NHE) inhibition reduces the hypertrophic response in animal models of left ventricular hypertrophy (LVH) and myocardial infarction. We examined the effect of chronic treatment with cariporide, a selective inhibitor of Na+/H+ exchanger isoform 1 (NHE‐1), on left ventricular (LV) systolic and diastolic function under pressure overload conditions. Male CD‐1 mice were randomized to receive either a control diet or an identical diet supplemented with 6000 p.p.m. of cariporide. Cardiac pressure overload was induced by thoracic aortic banding. LV dimension and systolic and diastolic function were assessed in sham and banded mice by echocardiography and cardiac catheterization 2 and 5 weeks after surgery. Histological analysis was also performed. After 2 weeks of pressure overload, the vehicle‐treated banded mice (Veh‐Bd) had enhanced normalized LV weight (about +50%) and normal chamber size and function, whereas cariporide‐treated banded mice (Car‐Bd) showed a preserved contractility and systolic function despite a marked attenuation of LVH. Diastolic function did not differ significantly among groups. After 5 weeks, the Veh‐Bd developed LV chamber enlargement and systolic dysfunction as evidenced by a 16% increase in LV end‐diastolic diameter, a 36% decrease in myocardial contractility, and a 26% reduction in percent fractional shortening. In contrast, Car‐Bd showed an attenuated increase in LV mass, normal chamber size, and a maintained systolic function. A distinct histological feature was that in banded mice, cariporide attenuated the development of cardiomyocyte hypertrophy but not the attendant myocardial fibrosis. In conclusion, the results of the present study indicate that (i) the hypertrophic response to pressure overload is dependent on NHE‐1 activity, and (ii) at the 5‐week stage, banding‐induced deterioration of LV performance is prevented by NHE‐1 inhibition.

Protection by Shear Stress From Collar-Induced Intimal Thickening Role of Nitric Oxide

Nitric oxide (NO) has potent relaxant and antiproliferative effects on vascular smooth muscle cells, which may represent an important antiatherosclerotic mechanism. Since one of the major stimuli for NO release is flow-related shear stress, we have investigated (1) the effect of increased shear stress on neointimal formation induced in the rabbit carotid artery by enclosing the vessel in a nonconstrictive silicone soft collar and (2) the role of NO in the antiproliferative effect of increased shear stress. Forty-three New Zealand White rabbits were used. High shear stress in the left common carotid artery (CCA) was induced by ligature of the contralateral right internal carotid artery; intimal thickening was produced by the positioning a nonconstrictive silicone soft collar around the left CCA. To evaluate the role of NO, N(G)-nitro-L-arginine methyl ester (L-NAME) was orally administered at a subpressor dose. In all rabbits, arterial blood pressure, heart rate, arterial diameters, and blood flow velocities of both CCAs were determined at days 0, 3, 7, and 14. At the end of the study, all rabbits were euthanized, and histological analyses were performed on both CCAs of each animal. The presence of the collar was associated with a marked degree of intimal hyperplasia (intimal/medial area ratio 29+/-3.0% in collared arteries compared with 3+/-0.7% in sham control [noncollared] arteries, P<0.001). The increase in blood flow almost completely inhibited neointimal formation and induced an increase in arterial diameter of approximately 30%. The effects of increased blood flow were reversed by the administration of L-NAME. In conclusion, we demonstrate that in collar-induced intimal thickening, a chronic increase in shear stress (1) almost completely inhibits intimal thickening, and (2) this protective effect is mediated by NO production.

ETAETB receptor antagonist Bosentan inhibits neointimal development in collared carotid arteries of rabbits

The contribution of endothelin to the genesis of neointimal development in collared rabbit carotid arteries, a widely accepted model of atherosclerosis, was investigated. Three sets of rabbits were studied. In the first group, a non-occlusive, biologically inert silastic collar was positioned around the right carotid artery of the rabbit. In another group, the application of the collar was accompanied by endothelial denudation via a Fogarty arterial balloon catheter, while the third group of animals underwent only endothelial denudation. After two weeks, intimal hyperplasia of a similar degree was observed in all groups. The administration of the nonselective ET(A)/ET(B) receptor antagonist Bosentan, significantly reduced both the neointimal area and the intima/media area ratio in all groups. However, the beneficial effects of Bosentan were less pronounced in balloon injured vessels than in collared ones. The results of the present study indicate that i) endothelin has a key role in the development of intimal hyperplasia following arterial collaring, ii) the contribution of endothelin to intimal hyperplasia is greater in collared arteries that in balloon injured ones, and iii) the nonselective ET(A)/ET(B) receptor antagonists are potential tools for the prevention of intimal hyperplasia.

Pressure overload causes cardiac hypertrophy in β1-adrenergic and β2-adrenergic receptor double knockout mice

Objective Cardiac hypertrophy arises as an adaptive response to increased afterload. Studies in knockout mice have shown that catecholamines, but not α1-adrenergic receptors, are necessary for such an adaptation to occur. However, whether β-adrenergic receptors are critical for the development of cardiac hypertrophy in response to pressure overload is not known at this time. Methods and results Pressure overload was induced by transverse aortic banding in β1-adrenergic and β2-adrenergic receptor double knockout (DβKO) mice, in which the predominant cardiac β-adrenergic receptor subtypes are lacking. Chronic pressure overload for 4 weeks induced cardiac hypertrophy in both DβKO and wild-type mice. There were no significant differences between banded mice in left ventricular weight to body weight ratio, in the left ventricular wall thickness, in the cardiomyocyte size or in the expression levels of the load-sensitive cardiac genes such as ANF and β-MHC. Additionally, the left ventricular systolic pressure, an index of afterload, and cardiac contractility, evaluated as dp/dtmax, the maximal slope of systolic pressure increment, and Ees, end-systolic elastance, were increased at a similar level in both wild-type and DβKO banded mice, and were significantly greater than in sham controls. Conclusion Despite chronic activation of the cardiac β-adrenergic system being sufficient to induce a pathological hypertrophy, we show that β1-adrenergic and β2-adrenergic receptors are not an obligatory component of the signaling pathway that links the increased afterload to the development of cardiac hypertrophy.

Propranolol causes a paradoxical enhancement of cardiomyocyte foetal gene response to hypertrophic stimuli.

Pathological cardiac hypertrophy is associated with the expression of a gene profile reminiscent of foetal development. The non selective β‐adrenoceptor antagonist propranolol is able to blunt cardiomyocyte hypertrophic response in pressure‐overloaded hearts. It remains to be determined whether propranolol also attenuates the expression of hypertrophy‐associated foetal genes.

Endothelin and mechanical properties of the carotid artery in Wistar-Kyoto and spontaneously hypertensive rats.

The aim of this study is to evaluate the role of endothelin in the control of the static mechanical properties of in vitro carotid arteries from 14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). An in vitro preparation in which the artery was allowed to longitudinally elongate similarly to the in situ carotid artery was employed. The diameter of in vitro carotid arteries subjected to static pressures (from 25 to 200 mmHg in 25 mmHg steps) was determined by videomicroscopy and computer-assisted image analysis, the cross-sectional compliance- and distensibility-pressure curves being then derived. The role of endothelin was assessed by incubating carotid arteries with the selective ETA and ETB endothelin receptor antagonists BQ123 and BQ788, respectively. These effects were compared with those observed under control conditions, as well as with those following complete abolition of vascular smooth muscle tone by potassium cyanide (KCN). Carotid diameter was significantly larger, and compliance and distensibility significantly smaller, in SHR compared to WKY rats. Local incubation with BQ123 was associated with significant dilations as well as significant increases in cross-sectional compliance and distensibility in both strains. This was even more pronounced with KCN, while BQ788 had no effect. The results of the present study suggest that: (i) endothelin exerts a tonic stiffening effect on the in vitro common carotid artery; (ii) this effect is mediated via the ETA endothelin receptor, and (iii) the stiffening effect of endothelin is exerted to a similar extent in the carotid arteries of normotensive WKY and SHR rats.

Effects of cholinergic drugs on neocortical EEG and flash-visual evoked potentials in the mouse.

The effects of single intraperitoneal injection of two cholinesterase inhibitors, physostigmine (PHY; 0.01, 0.025, 0.05, 0.1, 0.2 mg/kg) and heptylphysostigmine (HEP; 0.5, 2, 6 mg/kg) on electroencephalographic (EEG) activity and flash visual evoked potentials (f-VEP) in the occipital cortex were compared in DBA/2 mice. EEG spectral analysis of awake periods showed that PHY at all doses and HEP at 2 mg/kg induced an increase of power in the 4.25- to 7-Hz frequency band. Furthermore, PHY at the higher doses and HEP at all doses induced a decrease of power in the 7.25- to 12-Hz frequency band, while the lower doses of PHY (0.01, 0.025 mg/kg) produced an increase of this band. EEG effects elicited by the two drugs were similar, when doses displaying analogous biochemical effects (acetylcholinesterase inhibition) were used (i.e. 0.01 and 0.025 mg/kg of PHY versus 0.5 and 2 mg/kg of HEP). PHY and HEP induced similar changes in f-VEPs. Amplitudes of early and late components (P1N1, N1P2, P4N4 and particularly N1P3) were enhanced, while amplitudes of middle components were depressed after all doses. The peak latency measures were generally delayed, even though, after the lower doses, a trend to a latency reduction was evident in late components. This finding might indicate a possible effect on stimulus speed diffusion by ‘low therapeutic’ doses, analogous to the ones used in men. Our data show that both drugs are effective in modifying EEG and f-VEP parameters connected with brain cholinergic function, although in a very narrow dose range.

Effects of low doses of physostigmine on avoidance learning and EEG in two strains of mice

The effects of the cholinomimetic drug, physostigmine (0, 0.01, 0.025, 0.05 and 0.1 mg/kg, i.p.), on shuttle-box avoidance learning and electroencephalographic (EEG) activity were investigated, in two separate studies, in mice belonging to the inbred C57BL/6 (C57) and DBA/2 (DBA) strains. The results of the behavioral investigation showed a consistent, significant enhancement of avoidance performance, on the whole of 5 daily training sessions, in C57 mice treated with the lowest dose (0.01 mg/kg) and in DBA mice treated with the highest doses (0.05 and 0.1 mg/kg) of the drug. Doses higher than 0.01 mg/kg, in C57 mice, and lower than 0.05 mg/kg, in DBA mice, had no significant effect. The avoidance improvements induced by physostigmine cannot be ascribed to general behavioral activation, since the doses that increased avoidance responses did not affect or even depressed spontaneous locomotor activity. The same doses of treatment which increased avoidance responding, also induced, in the same strains, consistent enhancement of 4-7 Hz (theta) EEG band power and decrease of 7-12 Hz (alpha) band power. Results suggest that the effects induced by physostigmine on the EEG and on the shuttle-box performance of mice are related to the same neurochemical systems, and are dependent upon the interaction of the dose with specific strain sensitivity.