L-Arginine and Taurisolo® Effects on Brain Hypoperfusion-Reperfusion Damage in Hypertensive Rats.

Acute and chronic hypertension causes cerebral vasculopathy, increasing the risk of ischemia and stroke. Our study aimed to compare the effects of arterial pressure reduction on the pial microvascular responses induced by hypoperfusion and reperfusion in spontaneously hypertensive Wistar rats, desamethasone-induced hypertensive Wistar rats and age-matched normotensive Wistar rats fed for 3 months with a normal diet or normal diet supplemented with L-arginine or Taurisolo® or L-arginine plus Taurisolo®. At the end of treatments, the rats were submitted to bilateral occlusion of common carotid arteries for 30 min and reperfusion. The microvascular parameters investigated in vivo through a cranial window were: arteriolar diameter changes, permeability increase, leukocyte adhesion to venular walls and percentage of capillaries perfused. Hypoperfusion-reperfusion caused in all rats marked microvascular changes. L-arginine treatment was effective in reducing arterial blood pressure causing vasodilation but did not significantly reduce the damage induced by hypoperfusion-reperfusion. Taurisolo® treatment was less effective in reducing blood pressure but prevented microvascular damage from hypoperfusion-reperfusion. L-arginine plus Taurisolo® maintained blood pressure levels within the physiological range and protected the pial microcirculation from hypoperfusion-reperfusion-induced microvascular injuries. Therefore, the blood pressure reduction is not the only fundamental aspect to protect the cerebral circulation from hypoperfusion-reperfusion damage.

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.

Effects of a catechins-enriched diet associated with moderate physical exercise in the prevention of 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.

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.

Lumbar spinal cord neurons putatively involved in ejaculation are sexually dimorphic in early postnatal mice.

A crucial role in ejaculation is thought to be played by a population of lumbar spino-thalamic neurons (LSt), which express galanin and other neuropeptides. In rats, these neurons are activated with ejaculation and their lesion selectively abolishes ejaculation but not other mating behaviors. Consistently with their role, in adult rats and humans, LSt neurons are sexually dimorphic, being more numerous in males. Here we examined whether sexual dimorphism arises early in development, using a transgenic mouse line in which the expression of fluorescent protein is driven by the galanin promoter. We focused on postnatal day 4, shortly after a transient perinatal androgen surge in males that could play an organizational role in LSt development. We found a population of brightly fluorescent neurons organized in bilateral columns dorsolateral to the central canal in segments L1-L5, the expected location of the LSt group. Their number was close to that of adult preparations and significantly greater in male than in female siblings (+19%; CI95% : +13% to +27%; p < .01). This was not due to a generalized higher galanin expression in the male since fluorescent L4 DRG neurons, innervating the hindlimbs and lower back, were not significantly dimorphic (-4%; CI95% : -10% to +8%; p = .92). Unexpectedly, we found in cervical segments a population of fluorescent neurons having a location relative to the central canal similar to the LSt. Thus, the LSt group is sexually dimorphic soon after birth. However, it is possible that only a subset of its neurons participate in the control of ejaculation.

Effects of Mandibular Extension on Pial Arteriolar Diameter Changes in Glucocorticoid-Induced Hypertensive Rats.

Previously, in normotensive rats, it has been observed that a repetitive sub-maximal mouth opening (mandibular extension, ME) obtained by placing a home-made U-shaped dilator between the superior and inferior dental arches of the rat caused modulation of pial arteriolar tone. The present study was aimed to characterize pial microcirculation in two different cortical brain regions and to assess the hemodynamic effects of a single or double ME on pial arteriolar rhythmic diameter changes in rats rendered hypertensive by dexamethasone administrations. Cranial windows were prepared on parietal and frontal region. Pial arterioles were classified by Strahler method in five orders by in vivo fluorescence microscopy technique associated with a computerized system that permits off-line measurements of arteriolar diameter changes. Two 10 min ME at 10 min interval were applied; then the animals were monitored for further 240 min. Dexamethasone-treated rats exhibited a marked arterial rarefaction and asymmetry of bifurcation in the pial microvascular networks more evident in the frontal region. Starting from ME1, in both cortical areas, the arterioles dilated, and the vasodilation became significant compared to baseline after ME2 for the entire observation period. The spectral analysis carried out on order 2 arteriolar diameter change tracings, showed that double ME increased the spectral density of the frequency components related to endothelial, neuronal and myogenic activities in both the cortical regions studied. In conclusion, double ME has a generalized effect in the cortical areas by restoring the physiological vasomotion of the pial arterioles that was severely impaired by the experimentally hypertension.

Renin-Angiotensin System Responds to Prolonged Hypotensive Effect Induced by Mandibular Extension in Spontaneously Hypertensive Rats.

There is an ongoing interest in the renin-angiotensin system (RAS) contribution either to pathological mechanisms leading to hypertension (mainly regarding the ACE/AngII/AT1R axis), or, to RAS protective and pro-regenerative actions, primarily ascribed to the mediation of the AT2R and the MAS1 receptor. In the present study, we evaluated the modulation of gene expression and protein levels of "deleterious" (ACE/AngII/AT1R) and "protective" [ACE/AngII/AT2R and ACE2/Ang(1-7)/MAS1 arms] RAS components in parietal and frontal areas of cerebral cortex of spontaneously hypertensive rats (SHRs), after two periods of mandibular extensions (MEs). Blood pressure, BP and heart rate, HR were also measured. While no significant changes in BP and HR were present in the sham operated (SO) group, in rats after two MEs (2-ME rats), BP displayed a marked decrease (p < 0.001) at ME2, and remained then stably low for the subsequent observation period. In gene expression analysis, in SHRs undergoing two MEs, either in parietal or frontal cortex, we did not observe any significant variation of AT2R and ACE2 with respect to SO rats. In contrast, we observed a decrease in Mas1 gene expression in parietal area (p < 0.01) and an increase in frontal region (p < 0.01). AT1R and ACE gene expression was significantly higher in 2-ME rats than SO in parietal cortex (p < 0.05) but no difference was observed in the frontal area. Concerning protein levels, in parietal area, AT1R and AT2R did not change whereas MAS1 significantly decreased in 2-ME rats (p < 0.05). In frontal area, both AT1R and AT2R significantly decreased in 2-ME rats (p < 0.05), whereas MAS1 did not significantly change. Gene expression analysis in normotensive (NT) rats revealed the non-detectability of AT1R in both parietal and frontal zone. In parietal area, AT2R (p < 0.0001) and Mas1 (p < 0.01) were significantly decreased in 2-ME NT rats, when compared to SO, and ACE and ACE2 resulted not detectable whereas there was some expression of these genes after 2-ME procedure. In conclusion, our data in rat models indicated that a 2-ME procedure induced a hypotensive response and that a modulation of gene expression and protein levels of RAS components occurred in different cerebral cortex areas.

Evidence in hypertensive rats of hypotensive effect after mandibular extension.

Previous studies in anesthetized normotensive rats demonstrated that a single mouth opening for 10 min obtained by an ad hoc dilator (mandibular extension [ME]) produced a blood pressure reduction by about 20 mmHg lasting for about 2 h and that once-repeated ME prolonged this effect. We here describe these effects in hypertensive rats. Mean (intra) arterial blood pressure (MABP) and heart rate (HR) was followed for up to a maximum of 470 min after single or repeated 10 min-lasting ME in two groups of anesthetized, male, 6-9 months old hypertensive rats. In one group, hypertension was induced by dexamethasone (20 µg/kg/day, subcutaneously for 7 days; Dex-HT); the other group was spontaneously hypertensive rats (SHR). Studies were done, in Dex-HT rats, after only surgical procedures (no ME, sham-operated rats), single ME, early repeated (after 10 min) ME (ER-ME) and late repeated (after 160 min) ME (LR-ME) and, in SHR, after only surgical procedures and ER-ME. One-way ANOVA for repeated measures revealed no significant effect on MABP and HR in sham-operated groups. In Dex-HT rats, single ME was followed by a significant MABP decline by 25 mmHg, lasting for 100 min; ER-ME and LR-ME were followed by an even greater significant MABP decline by 40 mmHg, which outlasted the experimental observation period. In SHR, ER-ME gave similar results as in Dex-HT rats. HR significantly declined in all, except sham-operated groups. In conclusions, ME is followed by a prolonged MABP decline also in hypertensive rats. This effect is even more pronounced, in length and magnitude, after repeated ME.

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.

Modulation of gene expression in contextual fear conditioning in the rat.

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning.

Differentially expressed genes in Hirudo medicinalis ganglia after acetyl-L-carnitine treatment.

Acetyl-L-carnitine (ALC) is a naturally occurring substance that, when administered at supra-physiological concentration, is neuroprotective. It is involved in membrane stabilization and in enhancement of mitochondrial functions. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer's disease and painful neuropathies. ALC is known to improve the cognitive capability of aged animals chronically treated with the drug and, recently, it has been reported that it impairs forms of non-associative learning in the leech. In the present study the effects of ALC on gene expression have been analyzed in the leech Hirudo medicinalis. The suppression subtractive hybridisation methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the leech nervous system after ALC treatment. The method detects differentially but also little expressed transcripts of genes whose sequence or identity is still unknown. We report that a single administration of ALC is able to modulate positively the expression of genes coding for functions that reveal a lasting effect of ALC on the invertebrate, and confirm the neuroprotective and neuromodulative role of the substance. In addition an important finding is the modulation of genes of vegetal origin. This might be considered an instance of ectosymbiotic mutualism.

Lipofuscin accumulation and gene expression in different tissues of mnd mice.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage diseases characterized by neurological impairment and blindness. NCLs are almost always due to single mutations in different genes (CLN1-CLN8). Ubiquitous accumulation of undigested material and of a hydrophobic inner mitochondrial membrane protein, the subunit c of mitochondrial ATP synthase, has been described. Although protein mutation(s) in the endoplasmic reticulum-lysosomes axis can modify the trafficking and the recycling of different molecules, one of the upstream targets in these diseases may be represented by the balance of gene expression. To understand if and how neurons modify the levels of important genes during the first phases of the disease, it is important to characterize the mechanisms of neurodegeneration. Due to the impossibility of performing this analysis in humans, alternative models of investigation are required. In this study, a mouse model of human NCL8, the mnd mouse has been employed. The mnd mice recapitulate many clinical and histopathological features described in NCL8 patients. In this study, we found an altered expression of different genes in both central and peripheral organs associated with lipopigment accumulation. This is a preliminary approach, which could also be of interest in providing new diagnostic tools for NCLs.

Modulation of myelin basic protein gene expression by acetyl-L-carnitine.

Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring molecule which plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions, demonstrating efficacy and high tolerability in the treatment of neuropathies of various etiologies. ALC is a molecule of considerable interest for its clinical application in various neural disorders, although little is known regarding its effects on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after chronic ALC treatments. We provided evidence for a downregulation of the expression of all of the isoforms of myelin basic protein gene following prolonged ALC treatment, indicating a possible role in the modulation of myelin basic protein turnover, stabilizing and maintaining myelin integrity.

Cytoprotective effect of acetyl-L-carnitine evidenced by analysis of gene expression in the rat brain.

Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring substance that when administered at supraphysiological concentrations is neuroprotective. ALC plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions. ALC has demonstrated efficacy and high tolerability in the treatment of neuropathies of various etiologies, and it is a molecule of considerable interest for its clinical application in various neural disorders, such as Alzheimer's disease and painful neuropathies, although little is known regarding the effects of ALC on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after a chronic ALC treatment. In the present paper, we provide evidences for the up-regulation of the expression of prostaglandin D(2) synthase, brain-specific Na(+)-dependent inorganic phosphate transporter, and cytochrome b oxidase, bc1 complex induced in the rat brain by ALC. On the contrary, ALC treatment down-regulates the expression of the gene of ferritin-H. Altogether, these results suggest that ALC might play a cytoprotective role against various brain stressors.

Up-regulation of kinesin light-chain 1 gene expression by acetyl-L-carnitine: therapeutic possibility in Alzheimer's disease.

We investigated the effects of acetyl-l-carnitine on gene expression by means of the suppression subtractive hybridization method. The approach gives the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with acetyl-l-carnitine for 21 days. We observed that acetyl-l-carnitine increases the light-chain subunit of kinesin-1 gene expression. Recent evidences reported a link between kinesin-1 light-chain and Alzheimer's disease. Pathological hallmarks of Alzheimer's disease are potentially linked to alterations of the axonal compartments. Amyloid-beta peptide is a principal component of senile plaques and is considered to be central in the pathogenesis of the disease. The fast anterograde axonal transport of amyloid-beta peptide is mediated by direct binding to the light-chain subunit of kinesin-1. In this scenario, our results are of relevant importance for possible therapeutic intervention, suggesting a pathway for the treatment of Alzheimer's disease.