Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Low-Frequency Components in Rat Pial Arteriolar Rhythmic Diameter Changes.

This study aimed to analyze the frequency components present in spontaneous rhythmic diameter changes in rat pial arterioles. Pial microcirculation was visualized by fluorescence microscopy. Rhythmic luminal variations were evaluated via computer-assisted methods. Spectral analysis was carried out on 30-min recordings under baseline conditions and after administration of acetylcholine (Ach), papaverine (Pap), Nω-nitro-L-arginine (L-NNA) prior to Ach, indomethacin (INDO), INDO prior to Ach, charybdotoxin and apamin, and charybdotoxin and apamin prior to Ach. Under baseline conditions all arteriolar orders showed 3 frequency components in the ranges of 0.0095-0.02, 0.02-0.06, and 0.06-0.2 Hz, another 2 in the ranges of 0.2-2.0 and 2.5-4.5 Hz, and another ultra-low-frequency component in the range of 0.001-0.0095 Hz. Ach caused a significant increase in the spectral density of the frequency components in the range of 0.001-0.2 Hz. Pap was able to slightly increase spectral density in the ranges of 0.001-0.0095 and 0.0095-0.02 Hz. L-NNA mainly attenuated arteriolar responses to Ach. INDO prior to Ach did not affect the endothelial response to Ach. Charybdotoxin and apamin, suggested as endothelium-derived hyperpolarizing factor inhibitors, reduced spectral density in the range of 0.001-0.0095 Hz before and after Ach administration. In conclusion, regulation of the blood flow distribution is due to several mechanisms, one of which is affected by charibdotoxin and apamin, modulating the vascular tone.

Evidence in the human of a hypotensive and a bradycardic effect after mouth opening maintained for 10 min.

PURPOSE: We have recently shown that in humans submaximal mouth opening associated with partial masticatory movements for 10 min is followed by a small but significant and prolonged reduction of blood pressure and heart rate. We here report the effects of a fixed mouth opener. METHODS: In 22 seated normotensive volunteers the effect on blood pressure and heart rate was studied in randomized order after fixed mandibular extension and after a control procedure consisting in keeping a stick between the incisor teeth (both for 10 min). Automated recordings every 10 min were done for 40 min before and 120 min following the procedure. RESULTS: Two-way ANOVA for repeated measures on absolute values (actual recordings) and on changes from baseline revealed that, compared to controls, systolic, diastolic and mean blood pressure and heart rate were significantly lower after mandibular extension. Compared to controls, mandibular extension induced an average blood pressure drop of 2.88 mmHg (systolic), 2.55 mmHg (diastolic) and 2.42 mmHg (mean) over the entire observation period. The average decline over the central part of the observation period (30th to 80th min) was, respectively, of 3.62, 3.70 and 3.61 mmHg. The decrements of heart rate were of 2.11 and 2.66 beats per min. All these differences were statistically significant. The hypotensive and bradycardic responses persisted for 70-120 min. CONCLUSIONS: This study shows that, in normotensives, a single fixed submaximal mouth opening for 10 min is followed by prolonged albeit small reductions of blood pressure and heart rate.

Effects of oleuropein and pinoresinol on microvascular damage induced by hypoperfusion and reperfusion in rat pial circulation.

OBJECTIVE: The present study was aimed to assess the in vivo acute effects of oleuropein or/and pinoresinol, polyphenols widely diffused in natural sources, on rat pial microvascular responses during transient BCCAO and reperfusion. METHODS: Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified into five orders of branching. Capillaries were assigned order 0, the smallest arterioles order 1 and the largest ones order 5. RESULTS: Rats subjected to BCCAO and reperfusion showed: arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction in capillary perfusion. Pretreatment with oleuropein or pinoresinol, a higher dose before BCCAO determined dilation in all arteriolar orders RE. Microvascular leakage was reduced as well as leukocyte adhesion and ROS formation, while capillary perfusion was protected. Inhibition of endothelium nitric oxide synthase prior to oleuropein or pinoresinol reduced the effect of these polyphenols on pial arteriolar diameter and leakage. These substances, administered together, prevented microvascular damage to a larger extent. CONCLUSION: Oleuropein and pinoresinol were both able to protect pial microcirculation from I-reperfusion injury, to increase nitric oxide release and to reduce oxidative stress preserving pial blood flow distribution.

Effects of Citrus Flavonoids Against Microvascular Damage Induced by Hypoperfusion and Reperfusion in Rat Pial Circulation.

OBJECTIVE: The aim of the present study was to investigate the in vivo protective effects of hesperidin or diosmin or apigenin on damage induced by transient BCCAO and reperfusion. METHODS: Rat pial microcirculation was observed through a closed cranial window, using fluorescence microscopy. Pial arterioles were classified in five orders according to the Strahler's method. RESULTS: After 30 BCCAO and 60 minutes reperfusion, rats showed decreased arteriolar diameter, microvascular leakage, leukocyte adhesion, and reduction in capillary perfusion. Hesperidin and diosmin abolished the reduction in arteriolar diameter, while higher dose apigenin induced dilation by 21.7 ± 2.0% in order three arterioles RE. Nitric oxide synthase inhibition attenuated significantly hesperidin or diosmin or apigenin's effects on arteriolar diameter. Moreover, all these substances reduced microvascular leakage as well as leukocyte adhesion in dose-related manner, while capillary perfusion was protected. Furthermore, reduction in infarcted area and decrease in ROS production were observed. CONCLUSIONS: Hesperidin, diosmin, and apigenin showed dose-related protective effects on hypoperfusion-reperfusion injury, causing nitric oxide release and attenuating tissue edema and leukocyte adhesion.

Remodeling of Cerebral Microcirculation after Ischemia-Reperfusion.

Clinical and experimental studies have been focused on the pathophysiological mechanisms induced by brain ischemia-reperfusion injury. Recovery events, such as neurogenesis, angiogenesis and the growth of new blood vessels from the preexisting vascular tree, have been intensively studied in the last decades to clarify the vascular remodeling crucial for stroke outcome. This review aims to discuss the cerebral microcirculation remodeling induced by ischemia-reperfusion and the mechanisms involved in angiogenesis and vasculogenesis. The first in vivo observations were focused on anastomotic shunting of cerebral blood flow (CBF) in experimental and clinical models. Thereafter, vascular remodeling induced by cerebral ischemia-reperfusion was reported in mice and rats. Successively, other studies have assessed that within 30 days of middle cerebral artery (MCA) occlusion in rats, there is an increase in CBF and recovery from stroke. Recently, rats submitted to transient MCA occlusion showed pial microcirculation remodeling with the formation of new arterioles sprouting from penumbra vessels and overlapping the ischemic core. This review focuses on the production and/or activation of vasculotrophic factors able to trigger and facilitate microvascular remodeling. Vascular endothelial growth factor and endothelium-released nitric oxide appear to be the main factors involved in the formation of new vessels during microvascular remodeling. These studies are fundamental for consequent interventions on molecular targets, useful for fostering vascular remodeling and the recovery of functions.

Factors and Mechanisms of Thyroid Hormone Activity in the Brain: Possible Role in Recovery and Protection.

Thyroid hormones (THs) are essential in normal brain development, and cognitive and emotional functions. THs act through a cascade of events including uptake by the target cells by specific cell membrane transporters, activation or inactivation by deiodinase enzymes, and interaction with nuclear thyroid hormone receptors. Several thyroid responsive genes have been described in the developing and in the adult brain and many studies have demonstrated a systemic or local reduction in TH availability in neurologic disease and after brain injury. In this review, the main factors and mechanisms associated with the THs in the normal and damaged brain will be evaluated in different regions and cellular contexts. Furthermore, the most common animal models used to study the role of THs in brain damage and cognitive impairment will be described and the use of THs as a potential recovery strategy from neuropathological conditions will be evaluated. Finally, particular attention will be given to the link observed between TH alterations and increased risk of Alzheimer's Disease (AD), the most prevalent neurodegenerative and dementing condition worldwide.

The Anti-Inflammatory and Antioxidant Properties of Acebuche Oil Exert a Retinoprotective Effect in a Murine Model of High-Tension Glaucoma.

Glaucoma is characterized by cupping of the optic disc, apoptotic degeneration of retinal ganglion cells (RGCs) and their axons, and thinning of the retinal nerve fiber layer, with patchy loss of vision. Elevated intraocular pressure (IOP) is a major risk factor for hypertensive glaucoma and the only modifiable one. There is a need to find novel compounds that counteract other risk factors contributing to RGC degeneration. The oil derived from the wild olive tree (Olea europaea var. sylvestris), also called Acebuche (ACE), shows powerful anti-inflammatory, antioxidant and retinoprotective effects. We evaluated whether ACE oil could counteract glaucoma-related detrimental effects. To this aim, we fed mice either a regular or an ACE oil-enriched diet and then induced IOP elevation through intraocular injection of methylcellulose. An ACE oil-enriched diet suppressed glaucoma-dependent retinal glia reactivity and inflammation. The redox status of the glaucomatous retinas was restored to a control-like situation, and ischemia was alleviated by an ACE oil-enriched diet. Notably, retinal apoptosis was suppressed in the glaucomatous animals fed ACE oil. Furthermore, as shown by electroretinogram analyses, RGC electrophysiological functions were almost completely preserved by the ACE oil-enriched diet. These ameliorative effects were IOP-independent and might depend on ACE oil's peculiar composition. Although additional studies are needed, nutritional supplementation with ACE oil might represent an adjuvant in the management of glaucoma.

ß3-Adrenoceptor as a new player in the sympathetic regulation of the renal acid-base homeostasis.

Efferent sympathetic nerve fibers regulate several renal functions activating norepinephrine receptors on tubular epithelial cells. Of the beta-adrenoceptors (ß-ARs), we previously demonstrated the renal expression of ß3-AR in the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the collecting duct (CD), where it participates in salt and water reabsorption. Here, for the first time, we reported ß3-AR expression in the CD intercalated cells (ICCs), where it regulates acid-base homeostasis. Co-localization of ß3-AR with either proton pump H+-ATPase or Cl-/HCO3 - exchanger pendrin revealed ß3-AR expression in type A, type B, non-A, and non-B ICCs in the mouse kidney. We aimed to unveil the possible regulatory role of ß3-AR in renal acid-base homeostasis, in particular in modulating the expression, subcellular localization, and activity of the renal H+-ATPase, a key player in this process. The abundance of H+-ATPase was significantly decreased in the kidneys of ß3-AR-/- compared with those of ß3-AR+/+ mice. In particular, H+-ATPase reduction was observed not only in the CD but also in the TAL and DCT, which contribute to acid-base transport in the kidney. Interestingly, we found that in in vivo, the absence of ß3-AR reduced the kidneys' ability to excrete excess proton in the urine during an acid challenge. Using ex vivo stimulation of mouse kidney slices, we proved that the ß3-AR activation promoted H+-ATPase apical expression in the epithelial cells of ß3-AR-expressing nephron segments, and this was prevented by ß3-AR antagonism or PKA inhibition. Moreover, we assessed the effect of ß3-AR stimulation on H+-ATPase activity by measuring the intracellular pH recovery after an acid load in ß3-AR-expressing mouse renal cells. Importantly, ß3-AR agonism induced a 2.5-fold increase in H+-ATPase activity, and this effect was effectively prevented by ß3-AR antagonism or by inhibiting either H+-ATPase or PKA. Of note, in urine samples from patients treated with a ß3-AR agonist, we found that ß3-AR stimulation increased the urinary excretion of H+-ATPase, likely indicating its apical accumulation in tubular cells. These findings demonstrate that ß3-AR activity positively regulates the expression, plasma membrane localization, and activity of H+-ATPase, elucidating a novel physiological role of ß3-AR in the sympathetic control of renal acid-base homeostasis.

Effects of physical exercise associated with a diet enriched with natural antioxidants on cerebral hypoperfusion and reperfusion injury in spontaneously hypertensive rats.

Oxidative stress is implicated in the pathogenesis of arterial hypertension. The reduction in the bioavailability of nitric oxide (NO) causes endothelial dysfunction, altering the functions of cerebral blood vessels. Physical exercise and intake of antioxidants improve the redox state, increasing the vascular NO production and/or the decrease in NO scavenging by reactive oxygen species (ROS). The present study was aimed at assessing the effects of physical exercise associated with a diet enriched with antioxidants from the Annurca apple in preventing the microvascular damage due to cerebral hypoperfusion and reperfusion injury in spontaneously hypertensive rats (SHRs). The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed cranial window. As expected, SHRs subjected to physical exercise or an antioxidants-enriched diet showed a reduction of microvascular permeability, ROS formation, and leukocyte adhesion to venular walls, with a major effect of the antioxidants-enriched diet, when compared to untreated SHRs. Moreover, capillary perfusion was preserved by both treatments in comparison with untreated SHRs. Unexpectedly, the combined treatments did not induce higher effects than the single treatment. In conclusion, our results support the efficacy of physical activity or antioxidant supplement in reducing the microvascular alterations due to hypertension and ascribe to an antioxidants-enriched diet effective microvascular protection in SHRs.

Prolonged hypotensive and bradycardic effects of passive mandibular extension: evidence in normal volunteers.

Various procedures involving stimulations of facial regions are known to induce so-called trigemino-cardiac reflexes that entail a decrease of heart rate and blood pressure. We here report the effects of a specific stimulation that consists in a submaximal passive mandibular extension obtained by means of a dilatator applied for 10 minutes between the upper and lower incisor teeth, associated with partial active masticatory movements. Blood pressure and heart rate were determined in 18 young normal volunteers by Omron M4, before (20 minutes), during (10 minutes) and after mandibular extension (80 minutes) and under control conditions (same overall duration without stimulation). While control values remained stable, mandibular extension was followed by a progressive decline of both blood pressure (up to about 12/11 mmHg) and heart rate (up to about 13 bpm), statistically confirmed by ANOVA both on absolute values and on changes from basal values. The decline of systolic blood pressure and heart rate significantly correlated with basal values. The present findings indicate that submaximal opening of the mouth, associated to partial masticatory movements, induces a prolonged reduction of blood pressure and heart rate in normotensive volunteers.

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.

Rat pial microvascular responses to melatonin during bilateral common carotid artery occlusion and reperfusion.

The present study assessed the in vivo rat pial microvascular responses induced by melatonin during brain hypoperfusion and reperfusion (RE) injury. Pial microcirculation of male Wistar rats was visualized by fluorescence microscopy through a closed cranial window. Hypoperfusion was induced by bilateral common carotid artery occlusion (BCCAO, 30 min); thereafter, pial microcirculation was observed for 60 min. Arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (PCL), and capillary red blood cell velocity (V(RBC) ) were investigated by computerized methods. Melatonin (0.5, 1, 2 mg/kg b.w.) was intravenously administered 10 min before BCCAO and at the beginning of RE. Pial arterioles were classified in five orders according to diameter, length, and branchings. In control group, BCCAO caused decrease in order 2 arteriole diameter (by 17.5 ± 3.0% of baseline) that was reduced by 11.8 ± 1.2% of baseline at the end of RE, accompanied by marked leakage and leukocyte adhesion. PCL and capillary V(RBC) decreased. At the end of BCCAO, melatonin highest dosage caused order 2 arteriole diameter reduction by 4.6 ± 2.0% of baseline. At RE, melatonin at the lower dosages caused different arteriolar responses. The highest dosage caused dilation in order 2 arteriole by 8.0 ± 1.5% of baseline, preventing leakage and leukocyte adhesion, while PCL and V(RBC) increased. Luzindole (4 mg/kg b.w.) prior to melatonin caused order 2 arteriole constriction by 12.0 ± 1.5% of baseline at RE, while leakage, leukocyte adhesion, PCL and V(RBC) were not affected. Prazosin (1 mg/kg b.w.) prior to melatonin did not significantly change melatonin's effects. In conclusion, melatonin caused different responses during hypoperfusion and RE, modulating pial arteriolar tone likely by MT1 and MT2 melatonin receptors while preventing blood-brain barrier changes through its free radical scavenging action.

Thyroid hormone deiodinases response in brain of spontaneausly hypertensive rats after hypotensive effects induced by mandibular extension.

PURPOSE: The deiodinases activate or inactivate the thyroid hormones (TH) in virtually all tissues in both physiological and pathological conditions. The three deiodinases, DIO1, DIO2, and DIO3, have different catalytic functions and regulate TH tissue distribution. The aim of the present study was to evaluate the modulation of gene expression of the deiodinases and TH transporters and protein levels of DIO1 in parietal and frontal areas of cerebral cortex of spontaneously hypertensive rats (SHRs), after two successive mandibular extensions (ME). METHODS: ME was performed on anesthetized rats by a dilatator appropriately designed and real-time PCR and western blotting techniques were employed for gene expression and protein level study. RESULTS: Mean blood pressure (MBP) significantly decreased in 2ME-treated rats when compared to sham-operated rats (p < 0.001) and this decrease lasted for the entire observation period. In gene expression analysis, in 2ME-treated rats we did not observe any significant variation of DIO1 and DIO3 with respect to the sham-operated rats. Differently, DIO2 gene expression significantly increased in frontal area of 2ME-treated rats, with respect to sham-operated rats (p < 0.01). Furthermore, in parietal area, protein levels of DIO1 in 2ME-treated rats were significantly higher than in sham-operated rats (p < 0.01). Moreover MCT8 and OATP1C1 both resulted significantly higher (p < 0.05 and p < 0.001) in sham frontal cortex. CONCLUSION: In summary, our data on SHRs, while confirming the hypotensive effect of two MEs, show that the treatment also solicits the three deiodinases production in the cerebral cortex.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Geometric Features of the Pial Arteriolar Networks in Spontaneous Hypertensive Rats: A Crucial Aspect Underlying the Blood Flow Regulation.

BACKGROUND: Several studies indicate that hypertension causes major changes in the structure of the vessel wall by affecting the regulation of blood supply to the tissues. Recently, it has been observed that capillary blood flow is also considerably influenced by the structural arrangement of the microvascular networks that undergo rarefaction (reduction of the perfused vessel number). Therefore, this study aimed to assess the geometric arrangements of the pial arteriolar networks and the arteriolar rhythmic diameter changes in spontaneously hypertensive rats (SHRs). METHODS: Fluorescence microscopy was utilized to observe in vivo the pial microcirculation through a closed cranial window. Pial arterioles were classified according to Strahler's method. The arteriolar rhythmic diameter changes were evaluated by a generalization short-time Fourier transform. RESULT: Young SHRs showed four orders of vessels while the adult ones only three orders. The diameter, length, and branching number obeyed Horton's law; therefore, the vessels were distributed in a fractal manner. Larger arterioles showed more asymmetrical branches than did the smaller ones in young SHRs, while in adult SHRs smaller vessels presented asymmetrical branchings. In adult SHRs, there was a significant reduction in the cross-sectional area compared with the young SHRs: this implies an increase in peripheral resistance. Young and adult age-matched normotensive rats did not show significant alterations in the geometric arteriolar arrangement with advancing age, both had four orders of arteriolar vessels, and the peripheral resistance did not change significantly. Conversely, the frequency components evaluated in arteriolar rhythmic diameter changes of young and adult SHRs showed significant differences because of a reduction in the frequency components related to endothelial activity detected in adult SHRs. CONCLUSION: In conclusion, hypertension progressively causes changes in the microarchitecture of the arteriolar networks with a smaller number of vessels and consequent reduced conductivity, characteristic of rarefaction. This was accompanied by a reduction in the formation and release of independent and dependent - endothelial nitric oxide components regulating arterial vasomotion.

The Effects of Angiotensin II or Angiotensin 1-7 on Rat Pial Microcirculation during Hypoperfusion and Reperfusion Injury: Role of Redox Stress.

Renin-angiotensin systems produce angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7), which are able to induce opposite effects on circulation. This study in vivo assessed the effects induced by Ang II or Ang 1-7 on rat pial microcirculation during hypoperfusion-reperfusion, clarifying the mechanisms causing the imbalance between Ang II and Ang 1-7. The fluorescence microscopy was used to quantify the microvascular parameters. Hypoperfusion and reperfusion caused vasoconstriction, disruption of blood-brain barrier, reduction of capillary perfusion and an increase in reactive oxygen species production. Rats treated with Ang II showed exacerbated microvascular damage with stronger vasoconstriction compared to hypoperfused rats, a further increase in leakage, higher decrease in capillary perfusion and marker oxidative stress. Candesartan cilexetil (specific Ang II type 1 receptor (AT1R) antagonist) administration prior to Ang II prevented the effects induced by Ang II, blunting the hypoperfusion-reperfusion injury. Ang 1-7 or ACE2 activator administration, preserved the pial microcirculation from hypoperfusion-reperfusion damage. These effects of Ang 1-7 were blunted by a Mas (Mas oncogene-encoded protein) receptor antagonist, while Ang II type 2 receptor antagonists did not affect Ang 1-7-induced changes. In conclusion, Ang II and Ang 1-7 triggered different mechanisms through AT1R or MAS receptors able to affect cerebral microvascular injury.

Protective effects of Lactobacillus paracasei F19 in a rat model of oxidative and metabolic hepatic injury.

The liver is susceptible to such oxidative and metabolic stresses as ischemia-reperfusion (I/R) and fatty acid accumulation. Probiotics are viable microorganisms that restore the gut microbiota and exert a beneficial effect on the liver by inhibiting bacterial enzymes, stimulating immunity, and protecting intestinal permeability. We evaluated Lactobacillus paracasei F19 (LP-F19), for its potential protective effect, in an experimental model of I/R (30 min ischemia and 60 min reperfusion) in rats fed a standard diet or a steatogen [methionine/choline-deficient (MCD)] diet. Both groups consisted of 7 sham-operated rats, 10 rats that underwent I/R, and 10 that underwent I/R plus 8 wk of probiotic dietary supplementation. In rats fed a standard diet, I/R induced a decrease in sinusoid perfusion (P < 0.001), severe liver inflammation, and necrosis besides an increase of tissue levels of malondialdehyde (P < 0.001), tumor necrosis factor-alpha (P < 0.001), interleukin (IL)-1beta (P < 0.001), and IL-6 (P < 0.001) and of serum levels of transaminase (P < 0.001) and lipopolysaccharides (P < 0.001) vs. sham-operated rats. I/R also induced a decrease in Bacterioides, Bifidobacterium, and Lactobacillus spps (P < 0.01, P < 0.001, and P < 0.001, respectively) and an increase in Enterococcus and Enterobacteriaceae (P < 0.01 and P < 0.001, respectively) on intestinal mucosa. The severity of liver and gut microbiota alterations induced by I/R was even greater in rats with liver inflammation and steatosis, i.e., MCD-fed animals. LP-F19 supplementation significantly reduced the harmful effects of I/R on the liver and on gut microbiota in both groups of rats, although the effect was slightly less in MCD-fed animals. In conclusion, LP-F19 supplementation, by restoring gut microbiota, attenuated I/R-related liver injury, particularly in the absence of steatosis.

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.

The Pomace Extract Taurisolo Protects Rat Brain From Ischemia-Reperfusion Injury.

Taurisolo® is a pomace extract from Aglianico Grapes, a wine cultivar native to Campania (Southern Italy). It exhibits a very high polyphenolic content and, consumed as a nutraceutical, is effective in reducing the level of Trimethylamine N-oxide (TMAO), a cardiovascular disease risk factor marker. We here show the effects of Taurisolo® on rat brain microvascular alterations induced by a diminution in cerebral blood flow (CBFD) for 30 min, due to bilateral common carotid artery occlusion, and subsequent blood flow restoration (CBFR) for 60 min. The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed window implanted into the skull bone. The rat pial arterioles were classified according to Strahler's ordering scheme, from smaller penetrating arterioles up to the larger ones. Western blotting analysis and mass spectrometry (MS)-based metabolomics were used to investigate the expression of endothelial nitric oxide synthase (eNOS) or the presence of peroxidized cardiolipin and several inflammatory mediators, respectively. Radical Oxygen Species (ROS) formation and neuronal loss were assessed. In rats CBFD and CBFR caused a decrease in arteriolar diameter, increase in fluorescent leakage and in adhesion of leukocytes to venular walls, reduction in the length of perfused capillaries and increment of ROS formation with large infarct size. Taurisolo®, intravenously or orally administered, induced pial arteriolar dilation (up to >30% of baseline), prevented fluorescent leakage, adhesion of leukocytes, ROS formation, while facilitated capillary perfusion and significantly reduced infarct size. These effects were accompanied by an increase in eNOS expression. Mass-spectrometry metabolomics analysis detected a marked decrease in the amount of peroxidized cardiolipin and pronounced reduction in pro-inflammatory prostaglandins and thromboxane Txb2. Altogether, these results extend the nutraceutical potential of Taurisolo® and suggest their eligibility for preventing brain damage due to ischemia-reperfusion injury.