Frequently applied ketamine, medetomidine and thiopental anaesthesia induces high mortality in Wistar rats.

OBJECTIVE: Fatal reactions to the combination of ketamine-medetomidine and thiopental in Wistar rats are described in two different models of orthodontic tooth movement. MATERIALS AND METHODS: Thirty male rats were divided into two groups that required repeated anaesthesia during a 42-day study period, once a week or more frequently depending on the experimental group. The combination of ketamine [50 mg/kg body weight (b.w.)] and medetomidine (67 µg/kg b.w.) was administered intraperitoneally. Thiopental (8.3 mg/kg b.w.) was administered intraperitoneally 5 minutes later, barring any observable adverse reactions to the anaesthesia. RESULTS: Twelve animals died, though none during the first two procedures. Three animals died shortly after the administration of a ketamine-medetomidine combination, and the remainder died 10-25 minutes later. Only four of the affected animals received thiopental before their death on a particular day. As ten rats died in the more frequently anaesthetized group, repeated anaesthesia was suspected to be the cause of the increased mortality. CONCLUSIONS: Obstruction of the respiratory airways by saliva with subsequent suffocation may have been one of the causes of death, as it appeared in all the affected animals. Although the combination of ketamine and an alpha-2 adrenergic agonist is generally considered to be safe in rats, we propose that studies utilizing protocols requiring repeated anaesthesia adhere to a minimum safety period of 8.5 days between anaesthesia events. Alternative anaesthetic protocols should be employed if adherence to this is not possible due to the nature of the study.

Periodontal ligament regulatory role in experimental diabetic rat model of periodontium remodelling.

BACKGROUND: Diabetes, among multiple systemic harmful health issues, also may deteriorate normal regenerative and reparative functions of periodontium. The aim of this research was to study the role of periodontal ligament in tissue remodelling under the orthodontic appliance stimulation in two rat experimental models (healthy Wistar rats and Goto-Kakizaki, rodent model of non-obese type 2 diabetes). MATERIALS AND METHODS: Four groups of rats were defined: Wistar (WI; n = 8) and Goto-Kakizaki (GK; n = 8) control groups without orthodontic appliances, and Wistar (n = 16) and Goto-Kakizaki (n = 16) appliance groups with orthodontic appliances. After 42 days, rats were sacrificed and histopathology descriptive analysis about periodontal ligament and adjacent structures was performed as well as cellularity of periodontal ligament and Kappa curvature of tooth roots were measured. RESULTS: Goto-Kakizaki control rats showed statistically significantly higher cellularity in comparison with Wistar control rats (p < 0.001). Both applied groups (WI 44.63 ± 6.68; GK 79.58 ± 10.06) also showed statistically significantly higher cellularity (p < 0.001) in comparison with control groups (WI 34.48 ± 6.92; GK 45.31 ± 11.18). Applied groups (WI 0.197 ± 0.2; GK 0.126 ± 0.083) had statistically significant higher values of Kappa curvature in comparison with control groups (WI 0.023 ± 0.011; GK 0.037 ± 0.011) (WI appliance vs. WI control: p < 0.001; GK appliance vs. GK control: p < 0.05). Agitated periodontal ligament caused different degrees of cementoclasia and additionally dentinoclasia, altering the natural root curvature. CONCLUSIONS: Although not significantly different (WI and GK appliance groups) according to degree of molar roots odontoclasia, higher cellularity of agitated GK periodontal ligament could be influencing factor for, as previously reported, elevated osteoclast mobilization and possible prolonged periodontium reconstitution.

The role of endothelin B receptor in bone modelling during orthodontic tooth movement: a study on ETB knockout rats.

The endothelin system has an important role in bone modelling during orthodontic tooth movement (OTM); however, little is known about the involvement of endothelin B receptors (ETB) in this process. The aim of this study was to evaluate the role of ETB in bone modelling during OTM using ETB knockout rats (ETB-KO). Thirty-two male rats were divided into 4 groups (n = 8 per group): the ETB-KO appliance group, ETB-KO control group, wild type (ETB-WT) appliance group, and ETB-WT control group. The appliance consisted of a super-elastic closed-coil spring placed between the first and second left maxillary molar and the incisors. Tooth movement was measured on days 0 and 35, and maxillary alveolar bone volume, osteoblast, and osteoclast volume were determined histomorphometrically on day 35 of OTM. Next, we determined the serum endothelin 1 (ET-1) level and gene expression levels of the osteoclast activity marker cathepsin K and osteoblast activity markers osteocalcin and dentin matrix acidic phosphoprotein 1 (DMP1) on day 35. The ETB-KO appliance group showed significantly lower osteoblast activity, diminished alveolar bone volume and less OTM than the ETB-WT appliance group. Our results showed that ETB is involved in bone modelling in the late stage of OTM.

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins.

BACKGROUND AND AIMS: Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. METHODS AND RESULTS: Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. CONCLUSION: Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Expression levels of endothelin-1, endothelin-2, and endothelin-3 vary during the initial, lag, and late phase of orthodontic tooth movement in rats.

Endothelins (ET)-1, ET-2, and ET-3 are one group of cytokines likely to be released during orthodontic tooth movement (OTM). Therefore, the expression of ET levels was investigated to determine the importance and involvement of isopeptides during the several phases of OTM. Thirty-two male Wistar rats (12-13 weeks old) were divided into four groups of eight: control, 14, 28, and 42 day groups. Tooth movement was induced by a closed-coil spring inserted between the upper left first molar and the upper incisors. The distance between the teeth was measured on days 0, 2, 7, 14, 21, 28, 35, and 42 using a digital calliper. The rate of tooth movement was calculated. The animals were sacrificed on days 14, 28, and 42 and gene expression levels of all three ET were determined using reverse transcription polymerase chain reaction. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests. The distance between the teeth decreased in all appliance groups (P < 0.001). The rate of tooth movement was 0.20 +/- 0.02, 0.03 +/- 0.01, and 0.06 +/- 0.02 mm/day between days 0-2, 3-21, and 22-42, respectively. On day 14, gene expression levels for ET-1 (P < 0.05) and ET-3 (P < 0.001) increased compared with day 0. On day 28, a downregulation of ET-3 was observed when compared with day 0 (P < 0.001). On day 42, ET-1 (P < 0.001) and ET-3 (P < 0.01) gene expression levels were strongly upregulated, while ET-2 gene expression level was downregulated (P < 0.01) when compared with day 0. ET-1 and ET-3, but not ET-2, are involved in all three phases of OTM, and ET-1 seems to be the predominant form in the late phase of OTM.

Orthodontic force decreases the eruption rate of rat incisors.

The aim of this study was to determine whether a force applied in an antero-posterior direction would adequately reduce incisor eruption. This is needed to achieve a constant direction of force which is one of the demands for a good model for studying orthodontic tooth movement. Twenty male Wistar rats aged 11-12 weeks were divided into two equal groups: in the appliance group, a superelastic closed coil spring (25 cN) was placed between the upper left first molar and the incisors. The control group consisted of animals without an appliance. In both groups, cuts were created on the labial surfaces of the upper and lower incisors. The distance from the gingival reference point to the midpoint of the cut was measured for 10 days at 2 day intervals. Upper incisor inclination was determined as the distance from the most mesial point of the upper left first molar to the incisal edge of the ipsilateral incisor on days 0 and 10. Statistical analysis was carried out using two-way analysis of variance and a Bonferroni post- test to estimate reliability. The eruption rates of the maxillary incisors in the appliance group were significantly decreased when compared with the control group during the whole experiment. In the appliance group, the eruption rates of the mandibular incisors were decreased more than those of the maxillary incisors (P<0.01). There was no difference in incisor inclination between the appliance and control groups on day 10 (P=0.81). The applied force of 25 cN in an antero-posterior direction diminished incisor eruption to a level which enabled a constant direction of orthodontic force for 10 days.

The effects of TBC3214, a selective endothelin ETA receptor antagonist, on orthodontic tooth movement in rats.

Many chemical messengers are involved in the process of alveolar bone and periodontal ligament remodelling during orthodontic tooth movement. Among them is probably endothelin-1 (ET-1). Its role in this process has been partly explained using tezosentan, which affects endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Tezosentan enhances orthodontic tooth movement. The aim of this study was to determine the possible effects of a highly selective ET(A) antagonist on orthodontic tooth movement in rats. Thirty male Wistar rats, 11-12 weeks of age, divided into three equal groups. In group I, a closed-coil spring was used and they were treated daily with 15 mg/kg body weight of TBC3214, a highly selective ET(A) antagonist. A closed-coil spring was also used in group II and the animals were treated daily with a placebo. Group III were treated daily with a placebo. The coil spring delivered a force of 25 cN and was attached between the upper left first molar and upper left incisor. The distance between the teeth was measured with a digital calliper (accuracy +/- 0.01 mm) on days 0, 7, 14, 21, 24, 32, 37, and 40. The differences in the distance between the teeth were calculated to determine the amount of tooth movement. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests. The distance between the upper left first molar and the upper left incisor decreased in groups I and II. In group I, tooth movement was significantly less on days 32 and 37 (P < 0.01) and on day 40 (P < 0.001) compared with group II. In group III, the distance between the teeth increased during the study (P < 0.001). In animals treated daily with TBC3214, tooth movement was significantly less compared with the animals treated with a placebo. It is concluded that ET-1, which is the predominant form of endothelin isopeptides, is involved in orthodontic tooth movement in rats, probably by enhancing bone resorption via ET(A) receptors.

Nicardipine dose dependently reduces the effect of equinatoxin II on coronary flow in isolated rat heart.

Equinatoxin II (EqT II) decreases coronary flow in guinea-pig and rat heart. It increases cytosolic Ca2+ activity and is supposed to form cation selective membrane pores. In the present study we tried to evaluate the influence of the L-type Ca2+ channel antagonist nicardipine upon the vasoconstrictory effect of EqT II on coronary vessels in isolated heart of the rat. Coronary flow was measured during the experiment. Ten min after bolus injection of EqT II the coronary flow decreased to 17.2 +/- 4.3% in experiments without nicardipine, and to 39.4 +/- 4.5% in the group with 0.1 microM nicardipine (p < 0.05). Our results indicate the possibility that L-type Ca2+ channels could be involved in the effects of EqT II on coronary flow in the rat heart.

Lacidipine decreases the honeybee venom-induced vasoconstriction of the isolated porcine coronary artery.

The venom of the honeybee Apis mellifera induces cardiovascular dysfunction. As its effects on coronary arteries have not yet been described, we studied the effects of the whole honeybee venom (non-volatile part) in the isolated porcine left anterior descending coronary artery (LAD) and the influence of L-type Ca2+ channel blocker, lacidipine, upon the venom effects in LAD. The venom produced concentration dependent contractions (7-70 microg/ml) of the porcine LAD; maximal effect of the venom was approximately the same as the effect of 30 mM KCl. Lacidipine concentration dependently (0.1-10 microM) and significantly (P < or = 0.05) decreased the venom-induced vasoconstriction. The results indicate the involvement of L-type Ca2+ channels in coronary contraction, induced by bee venom.

Thermal conductivity of the porcine heart tissue.

The porcine heart was used as a model for studying the thermal changes in myocardium at cooling and re-warming during open heart surgery. A section of the heart septum was excised and tissue was cut into two similar square slices. The same shape of the tissue, cut from the surface from the upper lateral wall of the left ventricle, covered with epicardium and fat, was taken for another measurement. A thin (<0.5 mm) square thermal source of the same length of the side as the tissue samples was put between the two slices of tissue. This set was placed in the middle of two identical copper cylinders (2r=50 mm, height=55 mm) used to keep the outer side of the specimen at controlled room temperature. Thermal conductivity of the heart tissue was determined at controlled thermal power, and known difference of the temperature at the edge of the tissue and at the middle of the heater, when steady state was reached. Thermal conductivity calculated from the temperature difference and the geometry of heater and samples was 0.75 W/m.K for septal heart tissue, and 0.60 W/m.K for the lateral wall ventricle tissue with epicardium and fat.

Nicardipine dose-dependently reduces the effect of equinatoxin II on coronary flow in isolated porcine heart.

Death after i.v. administration of equinatoxin II (EqT II) has been attributed to the circulatory failure resulting from cardiotoxic effects. The mechanism of action is unknown. The aim of the present work was to study the effects of the toxin on vascular tone in the isolated porcine coronary artery and on coronary flow in the isolated pig heart. EqT II caused concentration-dependent contractions of rings of the isolated epicardial porcine coronary artery with an EC50 value of 89+/-5 nM (n=5-6) and maximal effect of about 140% of the contraction induced by 20 nM KCl. On Langendorffs porcine heart preparation EqT II caused a dose-dependent decrease of coronary flow. At EqT II doses lower than 0.05 micromol/100 g of heart weight there were no measurable effects of the toxin. At dose 0.5 micromol/100 g the toxin decreased coronary flow to less than 9.8+/-2.5% of the control value. The constrictory effect of the toxin on isolated porcine coronary arteries was diminished by the L-type calcium channel antagonist nicardipine (NC). NC in 1 microM concentration almost completely abolished the effect of EqT II on coronary flow. Our results confirmed involvement of L-type calcium channels in the vasoconstrictory effects of EqT II on epicardial coronary arteries.

Action of mibefradil and lacidipine on the isolated human anterior tibial artery.

In isolated human anterior tibial artery the effects of two different types of calcium channel antagonists, mibefradil (a selective T-type Ca2+ channel antagonist) and lacidipine (a 1,4 dihydropyridine Ca2+ channel antagonist, acting at L- and T-type, but binds preferentially at L-type Ca2+ channels) were compared. Both drugs reduced the contractions of isolated arteries induced by 60 mM KCl. The potency (IC50) of mibefradil was 6.5 microM and of lacidipine 82.4 nM. The potencies of both Ca2+ channel antagonists differed significantly (p<0.001 at 0.1 and 1 microM; p<0.01 at 10 microM). Lacidipine was 79-times more effective than mibefradil in reducing the vasoconstriction in isolated human anterior tibial artery. One of the reasons for higher potency of lacidipine could be a higher density of L-type than of T-type Ca2+ channels in tissue of the human anterior tibial artery.

Lowering of the coronary flow in isolated rat heart by equinatoxin II depends upon extracellular Ca2+ concentration.

Equinatoxin II (EqT II) is a basic polypeptide toxin from the sea anemone Actinia equina (L.). Its LD50 in mice is 33 g/kg. The cause of death after intravenous application has been attributed to the circulatory failure resulting in the cardiotoxic effects. In Langendorff's rat and guinea-pig heart preparations EqT II caused dose dependent decrease in the coronary flow (CF). Morphologic changes of different cell cultures incubated with EqT II are the result of Ca2+ entry through the newly formed discrete pores. Pores in the cell membranes are composed of the toxin oligomeres. In the present study we tried to evaluate the dependence of vasoconstrictor effects of EqT II on isolated rat hearts upon the Ca2+ concentration in the perfusion solution. EqT II did not affect the CF in the group without Ca2+. The strongest effect was observed in the group with 1.5 mM Ca2+ where the CF decreased to 7.7+/-7%. The results of our experiments indicate that the effects of EqT II on CF depend on Ca2+ concentration in the extracellular solution.

Effects of equinatoxin II from Actinia equina (L.) on isolated rat heart: the role of direct cardiotoxic effects in equinatoxin II lethality.

Equinatoxin II is a lethal basic protein isolated from the sea anemone Actinia equina (L.) with LD50 in mice 35 microg/kg. The putative cause of death is cardiorespiratory arrest, but the mechanism of cardiotoxicity is poorly understood. It is not clear whether the toxin injected intravenously into an experimental animal reaches the heart in a concentration sufficient to cause direct effects on the heart. Therefore experiments were performed on rats and on isolated rat hearts in order to investigate the possible direct cardiotoxic effects of the toxin. For this reason the hearts were perfused with different concentrations of the toxin and with the effluent from the lungs collected during perfusion of the lungs with equinatoxin II. The results revealed the clear dose-dependent, direct cardiotoxic effects of the toxin and of the effluent from the lungs on Langendorff's heart preparations. The threshold concentration of equinatoxin II causing a drop in the perfusion rate, decreased left ventricular pressure, arrhythmia and increased LDH release, was found to be around 0.1 to 1 nM. With 10 nM equinatoxin II the left ventricular pressure dropped to 14+/-11% of the control, and the coronary flow to 9+/-3%. These effects were followed by arrhythmia and cardiac arrest. The concentration of equinatoxin recovered from the lungs after the perfusion with 100 nM equinatoxin II ranged between 0.8 and 5 nM. The results indicate that direct cardiotoxic effects of equinatoxin II play an important role in the lethal effects of the toxin.

Effects of chloropyramine and famotidine on postischaemic and posthypoxic myocardial damage in isolated rat hearts.

In isolated rat hearts effects of chloropyramine (CP), histamine H1 antagonist, and famotidine (FA), H2 antagonist, upon two different myocardial injuries, ischaemia-reperfusion and hypoxia-reoxygenation were studied. In both types of injury the effects of drugs were seen mainly during reperfusion and reoxygenation, respectively. During reperfusion neither CP nor FA influenced amplitude of contractions, but CP lowered heart rate, +dp/dtmax and coronary flow. During reoxygenation CP and FA lowered early posthypoxic contractions, whereas CP decreased and FA increased heart rate. CP and FA did not significantly influence the post-ischaemic and posthypoxic lactate dehydrogenase (LDH) release. Present results indicate the existence of H1 and H2 receptors in rat heart as well as their involvement in both types of studied injuries.