László Dézsi

Evidence for the expression of cyclooxygenase-2 enzyme in periodontitis.

We investigated the role of the inducible isoform of cyclooxygenase (COX-2) in a rat model of periodontitis using a selective COX-2 inhibitor NS-398. Periodontitis was produced by a silk ligature placed around the lower left 1st molar. Animals were treated with NS-398 (3 mg kg(-1) i.p., 2 times per day for 7 days) or vehicle. At Day 8, the gingivomucosal tissues encircling the mandibular 1st molars were removed on both sides for COX-2 immunohistochemistry, measurement of plasma extravasation by the Evans blue technique, and alveolar bone loss by videomicroscopy. Immunohistochemical analysis revealed numerous strongly COX-2-positive cells in the subepithelial tissues in the ligated side and only a few COX-2-reactive cells in the contralateral (control) side. Ligation significantly increased Evans blue extravasation in the gingivomucosal tissue and alveolar bone destruction compared to the control side. NS-398 treatment significantly reduced the plasma extravasation and alveolar bone resorption of the ligated side compared to vehicle administration. The present results suggest that COX-2 is induced by periodontitis, and plays an important role in gingival inflammation and alveolar bone destruction. In a previous study (Br J Pharmacol 1998;123:353-60) we found the expression of the inducible isoform of nitric oxide synthase in this model. Therefore, based on our own data and the literature, we propose that selective inhibition of these inducible enzymes might be a basis for adjunctive therapy, or new therapeutic approaches in periodontitis.

Acute improvement in histological outcome by MK-801 following focal cerebral ischemia and reperfusion in the cat independent of blood flow changes.

The present study reports on the acute effects of MK-801 on the histopathological outcome and blood flow changes during focal cerebral ischemia and reperfusion. In addition, acute changes in the EEG and blood pressure are also reported. In 16 halothane-anesthetized cats, the left middle cerebral artery (MCA) was occluded for 2 h followed by 4 h of reperfusion. Thirty minutes after the onset of ischemia, eight animals were treated with 1 mg/kg of MK-801, while eight animals received saline. Blood flow from the peripheral MCA territory was measured with H2 clearance. There was a comparable reduction in blood flow (down to 20% of control) in the ischemic gyri of the two groups followed by a partial recovery after recirculation. There was a similar decrease in the EEG amplitude over the ischemic central MCA territory in the treated and the untreated group. Treatment with MK-801 induced a burst suppression in the EEG and a transient drop (11.4 ± 6.5 mm Hg) in the mean arterial pressure. The volume of early ischemic damage decreased by one-third in the MK-801-treated group compared to the untreated one, both in the total hemisphere (from 29 ± 10 to 20 ± 5%) and in the hemispheric cortex (range 36 ± 8 to 24 ± 13%). A major fraction of this improvement was localized to the middle and posterior parietal (mainly perifocal) regions of the MCA territory. These results show that in our model, MK-801 improves histopathological outcome despite the lack of apparent effect on the cortical blood flow, and an adverse effect on the systemic blood pressure. This is the first report that describes data on a reproducible model of reperfusion after temporary occlusion of the MCA in a cat, extending the findings of the Glasgow group, who observed similar neuroprotection in models of permanent MCA occlusion.

Prolonged effects of MK-801 in the cat during focal cerebral ischemia and recovery: Survival, EEG activity and histopathology

Previously we reported an improvement in histological outcome in cats treated with MK-801 shortly after the induction of temporary middle cerebral artery occlusion, and examined after 2 h of ischemia followed by 4 h of reperfusion. This study investigates the prolonged effects of the same drug treatment. Focal cerebral ischemia was produced in 34 cats by temporary occlusion of the left middle cerebral artery for 2 h. Stroke severity was determined using the ratio of the EEG amplitude from the ipsilateral to that of the contralateral hemisphere. Thirty minutes after the onset of ischemia, cats were treated i.v. with either 1 mg/kg MK-801 or saline. Electrocortical activity of the animals who survived were followed for 6 days postocclusion at which point they were sacrificed for histopathological analysis. Twelve of the animals died during recovery, of which 4 were MK-801 treated, and 8 were saline controls. The EEG ratios in the non-surviving animals were more depressed than in the animals that survived, whereas the depression in the EEG amplitude in both the treated and the control surviving animals was equal. Among the survivors no reduction in infarct size with MK-801 treatment was observed. Thus treatment with MK-801 in the middle cerebral artery occlusion model in the cat leads to a significant increase in the rate of survival (P < 0.05), but no prolonged improvement in late histopathology, in contrast with acute histological findings using this model. MK-801 treatment may be shifting the stroke model towards the survival of animals with larger infarcts. Histological recovery during prolonged reperfusion may eliminate the early neuroprotective effects seen with MK-801 treatment.

Features of complement activation-related pseudoallergy to liposomes with different surface charge and PEGylation: Comparison of the porcine and rat responses

Pigs are known to provide a sensitive model for studying complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2 kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~0.01 and ~22 mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1 mg/kg, while the reaction they caused in rats was mild even at 300 mg PL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2-3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG-dependent tachyphylaxis and the massive reactivity of Chol-PEG liposomes remain unclear.

Antinociceptive desensitizing actions of TRPV1 receptor agonists capsaicin, resiniferatoxin and N-oleoyldopamine as measured by determination of the noxious heat and cold thresholds in the rat.

Agonists of the TRPV1 receptor excite TRPV1‐expressing polymodal nociceptors that is followed after higher doses by a state of diminished responsiveness called desensitization which ensues at two levels: (i) diminished responsiveness of the ion channel (TRPV1 receptor desensitization); (ii) diminished responsiveness of the nerve endings to all stimuli including noxious heat. The aim was to compare these desensitizing actions of TRPV1 agonists in the rat by measuring with an incremental hot/cold plate the noxious heat and cold thresholds, i.e. the lowest hot and highest cold plate temperature, respectively, that evokes nocifensive behaviour. Capsaicin (3.3–1000 nmol) or resiniferatoxin (0.016–0.5 nmol) applied intraplantarly evoked a sustained dose‐dependent elevation of the noxious heat threshold lasting for 2–11 days. N‐oleoyldopamine failed to elevate the heat threshold. The noxious cold threshold was decreased by capsaicin or resiniferatoxin with a recovery within 2–4 days. The diminished acute nocifensive and heat threshold‐lowering effects of resiniferatoxin or N‐oleoyldopamine by pretreatment with doses that failed to elevate the heat threshold and to alter the nocifensive action of the TRPA1 activator formaldehyde, were taken as indication of TRPV1 receptor desensitization. In conclusion, using measurement of threshold temperatures eliciting nocifensive reactions in rats both in the hot and cold range revealed that capsaicin and RTX impair thermosensation in both noxious ranges due to a functional desensitization of peripheral terminals of TRPV1‐expressing sensory neurons responsible for noxious heat and cold responsiveness. This could be differentiated from desensitization of TRPV1 receptor evoked by lower doses of resiniferatoxin or N‐oleoyldopamine.

Pharmacologic Inhomogeneity Between the Reactivity of Intramural Coronary Arteries and Arterioles

We hypothesized that because of their size, anatomic location, and hemodynamic function, coronary arteries and arterioles would respond differently to vasoactive substances. Intramural arteries (281.7 ± 23.1 &mgr;m) and arterioles (77.3 ± 6.6 &mgr;m) of the left anterior descending coronary of rats were isolated and cannulated. Spontaneous tone was lower in arteries than in arterioles (81.1 ± 5.7 vs. 53.0 ± 3.9% of passive diameter, p < 0.05 at 60 mm Hg intraluminal pressure). Arterial tone was adjusted by the thromboxane receptor agonist U46619 (5 × 10 −8M) to reach an active tone close to that of arterioles. Bradykinin elicited dilations in both types of vessels. Acetylcholine (10 −6 –10 −5M) dilated arteries (by 42.6 ± 11.5 &mgr;m) but constricted arterioles (by 16.4 ± 9.3 &mgr;m). Sodium nitroprusside and adenosine elicited significantly greater dilations in arterioles than in arteries (by 7.9 and 11.9%, respectively, p < 0.05), whereas dilations to norepinephrine were similar. Inhibition of nitric oxide synthesis caused a significantly smaller constriction in arteries (10.2 ± 3.31%) than in arterioles (31.6 ± 6.9%) and completely blocked bradykinin-and acetylcholine-induced dilations, whereas it did not affect dilations to sodium nitroprusside, adenosine, and norepinephrine. Compared with arteries, arterioles have a greater spontaneous tone and enhanced nitric oxide modulation of basal tone and exhibit greater responsiveness to nitric oxide and adenosine. In addition, nitric oxide synthase is activated differently by pharmacologic stimuli in these segments. The qualitative and quantitative differences among vasoactive responses of coronary arteries and arterioles demonstrated in this study suggest segment-specific roles for endothelial and metabolic factors in regulation of coronary vascular resistance.

Effect of transient receptor potential vanilloid 1 (TRPV1) receptor antagonist compounds SB705498, BCTC and AMG9810 in rat models of thermal hyperalgesia measured with an increasing-temperature water bath

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.

Segmental Differences in Geometric, Elastic and Contractile Characteristics of Small Intramural Coronary Arteries of the Rat

The depedence of elastic and contractile properties on the caliber of small intramural coronary arteries was investigated in the rat in vitro. Different segments of the left anterior descending coronary artery branching system were prepared for microarteriography. The segments were cannulated at both ends, immersed in oxygenated normal Krebs Ringer (nKR) solution. Intraluminal pressure was changed at a rate of about 0.5 mm Hg/s between 0 and 150 mm Hg in repeated cycles. The outer diameter was continuously measured with microangiometry. Pressure-diameter curves were recorded after preconditioning pressure cycles in nKR, with PGF2α in the bath (7.5 × 10–6 M), and in maximal relaxation with papaverine (2.8 × 10–4 M). Biomechanical parameters were computed for vessels grouped according to their calibers (inner diameters: 50–150, 150–250, 250–350, >350 µm). Distensibility and contractility decreased with increasing caliber of the vessels, while the elastic modulus increased. Spontaneous tone was (at 100 mm Hg in mechanically preconditioned vessels) 18.8 ± 4.5, 8.4 ± 4.4, 9.7 ± 3.7 and 8.3 ± 3.8% in the four groups, respectively. PGF2α contraction was maximal around the 300-µm caliber. Our study is the first direct demonstration that intramural small coronary arteries exhibit characteristic variability in their elastic and contractile properties as a function of their caliber. Such differences may be important in segmentally specific control processes of the coronary microcirculation.

Nanoparticles for intravascular applications: physicochemical characterization and cytotoxicity testing

AIM We report the physicochemical analysis of nanosystems intended for cardiovascular applications and their toxicological characterization in static and dynamic cell culture conditions. METHODS Size, polydispersity and ζ-potential were determined in 10 nanoparticle systems including liposomes, lipid nanoparticles, polymeric and iron oxide nanoparticles. Nanoparticle effects on primary human endothelial cell viability were monitored using real-time cell analysis and live-cell microscopy in static conditions, and in a flow model of arterial bifurcations. RESULTS & CONCLUSIONS The majority of tested nanosystems were well tolerated by endothelial cells up to the concentration of 100 μg/ml in static, and up to 400 μg/ml in dynamic conditions. Pilot experiments in a pig model showed that intravenous administration of liposomal nanoparticles did not evoke the hypersensitivity reaction. These findings are of importance for future clinical use of nanosystems intended for intravascular applications.

Effect of a neuroprotective drug, eliprodil on cardiac repolarisation: importance of the decreased repolarisation reserve in the development of proarrhythmic risk

The aim of this study was to analyse the effects of eliprodil, a noncardiac drug with neuroprotective properties, on the cardiac repolarisation under in vitro circumstances, under normal conditions and after the attenuation of the ‘repolarisation reserve’ by blocking the inward rectifier potassium current (IK1) current with BaCl2. In canine right ventricular papillary muscle by applying the conventional microelectrode technique, under normal conditions, eliprodil (1 μM) produced a moderate reverse rate‐dependent prolongation of the action potential duration (7.4±1.5, 8.9±2.1 and 9.9±1.8% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=9). This effect was augmented in preparations where IK1 was previously blocked by BaCl2 (10 μM). BaCl2 alone lengthened APD in a reverse frequency‐dependent manner (7.0±1.3, 14.2±1.6 and 28.1±2.1% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). When eliprodil (1 μM) was administered to these preparations, the drug induced a marked further lengthening relative to the APD values measured after the administration of BaCl2 (12.5±1.0, 17.6±1.5 and 20.5±0.9% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). In the normal Langendorff‐perfused rabbit heart, eliprodil (1 μM) produced a significant QTc prolongation at 1 Hz stimulation frequency (12.7±1.8%, n=9). After the attenuation of the ‘repolarisation reserve’ by the IK1 blocker BaCl2 (10 μM), the eliprodil‐evoked QTc prolongation was greatly enhanced (28.5±7.9%, n=6). In two out of six Langendorff preparations, this QTc lengthening degenerated into torsade de pointes ventricular tachycardia. Eliprodil significantly decreased the amplitude of rapid component of the delayed rectifier potassium current (IKr), but slow component (IKs), transient outward current (Ito) and IK1 were not considerably affected by the drug when measured in dog ventricular myocytes by applying the whole‐cell configuration of the patch‐clamp technique. The results indicate that eliprodil, under normal conditions, moderately lengthens cardiac repolarisation by inhibition of IKr. However, after the attenuation of the normal ‘repolarisation reserve’, this drug can induce marked QT interval prolongation, which may result in proarrhythmic action.