Novel Insights into Beta 2 Adrenergic Receptor Function in the rd10 Model of Retinitis Pigmentosa

Background: In retinitis pigmentosa (RP), inherited rod death is followed by cone loss and blindness. Why cones die is still a matter of consideration. Here, we investigate the pathogenic role of the sympathetic transmission in the rd10 mouse model of RP. Methods: Retinal levels of beta adrenergic receptor (BAR) 2 and norepinephrine (NE) were measured. After administration of the BAR1/2 blocker propranolol or the hypoxia-inducible factor (HIF)-1 activator dimethyloxalylglycine (DMOG), retinal levels of HIF-1α, BAR2 or proteins involved in BAR2 desensitization were also measured. In DMOG treated mice, expression and localization of BAR2, inflammatory markers and cone arrestin were determined. Finally, rd10 mice were subjected to electroretinogram (ERG) analysis to assess rod and cone function. Results: In the rd10 retina, BAR2 overexpression and NE accumulation were found, with BAR2 immunoreactivity localized to Müller cells. BAR2 overexpression was likely due to desensitization defects. Upregulated levels of BAR2 were drastically reduced by propranolol that also restored desensitization defects. Due to the low level of HIF-1 consequent to the hyperoxic environment in the rd10 retina, we hypothesized a link between HIF-1 and BAR2. HIF-1α stabilization with DMOG resulted in i. increased HIF-1α accumulation, ii. decreased BAR2 levels, iii. restored desensitization processes, iv. reduced expression of inflammatory markers and v. increased cone survival without improved retinal function. Conclusions: Our results support a pathogenic role of the sympathetic system in RP that might help to understand why rd10 mice show a positive response to BAR blockers

Effects of a catechins‑enriched diet associated with moderate physical exercise in the preventionof hypertension in spontaneously hypertensive rats

Hypertension represents the main risk factor for the onset of cardiovascular diseases. Pharmacological treatments to control hypertension have been associated with new treatments involving physical activity and/or the intake of natural components (nutraceuticals). We here report the effects produced by a combination of a natural component (catechins) and a moderate exercise program on the development of hypertension in spontaneous hypertensive rats compared with those of each individual treatment. Arterial blood pressure and heart rate were measured with a non-invasive method in 28 rats randomly assigned to four groups: rats subjected to moderate physical exercise; rats with a catechins-enriched diet; rats subjected to moderate physical exercise combined with a catechins-enriched diet; control, untreated-rats left to age. All treatments were applied for 6 weeks. The statistical analysis revealed that the three treatments significantly reduced the weekly increase in arterial blood pressure observed in control rats (SBP, P < 0.0001; DBP, P = 0.005). However, the reduction of arterial blood pressure induced by combined treatments was not higher than that induced by the single treatment, but more prolonged. All treatments showed strong antioxidative properties. Our data show that physical activity and a diet enriched with catechins individually have an important hypotensive effect, while the association did not produce a higher hypotensive effect than the single treatment, even if it was able to decrease blood pressure for a longer time. These findings have important implications for developing a protocol to apply in novel hypertension prevention procedures

The Hypotensive and bradycardic effects of mouth opening: evidence in an animal model.

Objective Previous studies in normotensive anesthetized rats (Lapi Arch.Ital.Biol 151:11-23,2013) showed that peripheral stimulation of the trigeminal nerve induced by submaximal mouth opening (mandibular extension, ME) caused prolonged (at least 80min) bradycardia, hypotension and cerebral hemodynamic changes (pial arterioles showed a characteristic response pattern consisting in a significant constriction during ME followed by a dilatation for the entire remaining observation time). Design and method In this study we assessed the in vivo effects of ME on HR, MABP and pial microcirculation in hypertensive rats. Experiments were performed in male Wistar rats weighing 250-300g (n = 8). Hypertension was induced by intraperitoneal daily injection of dexamethasone (0.03mg/kg/day) for 10 days. ME was obtained by inserting an ad hoc developed retractor between the dental arches. HR and MABP were recorded by ECG and a catheter placed in the left femoral artery and measured by a computer-assisted system. Pial arterioles were observed through a closed cranial window implanted above the left parietal cortex and visualized by an in vivo fluorescence microscopy technique to assess vessel diameter changes before (baseline), during 10min ME and thereafter until 160min. Arteriolar diameters were measured with a computer-assisted method (MIP Image program, frame by frame). Results In sham-treated (no ME) hypertensive rats (n=3) HR, MABP and pial microcirculation did not change during whole observation period. Hypertensive rats subjected to ME (n=5) showed a significant decrease of HR and MABP. HR declined by 42bpm, (p<0.01) starting from 60 min after ME up to 160min, while MABP by 18mmHg (p<0.05) starting from 20min after ME up to 100min, compared with baseline. Pial arterioles exhibited a biphasic response: the arteriolar diameter decreased by 2.94&#956;m (p<0.05) during ME, afterwards it significantly increased by 3.46&#956;m (p<0.01) starting from 20min after ME; this vasodilatation lasted for the whole observation period. Conclusions Our results suggest that ME is able to exert profound and prolonged regulatory effects on systemic arterial blood pressure and pial arteriolar tone in hypertensive rats

Effects of Propionyl-L-Carnitine on Ischemia–Reperfusion Injury in Hamster Cheek Pouch Microcirculation

Background and purpose Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia–reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. Methods The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (VRBC) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2′-7′-dichlorofluorescein (DCF), respectively. Results In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and VRBC decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. Conclusions pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase

Protective Effects of Quercetin on Rat Pial Microvascular Changes during Transient Bilateral Common Carotid Artery Occlusion and Reperfusion

The aim of this study was to assess the in vivo effects of quercetin on pial microvascular responses during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. Capillaries were assigned order 0, the smallest arterioles order 1, and the largest ones order 5. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease (by 14.5 ± 3.3% of baseline in order 2), microvascular leakage [0.47 ± 0.04, normalized gray levels (NGL)], leukocyte adhesion in venules (9 ± 2/100 μm venular length, v.l./30 s), and reduction of capillary perfusion (by 40 ± 7% of baseline). Moreover, at the end of BCCAO and reperfusion there was a significant increase in reactive oxygen species (ROS) formation when compared with baseline. Quercetin highest dose determined dilation in all arteriolar orders (by 40 ± 4% of baseline in order 2) and prevented microvascular permeability (0.15 ± 0.02 NGL), leukocyte adhesion (3 ± 1/100 μm v.l./30 s) as well as ROS formation, while capillary perfusion was protected. Inhibition of endothelial nitric oxide synthase (NOS) prior to quercetin reduced arteriolar dilation (order 2 diameter increase by 10.3 ± 2.5% of baseline) and caused permeability increase (0.29 ± 0.03 NGL); inhibition of neuronal NOS or inducible NOS did not affect quercetin-induced effects. Inhibition of guanylyl cyclase prior to quercetin reversed the quercetin’s effects on pial arteriolar diameter and leakage. In conclusion, quercetin was able to protect pial microcirculation from ischemia–reperfusion damage inducing arteriolar dilation likely by nitric oxide release. Moreover, quercetin scavenger activity blunted ROS formation preserving the blood–brain barrier integrity

Long term remodeling of rat pial microcirculation after transient middle cerebral artery occlusion and reperfusion.

Objective: The aim of this study was to assess the in vivo structural and functional remodeling of pial arteriolar networks in the ischemic area of rats submitted to transient middle cerebral artery occlusion (MCAO) and different time intervals of reperfusion. Methods and results: Two closed cranial windows were implanted above the left and right parietal cortex to observe pial microcirculation by fluorescence microscopy. The geometric characteristics of pial arteriolar networks, permeability increase, leukocyte adhesion and capillary density were analyzed after 1 h or 1, 7, 14 or 28 days of reperfusion. MCAO and 1-hour reperfusion caused marked microvascular changes in pial networks. The necrotic core was devoid of vessels, while the penumbra area presented a few arterioles, capillaries and venules with severe neuronal damage. Penumbra microvascular permeability and leukocyte adhesion were pronounced. At 7 days of reperfusion, new pial arterioles were organized in anastomotic vessels, overlapping the ischemic core and in penetrating pial arterioles. Vascular remodeling caused different arteriolar rearrangement up to 28 days of reperfusion and animals gradually regained their motor and sensory functions. Conclusions: Transient MCAO-induced pial-network remodeling is characterized by arteriolar anastomotic arcades. Remodeling mechanisms appear to be accompanied by an increased expression of nitric oxide synthases

Hypotensive and bradycardic effects of mouth opening: evidence in the human

Objective Previous studies in normotensive anesthetized rats (Lapi Arch.Ital.Biol 151:11-23,2013) showed that peripheral stimulation of the trigeminal nerve induced by submaximal mouth opening (mandibular extension, ME) caused prolonged (at least 80min) bradycardia, hypotension and cerebral hemodynamic changes (pial arterioles showed a characteristic response pattern consisting in a significant constriction during ME followed by a dilatation for the entire remaining observation time). Design and method In this study we assessed the in vivo effects of ME on HR, MABP and pial microcirculation in hypertensive rats. Experiments were performed in male Wistar rats weighing 250-300g (n = 8). Hypertension was induced by intraperitoneal daily injection of dexamethasone (0.03mg/kg/day) for 10 days. ME was obtained by inserting an ad hoc developed retractor between the dental arches. HR and MABP were recorded by ECG and a catheter placed in the left femoral artery and measured by a computer-assisted system. Pial arterioles were observed through a closed cranial window implanted above the left parietal cortex and visualized by an in vivo fluorescence microscopy technique to assess vessel diameter changes before (baseline), during 10min ME and thereafter until 160min. Arteriolar diameters were measured with a computer-assisted method (MIP Image program, frame by frame). Results In sham-treated (no ME) hypertensive rats (n=3) HR, MABP and pial microcirculation did not change during whole observation period. Hypertensive rats subjected to ME (n=5) showed a significant decrease of HR and MABP. HR declined by 42bpm, (p<0.01) starting from 60 min after ME up to 160min, while MABP by 18mmHg (p<0.05) starting from 20min after ME up to 100min, compared with baseline. Pial arterioles exhibited a biphasic response: the arteriolar diameter decreased by 2.94&#956;m (p<0.05) during ME, afterwards it significantly increased by 3.46&#956;m (p<0.01) starting from 20min after ME; this vasodilatation lasted for the whole observation period. Conclusions Our results suggest that ME is able to exert profound and prolonged regulatory effects on systemic arterial blood pressure and pial arteriolar tone in hypertensive rats

Repeated mandibular extension in rat: A procedure to modulate the cerebral arteriolar tone

Previous data have shown both in the rat and in the human that a single mandibular extension lasting 10 min induces a significant important and prolonged reduction in blood pressure and heart rate, affecting also rat pial microcirculation by the release of endothelial factors. In the present work, we assessed whether repeated mandibular extension could further prolong these effects. We performed two mandibular extensions, the second mandibular extension being applied 10 min after the first one. The second mandibular extension produced a reduction in blood pressure and heart rate for at least 240 min. As in the case of a single mandibular extension, pial arterioles dilated persisting up to 140 min after the second extension. Spectral analysis on 30 min recordings under baseline conditions and after repetitive mandibular extensions showed that the pial arterioles dilation was associated with rhythmic diameter changes sustained by an increase in the frequency components related to endothelial, neurogenic, and myogenic activity while a single mandibular extension caused, conversely, an increase only in the endothelial activity. In conclusion, repetitive mandibular extension prolonged the effects of a single mandibular extension on blood pressure, heart rate and vasodilation and induced a modulation of different frequency components responsible of the pial arteriolar tone, in particular increasing the endothelial activity

Increased efficacy of dietary supplement containing wax ester-rich marine oil and xanthophylls in a mouse model of dry macular degeneration

Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy

Trigeminocardiac reflex by mandibular extension on rat pial microcirculation: Role of nitric oxide

In the present study we have extended our previous findings about the effects of 10 minutes of passive mandibular extension in anesthetized Wistar rats. By prolonging the observation time to 3 hours, we showed that 10 minutes mandibular extension caused a significant reduction of the mean arterial blood pressure and heart rate respect to baseline values, which persisted up to 160 minutes after mandibular extension. These effects were accompanied by a characteristic biphasic response of pial arterioles: during mandibular extension, pial arterioles constricted and after mandibular extension dilated for the whole observation period. Interestingly, the administration of the opioid receptor antagonist naloxone abolished the vasoconstriction observed during mandibular extension, while the administration of Nω-Nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, abolished the vasodilation observed after mandibular extension. Either drug did not affect the reduction of mean arterial blood pressure and heart rate induced by mandibular extension. By qRT-PCR, we also showed that neuronal nitric oxide synthase gene expression was significantly increased compared with baseline conditions during and after mandibular extension and endothelial nitric oxide synthase gene expression markedly increased at 2 hours after mandibular extension. Finally, western blotting detected a significant increase in neuronal and endothelial nitric oxide synthase protein expression. In conclusion mandibular extension caused complex effects on pial microcirculation involving opioid receptor activation and nitric oxide release by both neurons and endothelial vascular cells at different times