Comparison of two methods (left carotid artery and abdominal aorta) for surgical implantation of radiotelemetry devices in CD-1 mice.

The goal of this study was to compare two surgical methods, the left carotid (LC) and the abdominal aorta (AA), for mouse instrumentation with telemetry devices, to determine the best method for measuring cardiovascular (CV) parameters by radiotelemetry in freely moving mice. Surgery success rate, postsurgical recovery rate, clinical parameters, CV data (baseline and response to nicotine) and circadian rhythm measurements were compared between these techniques. Brains of LC-implanted mice were evaluated for potential ischaemia by direct observation of the Circle of Willis anatomy and histopathology. For this purpose, a total of 31 CD-1 male mice were instrumented with PA C20 devices (10 with LC and 21 with AA). Mortality, morbidity, physical examination, body weight (BW), water and food consumption (W/FC), mean blood pressure (MBP) and heart rate (HR) were monitored daily during the recovery period (10 days). CV baseline data were recorded continuously during two periods of four days, and finally, both LC- and AA-implanted mice received an acute subcutaneous administration of 1 mg/kg nicotine; BP and HR were recorded during 5 h after nicotine administration. Results showed that, in LC-implanted mice, 80% survived surgery and recovered well. In contrast, only 57% of mice implanted with the AA technique survived surgery and some presented lethal complications. Both techniques had similar recovery times for BW and W/FC, comparable return to normal circadian rhythm (day 6 post-surgery) and similar CV baseline values. No significant differences were observed in CV response to nicotine between both groups of implanted CD-1 mice. No histopathological changes suggestive of ischaemia were noted in the brain of mice implanted in the LC. Six out of the eight LC-implanted mice remained in good health and had good pressure signal for at least 100 days post-surgery, while most of the AA-implanted mice lost the signal pressure within 14-49 days post-surgery. In conclusion, we believe that LC implantation in mice is superior to the AA technique and is more appropriate for long-term telemetry studies, especially for smaller (transgenic) animals.

A novel and simple method for endotracheal intubation of mice.

Endotracheal intubation in mice is necessary for experiments involving intratracheal instillation of various substances, repeated pulmonary function assessments and mechanical ventilation. Previously described methods for endotracheal intubation in mice require the use of injection anaesthesia to immobilize the animal during the intubation procedure or the use of a volatile anaesthetic prior to intubation for immobilization. With these methods, the control of anaesthetic depth during the intubation procedure is absent. We describe a method for simple and rapid intratracheal intubation in mice for mechanical ventilation, using a self-built plastic support to facilitate the intubation procedure. General anaesthesia is maintained by means of inhalation through a non-rebreathing circuit connected to the plastic support. This set-up gives the operator control of anaesthetic depth and sufficient time to perform the intubation procedure. A purpose-made laryngoscopic blade is used to facilitate the intubation tube entering the trachea. The blade of the purpose-made laryngoscope is constructed as a retraction guide and is curved for easy handling. Under direct vision, the epiglottis is gently lifted by the laryngoscopic blade while the intubation tube is pushed into the trachea. Following this novel intubation technique, we were able to mechanically ventilate mice for at least 2 h without severely disturbing blood gases. Histological evaluation of the lungs and microscopic evaluation of the trachea and larynx showed no signs of trauma related to the intubation technique or mechanical ventilation.

Presynaptic muscarinic receptors inhibiting endogenous noradrenaline release in the portal vein of the freely moving rat.

1. In the portal vein of the freely moving unanaesthetized rat, the existence of presynaptically located inhibitory muscarinic receptors was investigated by use of the muscarinic agonist methacholine (MCh) 2. Infusion of MCh (0.3 micrograms min-1) did not significantly inhibit the endogenous noradrenaline (NA) overflow in portal plasma. However, after inducing high intra-synaptic concentrations of NA by blocking the presynaptic alpha 2-adrenoceptors with yohimbine (1 mg kg-1), MCh (0.3 microgram min-1) was able to reduce the yohimbine-induced enhanced NA overflow by 38%. 3. The MCh-induced inhibition was almost completely abolished after blockade of the presynaptic muscarinic receptors with atropine (0.6 mg kg-1). 4. During electrical stimulation of the portal vein nervous plexus the evoked NA overflow was strongly inhibited (95%) during MCh-infusion (0.3 microgram min-1). Again atropine (0.6 mg kg-1) was able to reverse this inhibition. 5. These results show the existence of presynaptic muscarinic receptors inhibiting endogenous NA overflow from the portal vein nervous plexus under conditions of enhanced sympathetic activity in the freely moving rat.

Characterization of presynaptic vascular muscarinic receptors inhibiting endogenous noradrenaline overflow in the portal vein of the freely moving rat.

1. In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically-evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2. By use of pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and AF-DX 116 as M1-, M3-, and M2-selective antagonists respectively, the MCh (0.1 microM)-induced inhibition of the electrically-evoked NA overflow could be reversed to the control stimulation value dose-dependently. 3. The potency order of the antagonists was: 4-DAMP greater than AF-DX 116 greater than pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4. From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of conscious unrestrained rats are of the cardiac M2-subtype.

Dysfunctional presynaptic alpha 2-adrenoceptors expose facilitatory beta 2-adrenoceptors in the vasculature of spontaneously hypertensive rats.

Previous studies on spontaneously hypertensive rats (SHR) have yielded inconsistent information about functional aberrations of the presynaptic alpha 2- and beta 2-adrenoceptor-mediated modulation of sympathetic neurotransmitter release. In the present investigation we studied the capacity of presynaptic beta 2-adrenoceptors that enhance noradrenaline (NA) release in the portal vein of freely moving, unanesthetized SHR and normotensive Wistar rats (WR) using the beta 2-selective agonist fenoterol. The results show that the presynaptic beta 2-adrenoceptor population in SHR responds to significantly lower dosages of fenoterol than that in WR. The reason for this enhanced action, however, could not be attributed to the beta 2-adrenoceptor itself, nor to a diminished neuronal uptake of NA, but to a diminished responsiveness of the presynaptic alpha 2-adrenoceptor. Stimulation of presynaptic alpha 2-adrenoceptors with oxymetazoline (45 micrograms/min) decreased basal NA levels by 46% in WR and by 3% in SHR. Blockade of alpha 2-adrenoceptors, using 0.5 mg/kg yohimbine, induced a 4.86-fold rise in the basal NA level in WR but only a 1.89-fold rise in SHR. A subsequent dose of fenoterol, however, resulted in a further 2.5- and 2.6-fold rise in WR and SHR, respectively, indicating that there is a normal presynaptic beta 2-adrenoceptor population in the vasculature of SHR.

Characterization of presynaptic beta-adrenoceptors facilitating endogenous noradrenaline release in the portal vein of permanently cannulated, freely moving rats.

We investigated the nature of presynaptic beta-adrenoceptors and the possible heterogeneity of these receptors in the portal vein nervous plexus of freely moving unanesthetized rats using the differential blockade technique with CGP 20712A as a highly beta 1-selective antagonist, ICI 118,551 as a very beta 2-selective antagonist and fenoterol and endogenous NA as beta 2- and beta 1-selective agonists respectively. The fenoterol (0.25 mg/kg)-induced increase of the basal NA level (290%) was dose dependently decreased by 0.1, 0.3 and 1.0 mg/kg ICI 118,551, but was not affected by a high dose (3.0 mg/kg) of CGP 20712A. During electrical stimulation (2 Hz, 3 ms, 5 mA) of the portal vein nervous plexus, 0.25 mg/kg fenoterol induced a 2.1-fold increase in NA overflow compared to the control stimulation value. ICI 118,551 was also able to decrease the fenoterol-induced enhancement during stimulation. During stimulation in the presence of CGP 20712A and fenoterol, the control stimulation value was not significantly decreased. Pretreatment with yohimbine (0.5 mg/kg) was used to create a strong beta 1-stimulus by raising the intra-synaptic NA level through blockade of the inhibitory alpha 2-adrenoceptors. Under these conditions, CGP 20712A did not deminish the yohimbine-induced enhancement of the stimulus evoked NA overflow, clearly indicating the absence of the beta 1-adrenoceptor subtype. ICI 118,551 (0.1 mg/kg) was also unable to influence the evoked NA overflow under these conditions, implying that, even at high concentrations, NA is not able to facilitate its own release by stimulation of the presynaptic beta 2-adrenoceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Pronounced facilitation of endogenous noradrenaline release by presynaptic beta 2-adrenoceptors in the vasculature of freely moving rats.

The facilitation of the noradrenaline (NA) overflow by stimulation of the presynaptic beta-adrenoceptor of the rat portal vein was investigated, using the freely moving unanesthetized permanently cannulated rat as a model. The beta 2-selective agonist fenoterol caused a maximal enhancement of about 300% of the basal NA level at a dose of 0.5 mg/kg. Following administration of cocaine (2.5 mg/kg plus 0.05 mg/kg/min) basal NA levels increased to 150% whereas combination of cocaine and fenoterol results in a dose dependant rise up to over 560% of the basal level (at a fenoterol dosage of 0.5 mg/kg). Blockade of the alpha 2-adrenoceptors with yohimbine (0.5 mg/kg) which enhances the NA level to 486%, followed by 0.125 mg/kg fenoterol results in a further 2.53-fold rise to more than 1,200% of the basal level, indicating the pronounced counterregulatory role of the presynaptic alpha 2-adrenoceptor. After ganglionic blockade with hexamethonium (3 mg/kg plus 6 mg/kg/h) the effect of yohimbine (0.5 mg/kg) alone was diminished to 162%, but the additional facilitatory effect of 0.125 mg/kg fenoterol still was 1.82-fold, to 294% of the basal level. Combination of cocaine (2.5 mg/kg plus 0.05 mg/kg/min), yohimbine (0.5 mg/kg) and fenoterol (0.125 mg/kg) induced a rise to over 9,000 pg/ml NA (about 40-fold of the basal NA level). During electrical stimulation (2 Hz, 3 ms, 5 mA) of the local portal vein nervous plexus, the role of the inhibitory alpha 2-adrenoceptor becomes even more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine stress response in jugular-vein cannulated rats upon multiple exposure to either diethyl-ether, halothane/O2/N2O or sham anaesthesia.

The main objective of this study was to assess the endocrine stress response to multiple anaesthesia followed by sham anaesthesia in order to detect any memory effects. For this purpose, jugular-vein cannulated rats were subjected to either sham, diethyl-ether or halothane/O2/N2O anaesthesia, and their plasma ACTH, corticosterone, glucose, adrenaline and noradrenaline levels measured. The study had three separate experiments, each consisting of a control and treatment group. In two experiments, the rats were exposed to high or low concentrations (40-15%) of diethyl ether, using either a jar containing cotton soaked in diethyl ether or a vaporizer. In the third experiment, rats were exposed to halothane/O2/N2O. Control animals underwent sham anaesthesia. Blood samples were taken 6 min before and at 5, 15 and 55 min after starting the exposure (t = 0 min). For each variable, the dt5 (level at t = 5 min minus that at t = -6 min) and the cumulative levels over the one-hour period as determined by the area under the curve (AUC) were calculated. Further, the peak levels (Cmax) were determined. The mean time needed to induce anaesthesia was 68, 121 and 55 s for exposure to high and low concentrations of diethyl ether and to halothane/O2/N2O, respectively. Increased noradrenaline and adrenaline dt5 levels were observed only after the first exposure to the high concentration of diethyl ether. Multiple anaesthesia sessions using either diethyl ether or halothane/O2/N2O did not clearly influence adrenaline and noradrenaline levels. Diethyl ether induced a sharp rise in plasma ACTH and glucose levels, irrespective of the concentration used. The response of the ACTH and glucose was similar for single and multiple exposure. An increased response of ACTH, corticosterone and glucose to sham anaesthesia following multiple induction of anaesthesia was observed for the high concentration of diethyl ether only. Halothane/O2/N2O raised plasma glucose without differences between single and multiple anaesthesia sessions. Upon sham anaesthesia following multiple exposures to halothane/O2/N2O, glucose levels were significantly increased. This study indicates that repeated anaesthesia in rats can elicit an increased stress response during subsequent handling and change of environment.

The use of radiotelemetry in small laboratory animals: recent advances.

Radiotelemetry provides an alternative means of obtaining physiological measurements from awake and freely moving laboratory animals, without introducing stress artifacts. For researchers, especially those in the fields of pharmacology and toxicology, the technique may provide a valuable tool for predicting the effectiveness and safety of new compounds in humans. In light of studies described in the literature, it is concluded that there is ample evidence that the use of radiotelemetry for measuring blood pressure, cardiac activity, heart rate, body temperature, and locomotor activity in rodents has been validated sufficiently. Today, this technology is an important tool for the stress-free collection of these physiologic data in small rodents, including mice.

The PVC-rat and other alternatives in microsurgical training.

The number of animals used in educational training programs in experimental microsurgery can be reduced by using artificial devices such as the anastomoses device and the MD PVC-Rat. Such in vitro methods allow development of technical skills, making the transition to in vivo models much easier.

A new technique for the study of vascular presynaptic receptors in freely moving rats.

A technique is described in which a silicon catheter is utilized for permanent catheterization of the portal vein, and curved platinum stimulation electrodes around this vein are used for the stimulation of the local nervous plexus. The method has a success ratio of over 70% during a period of more than 2 mo. Five animals were used in experiments over a period longer than 5 mo. Using this technique, we investigated the influence of variation of current density, pulse duration, and frequency of stimulation on the electrically evoked release of norepinephrine in portal blood. Enhancement of all stimulation parameters proportionally increased the evoked norepinephrine release. The alpha 2-adrenoceptor antagonist yohimbine was found to increase basal norepinephrine levels 4.43-fold and facilitated the evoked release additionally with a factor of 4.70. The results show that noradrenergic neurotransmission in the portal vein nervous plexus is controlled presynaptically by a strong local alpha 2-adrenergic autoinhibition. The method offers the means to explore locally the presynaptic modulation of endogenous neurotransmitter release in unrestrained unanesthetized animals.

Influence of the baroreceptor reflex on the modulation of noradrenaline overflow through prejunctional receptors in the portal vein of freely moving rats.

1. The effects of alterations in mean arterial blood pressure (MAP), as induced by vasoactive drugs, on heart rate (HR), basal noradrenaline concentration and electrically evoked noradrenaline overflow and on blood flow in the portal vein of freely moving rats, were investigated. 2. By infusion of sodium nitroprusside or phenylephrine (0.5, 1.0 and 2.5 micrograms kg-1 min-1), MAP was altered over a range of 50 to 150 mmHg. The resulting changes in HR showed a sigmoidal relationship with MAP. Noradrenaline overflow increased linearly when MAP was decreased; when MAP was increased, however, noradrenaline levels only decreased to 70% and reached a plateau from 125 mmHg onwards. 3. Nitroprusside (2.5 micrograms kg-1 min-1) and fenoterol (0.25 mg kg-1) decreased MAP to the same extent (-46 mmHg). HR and basal noradrenaline concentration, however, were increased to a higher extent by fenoterol (+192 beats min-1; +373 pg ml-1, respectively) than by nitroprusside (+78 beats min-1; +206 pg ml-1, respectively). Electrically evoked overflow was not changed at all after nitroprusside, whereas fenoterol induced an increase to 206% of control. 4. Phenylephrine (2.5 micrograms kg-1 min-1) and angiotensin II (1 microgram kg-1 min-1) increased MAP to the same extent (to 155 and 161 mmHg, respectively). Basal noradrenaline concentration decreased by 30% after phenylephrine, whereas angiotensin II increased noradrenaline levels to 226% of control. Evoked noradrenaline overflow was not changed after phenylephrine but was increased to 204% of control after angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

Technique for creating a permanent cecal fistula in the rat.

A method was developed to surgically implant a silicone/stainless steel fistula-cannula in the cecum of freely moving rats. The specially designed fistula-cannula allows sampling of cecal contents at any time without disturbing the physiologic functions of the intestinal tract, as evaluated by observation of general well-being and postmortem macroscopic inspection. The fistula-cannula was implanted in 12 male rats under general anesthesia. The animals remained in good health during the postoperative period lasting up to 9 weeks. They gained weight normally compared with a control group (n = 8) that had not undergone surgery. Samples of 0.2 to 1.0 g of contents could be collected with a microspatula, without the need to anesthetize the animal. The model provides a useful system for sampling cecal contents without the need to sacrifice the animal. Because it allows cross-over studies to be carried out, this approach may result in significantly reducing the number of animals required for digestive studies.

Sympathoadrenal influence on glucose, FFA, and insulin levels in exercising rats.

The effects of sympathoadrenal manipulations on the exercise-induced alterations in blood glucose, plasma free fatty acids (FFA), and insulin were investigated in intact and adrenodemedullated rats. Exercise consisted of strenuous swimming against a countercurrent for 15 min. Before, during, and after swimming, blood samples were taken through a permanent heart catheter. Adrenodemedullation (Adm) markedly reduced the exercise-induced increase in both glucose and FFA. This effect was counteracted by intravenous infusion of epinephrine (E, 20 ng/min). Intravenous infusion of 50 ng E/min into Adm rats caused an exaggerated increase in glucose. In two additional experiments 1) specific adrenoceptor agonists and antagonists were administered to exercising intact and Adm rats, and 2) E or norepinephrine (NE; 20 ng/min) was infused into intact resting rats. The results suggest that E from the adrenal medulla directly affects glucose and insulin but not FFA concentrations in the blood. NE released from peripheral sympathetic nerve endings probably acts in two different ways: as neurotransmitter on liver and pancreas and as a hormone on adipose tissue.

Adrenal and sympathetic catecholamines in exercising rats.

The effects of adrenodemedullation and/or adrenoceptor agonists and antagonists on plasma epinephrine (E) and norepinephrine (NE) concentrations during exercise were investigated in rats. Exercise consisted of strenuous swimming against a countercurrent for 15 min in a pool with water of 33 degrees C. Before, during, and after swimming, blood samples were taken through a permanent heart catheter. E was not detectable in plasma of adrenodemedullated (Adm) rats. A marked reduction in the normal exercise-induced increase in plasma NE concentrations occurred in both Adm rats as well as in intact rats injected with the beta 2-selective adrenoceptor antagonist ICI 118551. Intravenous infusion of either E or the beta 2-selective agonist fenoterol restored the increase in plasma NE in Adm rats. Injection of the alpha 2-selective antagonist yohimbine in combination with infusion of the beta 2-selective agonist fenoterol into Adm rats caused an enormous increase in plasma NE. It is concluded that all NE in plasma as released during exercise originates from the peripheral nerve endings of the sympathetic nervous system. Adrenal E influences the release of NE via activation of presynaptic beta 2-adrenoceptors.

A novel method to assess airway function parameters in chronically instrumented, unrestrained guinea-pigs.

A new method has been developed to measure airway functions in unanaesthetized, unrestrained guinea-pigs. The technique utilizes a specially designed pneumotachograph that is placed inside the trachea, and a saline-filled balloon, placed inside the pleural cavity. The pneumotachograph consists of a stainless steel cylinder with coaxial and perpendicular tubes attached to it, measuring the total pressure and the lateral pressure in the trachea, respectively. Via air-filled silicon conducting tubes, subcutaneously driven and permanently attached to the neck of the animal, the pressures are fed into a differential pressure transducer, yielding a pressure difference proportional to the airflow in the trachea. Via a saline-filled tubing, the pleural balloon is similarly attached to the neck of the animal, and pleural pressure (Ppl) is measured using a second pressure transducer. These data permit calculation of airway functions in conscious, unstressed animals. Control values for airway resistance (RAW), Ppl, tidal volume and respiratory frequency are all in the range of results reported previously for this species. A very significant correlation between RAW and Ppl (P < 0.001) was observed, indicating that sole Ppl-measurement can be used as a relatively simple and sensitive method to assess bronchial obstructive reactions in unrestrained guinea-pigs. Using a specially designed provocation cage, which allowed the animals to remain in a stress-free, unrestrained condition, the method has been successfully applied for the assessment of the dose-dependent bronchoconstrictor sensitivity to histamine as well as to allergen-induced early and late phase airway reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of a new system for the automatic registration of behaviour in mice and rats.

A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) for the automatic registration of different behavioural elements of mice and rats was validated. The LABORAS sensor platform records vibrations evoked by animal movements and the LABORAS software translates these into the corresponding behaviours. Data obtained by using LABORAS were compared with data from conventional observation methods (observations of videotapes by human observers). The results indicate that LABORAS is a reliable system for the automated registration of eating, drinking, grooming, climbing, resting and locomotion of mice during a prolonged period of time. In rats, grooming, locomotion and resting also met the pre-defined validation criteria. The system can reduce observation labour and time considerably.