Cerebral cortex blood flow and vascular smooth muscle contractility in a rat model of ischemia: a correlative laser Doppler flowmetric and scanning electron microscopic study

The present study was undertaken to ascertain the role of smooth muscles and pericytes in the microcirculation during hyperperfusion and hypoperfusion following ischemia in rats. Paired external carotids, the pterygopalatine branch of the internal carotids and the basilar artery were exposed and divided. Reversible inflatable occluders were placed around the common carotids. After 24 h, the unanesthetized rat underwent 10-min ischemia by inflating the occluders. Continuous cortical cerebral blood flow (c-CBF) was monitored by laser Doppler flowmetry. The measured c-CBF was below 20% of control (P < 0.001) during ischemia. A c-CBF of 227.5 ± 54.1% (P < 0.001) was obtained during reperfusion hyperemia. A c-CBF of 59.7 ± 8.8% (P < 0.001) occurred at the nadir of postischemic hypoperfusion, and this was followed by a second hyperemia. The cytoarchitecture of the vascular smooth muscles and pericytes was assessed by scanning electron microscopy. Samples were prepared using a KOH-collagenase digestion method. In control rats, arteriolar muscle cells showed smooth surfaces. Capillary pericytes were closely apposed to the endothelium. Immediately after reperfusion, transverse membrane creases were observed on the smooth muscle surfaces. During maximal hyperemia the creases disappeared. When c-CBF started to decrease the creases became visible again. Throughout the postischemic hypoperfusion the creases remained. Capillary endothelial walls became tortuous in the late phase of hypoperfusion. During the second hyperemia most arteriolar muscle cells showed smooth surfaces. Some pericytes appeared to have migrated from the vascular wall. The morphological changes of smooth muscle membranes suggest that they are related to specific perfusional disturbances during ischemia and reperfusion. Received: 2 July 1996 / Revised, accepted: 24 September 1996     

Changes in guanylate cyclase activity in arteriolar smooth muscle cells and hemodynamics after ischemia-reperfusion in rats

Participation of nitric oxide (NO) and hydroxyl radicals in the pathogenesis of hemodynamic alterations after postischemic recirculation were examined by measuring cerebral blood flow (CBF) and estimating guanylate cyclase activities in arteriolar smooth muscle cells using a reversible 2-h thread occlusion model in rats and an electron microhistochemical technique. In the reversible 2-h ischemia model, guanylate cyclase activity in the arteriolar smooth muscle cells increased at the peak of hyperemia and decreased during postischemic hypoperfusion. Administration of N ^ω -nitro-l-arginine (L-NNA), a NO synthase inhibitor, in this model decreased infarct volume and completely inhibited both hyperemia and guanylate cyclase activation at hyperemia. Administration of 1,2-bis(nicotinamido)-propane (AVS), a free radical scavenger, affected neither CBF nor guanylate cyclase activity during hyperemia despite a significant reduction in infarct volume. Administration of L-NNA and AVS significantly suppressed the decrease in CBF during postischemic hypoperfusion and the effect of AVS was greater than that of L-NNA. Although continuous infusion of sodium nitroprusside (SNP) following postischemic hypoperfusion in the reversible 2-h ischemia rats without treatment with L-NNA and AVS did not alter either CBF or guanylate cyclase activity, it significantly elevated both CBF and guanylate cyclase activities in rats administered L-NNA and AVS. The responses of CBF and guanylate cyclase to SNP were also greater in AVS- than L-NNA-treated rats. These results suggest that a physiological vasodilative mechanism is involved in the induction of postischemic hyperemia through the NO-guanylate cyclase pathway in arteriolar smooth muscle cells. Both NO-related and non-related radicals are involved in the pathogenesis of postischemic delayed hypoperfusion through the loss of arteriolar smooth muscle relaxation capability. Received: 2 November 1998 / Revised, accepted: 29 March 1999     

Mild intraischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats

Oxidative damage by free radicals has been proposed as a mechanism of cerebral injury due to ischemia and reperfusion. Hypothermia protects against ischemic necrosis; however, its effect on oxidative stress has not been investigated. In this study, the effects of hypothermia on oxidative stress were studied by determining consumption of endogenous antioxidants after temporary focal ischemia in rats. Thirty-two Sprague-Dawley rats anesthetized with 1.5% isoflurane underwent 3 h of middle cerebral artery occlusion under hypothermic (33°C) or normothermic (37°C) conditions followed by 3 h of normothermic reperfusion. In the first study (n = 8per group), intraischemic hypothermia suppressed the reduction of tissue concentrations of endogenous antioxidants, ascorbate (P≤ 0.05), and glutathione (P≤ 0.05) in ischemic cortex but not in caudoputamen. In a parallel study (n = 8per group), hypothermia reduced tissue damage in ischemic frontoparietal cortex (P ≤ 0.05), but not in caudoputamen. Laser-Doppler estimates of cortical blood flow showed that intraischemic hypothermia significantly attenuated early postischemic hyperperfusion (P ≤ 0.01) and delayed postischemic hypoperfusion (P ≤ 0.01). These results demonstrate that intraischemic mild hypothermia reduces oxidative stress and cell injury after prolonged focal ischemia followed by reperfusion. The reduction of oxidative stress by hypothermia may be related indirectly to attenuation of postischemic blood flow changes.     

Protein kinase C delta modulates endothelial nitric oxide synthase after cardiac arrest

We previously showed that inhibition of protein kinase C delta (PKCδ) improves brain perfusion 24 hours after asphyxial cardiac arrest (ACA) and confers neuroprotection in the cortex and CA1 region of the hippocampus 7 days after arrest. Therefore, in this study, we investigate the mechanism of action of PKCδ-mediated hypoperfusion after ACA in the rat by using the two-photon laser scanning microscopy (TPLSM) to observe cortical cerebral blood flow (CBF) and laser Doppler flowmetry (LDF) detecting regional CBF in the presence/absence of δV1-1 (specific PKCδ inhibitor), nitric oxide synthase (NOS) substrate (L-arginine, L-arg) and inhibitor (N^ω-Nitro-L-arginine, NLA), and nitric oxide (NO) donor (sodium nitroprusside, SNP). There was an increase in regional LDF and local (TPLSM) CBF in the presence of δV1-1+L-arg, but only an increase in regional CBF under δV1-1+SNP treatments. Systemic blood nitrite levels were measured 15 minutes and 24 hours after ACA. Nitrite levels were enhanced by pretreatment with δV1-1 30 minutes before ACA possibly attributable to enhanced endothelial NOS protein levels. Our results suggest that PKCδ can modulate NO machinery in cerebral vasculature. Protein kinase C delta can depress endothelial NOS blunting CBF resulting in hypoperfusion, but can be reversed with δV1-1 improving brain perfusion, thus providing subsequent neuroprotection after ACA.     

An investigation on the role of nitric oxide in the modulation of the binding characteristics of various radioligands of GABAA receptors by 5α-pregnan-3α-ol-20-one in the rat brain regions

Chronic administration of N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of nitric oxide synthase, diminished the ability of 5α-pregnan-3α-ol-20-one, a neurosteroid, to potentiate the [³H]muscimol (5 nM) binding in the rat hippocampus but not in the cerebellum or cerebral cortex. This effect of NAME was stereospecific and susceptible to reversal by the pre-treatment of rats with -arginine. However, chronic administration of -NAME did not affect the modulation of the [³H]flunitrazepam (2 nM) or [³⁵S]TBPS (4 nM) binding by the neurosteroid in any of the brain regions investigated in this study. These results suggest that nitric oxide may be involved in some of the effects of neurosteroids in hippocampus.     

Effects of combined postischemic hypothermia and delayed N-tert-butyl-a-pheylnitrone (PBN) administration on histopathological and behavioral deficits associated with transient global ischemia in rats

Previous cerebral ischemia studies have reported the limitations of restricted periods of postischemic hypothermia in producing long-term neuroprotection. The present experiment attempts to determine whether delayed treatment with the free radical scavenger N-tert-butyl-a-phenylnitrone (PBN) is protective at 2 months following transient global forebrain ischemia, and whether additive effects can be observed when PBN is administered in combination with moderate hypothermia. For this aim rats were subjected to 10 min of two-vessel forebrain ischemia followed by (a) 3 h of postischemic normothermia (37°C); (b) 3 h of postischemic hypothermia (30°C); (c) normothermic procedures combined with delayed injections of PBN (100 mg/kg) on days 3, 5 and 7 post-insult; (d) postischemic hypothermia combined with delayed PBN treatment; or (e) sham procedures. Outcome measures included cognitive behavioral testing and quantitative histopathological analysis at 2 months. Postischemic PBN injections induced a systemic hypothermia (1.5°C–2.0°C) that lasted for 2–2.5 h. Water maze testing revealed significant performance deficits relative to shams in the normothermic ischemic group, with the postischemic hypothermia and PBN groups showing intermediate values. A significant attenuation of cognitive deficits was observed in the animal group receiving the combination postischemic hypothermia and delayed PBN treatment. Quantitative CA1 hippocampal cell counts indicated that each of the ischemia groups exhibited significantly fewer viable CA1 neurons compared to sham controls. However, in rats receiving either delayed PBN treatment or 3 h of postischemic hypothermia, significant sparing of CA1 neurons relative to the normothermic ischemia group was observed. These data indicate that hypothermia combined with PBN treatment provides long-term cognitive improvement compared to nontreatment groups. PBN-induced mild hypothermia could contribute to the neuroprotective effects of this pharmacological strategy.     

Morphine tolerance does not develop in mice treated with endothelin-A receptor antagonists

Long-term use of morphine leads to development of antinociceptive tolerance. We provide evidence that central endothelin (ET) mechanisms are involved in development of morphine tolerance. In the present study, we investigated the effect of ET(A) receptor antagonists, BQ123 and BMS182874, on morphine antinociception and tolerance in mice. Mechanism of interaction of ET(A) receptor antagonists with morphine was investigated. BQ123 (3 microg, i.c.v.) and BMS182874 (50 microg, i.c.v.) significantly enhanced antinociceptive effect of morphine (P < 0.05), through an opioid-mediated effect. Treatment with a single dose of BQ123 (3 microg, i.c.v.) reversed tolerance to morphine antinociception in morphine-tolerant mice. BQ123 or BMS182874 did not affect naloxone binding in the brain. Therefore, ET(A) receptor antagonists did not bind directly to opioid receptors. [35S]GTPgammaS binding was stimulated by morphine and ET-1 in non-tolerant mice. Morphine- and ET-1-induced GTP stimulation was significantly lower (P < 0.05) in morphine-tolerant group (33% and 42%, respectively) compared to control group. BQ123 and BMS182874 did not activate binding in non-tolerant mice. BQ123 and BMS182874 significantly increased G protein activation in morphine-tolerant mice (96% and 86%, respectively; P < 0.05). These results provide evidence that uncoupling of G protein occurs in morphine-tolerant mice, and ET(A) antagonists promote coupling of G protein to its receptors, thereby restoring antinociceptive effect. These findings indicate that ET(A) receptor antagonists potentiate morphine antinociception and reverse antinociceptive tolerance in mice, through their ability to couple G proteins to opioid receptors.     

A novel type of calcium channel sensitive to ω-agatoxin-TK in cultured rat cerebral cortical neurons

We characterized the electrophysiological properties of calcium channels in cultured rat cerebral cortical neurons using ω-agatoxin-TK (ω-Aga-TK) by a patch-clamp technique. Two types of slowly inactivating calcium channels sensitive to ω-Aga-TK were detected. The first type showed high sensitivity to ω-Aga-TK and low recovery from the ω-Aga-TK-induced blockade during washout, corresponding to the P-type channel. The second type showed low sensitivity to ω-Aga-TK and high recovery, resembling the Q-type channel, although it was distinct from the Q-type in terms of slower inactivation kinetics. We designate this channel as Q_L-type (long-lasting Q channel). The ω-Aga-TK-sensitive calcium channels involved in the glutamatergic synaptic transmission were also divided into two types based on the sensitivity to ω-Aga-TK and reversibility of ω-Aga-TK-induced blockade. We conclude that the Q_L-type is a novel type of channel, and that both P-type and Q_L-type channels play a significant role in the cerebral cortical synaptic transmission.     

Indications of pre- and post-synaptic 5-HT1A receptor interactions in feeding behavior and neuroendocrine regulation

This bipartite study uses behavioral and biochemical means to explore the involvement of both pre- and post-synaptic 5-HT_1A receptors in the control of food intake and neuroendocrine regulation. In the pharmacological study, the administration of the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 60 μg/kg b.wt., i.p.) to rats caused a significant increase in 2 h intake of a high carbohydrate (CARB)/sugar diet (P < 0.05) during the relatively inactive feeding period of the late light cycle. No significant change was detected in the intake of Purina laboratory chow at 2 h, or of the intake of either diet at 4 h and 24 h after 8-OH-DPAT administration. Injection of 8-OH-DPAT induced a drop in insulin levels in rats maintained on high CARB/sugar diets only (−90%; P<0.05). It also caused an increase in circulating glucose levels in both high CARB/sugar (240%; P<0.01) and chow fed (123%; P<0.05) rats; it did so more intensely in high CARB/sugar-fed rats. In the biochemical study, radioligand binding techniques were used to assess 5-HT_1A receptor density in the hypothalamus, as well as the relationship between 5-HT_1A receptors and circulating levels of insulin and glucose. Chronic and acute administration (25 mg/kg b.wt./5 injections, and 50 mg/kg b.wt., respectively, i.p.) of the potent hypoglyce mic agent tolbutamide (TOL) caused a significant increase in 5-HT_1A receptor density (+243% and +132.6%, respectively; P<0.05) in the medial hypothalamus but not in the lateral hypothalamus, as compared to vehicle-treated rats. Chronic glucose replacement therapy showed a trend towards reversing the depressed circulating glucose levels as well as the medial hypothalamic 5-HT_1A receptor density to control levels. These studies indicate that the pre-synaptic mechanism of 8-OH-DPAT-induced hyperphagia may require specific circulating levels of insulin and glucose, which are regulated via post-synaptic 5-HT_1A receptors.     

Behavioral evidence for a modulating role of σ ligands in memory processes. II. Reversion of carbon monoxide-induced amnesia

This study examined the effect of low doses of σ ligands amnesia induced in mice by successive carbon monoxide (CO) exposure. Mice were exposed three consecutive times to CO (10 ml/min, 30–50 s) at 38°C. Spatial working memory impairment was investigated 5 days later by monitoring spontaneous alternation behavior in a Y-maze. Delayed amnesia was examined 7 days after CO exposure by using a step-down passive avoidance test. The preadministration of the σ ligand 1,3-di-(2-tolyl)guanidine (DTG), at doses of 1 to 1000 μg/kg, s.c., 30 min before CO exposure did not affect the resulting amnesia in either test. However, when administered 30 min before the test, i.e., 5 or 7 dats after CO exposure, this agent completely reversed the CO-induced decrease in alternation performance, at doses of 10 to 100 μg/kg. The same effect was observed with (+)-N-allylnormetazocine ((+)-SKF 10,047), at doses of 100 to 300 μg/kg, but not with (-)-SKE 10,047. DTG, at the same dose range that reversed the decrease in alternation, also totally reversed the CO-induced decrease in step-down latency in the passive avoidance test. The curve for these effects was bell-shaped; the effects were not observed at the dose of 1 mg/kg. Moreover, α-(-4-fluorophenyl-2-pyrimidinyl)-1-piperazine butanol (BMY 14802), a putative σ antagonist (1–10 mg/kg i.p), did not affect CO-induced amnesia, but when simultaneously administered with DTG, it completely prevented its effect in both tests. These findings indicate that σ₁ sites may mediate a complete but bell-shaped reversion of CO-induced amnesia. The exact mechanisms remain to be determined, but they may involve the modulation of both N-methyl-d-aspartate and cholinergic nicotinic systems.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Neuroprotective effect of remacemide hydrochloride in focal cerebral ischemia in the cat

The neuroprotective effects of remacemide hydrochloride ((±)-2-amino-N-(1-ethyl-1,2-diphenylethyl)acetamide hydrochloride) have been assessed with permanent occlusion of one middle cerebral artery in chloralose-anesthetized cats in which key physiologic variables have been monitored throughout the post-ischemic period. An infusion of remacemide hydrochloride (278 μg/kg/min; total dose 25 mg/kg) initiated 90 min prior to middle cerebral artery occlusion and discontinued at occlusion, reduced significantly (P < 0.02) the volume of ischemic damage (from 2505 ± 454 mm³ of vehicle-treated cats to 1266 ± 54 mm³ of remacemide hydrochloride-treated cats).     

Temporal profile of interneuron and pyramidal cell protein synthesis in rat hippocampus following cerebral ischemia

Summary Cellular protein synthesis was investigated in the rat hippocampus 2–100 h following 20 min of cerebral ischemia induced by four-vessel occlusion. [³H]-Phenylalanine was retrogradely infused through the external carotid artery for 30 min. This method limited the distribution of the tracer to one hemisphere and required 1/50th of the tracer amount used for intravenous tracer infusion. Cellular [³H]phenylalanine incorporation was examined in hematoxyline and eosin-stained sections coated with nuclear emulsion. A score for relative protein synthesis was estimated from counts of silver grains across neuron somata with undamaged morphology. Shortly after ischemia a generalized complete arrest of protein synthesis was observed. In CA1 pyramidal cells, this was followed by a transient incomplete regeneration (9–20 h) and later (46–100 h) persistent cessation of protein synthesis. By contrast protein synthesis in interneurons, CA3c pyramidal cells and granule cells recovered to preischemic levels 9–100 h after ischemia, as did the CA3ab pyramidal cells 46–100 h postischemia. Moreover, eosinophilic cell changes were seen in hilar and CA3c neurons at all postischemic stages and in CA1 pyramidal cells 46–72 h after ischemia. [³H]Phenylalanine incorporation was absent in neurons demonstrating eosinophilic cell changes. From the rapid recovery of protein synthesis in hippocampal interneurons, we conclude that changes in interneuronal protein synthesis per se are not involved in the pathophysiology of the delayed ischemic CA1 pyramidal cell death.Supported by research grants from The Danish Research Council and the Danish Biotechnology Program     

The anti-amnesic effects of sigma1 (σ1) receptor agonists confirmed by in vivo antisense strategy in the mouse

The sigma₁ (σ₁) receptor cDNA was recently cloned in several animal species, including the mouse. In order to firmly establish the implication of σ₁ receptors in memory, a phosphorothioate-modified antisense oligodeoxynucleotide (aODN) targeting the σ₁ receptor mRNA and a mismatched analog (mODN) were administered intracerebroventricularly for 3 days in mice. Scatchard analyses of in vitro (+)-[³H]SKF-10,047 binding to σ₁ sites showed that B_max values were significantly decreased in the hippocampus (−58.5%) and cortex (−38.1%), but not in the cerebellum, of aODN treated mice, as compared to saline- or mODN-treated animals. In vivo binding levels were also significantly decreased after aODN treatment in the hippocampus and cortex but not in the cerebellum. The anti-amnesic effects of the selective σ₁ agonists PRE-084 or SA4503 were evaluated against the learning impairments induced by dizocilpine or scopolamine, respectively, using spontaneous alternation behavior and passive avoidance task. The anti-amnesic effects of PRE-084 or SA4503, observed after saline- or mODN-treatment, were blocked after aODN administration. These observations bring a molecular basis to the modulatory role of σ₁ receptors in memory processes.     

Role of nitric oxide in regulation of cerebral microvascular tone and autoregulation of cerebral blood flow in cats

The role of nitric oxide in the regulation of cerebrocortical microvascular tone and autoregulation of cerebral blood flow (CBF) was examined in 24 anesthetized cats. The local cerebral blood volume (CBV), mean transit time of blood (MTT), and CBF in the cortex were measured by our photoelectric method. CBV represents the cumulative dimensions of the cerebral microvessels. Intravenous injection of 0.35–0.7 mg/kg/minN^G-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide synthesis, significantly increased mean arterial blood pressure (MABP; 8.4–14.1%,P < 0.01), decreased CBV (15.2–28.7%,P < 0.01), and decreased CBF (20.0–29.8%,P < 0.01) in a dose-related manner. The changes in MABP, CBV, and CBF elicited byl-NMMA were inhibited (P < 0.05) by simultaneous infusion of 35 mg/kg/minl-arginine. Autoregulation of CBF was examined during controlled hypotension of −30 to −40 mmHg (artificial bleeding) and recovery of blood pressure (reinfusion of blood). Although CBF remained constant with blood pressure changes in the control state (ΔCBF/ΔMABP of 0.037±0.155 with hypotension), CBF became dependent on blood pressure changes (ΔCBF/ΔMABP of 0.478±0.135, P < 0.05) during infusion of 0.35 mg/kg/minl-NMMA. It is concluded that nitric oxide participates in both the regulation of basal tone of cerebral microvessels and the autoregulation of CBF.     

Striatal dopamine transporter in different disability stages of Parkinson's disease studied with [I]β-CIT SPECT

Six healthy controls and eighteen patients with Parkinson's disease in different disability stages were studied with SPECT using [¹²³I]β-CIT to label the striatal dopamine transporter. The mean uptake of [¹²³I]β-CIT in the putamen was reduced to 54% of the control mean and to 65% of the average control value in the caudate nucleus. In patients with totally, or predominantly unilateral symptoms the reduction was greater on the side opposite to the predominant symptoms (to 56% of the control mean in the contralateral putamen and to 77% in the ipsilateral putamen).There was a significant negative correlation between [¹²³I]β-CIT uptake in the putamen and the Hoehn and Yahr stage (r = -0.81, p < 0.0001). An analysis of covariance was performed using age and disease duration as covarianes, and the correlation between putaminal [¹²³I]β-CIT uptake and parkinsonian disability according to the Hoehn and Yahr stage remained significant (r = -0.75, P = 0.02). A similar correlation was seen in the caudate nucleus (r = −0.79, p < 0.0001) between the uptake of [¹²³I]β-CIT and the Hoehn and Yahr stage. The correlation also remained significant after correction for the duration of disease and age of the patients (r= -0.76, P = 0.02).The present results show that [1231],Q-CIT SPECT is a useful method to study the function of presynaptic dopaminergic terminals in PD, and might be used in the early diagnosis and follow-up of the disease.     

Angiotensin-(1–7) causes endothelium-dependent relaxation in canine middle cerebral artery

The heptapeptide, angiotensin-(1–7), is an active member of the renin–angiotensin system. The present study was designed to characterize the role of endothelium in relaxations of large cerebral arteries to angiotensin-(1–7). Rings of canine middle cerebral arteries were suspended in organ chambers for isometric force recording. The levels of cyclic guanosine 3′,5′-monophosphate (cGMP) were assessed by radioimmunoassay. During contraction to uridine 5′-triphosphate (UTP, 3×10⁻⁶ to 10⁻⁵ mol/l), angiotensin-(1–7) (10⁻⁹ to 3×10⁻⁵ mol/l) caused concentration-dependent relaxations in arteries with endothelium, but not in endothelium-denuded vessels. Angiotensin-(1–7) significantly increased formation of cGMP. Nitric oxide synthase inhibitor, N-ω-nitro- -arginine methyl ester ( -NAME, 3×10⁻⁴ mol/l), and selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ, 3×10⁻⁶ mol/l), abolished angiotensin-(1–7)-induced relaxations. Angiotensin receptor antagonists, losartan (10⁻⁵ mol/l), PD 123 319 (10⁻⁵ mol/l), [Sar¹,Thr⁸]-angiotensin II (10⁻⁵ mol/l) [Sar¹,Val⁵,Ala⁸]-angiotensin II (10⁻⁵ mol/l) or [7- -Ala]-angiotensin 1–7 (10⁻⁶ mol/l) did not affect these relaxations. However, angiotensin-converting enzyme inhibitor, captopril (10⁻⁵ mol/l) augmented relaxations to angiotensin-(1–7). Finally, bradykinin B₂ receptor antagonist, [ -Arg⁰,Hyp³,Thi⁵, -Tic⁷,Oic⁸]-bradykinin (HOE 140, 5×10⁻⁸ mol/l) significantly reduced the effect of angiotensin-(1–7), while bradykinin B₁ receptor antagonist, des-Arg⁹, [Leu⁸]-bradykinin (6×10⁻⁹ mol/l) did not influence the vascular response to the heptapeptide. These findings indicate that (1) angiotensin-(1–7) produces relaxation of canine middle cerebral arteries by the release of nitric oxide from endothelial cells, (2) angiotensin receptors do not mediate endothelium-dependent relaxations to the heptapeptide, and (3) this effect appears to be dependent on activation of local production of kinins. Our studies support the concept that angiotensin-(1–7), as a natural vasodilator hormone, may counterbalance the hemodynamic actions of angiotensin II.     

Expression of Ca-dependent (classical) PKC mRNA isoforms after transient cerebral ischemia in gerbil hippocampus

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (α, βI and γ) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one γ isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60–65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that γ PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.     

Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states

Neuropathic pain is the most difficult type of pain to treat. The P2X₃ receptors play a crucial role in facilitating pain transmission at peripheral and spinal sites. The present research investigated the effects of tetramethylpyrazine (TMP) on the primary afferent transmission induced by P2X₃ receptor in neuropathic pain states. Chronic constriction injury (CCI) model was adopted. Sprague–Dawley male rats (n = 30) had been randomly divided into normal saline (sham + NS) group (I), TMP group (II), sham group (III), CCI + TMP group (IV), and CCI group (V). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X₃ immunoreactivity in L4/L5 dorsal root ganglion (DRG) and spinal cord was detected by immunohistochemistry. The mechanical withdrawal threshold and thermal withdrawal latency in group V were lower than those in groups I–III or IV (p < 0.05), while P2X₃ receptor expression of L4/L5 DRG and spinal cord in group V was higher than those in groups I–III (p < 0.01) or group IV (p < 0.05). The mechanical withdrawal threshold, thermal withdrawal latency and P2X₃ immunoreactivity of L4/L5 DRG and spinal cord in group IV showed no significant difference compared with those in groups I, II or III (p > 0.05). The amplitudes of the currents in group V (CCI) were much larger than those obtained in other groups after application of same concentration adenosine 5′-triphosphate disodium (ATP) (p < 0.01). α,β-Methylene-ATP (α,β-meATP)-activated currents in DRG neurons of CCI rats were more obvious than those obtained in other group rats (p < 0.01). The results showed that TMP may inhibit the primary afferent transmission of neuropathic pain induced by P2X₃ receptor.     

Congenital cerebral venous dysgenesis

This report describes a rare case of primary cerebral venous dysgenesis in a 3-year-old child with development retardation. Angiography resulted in nonvisualization not only of deep cerebral veins but also of superficial cerebral veins. In computed tomography and in magnetic resonance imaging the collateral venous circulation appeared as a strange configuration in the pineal region. Single photon emission computed tomography using N-isopropyl-p-[I-123]-iodoamphetamine revealed decreased regional cerebral blood flow in the basal gangalia and thalamus, but cerebral infarction was not detected in the area. These features indicate that in this case, dysgenesis of deep cerebral veins, which probably occurred during prenatal life, had caused hypoperfusion in the deep cerebral regions.