Enrique Leo Portiansky

Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na+/H+ exchanger

OBJECTIVE Experiments were performed to examine the effect of chronic inhibition of the Na(+)/H(+) exchanger isoform-1 (NHE-1) on cardiac hypertrophy of spontaneously hypertensive rats (SHR). METHODS SHR were orally treated during 1 month with two different doses (0.3 and 3.0 mg/kg/day) of the NHE-1 inhibitor, cariporide, or nifedipine (10.0 mg/kg/day). RESULTS The two doses of cariporide did not differ in their effects after 1 month of treatment, since both induced a slight decrease in systolic blood pressure (SBP) of approximately 6 mmHg and regression of the heart weight to body weight ratio (mg/g) from 3.28+/-0.05 to 3.04+/-0.05 (0.3 mg) and 2.99+/-0.10 (3.0 mg, P<0.05). Nifedipine, given for the same period, produced similar reduction in the hypertrophy index (3.03+/-0.05), but with a much greater decrease in arterial pressure (35.6+/-7.4 mmHg). Chronic treatment with cariporide induced a complete regression of the augmented cross sectional area of left ventricular myocytes without significant changes in collagen content, serum procollagen 1 propeptide levels or myocardial distensibility. CONCLUSIONS NHE inhibition represents a novel approach to induce regression of pathological hypertrophy of the heart. The finding can be rationalized mechanistically by previous in vitro studies suggesting a role of the NHE in the development of myocardial hypertrophy.

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene), mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.

Regression of Hypertensive Myocardial Fibrosis by Na+/H+ Exchange Inhibition

Abstract—We have recently reported that the inhibition of the Na+/H+ exchanger (NHE) during 1 month in spontaneously hypertensive rats (SHR) is followed by regression of cardiomyocyte hypertrophy but not of myocardial fibrosis. The aim of this study was to evaluate whether a treatment of longer duration could reduce myocardial fibrosis and stiffness. SHR received 3.0 mg/kg per day of the specific NHE-1 inhibitor cariporide; the effect on cardiomyocyte cross-sectional area, myocardial collagen volume fraction, collagen synthesis, and myocardial stiffness (length-tension relation in left papillary muscles) was evaluated at several time points (after 1, 2, or 3 months). A slight decrease of ≈5 mm Hg in systolic blood pressure was observed after 1 month of treatment with no further changes. After 2 and 3 months of treatment, the size of cardiomyocytes remained within normal values and myocardial fibrosis progressively decreased to normal level. Accordingly, myocardial stiffness and the serum levels of the carboxyterminal propeptide of procollagen type I, a marker of collagen type I synthesis, were normalized after 3 months. Left ventricular weight decreased from 910±43 (in untreated SHR) to 781±21 mg (treated SHR) after 3 months of treatment. No difference in body weight between treated and untreated SHR was observed after this period of treatment. The present data allow us to conclude that in the SHR the administration of an NHE-1 inhibitor for 2 or 3 months leads to the normalization of collagen type I synthesis, myocardial collagen volume fraction, and stiffness.

DOPAMINERGIC MESENCEPHALIC SYSTEMS AND BEHAVIORAL PERFORMANCE IN VERY OLD RATS

Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinsons disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4-6 months), old (22-24 months) and senile (30-32 months) Sprague-Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.

Morphological characterization of the female prostate (Skene's gland or paraurethral gland) of Lagostomus maximus maximus

The Skenes (paraurethral) gland is the histologic homologue to the male prostate. Much experimental work has been done on women and on various rodents. In this study we describe for the first time the anatomical and histological characteristics of the paraurethral gland in the plain viscacha (Lagostomus maximus maximus). This gland is formed by tubuloalveolar adenomers surrounded by connective tissue and smooth muscle fibers. The adenomers are lined by cuboidal or columnar epithelium. In the lumen of the adenomers, PAS positive secretions could be detected. The conduits are lined by bi-stratified epithelium. In some aspects this gland is similar to that of other mammals.

Impact of very old age on hypothalamic dopaminergic neurons in the female rat: A morphometric study

Dopaminergic neurons of the A12 (tuberoinfundibular dopaminergic system) and A14 (periventricular dopaminergic system) hypothalamic areas exert a tonic inhibitory control of prolactin secretion. Tuberoinfundibular dopaminergic system neuron function is known to decline during aging in rats, but little is known about the impact of extreme age on neuron number and morphology in the two systems. We morphometrically assessed the neurons of the tuberoinfundibular dopaminergic system and the periventricular dopaminergic system in female rats 6 (young, Y), 24 (old, O), and 30–32 (senescent, S) months old. Serial coronal sections of fixed hypothalami were immunohistochemically labeled for tyrosine hydroxylase, and immunoreactive perikarya from the A12 and A14 areas were quantitatively characterized and compared among the three age groups. Radioimmunoassay was used to measure serum prolactin. The number of A12 tyrosine hydroxylase–immunoreactive perikarya showed a steady decline with age, whereas the number of A14 tyrosine hydroxylase–immunoreactive perikarya remained stable from young to old age but showed a sharp drop in the senescent rats. In the old rats, tyrosine hydroxylase–immunoreactive neuronal area (A12 = 135.37 and A14 = 158.79 μm2) was significantly higher than that of young (A12 = 72.56 and A14 = 99.7 μm2) and senescent animals (A12 = 95.5 and A14 = 106.5 μm2). Densitometric assessment of median eminence tyrosine hydroxylase immunoreactivity revealed a steady age‐related reduction of tyrosine hydroxylase content in the median eminence. Serum prolactin levels increased steadily with age. We conclude that, in the female rat, aging brings about a progressive loss of both tuberoinfundibular dopaminergic system and periventricular dopaminergic system neurons, which becomes more conspicuous at extreme ages. J. Comp. Neurol. 458:319–325, 2003.

Loss of NeuN immunoreactivity in rat spinal cord neurons during aging

A morphologic study of the impact of aging on neuron marker expression was performed in different segments of the rat spinal cord. Spinal cord specimens from young (5 months), middle-aged (12 months) and senile (32 months) female rats were assessed. We found a complete loss of neuron-specific nuclear protein (NeuN) immunoreactivity in cervical, thoracic and lumbar segments of the senile animals whereas neuron-specific enolase (NSE) immunoreactivity was comparable in young and senile rats. These findings in otherwise morphologically well preserved spinal cord neurons are of interest and reveal that NeuN may not be a reliable marker to identify neurons in the spinal cord of aging rats.

Silencing of NHE-1 blunts the slow force response to myocardial stretch

Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²⁺ transient of unclear mechanism. We proposed that Na⁺/H⁺ exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(H⁺) (H⁺ efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Morphometry of cervical segments grey matter in the male rat spinal cord

The cervical portion of the spinal cord is an area frequently affected by alterations of medical and veterinary importance. Since there is scarce quantitative anatomical data on this region, we undertook a morphometric study of the grey matter of all segments of the rat cervical spinal cord of male rats in order to generate reference patterns to be used in future experimental studies. Using image analysis software, the total spinal cord length and grey and white matter area of each segment was recorded. The morphometric characteristics of the neurones populating the laminae of the grey matter of the cervical segments was also recorded. Neurones were classified into small, medium and large sizes for each lamina and statistically compared. The present data fill an anatomical information gap by providing quantitative data about the normal anatomical features of the rat cervical cord. The anatomical data found could be used to better understand the physiological relevance of that region in the rat.