Protective effects of delapril combined with indapamide or hydrochlorothiazide in spontaneously hypertensive stroke-prone rats: a comparative dose-response analysis.

In previous articles, we have shown that the combination of the angiotensin-converting enzyme (ACE) inhibitor delapril (12 mg/kg/day) and the diuretic indapamide (1 mg/kg/ day) was able to prolong the life span significantly in salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Because this finding was partly dependent on the antagonism of salt-loading effects by pharmacologic induction of diuresis, which prevented any increase in blood pressure values, we decided to evaluate whether lower doses of the combination could be equally protective without changing the progression of hypertension. Thus, we studied several treatments with progressively lower doses of delapril (6, 3, or 1.5 mg/kg/day) combined with indapamide (0.5, 0.25, or 0.125 mg/kg/day) or hydrochlorothiazide (2.5, 1.25, or 0.625 mg/kg/day) in salt-loaded SHRsp. Salt-loaded untreated animals were considered to be the control group. In agreement with previous experiments, control rats reached 50% mortality approximately 7 weeks after the beginning of salt loading. The combination of delapril and hydrochlorothiazide at the two lowest doses was not able to delay animal death significantly, whereas treatment with delapril and indapamide at the lowest dose was effective (50% survival rate, 15 weeks). The groups treated with the highest dose of delapril and hydrochlorothiazide or with the intermediate or highest dose of delapril and indapamide did not reach 50% mortality by the end of the experiment, at 44 weeks of treatment (i.e., when animals reached age 1 year). Only the highest delapril and indapamide doses were able to increase diuresis, but for a relatively short period. None of the treatments was able to lower or control blood pressure levels adequately. Therefore, blood pressure levels by themselves were not predictive of rat mortality. In contrast, the maximal value of proteinuria in the weeks preceding death was inversely correlated with the survival time. In conclusion, this study shows that low doses of an ACE inhibitor in combination with a diuretic can be effectively protective in a model of severe hypertension, independent of any change in blood pressure levels.

Changes in nicotinic acetylcholine receptor subunit mRNAs and nicotinic binding in spontaneously hypertensive stroke prone rats.

We have studied nicotinic acetylcholine receptors in spontaneously hypertensive 'stroke prone' (SHsp) rats. We found a significant decrease in 125I-alpha-bungarotoxin binding and alpha7 subunit mRNA levels in cortical areas of the SHsp rats with respect to Wistar Kyoto (WKy) normotensive rats. Antihypertensive drug treatment counteracted these changes in cerebral cortex but not in hippocampus. No significant change was instead found in [3H]-epibatidine binding and alpha4 and beta2 subunit mRNA levels. SHsp rats showed decreased latency at the active avoidance test and transiently increased threshold at both hot-plate and tail-flick tests in comparison with WKy rats. None of these behavioral parameters was correlated with 125I-alpha-bungarotoxin binding in cortical areas. In conclusion, present data show a preferential impairment of alpha-bungarotoxin sensitive nAChRs in SHsp rats.

The emergence of the volume transmission concept.

Interneuronal communication in the central nervous system (CNS) have always been of basic importance for theories on the cerebral morphofunctional architecture. Our group has proposed that intercellular communication in the brain can be grouped into 2 broad classes based on some general features of the transmission: wiring (WT) and volume (VT) transmission. WT occurs via a relatively constrained cellular chain (wire), while VT consists of 3-dimensional diffusion of signals in the extracellular fluid (ECF) for distances larger than the synaptic cleft. Both morphological and functional evidence indicates that dopamine (DA) synapses in striatum are 'open' synapses, i.e., synapses which favor diffusion of the transmitter into the surrounding ECF and observations are compatible with the view that DA varicosities can synthesize, store and release DA for VT. The DAergic mesostriatal transmission has, therefore, been examined by several groups to give experimental support to VT. Moreover, due to its minor structural requirements, VT may become prevalent under some pathological conditions, e. g. Parkinson's disease. In animal models of DAergic pathway degeneration, it has been shown that a compensatory activation of surviving DA terminals may lead to a preferential potentiation of VT. WT and VT favor different and complementary types of computation. VT is markedly slower and less safe than WT, but has minor spatial constraints and allows the reach of a large number of targets. Models of neuronal systems integrating classical neuronal circuits and diffusible signals begin to show how WT and VT may interact in the neural tissue.

Short- and long-term changes in striatal neurons and astroglia after transient forebrain ischemia in rats.

BACKGROUND AND PURPOSE: The striatum is one of the regions most sensitive to transient forebrain ischemia. After 30-minute ischemia, areas of massive neuronal degeneration are clearly detectable a few hours after the insult and attain their maximal extension 24 hours after the insult. However, for most cellular and neurochemical parameters it is not known whether some recovery occurs at later times. We examined certain cell populations in the caudate putamen at different times after transient ischemia. METHODS: Adult male Sprague-Dawley rats were subjected to 30-minute forebrain ischemia (four-vessel occlusion model). Six experimental groups were considered: control animals and ischemic animals killed 4 hours, 1 day, 7 days, 40 days, and 8 months after reperfusion. Three striatal cell populations were examined by means of immunocytochemistry coupled to computer-assisted image analysis: vulnerable medium spiny neurons, resistant aspiny neurons, and reactive astrocytes, labeled for their content of dopamine- and cAMP-regulated phosphoprotein mr32 (DARPP-32), somatostatin and neuropeptide Y, and glial fibrillary acidic protein, respectively. RESULTS: (1) The area containing DARPP-32 immunoreactive neurons was markedly decreased (15% to 20% of control caudate putamen area) at 1 day after reperfusion and partially recovered at the following times (40% to 50% at 7 days and 50% to 60% at 40 days and 8 months after reperfusion). (2) The appearance of reactive astrocytes was precocious (4 hours to 1 day after ischemia) in the medial caudate putamen, the region in which DARPP-32 recovered within 40 days after ischemia, and late (7 to 40 days after ischemia) in the lateral caudate putamen, where no DARPP-32 recovery was detected. (3) Neuropeptide Y/somatostatin-containing neurons resisted the ischemic insult and could be detected in areas devoid of DARPP-32 immunoreactive neurons as long as 8 months after reperfusion. CONCLUSIONS: The present results show a marked recovery of DARPP-32-positive neurons within 40 days after 30-minute forebrain ischemia in the medial, but not the lateral, caudate putamen. Medial caudate putamen also contains a high density of reactive astrocytes on the first day after ischemia, suggesting that astrocytic support has an important role in the spontaneous recovery of ischemic neurons.

Spermidine/spermine N1-acetyltransferase mRNA levels show marked and region-specific changes in the early phase after transient forebrain ischemia.

Considerable evidence points to an involvement of natural polyamines (putrescine, spermidine and spermine) in trophic regulation of brain tissue. Spermidine/spermine N1-acetyltransferase is the key enzyme in the interconversion pathway which leads to the formation of spermidine and putrescine from spermine and spermidine, respectively. In the present paper we have studied using in situ hybridization histochemistry the levels of spermidine/spermine N1-acetyltransferase mRNA in the rat central nervous system after transient forebrain ischemia. In the first hours after the insult, a modest increase in spermidine/spermine N1-acetyltransferase mRNA levels was observed in ependymal cells and other non-neuronal cells of all telencephalic and diencephalic regions. In addition, major increases in spermidine/spermine N1-acetyltransferase mRNA levels were observed in regions selectively vulnerable to the ischemic insult, such as striatum, hippocampus and cerebral cortex, during the first day post-reperfusion. The time course and extent of labelling increase were subregion- and cell-specific. At the cellular level, the labelling appeared markedly increased in neurons (8-10 fold in ventromedial striatum and CA1 region) and, to a lesser extent, in non-neuronal cells. The increase in SSAT mRNA levels was not directly related to cell degeneration, as it was detected in both some vulnerable and some resistant cell populations. However, the peak increase of SSAT labelling was precocious in resistant neurons (such as those of ventromedial striatum and dentate gyrus granular layer) and delayed or very limited in vulnerable neurons (such as those of CA1 pyramidal layer and dorsolateral striatum). The increase in spermidine/spermine N1-acetyltransferase may contribute to the increase in putrescine and decrease in spermidine levels observed after ischemia and gives further support to the notion that polyamine metabolism in the early phase after lesion is oriented towards putrescine production. This phenomenon could be relevant in determining the prevalence of neurotrophic vs. neurotoxic effects of polyamines.

Ex vivo demonstration of nitric oxide in the rat brain: effects of intrastriatal endothelin-1 injection.

Nitric oxide (NO) is a novel transmitter with multiple functions in endothelium and neuronal tissue. In particular, it has been implicated in the pathogenesis of neurodegenerative diseases. The aim of the present study was to demonstrate the ex vivo detection of NO in basal conditions and after ET-1 intrastriatal injection by means of electron paramagnetic resonance (EPR) spectroscopy using locally injected hemoglobin (Hb) as a NO trapping agent. The extent of neostriatal damage after Hb and ET-1 injections was assessed by means of immunocytochemistry with a monoclonal antibody against dopamine and cAMP-phosphoprotein M(r) 32 (DARPP-32), which is considered a marker of striatal intrinsic neurons. In the absence of local Hb injection, no signal related to endogenous NO was detected in the neostriatum, suggesting that endogenous NO trapping agents are not sufficiently concentrated to allow NO detection with the present technique. Instead, 1 h after Hb injection, a clear nitrosyl-Hb signal can be detected in neostriatal homogenates. ET-1, a powerful vasoconstrictor agent, was used to cause neuronal loss in the neostriatum. No change in nitrosyl-Hb signal was observed in neostriatal 1 h after ET-1 injection, whereas an almost 3-fold increase in the signal intensity was present 24 h after ET-1 injection. The analysis of neostriatal damage showed that Hb injection did not cause either significant damage of striatal tissue or potentiation of ET-1-induced lesions. In conclusion, the present technique allows ex vivo detection of NO in the brain. The delayed increase in NO observed after ET-1 injection indicates that this molecule may participate in the development of slowly progressive neuronal damage occurring at late post-ischemic times.

Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes.

Synaptosomes obtained from rat striata lesioned by central injection of endothelin-1 (ET-1) were analyzed for the levels of lipid peroxidation products, the susceptibility to lipid peroxidation, the phospholipid and free fatty acid composition and the activity of Na+,K(+)-ATPase one hour after ET-1 treatment. The intrastriatal injection of ET-1 promoted an increase of endogenous thiobarbituric reactive substances (TBARS), as index of free radical mediated lipid damage, and a greater susceptibility to iron/ascorbate-induced lipid peroxidation. The pattern of free fatty acids showed a significant decrease of arachidonic and docosahexaenoic acid consequent to ET-1 treatment. The analysis of lipid composition showed a significant loss of phospholipids: among phospholipid species, sphingomyelin and phosphatidylethanolamine plasmalogen were particularly reduced by ET-1 treatment. The activity of membrane-bound Na+,K(+)-ATPase was also significantly reduced in synaptosomes obtained from ET-1 lesioned striata. Taken together these results indicate a significant modification of synaptosomal membrane of ET-1 treated rat striata, possibly due to a free radical mediated damage.

Diethyldithiocarbamate, a superoxide dismutase inhibitor, counteracts the maturation of ischemic-like lesions caused by endothelin-1 intrastriatal injection.

The effects of a focal lesion induced by endothelin-1 (ET-1, 0.8 microgram/0.8 microliter) on superoxide dismutase (SOD) were studied in the neostriatum of male rats. SOD activity was analyzed at several time intervals (5, 20, 60 min, 4, 24 h and 7 days) after the lesion. No significant changes were observed early after the injection, but SOD activity started to rise significantly at the 60-min time interval reaching a peak 24 h after the injection. In a second experiment the volume of ET-1-induced lesion was evaluated following treatments which induce variations of SOD activity. ET-1 caused a large lesion (9.20 +/- 1.32 mm3) in the neostriatum 24 h after the injection that was 3-fold greater than that observed 1 h after. Rats treated with the SOD inhibitor diethyldithiocarbamate showed a lesion equivalent to that observed 1 h after ET-1 injection, suggesting that SOD may be involved in the maturation of ET-1-induced neuronal damage.

Neuropeptide Y produces anxiolytic effects in spontaneously hypertensive rats.

The sedative and anxiolytic effects of intracerebroventricular administration of neuropeptide Y (NPY) were studied in spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto (WKy) rats using the two-compartment exploratory test, and in the open-field test after habituation. In the two-compartment tests, NPY produced anxiolytic effects by increasing the exploratory activity in SHR at a dose (0.25 nmol) lower than the minimal effective dose in WKy rats (1.25 nmol). In SHR, anxiolytic effects were observed for the whole NPY dose range (0.25-5.0 nmol), whereas in normotensive WKy rats the highest dose (5.0 nmol) failed to increase exploratory activity. The open-field test showed reduced locomotor activity and rearings in WKy rats when injected with 5.0 nmol NPY. These effects were not observed in SHR. The absence of sedative effects and the higher sensitivity to the anxiolytic effects of NPY in SHR are suggestive of a genetically determined difference in central NPY systems involved in behavioral adaptation that may be relevant for the development of hypertension.

Involvement of alpha 2-receptors in the analgesia induced by transient forebrain ischemia in rats.

Transient forebrain ischemia induced in rats by the four-vessel occlusion method produced analgesic effects in the hotplate test that persisted for 2 weeks. Ischemia-induced analgesia was attenuated by low doses of alpha 2-agonist clonidine (0.01-0.10 mg/kg, IP) and enhanced by low doses of alpha 2-antagonists yohimbine (1-2 mg/kg, IP) and idazoxan (0.25-1.00 mg/kg, IP) administration 7 days after ischemia. Ischemia-induced analgesia was not affected by methysergide, naloxone, propranolol, or phenoxybenzamine administered 7 days after ischemia, when motor control and arousal level of rats recovered to normal conditions. The enhanced response to yohimbine was antagonized by pretreatment with clonidine (0.75 mg/kg, IP) and naloxone (10 mg/kg, IP), suggesting the involvement of endogenous opioid peptides. The enhanced response to yohimbine was still present 2 months after ischemia, when preischemic hotplate threshold was restored. As alpha 2-agonists reduce and alpha 2-antagonists increase the outflow of central noradrenaline, it is suggested that activation of central noradrenergic systems is involved in the mediation of ischemia-induced analgesia.

Effects of polyamine synthesis blockade on neuronal loss and astroglial reaction after transient forebrain ischemia.

Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylornithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hr, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylornithine can reduce the increase of glial fibrillary acid protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylornithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylornithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that ornithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylornithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.

Increases in sulphated glycoprotein-2 mRNA levels in the rat brain after transient forebrain ischemia or partial mesodiencephalic hemitransection.

Sulphated glycoprotein-2, thought to be involved in programmed cell death in peripheral organs, has been detected at increased levels in brain degenerative states. In this paper we have investigated the regional and cellular expression of this protein during development of brain lesion. With this aim sulphated glycoprotein-2 mRNA levels were studied in models of ischemic (transient forebrain ischemia) or mechanical (partial mesodiencephalic hemitransection) brain injuries using in situ hybridization histochemistry. Marked increases in sulphated glycoprotein-2 mRNA were observed in lesioned brains in both models. In addition, we report a shift in the regional distribution of positive cells in both lesion models 1-7 days post-lesion. After transient forebrain ischemia, sulphated glycoprotein-2 mRNA increases were always localized in selectively vulnerable regions (caudate-putamen, hippocampal formation), showing a temporal change in the pattern of intraregional distribution from less to more lesioned parts. In the case of mechanical lesion at 1 day, increased labelling had a widespread distribution on the lesioned side and was also observed on the intact side near the midline. In contrast, at 7 days increased labelling was restricted to regions directly lesioned (either areas whose input and/or output connections were severed by the transection or areas which were directly affected by the mechanical lesion). Analysis at the cellular level revealed that at both time intervals and in both lesion models most cell bodies overlain by dense clusters of specific grains were non-neuronal cells. The distribution patterns and their change over time suggest that at least some of these cells are inflammatory and phagocytic cells. The majority of degenerating neuronal cells after ischemia did not show increased levels of sulphated glycoprotein-2 mRNA. However, seven days after hemitransection and at all time intervals after transient ischemia, some cells clearly identifiable as neurons exhibited increased sulphated glycoprotein-2 mRNA levels.

Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis.

Extracellular free radicals were detected in rat striatal perfusate samples by intracerebral microdialysis coupled to the spin trapping technique. Five Sprague-Dawley rats were subjected to 30 min of global ischemia followed by reperfusion; throughout the experimental period the intrastriatal dialysing probe was perfused with Ringer's solution containing the spin trap agent pyridyl-N-oxide-t-butylnitrone (100 mM) together with the iron chelating agent diethylentriaminepentacetic acid (100 microM). A radical adduct occurred during ischemia and early reperfusion, but not in basal conditions; the spin adduct was characterized as a carbon centered radical, consistent with the presence of an oxidative attack on membrane lipids. The direct evidence of the formation of free radicals supports the hypothesis that free radicals play a role in the pathogenesis of the histological damage during brain ischemia.

Microwave-cured tracheostoma vents.

This article describes a technique of making custom flexible and combined flexible/rigid tracheostoma vents. The combined flexible rigid tracheostoma vent provides a flexible material that is nonirritating in the peristomial region and maintains a patent tracheostoma by the rigidity of the hard acrylic resin section. The flexible tracheostoma vent can be easily inserted and is more comfortable than the rigid commercially available tracheostomy tube. The use of microwave-cured materials permits fabrication during a single visit. The steps involved in the fabrication of the tracheostoma vents are simple and require no elaborate laboratory equipment.

A homemade microwaveable denture reline jig.

This article describes a procedure for making a denture reline jig for processing denture base resin in a microwave oven. The jig is intended for use with the newer microwave curing denture base resins. The reline jig is economical to produce and is made from fiberglass liquid resin commonly used in automobile body repairs. The fiberglass liquid resin is strengthened by adding fillers of dental stone and chopped fiberglass strands.

A new model of focal brain ischemia based on the intracerebral injection of endothelin-1.

Endothelin-1 and its receptors are widely distributed in the brain of rodents and humans. In view of its potent and long-lasting vasoconstrictor activity, a role of endothelin-1 has been proposed in brain ischemia. In the present paper, the local injection of endothelin-1 was utilized to induce ischemia in rat striatum. An evaluation of the rostrocaudal extension of the lesion is reported. By using intracerebral microdialysis, a marked increase of lactate and dopamine, but not glutamate, was observed in this region upon endothelin-1 administration. Moreover, preliminary data reported show a protective effect of ganglioside treatment on endothelin-1 lesion of rat striatum. The characteristics of the present model of brain ischemia are discussed in comparison with well characterized models, such as the Pulsinelli's four vessel occlusion and the middle cerebral artery occlusion.

Decrease in mRNA levels but not in the density of D2 dopamine receptors in rat striatum after transient forebrain ischemia.

D2 dopamine receptor mRNA was analyzed by in situ hybridization histochemistry in rat striatum 7 days after transient forebrain ischemia. A patchy disappearance of the D2 receptor mRNA was observed in the dorsolateral striatum. In the same area, a disappearance of D1 binding sites occurred in the absence of significant changes in D2 receptor density. These results suggest that, although D2 receptors seem to be apparently unaffected after forebrain ischemia, a long-lasting impairment of their neosynthesis may be present in striatal D2 dopaminoceptive neurons.