Breathing and lung mechanics in hamsters: effect of pentobarbital anesthesia

An adaptation of the method reported by Skornik, Heimann, and Jaeger (Toxicol. Appl. Pharmacol. 59: 314-323, 1981) was used to evaluate pulmonary mechanics in intact awake hamsters. Lung volume changes were measured with a pressure plethysmograph, and pleural pressure was estimated by the use of a saline-filled esophageal catheter. We report data for normal awake hamsters studied at 18, 20, 22, 32, and 98 wk of age. Age-related differences were observed in tidal volume, dynamic compliance, and pulmonary resistance. To determine to what extent pulmonary mechanics are changed by anesthesia, hamsters were measured during spontaneous breathing while awake and while anesthetized. We found that anesthesia had a marked effect on the breathing pattern of normal hamsters. Twenty-five minutes after injection of pentobarbital sodium (70 mg/kg ip), tidal volume, dynamic compliance, pulmonary resistance, breathing frequency, and minute ventilation were 66, 40, 375, 60, and 41% of the corresponding awake values. Anesthesia always provoked a significant and dose-related decrease in tidal volume and an increase in respiratory period, together resulting in a profound decrease in minute ventilation. These significant differences from the awake values call into question the value of measurements in anesthetized animals. The methods described here yield reasonable and repeatable measurements and, because no anesthesia or surgery is required, they can be used in longitudinal studies when repeated measurements in the same animal over long periods of time can help define pathological changes or the effectiveness of various interventions.     

Effect of exercise on redistribution and clearance of inhaled particles from hamster lungs

Does exercise alter the redistribution and clearance of particles from the lungs? Sedentary hamsters and hamsters that were exercise trained by voluntary wheel running for the previous 5 wk were exposed to a 198Au-labeled aerosol for 25 min. Six trained and 6 sedentary animals were killed within 5 min after the exposure (day 0); the same number were killed 5 days later. The trained hamsters ran ad libitum during those 5 days. The lungs of all animals were excised, dried at total lung capacity, sliced into 1-mm-thick sections, and dissected into pieces that were counted for radioactivity and weighed. On day 0, trained hamsters had 80% more particles per milligram of lung than sedentary hamsters, although both were exposed under identical conditions of restraint. After five days, exercising hamsters cleared 38% of the particles present at day 0, whereas sedentary animals removed only 15%. Significant clearance was observed from the middle lung regions of sedentary hamsters and from all lung regions in exercising hamsters. We conclude that exercise can enhance the redistribution and clearance of particles from the lungs; the mechanisms responsible are as yet unclear.     

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 μg/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 μg/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 μg/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.     

Evaluation of a combination of tiletamine and zolazepam as an anesthetic for laboratory rodents

A combination of equal parts of tiletamine hydrochloride and zolazepam hydrochloride was evaluated as an injectable anesthetic for rats, mice, and hamsters. The drug produced satisfactory anesthesia and analgesia in rats when given either intraperitoneally or intramuscularly at concentrations of 20 or 40 mg/kg body weight. The length of anesthesia was dose dependent and was somewhat longer in females as compared to males, and inbreds compared to outbreds. Incisions through the peritoneum of anesthetized rats evoked little or no response, whereas cervical skin incisions evoked a slight response in many rats. Anesthesia without analgesia occurred in mice at dosages of 80 mg/kg body weight and higher, however, many animals developed respiratory distress and died at dosages of 100 to 160 mg/kg body weight. In hamsters, anesthesia but not analgesia occurred at drug concentrations of 50 to 80 mg/kg body weight. It was concluded that a tiletamine and zolazepam combination was an effective anesthetic for rats, but not for mice or hamsters.     

Effect of hypothermia on brain multi-parametric activities in normoxic and partially ischemic rats

Hypothermia, as well as anesthesia, are known to protect the brain against ischemia, hypoxia and other pathological damages. One of the mechanisms of this improvement could be by lowering brain function, and thereby lowering oxygen demand. We examined the effect of hypothermia on brain function and blood supply in awake and anesthetized rats and studied the interaction between partial ischemia and the responses to hypothermia. The brain function multiprobe (BFM) used enabled simultaneous measurements of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular K(+) concentration, DC potential and ECoG from the cerebral cortex in rats whose brain temperature was lowered by 5 degrees C. Hypothermia was induced in awake, anesthetized and brain ischemic-anesthetized rats. In anesthetized and ischemic-anesthetized rats, the time required for lowering the brain temperature by 5 degrees C was five times less than in the normal awake animals. No significant changes in CBF and NADH levels were found in response to hypothermia in the awake animals. In contrast, a significant decrease in extracellular K(+) concentration was recorded under hypothermia, probably due to the lower rate of depolarization. Hypothermia in anesthetized and in ischemic-anesthetized rats did not significantly affect the levels of mitochondrial NADH, CBF and extracellular K(+). Hypothermia under ischemia was expected to be more effective.     

Effect of papain-induced emphysema on permeability of rat lung to drugs.

To investigate the effect of a papain-induced emphysema-like condition on pulmonary absorption of drugs, rats were exposed to either papain aerosol or distilled water aerosol (control) intermittently for 2 wk, and rates of drug absorption from damaged and control lungs were compared. To measure absorption rates, 0.1 ml of drug solution (0.1-10 mM) was administered through a tracheal cannula to anesthetized animals, and after various times lungs were assayed for unabsorbed compound. In absorption experiments with the lipoid-insoluble compounds, mannitol, p-aminohippuric acid, and procaine amide ethobromide, all three drugs were absorbed from the lungs about twice as rapidly in papain-treated rats as in control. In contrast, procaine amide, a relatively lipoid-soluble drug, was absorbed at the same rate in both control and papain-treated animals. The results suggest that papain-induced lung damage increases the porosity of the pulmonary epithelium.     

Ventilatory failure induced by tracheal banding in the hamster.

Previous animal models of hypercapnic ventilatory failure are limited in that the resistive load has only been applied acutely and often in anesthetized animals. We therefore developed a chronic animal model of hypercapnic ventilatory failure by increasing airway resistance via tracheal banding over several days. To test the efficacy of this model, we compared arterial blood gases, pulmonary function, and internal area of the trachea 6 days after the banding or sham procedure in 20 hamsters. Six days later, banded animals had an increased airway resistance as indicated by a 66% reduction in internal cross-sectional area of the trachea and a 6.5-fold increase in pulmonary resistance compared with control hamsters. The increased airway resistance resulted in a severe respiratory acidosis and hypoxemia in the awake banded hamsters. Banded hamsters were also hyperinflated. This animal model will be useful for investigating the various mechanisms that contribute to hypercapnic ventilatory failure and interventions that may promote recovery.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B4 (LTB4) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB4 showed a pronounced neutrophil infiltration. These results confirm previous in vitro studies in which LTB4 was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A2.     

Chloral hydrate anesthesia alters the responsiveness of dorsal raphe neurons to psychoactive drugs

The effects of several psychoactive drugs on raphe unit activity in freely moving cats was compared with drug-induced effects in chloral hydrate anesthetized cats. The anesthesia greatly potentiated the depressant effects of LSD, phenoxybenzamine, clonidine, methiothepin, clozapine, and chlorimipramine on raphe units, but partially antagonized the depressant effects of diazepam. These results demonstrate that apparently discrepant reports of the affects of these drugs on raphe neurons in anesthetized rats versus freely moving cats are attributable to the use of anesthesia in rat studies. These data underscore the importance of conducting such drug studies in awake, freely moving animals, for which the results would be far more relevant to the issue of human drug use.     

Binocular Flash Suppression in the Primary Visual Cortex of Anesthetized and Awake Macaques

Primary visual cortex (V1) was implicated as an important candidate for the site of perceptual suppression in numerous psychophysical and imaging studies. However, neurophysiological results in awake monkeys provided evidence for competition mainly between neurons in areas beyond V1. In particular, only a moderate percentage of neurons in V1 were found to modulate in parallel with perception with magnitude substantially smaller than the physical preference of these neurons. It is yet unclear whether these small modulations are rooted from local circuits in V1 or influenced by higher cognitive states. To address this question we recorded multi-unit spiking activity and local field potentials in area V1 of awake and anesthetized macaque monkeys during the paradigm of binocular flash suppression. We found that a small but significant modulation was present in both the anesthetized and awake states during the flash suppression presentation. Furthermore, the relative amplitudes of the perceptual modulations were not significantly different in the two states. We suggest that these early effects of perceptual suppression might occur locally in V1, in prior processing stages or within early visual cortical areas in the absence of top-down feedback from higher cognitive stages that are suppressed under anesthesia.     

Investigation of Cross-Species Translatability of Pharmacological MRI in Awake Nonhuman Primate - A Buprenorphine Challenge Study

Background

Pharmacological MRI (phMRI) is a neuroimaging technique where drug-induced hemodynamic responses can represent a pharmacodynamic biomarker to delineate underlying biological consequences of drug actions. In most preclinical studies, animals are anesthetized during image acquisition to minimize movement. However, it has been demonstrated anesthesia could attenuate basal neuronal activity, which can confound interpretation of drug-induced brain activation patterns. Significant efforts have been made to establish awake imaging in rodents and nonhuman primates (NHP). Whilst various platforms have been developed for imaging awake NHP, comparison and validation of phMRI data as translational biomarkers across species remain to be explored.

Methodology

We have established an awake NHP imaging model that encompasses comprehensive acclimation procedures with a dedicated animal restrainer. Using a cerebral blood volume (CBV)-based phMRI approach, we have determined differential responses of brain activation elicited by the systemic administration of buprenorphine (0.03 mg/kg i.v.), a partial µ-opioid receptor agonist, in the same animal under awake and anesthetized conditions. Additionally, region-of-interest analyses were performed to determine regional drug-induced CBV time-course data and corresponding area-under-curve (AUC) values from brain areas with high density of µ-opioid receptors.

Principal Findings

In awake NHPs, group-level analyses revealed buprenorphine significantly activated brain regions including, thalamus, striatum, frontal and cingulate cortices (paired t-test, versus saline vehicle, p<0.05, n = 4). This observation is strikingly consistent with µ-opioid receptor distribution depicted by [6-O-[¹¹C]methyl]buprenorphine ([¹¹C]BPN) positron emission tomography imaging study in baboons. Furthermore, our findings are consistent with previous buprenorphine phMRI studies in humans and conscious rats which collectively demonstrate the cross-species translatability of awake imaging. Conversely, no significant change in activated brain regions was found in the same animals imaged under the anesthetized condition.

Conclusions

Our data highlight the utility and importance of awake NHP imaging as a translational imaging biomarker for drug research.     

豚鼠背侧海马锥体细胞自发放电特征

实验采用细胞外玻璃微电极采集豚鼠海马神经元放电信号,并将信号转化为峰峰间期(interspike interval,ISI)以研究麻醉和清醒状态海马锥体细胞自发放电线性和非线性特点。实验建立了豚鼠海马锥体细胞与中间神经元电生理鉴别标准;麻醉和清醒状态下豚鼠海马CA1和CA3区锥体细胞自发放电频率、时程、复杂度等无显著区别;麻醉组豚鼠海马锥体细胞ISI序列的复杂度小于清醒组,锥体细胞分型和ISI变异度等表现不同。实验表明,麻醉和清醒状态下豚鼠海马锥体细胞自发放电呈不同线性和非线性特征。传统和非线性研究手段的结合,可能较全面地反映海马锥体细胞自发放电特性。     

Sexual dimorphism and gonadal control of ultrasonic vocalizations in adult pine voles, Microtus pinetorum

We investigated the vocalizations produced by adult pine voles during various social interactions by presenting an experimental animal with either an anesthetized or awake (unanesthetized) conspecific. Ultrasonic vocalizations (USVs) occurred frequently during tests in which an awake male was present, but were rarely detected in tests involving only awake females, or when a female was presented with an anesthetized conspecific. Higher rates of USVs were produced when males were tested with a familiar female than when tested with an unfamiliar male or female. Equivalent rates were produced when males were presented with either anesthetized or awake animals, but female-soiled bedding failed to elicit USVs from males. Sonic vocalizations (SVs) were produced by both sexes and were associated with aggressive behavior, but occurred only in tests between awake, unfamiliar animals. Castration greatly reduced and testosterone therapy restored USVs emitted by males in response to anesthetized conspecifics. Our results suggest that (i) USVs are emitted predominantly by males; (ii) familiarity enhances USV response; (iii) SVs are produced during aggressive interactions; and (iv) androgens regulate the production of USVs by males. Possible roles for pine vole vocalizations are discussed.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Effects of aerosol histamine and carbachol on central and peripheral airflow resistance in sheep

Sixteen anesthetized artificially ventilated open-chest sheep were prepared with retrograde catheters to allow for measurement of dynamic compliance of the lungs (Cdyn), total airflow resistance of the lungs (RL), and central (Rc) and peripheral (Rp) airflow resistance. Twelve sheep received aerosol histamine and 12 sheep received aerosol carbachol. Eight sheep received and responded to both aerosol histamine and aerosol carbachol. Three sheep received both aerosol histamine and aerosol carbachol but failed to respond to both agents. Under base-line conditions, for the 16 sheep, 69% of total RL was located in the peripheral component, Rp, and 31% in the central component, Rc. Aerosol histamine caused only peripheral small airway changes while aerosol carbachol predominantly effected the central large airways. When aerosol histamine responsiveness, defined using Cdyn or Rp, was compared to aerosol carbachol responsiveness using Rc, a correlation was demonstrable (r = 0.84, n = 8, P less than 0.05). It is possible in sheep to cause relatively pure peripheral small airway and relatively pure central large airway changes by using different bronchoconstrictor agents. Aerosol histamine and aerosol carbachol responsiveness correlated with each other in these artificially ventilated anesthetized sheep.     

Detection of mediator-induced airway constriction by barometric plethysmography in mice

The barometric method has recently been employed to detect airway constriction in small animals. This study was designed to evaluate the barometric method to detect mediator-induced central and peripheral airway constriction in BALB/c mice. First, the central airway constrictor carbachol and the peripheral airway constrictor histamine were employed to induce airway constriction, which was detected by both the conventional body plethysmography and the barometric method in anesthetized mice. Second, bronchoconstriction induced by aerosolized carbachol or other mediators was detected with the barometric plethysmography in conscious, unrestrained mice. Carbachol inhalation caused about four-fold increase in pulmonary resistance (RL) and about two-fold increase in enhanced pause (Penh) in anesthetized mice. In contrast, in the same preparation, histamine aerosol induced a decrease in dynamic compliance (Cdyn), with no alteration in RL or Penh. In awake mice, carbachol and methacholine caused increases in Penh, frequency, and tidal volume (VT). On the other hand, histamine, histamine + bradykinin, and prostaglandin-D2 did not alter Penh but decreased VT in conscious mice. These data suggest that there was no sufficient evidence to indicate that Penh could be a good indicator of bronchoconstriction for the whole airways.     

Transoral tracheal intubation of rodents using a fiberoptic laryngoscope

A fiberoptic laryngoscope which allows direct visualization of the deep pharynx and epiglottis has been developed for transoral tracheal intubation of small laboratory mammals. The device has been employed in the intubation and instillation of a variety of substances into the lungs of rats, and with minor modification, has had similar application in mice, hamsters, and guinea pigs. The simplicity and ease of handling of the laryngoscope permits one person to intubate large numbers of enflurane anesthetized animals either on an open counter top or in a glove-box, as may be required for administration of carcinogenic materials. Instillation of 7Be-labeled carbon particles into the lungs of mice, hamsters, rats, and guinea pigs resulted in reasonably consistent interlobal distribution of particles for each test animal species with minimal tracheal deposition. However, actual lung tissue doses of carbon exhibited some species dependence.     

Effects of anesthesia on cystometry and leak point pressure of the female rat

Anesthetics operate by different mechanisms and are often used to perform urodynamics in animals. The objective of this study was to compare the effects of ketamine/xylazine and urethane anesthetics on filling, voiding, and leak point pressure (LPP) in female rats. Nineteen rats underwent awake cystometry 2 days after suprapubic bladder catheter implantation. Bladders were filled with saline (5 ml/hr), while bladder pressure was measured. Half the rats were then anesthetized with urethane i.p. and half were anesthetized with ketamine and xylazine i.p. (K/X). All rats then underwent cystometry and LPP testing under anesthesia. Spontaneous nonvoiding contractions were analyzed and capacity was determined by voiding or leakage. Capacity was significantly higher in awake rats (0.55 +/- 0.06 ml) than with either K/X (0.21 +/- 0.06 ml) or urethane (0.30 +/- 0.05 ml). The pressure just prior to voiding in awake cystometry (15.6 +/- 1.7 cm H2O) was not significantly different from that with either anesthetic (K/X: 10.1 +/- 1.0 cm H2O; urethane: 13.3 +/- 2.0 cm H2O). Spontaneous nonvoiding contractions occurred in 4 rats with urethane and 3 rats with K/X. The volume at which the first contraction occurred was significantly lower with K/X (0.05 +/- 0.02 ml) than urethane (0.19 +/- 0.04 ml). There was no significant difference in the frequency of spontaneous nonvoiding contractions between K/X (4.58 +/- 0.30/min) and urethane (5.16 +/- 2.66/min), nor was there a difference in LPP between anesthetics (K/X: 40.4 +/- 2.4 cm H2O; urethane: 36.2 +/- 3.9 cm H2O). The results suggest that urethane is preferable to K/X for anesthetized cystometry studies since it more closely simulates normal physiological responses.     

Experimental study of a gentamicin aerosol]

The study of gentamicin aerosol showed its relative innocuousness: it did not inhibit the growth and development of young animals, did not induce pathological changes in the upper respiratory tract, kidneys, liver, heart and spleen on its prolonged use. Pathohistological examination revealed slight irritating effect of the gentamicin aerosol in the lungs after its use in a dose of 8 or 25 mg/kg for 6 weeks. A procedure for investigating the effect of the aerosol on the activity of the trachea ciliated epithelium of warm blooded animals was developed. The gentamicin aerosols prepared from solutions of different concentrations (1 to 50 mg/ml) induced ingibition of the ciliated epithelium function at average from 15 to 35 per cent which was associated with the solution acidity (pH 4.54 to 4.82). Such a decrease in the function of the ciliated epithelium due to the antibiotic aerosol use was a factor prolonging the antibiotic retention time in the respiratory organs. It was found that aqueous solutions of drugs used for inhalation, such as ephedrin, euphelin, dimedrol, N-acetyl-L-cystein and others had no effect on the activity of gentamicin and may be used with it in a form of aerosols.     

Homeostatic regulation of sleep in arrhythmic Siberian hamsters

Sleep is regulated by independent yet interacting circadian and homeostatic processes. The present study used a novel approach to study sleep homeostasis in the absence of circadian influences by exposing Siberian hamsters to a simple phase delay of the photocycle to make them arrhythmic. Because these hamsters lacked any circadian organization, their sleep homeostasis could be studied in the absence of circadian interactions. Control animals retained circadian rhythmicity after the phase shift and re-entrained to the phase-shifted photocycle. These animals displayed robust daily sleep-wake rhythms with consolidated sleep during the light phase beginning about 1 h after light onset. This marked sleep-wake pattern was circadian in that it persisted in constant darkness. The distribution of sleep in the arrhythmic hamsters over 24 h was similar to that in the light phase of rhythmic animals. Therefore, daily sleep amounts were higher in arrhythmic animals compared with rhythmic ones. During 2- and 6-h sleep deprivations (SD), it was more difficult to keep arrhythmic hamsters awake than it was for rhythmic hamsters. Because the arrhythmic animals obtained more non-rapid eye movement sleep (NREMS) during the SD, they showed a diminished compensatory response in NREMS EEG slow-wave activity during recovery sleep. When amounts of sleep during the SD were taken into account, there were no differences in sleep homeostasis between experimental and control hamsters. Thus loss of circadian control did not alter the homeostatic response to SD. This supports the view that circadian and homeostatic influences on sleep regulation are independent processes.