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Alpha-chloralose (AC) is used as a rodenticide as well as an anesthetic agent in laboratory animals. It was previously also used as an avicide. Detection of AC in blood samples or in body tissues collected postmortem is key for the diagnosis of clinical cases and a requirement for surveillance of secondary toxicosis, including potential cases in wild animals. Reports on poisoning of humans and non-laboratory animals confirmed by the detection of AC or its metabolites are available, however poisoning of domestic animals are rarely available. Furthermore, reports on clinical cases in domestic animals rarely report quantifications of AC in blood or body tissues. The present study describes the validation of a quantitative ultra high performance liquid chromatography--tandem mass spectrometry (UHPLC--MS-MS) method that can be used in cases of suspected AC poisoning in cats. The validation study showed the method to be fit for purpose. In serum, the limit of quantification was 100 ng/mL and the limit of detection was 30 ng/mL. The new analytical method was applied on blood samples collected from 20 individual cats with a preliminary clinical diagnosis of acute AC poisoning. AC was confirmed in all 20 feline blood samples, and the concentration range of AC was 538–17,500 ng/mL. The quantitative method developed in this study was found to be a fast and selective method for confirmation of AC poisoning using blood samples from cats.

Spontaneous neural activity has been widely adopted to construct functional connectivity (FC) amongst distant brain regions. Although informative, the functional role and signaling mechanism of the resting state FC are not intuitive as those in stimulus/task-evoked activity. In order to bridge the gap, we investigated anesthetic modulation of both resting-state and sensory-evoked activities. We used two well-studied GABAergic anesthetics of varying dose (isoflurane: 0.5–2.0% and α-chloralose: 30 and 60 mg/kg∙h) and recorded changes in electrophysiology using a pair of laminar electrode arrays that encompass the entire depth of the bilateral somatosensory cortices (S1fl) in rats. Specifically, the study focused to describe how varying anesthesia conditions affect the resting state activities and resultant FC between bilateral hemispheres in comparison to those obtained by evoked responses. As results, isoflurane decreased the amplitude of evoked responses in a dose-dependent manner mostly due to the habituation of repetitive responses. However, α-chloralose rather intensified the amplitude without exhibiting habituation. No such diverging trend was observed for the spontaneous activity, in which both anesthetics increased the signal power. For α-chloralose, overall FC was similar to that obtained with the lowest dose of isoflurane at 0.5% while higher doses of isoflurane displayed increased FC. Interestingly, only α-chloralose elicited relatively much greater increases in the ipsi-stimulus evoked response (i.e., in S1fl ipsilateral to the stimulated forelimb) than those associated with the contra-stimulus response, suggesting enhanced neuronal excitability. Taken together, the findings demonstrate modulation of the FC profiles by anesthesia is highly non-linear, possibly with a distinct underlying mechanism that affects either resting state or evoked activities differently. Further, the current study warrants thorough investigation of the basal neuronal states prior to the interpretation of resting state FC and evoked activities for accurate understanding of neural signal processing and circuitry.

Background Alpha-chloralose (AC) is a compound known to be toxic to various animal species and humans. In 2018 and 2019 an increase in suspected cases of AC poisoning in cats related to the use of AC as a rodenticide was reported to national veterinary and chemical authorities in Finland, Norway and Sweden by veterinarians working in clinical practices in respective country. The aims of this study were to prospectively investigate AC poisoning in cats, including possible secondary poisoning by consuming poisoned mice, and to study metabolism and excretion of AC in cats through analysis of feline urine. Methods Data on signalment, history and clinical findings were prospectively collected in Finland, Norway and Sweden from July 2020 until March of 2021 using a questionnaire which the attending veterinarian completed and submitted together with a serum sample collected from suspected feline cases of AC-poisoning. The diagnosis was confirmed by quantification of AC in serum samples. Content of AC was studied in four feline urine samples, including screening for AC metabolites by UHPLC-HRMS/MS. Bait intake and amount of AC consumed by mice was observed in wild mice during an extermination of a rodent infestation. Results In total, 59 of 70 collected questionnaires and accompanying serum samples were included, with 127 to 70 100 ng/mL AC detected in the serum. Several tentative AC-metabolites were detected in the analysed feline urine samples, including dechlorinated and oxidated AC, several sulfate conjugates, and one glucuronic acid conjugate of AC. The calculated amount of AC ingested by each mouse was 33 to 106 mg with a mean of 61 mg. Conclusions Clinical recognition of symptoms of AC poisoning in otherwise healthy cats roaming free outdoors and known to be rodent hunters strongly correlated with confirmation of the diagnosis through toxicological analyses of serum samples. The collected feline exposure data regarding AC show together with the calculation of the intake of bait and subsequent AC concentrations in mice that secondary poisoning from ingestion of mice is possible. The results of the screening for AC metabolites in feline urine confirm that cats excrete AC both unchanged and metabolized through dechlorination, oxidation, glucuronidation and sulfatation pathways.

Objectives Most individuals with spinal cord injury have neurogenic bowel dysfunction, which includes slowed colonic motility and has a significant impact on their health and quality of life. Bowel management typically includes mechanical rectal distension to evoke a recto-colic reflex and promote bowel emptying. Electrical stimulation could replace this mechanical distension. The purpose of this study was to determine the feasibility of evoking colonic activity using electrical stimulation. Materials and methods Acute experiments were conducted in eight neural-intact cats under chloralose anesthesia. Patterned electrical stimulation was administered via electrodes placed on the surface of the distal colon or proximal colon. Distal and proximal colon pressures were measured using saline-filled balloon catheters. Outcome measures included peak colonic pressure; time to onset of response; and time to peak pressure compared to baseline without stimulation. Results Stimulation elicited colonic activity in all animals. Peak colon pressures were 15 ± 28 cmH2 O and were proportional to stimulation amplitudes. Time to onset and time to peak pressure were 13 ± 19 s and 37 ± 49 s, respectively, and were not significantly affected by stimulus parameters. Proximal colon stimulation only resulted in pressure responses from the proximal colon, but distal colon stimulation resulted in both proximal and distal responses in 40% of trials. Adding isoflurane anesthesia removed this proximal response to distal stimulation. Conclusions Distal colon stimulation evoked colonic activity. The dependence of this response on stimulation location and anesthesia suggests that responses were reflex mediated. Colonic stimulation may have the potential to improve colonic motility for individuals with neurogenic bowel dysfunction.

By reducing the cerebral blood flow and thereby increasing the resting deoxyhaemoglobin concentration, many human studies have shown that caffeine has a beneficial effect on enhancing the magnitude of blood-oxygenation-level-dependent (BOLD) responses. However, the effect of caffeine on BOLD responses in animals under anaesthesia has not been demonstrated. In this study, we aimed to determine the effect of systemic caffeine administration on BOLD responses in rats under alpha-chloralose. By applying electric whisker pad stimulation to male Sprague-Dawley rats, we performed fMRI measurements before and after the caffeine injection (40 mg/kg, n = 7) or an equivalent volume of saline (n = 6) at 7T. To understand the potential perturbation of animal physiology during stimulation, arterial blood pressure was measured in a separate group of animals (n = 3) outside the scanner. Caffeine significantly decreased baseline BOLD signals (p = .05) due to the increased deoxyhaemoglobin level. Both BOLD responses and t-values in the primary somatosensory cortex were significantly increased (both p .05). The blood pressure changed insignificantly (p .05). No significant differences in BOLD responses and t-values were observed in the control condition of saline injection (both p .05). These findings suggested that, although the cerebral activity was lower under alpha-chloralose anaesthesia, the higher level of deoxygemoglobin at the baseline under the caffeinated condition can benefit the magnitude of BOLD responses in rats. These findings suggest that animal models might serve as potential platforms for further caffeine-related fMRI research studies.

The aim of this study was to assess the effects of radiofrequency electromagnetic field (RF EMF) on heart rate variability (HRV) in rabbits with intensity slightly exceeding the limits for occupations. Totally 21 New Zealand white rabbits divided into two groups were used in this double-blind study. The first group of animals without general anesthesia was subjected to HRV examination under exposure to a device generated RF EMF source (frequency 1788 MHz, intensity 160 V/m, lasting 150 min.). The second group (premedications + α chloralose mg/kg) underwent the same protocol under the exposure to the real RF EMF signal from the base stations of mobile providers (frequency range 1805 – 1870 MHz - corresponding to the downlink signal of Slovak mobile providers, 160 V/m, 150 min., respectively). Individual 5 min records were used to analyze the HRV parameters: heart rate and root Mean Square of the Successive Differences (rMSSD) for time domain analysis and spectral powers in the low (LF-VFS) and high frequency (HF-VFS) bands for frequency domain analysis. Our study revealed the increased in HRV parameters (HF-HRV, rMSSD) associated with lower heart rate indicating increased cardiac vagal control under the exposure to RF EMF in experimental methods.

Objectives The aim of this study was to describe the clinical picture in cats with alpha-chloralose (AC) intoxication and to confirm AC in serum from suspected cases of AC poisoning. Methods Suspected cases of AC poisoning were identified in patient records from a small animal university hospital from January 2014 to February 2020. Clinical signs of intoxication described in respective records were compiled, the cats were graded into four intoxication severity scores and hospitalisation time and mortality were recorded. Surplus serum from select cases in late 2019 and early 2020 was analysed to detect AC with a quantitative ultra-high performance liquid chromatography tandem mass spectrometry analysis, and the AC concentration was compared with the respective cat’s intoxication severity score. Results Serum from 25 cats was available for analysis and AC poisoning was confirmed in all. Additionally, 78 cats with a clinical suspicion of AC intoxication were identified in the patient records, most of which presented from September to April. The most common signs of intoxication were ataxia, tremors, cranial nerve deficits and hyperaesthesia. The prevalence of clinical signs and intoxication severity differed from what has previously been reported, with our population presenting with less severe signs and no deaths due to intoxication. The majority had a hospitalisation time Conclusions and relevance This study describes the clinical signs and prognosis in feline AC intoxication. There were no mortalities in confirmed cases, indicating that AC-poisoned cats have an excellent prognosis when treated in a timely manner. Recognition of AC intoxication as a differential diagnosis for acute onset of the described neurological signs in areas where AC exposure is possible may influence clinical decision-making and help avoid excessive diagnostic procedures. A severe clinical picture upon presentation could be misinterpreted as a grave prognosis and awareness about AC poisoning may avoid unnecessary euthanasia.

Periaqueductal gray (PAG) is a midbrain region that projects to areas controlling behavioral and autonomic outputs and is involved in the behavioral and physiological components of defense reactions. Since Raphe Pallidus (RPa) is a medial medullary region comprising sympathetic premotor neurons governing heart function, it is worth considering the PAG-RPa path. We assessed: i) whether PAG projects to RPa; ii) the amplitude of cardiac responses evoked from PAG; iii) whether cardiovascular responses evoked from PAG rely on RPa. Experiments conducted in Wistar rats (±300 g) were approved by Ethics Committee CEUA-UFG (092/18). Firstly, (n = 3), monosynaptic retrograde tracer Retrobeads was injected into RPa; PAG slices were analyzed. Other two groups (n = 6 each) were anesthetized with urethane (1.4 g/kg) and chloralose (120 mg/kg) and underwent craniotomy, tracheostomy, catheterization of femoral artery and vein and of cardiac left ventricle. In one group, we injected the GABAA receptor antagonist, bicuculline methiodide (BMI – 40 pmol/100 nL) into lateral/dorsolateral (l/dl) PAG. Another group was injected (100nL) with the GABAA receptor agonist muscimol (20 mM) into RPa, 20 min before BMI into PAG. The results were: i) retrogradely labelled neurons were found in PAG; ii) PAG activation by BMI caused positive chronotropism and inotropism, which were accompanied by afterload increases; iii) RPa inhibition with Muscimol reduced heart rate, arterial and ventricular pressures; iv) the subsequent PAG activation still increased arterial pressure, cardiac chronotropy and inotropy, but these responses were significantly attenuated. In conclusion, PAG activation increases cardiac chronotropy and inotropy, and these responses seem to rely on a direct pathway reaching ventromedial medullary RPa neurons.

Purpose Sensitivity and specificity of blood oxygenation level–dependent (BOLD) functional MRI (fMRI) is sensitive to magnetic field strength and acquisition methods. We have investigated gradient‐echo (GE)‐ and spin‐echo (SE)‐BOLD fMRI at ultrahigh fields of 9.4 and 15.2 Tesla. Methods BOLD fMRI experiments responding to forepaw stimulation were performed with 3 echo times (TE) at each echo type and B 0 in α‐chloralose–anesthetized rats. The contralateral forelimb somatosensory region was selected for quantitative analyses. Results At 9.4 T and 15.2 T, average baseline T 2 * (n = 9) was 26.6 and 17.1 msec, whereas baseline T 2 value (n = 9) was 35.7 and 24.5 msec, respectively. Averaged stimulation‐induced ΔR 2 * was –1.72 s–1 at 9.4 T and –3.09 s–1 at 15.2 T, whereas ΔR 2 was –1.19 s–1 at 9.4 T and –1.97 s–1 at 15.2 T. At the optimal TE of tissue T 2 * or T 2, BOLD percent changes were slightly higher at 15.2 T than at 9.4 T (GE: 7.4% versus 6.4% and SE: 5.7% versus 5.4%). The ΔR 2 * and ΔR 2 ratio of 15.2 T to 9.4 T was 1.8 and 1.66, respectively. The ratio of the macrovessel‐containing superficial to microvessel‐dominant parenchymal BOLD signal was 1.73 to 1.76 for GE‐BOLD versus 1.13 to 1.19 for SE‐BOLD, indicating that the SE‐BOLD contrast is less sensitive to macrovessels than GE‐BOLD. Conclusion SE‐BOLD fMRI improves spatial specificity to microvessels compared to GE‐BOLD at both fields. BOLD sensitivity is similar at the both fields and can be improved at ultrahigh fields only for thermal‐noise–dominant ultrahigh‐resolution fMRI.

Acupuncture therapy has a positive role in the prevention and treatment of some related diseases by regulating autonomic nervous function. But it has been found in experimental researches that the modulatory effects of acupuncture on autonomic nervous system are not always consistent with each other. Because in the animal studies on the mechanism of acupuncture intervention, the anesthetic agent has to be used and definitely affects the activities of the autonomic nervous system while playing its pharmacological effects. Hence, it is very significant to explore the rational application of anesthetic agents and minimize their unfavorable impacts on the research outcomes. In the present paper, we make a retrospective analysis on (1) the effect of acupuncture intervention on activities of the autonomic nerve in animal models of myocardial ischemia, gastrointestinal mobility, and urinary system; (2) the effect of anesthetics as isoflurane, urethane, pentobarbital sodium, ketamine, α-chloralose, propofolum, etc. on activities of the autonomic nerve system. In terms of different anesthetic modes and various depths of anesthesia by using isoflurane inhalation, mixed solution of urethane and chloralose, etc., some approaches for assessing the state of anesthesia in accordance with the pupillary reflex, righting reflex, footboard reflex, swallowing reflex, heart rate, blood pressure, body temperature, respiration frequency, ventilation volume, oxygen saturation, partial pressure of carbon dioxide, neuromuscular blo-ckade, etc. in combination with the indexes directly and indirectly reflecting functions of the sympathetic and vagus nerves are proposed to analyze the impact of different anesthetic states on the therapeutic effect of acupuncture in various models of dysfunction of the autonomic nerve system. Under the circumstances, it is possible to provide a reference for rational use of anesthetic agents and dosages in the acupuncture research of autonomic nerve regulation.

Resting-state functional magnetic resonance imaging (rsfMRI) is a translational imaging method with great potential in several neurobiologic applications. Most preclinical rsfMRI studies are performed in anesthetized animals, but the confounding effects of anesthesia on the measured functional connectivity (FC) are poorly understood. Therefore, we measured FC under six commonly used anesthesia protocols and compared the findings with data obtained from awake rats. The results demonstrated that each anesthesia protocol uniquely modulated FC. Connectivity patterns obtained under propofol and urethane anesthesia were most similar to that observed in awake rats. FC patterns in the α-chloralose and isoflurane-medetomidine combination groups had moderate to good correspondence with that in the awake group. The FC patterns in the isoflurane and medetomidine groups differed most from that in the awake rats. These results can be directly exploited in rsfMRI study designs to improve the data quality, comparability, and interpretation, published version, peerReviewed

Systemic or central administration of 2-deoxy- d -glucose (2DG), a competitive inhibitor of glucose utilization, induces hypothermia in awake animals and humans. This response is mediated by the central nervous system, though the neural mechanism involved is largely unknown. In this study, I examined possible involvement of the forebrain, which contains the hypothalamic thermoregulatory center, and the medullary rostral raphe/parapyramidal regions (rRPa/PPy), which mediate hypoxia-induced heat-loss responses, in 2DG-induced hypothermia in urethane–chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. The intravenous injection of 2DG (250 mg kg −1 ) elicited an increase in tail skin temperature and decreases in body core temperature and the respiratory exchange ratio, though it did not induce any significant change in the metabolic rate. These results indicate that the hypothermic response was caused by an increase in heat loss, but not by a decrease in heat production and that it was accompanied by a decrease in carbohydrate utilization and/or an increase in lipid utilization as energy substrates. Complete surgical transection of the brainstem between the hypothalamus and the midbrain had no effect on the 2DG-induced hypothermic responses, suggesting that the hindbrain, but not the forebrain, was sufficient for the responses. However, pretreatment of the rRPa/PPy with the GABA A receptor blocker bicuculline methiodide, but not with vehicle saline, greatly attenuated the 2DG-induced responses, suggesting that the 2DG-induced hypothermia was mediated, at least in part, by GABAergic neurons in the hindbrain and activation of GABA A receptors on cutaneous sympathetic premotor neurons in the rRPa/PPy.

It has previously been proved that sodium nitrite, infused prior to coronary artery occlusion or before reperfusion, results in marked antiarrhythmic effect in anaesthetized dogs. We have now examined whether this protection involves the modulation of gap junction (GJ) function by nitric oxide (NO), derived from nitrite administration under ischaemic conditions. Two groups of chloralose and urethane anaesthetized dogs, each containing 13 animals, were subjected to a 25 min period occlusion of the left anterior descending (LAD) coronary artery, followed by reperfusion. One group was infused with sodium nitrite (0.2 μmol/kg/min, i.v.), the other group with saline 10 min prior to reperfusion. The severities of arrhythmias and of ischaemia (epicardial ST-segment, total activation time), parallel with changes in myocardial tissue impedance, a measure of electrical coupling of gap junctions, were assessed during the experiments. Compared to the controls, nitrite infusion administered prior to reperfusion significantly attenuated the severity of ischaemia, the ischaemia-induced impedance changes and, consequently, the severity of arrhythmias, occurring during the 1B phase of the occlusion, and increase survival following reperfusion (0% vs. 85%). It is concluded that the marked antiarrhythmic effect of sodium nitrite is partly due, to the preservation of the electrical coupling of GJs by NO.

Objective To investigate the use of a microelectrode array with a high spatial density of penetrating intrafascicular electrodes for selective recording of pudendal nerve activity evoked by a variety of genitourinary stimuli. Materials and Methods Felines were anesthetized with alpha-chloralose and high-density Utah slanted electrode arrays (48 microelectrodes; 200-μm spacing) were implanted into the pudendal nerve for acute experimentation. Neural activity was recorded during bladder filling, spontaneous reflexive distention-evoked bladder contractions, and tactile somatosensory stimulation. Results The intrafascicularly implanted pudendal nerve electrodes were able to selectively record neural activity that corresponded to various genitourinary stimuli. Across all seven experimental animals, a total of 10 microelectrodes recorded neural units that were selectively driven by bladder filling or distention-evoked bladder contractions. Twenty-two electrodes were selectively driven by tactile stimulation. Conclusion Microelectrode arrays implanted intrafascicularly into the pudendal nerve can be used to selectively record the neural responses that reflect bladder status and urogenital tactile stimulation. This work sets the stage for developing future implantable closed-loop neuroprosthetic devices for restoration of bladder function.

The frequent accompaniment of hypertension by orthostatic circulatory disorders prompted us to investigate the effect of repeated and sustained head-up and head-down tilt positions on cardiovascular responses in spontaneously hypertensive rats vs. Wistar rats using radiotelemetric implants. Repeated orthostasis caused a transient elevation in blood pressure (7.3±1.7 mmHg) and heart rate (39.7±10.5 BPM), while repeated antiorthostasis led only to reversible tachycardia (85.6±11.7–54.3±16.8 BPM) in spontaneously hypertensive rats. In contrast to the Wistar rats, sustained tilt failed to affect the blood pressure or heart rate in spontaneously hypertensive rats because the environmental stress of being placed in horizontal tilt cages prior to the sustained tilt test induced marked changes in cardiovascular parameters. Non-specific stress responses were eliminated both by the anxiolytic diazepam and a sub-anesthetic dose of chloralose. Unlike diazepam, chloralose amplified the orthostatic pressor responses in the Wistar rats. In contrast to diazepam preventing the pressor response and associated tachycardia in spontaneously hypertensive rats, chloralose elicited this effect during both sustained orthostasis (36.0±7.3 mmHg, 63.7±21.8 BPM) and antiorthostasis (42.9±10.9 mmHg, 82.8±25.4 BPM), with a reduced baroreflex sensitivity. However, during sustained orthostasis, removal of the vestibular input led to a depressor response with bradycardia (12.5±3.2 mmHg, 59.3±17.3 BPM), whereas antiorthostasis only reduced blood pressure (20.5±7.1 mmHg) in the spontaneously hypertensive rats. We conclude that repeated tilts induce a transient pressor response and/or tachycardia in spontaneously hypertensive rats. Cardiovascular parameters are suppressed by diazepam, whereas chloralose evokes both blood pressure and heart rate responses during sustained tilts, which are primarily elicited by baroreflex suppression in hypertension. Vestibular inputs support cardiovascular tolerance to sustained postural changes in a rat model of human ‘essential’ hypertension.

Background Mechanosensitive afferents innervating the bladder increase their firing rate as the bladder fills and pressure rises. However, the relationship between afferent firing rates and intravesical pressure is not a simple linear one. Firing rate responses to pressure can differ depending on prior activity, demonstrating hysteresis in the system. Though this hysteresis has been commented on in published literature, it has not been quantified. Results Sixty-six bladder afferents recorded from sacral dorsal root ganglia in five alpha-chloralose anesthetized felines were identified based on their characteristic responses to pressure (correlation coefficient ≥ 0.2) during saline infusion (2 ml/min). For saline infusion trials, we calculated a maximum hysteresis ratio between the firing rate difference at each pressure and the overall firing rate range (or Hmax) of 0.86 ± 0.09 (mean ± standard deviation) and mean hysteresis ratio (or Hmean) of 0.52 ± 0.13 (n = 46 afferents). For isovolumetric trials in two experiments (n = 33 afferents) Hmax was 0.72 ± 0.14 and Hmean was 0.40 ± 0.14. Conclusions A comprehensive state model that integrates these hysteresis parameters to determine the bladder state may improve upon existing neuroprostheses for bladder control.

Functional magnetic resonance imaging of mice requires that the physiology of the mouse (body temperature, respiration and heart rates, blood pH level) be maintained in order to prevent changes affecting the outcomes of functional scanning, namely blood oxygenation level dependent (BOLD) measures and cerebral blood flow (CBF). The anesthetic used to sedate mice for scanning can have major effects on physiology. While alpha chloralose has been commonly used for functional imaging of rats, its effects on physiology are not well characterized in the literature for any species. In this study, we anesthetized or sedated mice with isoflurane or alpha chloralose for up to two hours, and monitored physiological parameters and arterial blood gasses. We found that, when normal body temperature is maintained, breathing rates for both drugs decrease over the course of two hours. In addition, alpha chloralose causes a substantial drop in heart rate and blood pH with severe hypercapnia (elevated blood CO2) that is not seen in isoflurane-treated animals. We suggest that alpha chloralose does not maintain normal mouse physiology adequately for functional brain imaging outcome measures.

Despite positive metabolic effects, genetic deletion of protein phosphatase 1b (PTP1b) results in sympathetically mediated elevations in arterial pressure (AP) in mice. Because several PTP1b-regulated peptides also impair the baroreflex sensitivity (BRS) for control of heart rate (HR), we hypothesized that PTP1b in the solitary tract nucleus (NTS) participates in the maintenance of resting baroreflex function. To test this hypothesis, we performed acute bilateral microinjection of an allosteric PTP1b inhibitor (100 nM/120 nL) in the NTS of urethane/chloralose anesthetized Sprague-Dawley rats and assessed the BRS, responses to cardiac vagal chemosensitive fiber activation, and resting AP and HR before and after the injection. PTP1b inhibition impaired the BRS for bradycardia (n = 6; 0.93 ± 0.14 baseline vs. 0.48 ± 0.04 at 10 minutes vs. 0.49 ± 0.04 millisecond/mm Hg at 60 minutes; P0.01), with no significant effect on the BRS for tachycardia (0.30 ± 0.16 baseline vs. 0.24 ± 0.08 at 10 minutes vs. 0.24 ± 0.12 millisecond/mm Hg at 60 minutes). The reduced BRS for bradycardia was associated with a significant decrease in alpha-adrenergic responsiveness to phenylephrine at 60 minutes after PTP1b inhibition. Injection of the PTP1b inhibitor in the NTS elicited transient decreases in AP and HR in these animals. However, there was no effect of the inhibitor on depressor or bradycardic responses elicited by activation of cardiac vagal chemosensitive fibers, which converge with baroreceptor afferents in the NTS. These results suggest that PTP1b within the NTS may be a novel molecular mechanism for preservation of resting baroreflex function and provides further evidence for deleterious cardiovascular effects associated with PTP1b inhibition.

Nitric oxide (NO), a potent vasodilator and nontraditional neurotransmitter, is an important mediator of the changes in cerebral blood flow (CBF) associated with increased neuronal activity (neurovascular coupling). In the present work, we investigated the role of NO and of its newly recognized precursor, nitrite, in neurovascular coupling using a well-established rat model of somatosensory stimulation. Biological synthesis of NO of neuronal origin was inhibited pharmacologically. Following the initial uncoupling of neuronal and hemodynamic responses to somatosensory stimulation, the NO donor sodium nitroprusside, added within the range of physiological concentrations, significantly increased, but did not fully restore the functional CBF response. In contrast, nitrite at its physiological concentration fully recovered neurovascular coupling to its original magnitude. The magnitude of the effect is, however, dose-dependent. Sub-physiological concentrations of nitrite were not enough to entirely restore neurovascular coupling and supra-physiological concentrations acted more as a local vasodilator that changed resting CBF and interfered with the functional CBF response. These results suggest that nitrite can be efficiently converted into NO and utilized to support normal cerebrovascular physiology.

The effects of α-chloralose on presynaptic GABA(A) receptors were investigated with respect to spontaneous and evoked GABAergic transmission (sIPSCs and eIPSCs) in rat hippocampal CA1 pyramidal neurons. sIPSCs were recorded in mechanically dissociated CA1 neurons with intact GABAergic terminals, namely the "synaptic bouton preparation." eIPSCs were elicited by focal electrical stimuli of a single GABAergic bouton on an isolated CA1 neuron using the whole-cell patch recording configurations under voltage-clamp condition. We found that α-chloralose potentiated the exogenous GABA-induced Cl(-) response in a concentration dependent manner, and the drug itself induced Cl(-) response at high concentrations (>100 μM). α-Chloralose at low concentrations (3-10 μM) increased sIPSC frequency without affecting the current amplitude and kinetics, but prolonged the slow current decay time constant (τ(s)) at concentrations greater than 30 μM without changing either current amplitude or frequency. α-Chloralose at 10 μM enhanced amplitude of eIPSCs and decreased the failure rate (Rf), but at 30 μM decreased the amplitude and increased the Rf. Pretreatment with bumetanide, a blocker of NKCC-1, completely prevented the 30 μM α-chloralose-induced inhibition on eIPSC amplitude and Rf. These results suggest that α-chloralose activates GABA(A) receptors on GABAergic presynaptic nerve terminals and depolarizes the terminals, mediating presynaptic inhibition or autoregulation, in a concentration-dependent manner. In addition, α-chloralose at high concentrations activates not only extrasynaptic GABA(A) receptors on the postsynaptic soma membrane but also synaptic GABA(A) receptors resulting in prolongation of current decay phase. Thus α-chloralose induces complex and differential modulation of sIPSCs and eIPSCs in a concentration dependent manner.

Our previous studies showed that electrical stimulation of the nuclei ambiguous (NA) or dorsomotor nuclei of the vagus (DMV) complex in the brain stem of spontaneously breathing pond turtles (Cyclemys fiavomarginata), anesthetized with chloralose (4 mg/100 g) and urethane (40 mg/100 g), produced a marked slowing or even cessation of the heart rate, and resulted in an immediate fall of blood pressure. Results of the present study further demonstrated that the cardioinhibitory responses could also be elicited by microinjection of monosodium glutamate (0.2-20 nl, 50 mM) into the NA/DMV complex in turtles. A two-barrel glass micropipette held in a manipulator was connected to a pneumatic pressure pump for microinjection. The glutamate-induced cardioinhibitory responses could be significantly reduced in a dose-dependent manner by pretreatment with AP-5 (a NMDA receptor antagonist, at 1-8 nmole) or CNQX (a non-NMDA receptor antagonist; at 0.1-0.8 nmole) 20 min before glutamate administration. Histochemical verification by injecting horseradish peroxidase into the cervical vagus nerves revealed that retrogradely labeled glutamatergic neurons in the NA/DMV complex were observed. These results suggest that glutamatergic receptors in the caudal medulla may mediate vagal cardioinhibitory responses in the turtle.

Autoregulation, an important feature of the cerebral circulation, is affected in many diseases. Since genetically modified mice are a fundamental tool in biomedical research, including neuro(bio)logy also in this specie measurements of cerebral autoregulation (CA) are mandatory. However, this requires anesthesia that unfortunately significantly impacts cerebral perfusion and consequently might distort CA measurements directly or by altering arterial pCO(2). The latter can be avoided by artificial ventilation but requires several control measurements of blood gases, each consuming at least 100 μl of blood or 5% of a mouse's blood volume. To avoid such diagnostic hemorrhage, we systematically analyzed the effect of different common anesthetic protocols used for rodents in spontaneously breathing mice on CA measured with Laser speckle perfusion imaging. Halothane, Isoflurane and Pentobarbital abrogated CA and Ketamin/Xylazine as well as Chloralose had a moderate reproducibility. In contrast, the rather rarely used anesthetic Ethomidate applied in low doses combined with local anesthetics had the best reproducibility. Although with this anesthesia the lower CA limit was lower than with Ketamin/Xylazine and Chloralose as reported in the handful of papers so far dealing with CA in mice, we suggest Ethomidate as the anesthetic of choice for CA measurements in spontaneously breathing mice.

Most studies of the effect of cocaine on brain activity in laboratory animals are preformed under anesthesia, which could potentially affect the physiological responses to cocaine. Here we assessed the effects of two commonly used anesthetics [alpha-chloralose (alpha-CHLOR) and isofluorane (ISO)] on the effects of acute cocaine (1 mg/kg i.v.) on cerebral blood flow (CBF), cerebral blood volume (CBV), and tissue hemoglobin oxygenation (S(t)O(2)) using optical techniques and cocaine's pharmacokinetics (PK) and binding in the rat brain using (PET) and [(11)C]cocaine. We showed that acute cocaine at a dose abused by cocaine abusers decreased CBF, CBV and S(t)O(2) in rats anesthetized with ISO, whereas it increased these parameters in rats anesthetized with alpha-CHLOR. Importantly, in ISO-anesthetized animals cocaine-induced changes in CBF and S(t)O(2) were coupled, whereas for alpha-CHLOR these measures were uncoupled. Moreover, the clearance of [(11)C]cocaine from the brain was faster for ISO (peak half-clearance 15.8 +/- 2.8 min) than for alpha-CHLOR (27.5 +/- 0.6 min), and the ratio of specific to non-specific binding of [(11)C]cocaine in the brain was higher for ISO- (3.37 +/- 0.32) than for alpha-CHLOR-anesthetized rats (2.24 +/- 0.4). For both anesthetics, cocaine-induced changes in CBF followed the fast uptake of [(11)C]cocaine in the brain (peaking at approximately 2.5-4 min), but only for ISO did the duration of the CBV and S(t)O(2) changes correspond to the rate of [(11)C]cocaine's clearance from the brain. These results demonstrate that anesthetics influence cocaine's hemodynamic and metabolic changes in the brain, and its binding and PK, which highlights the need to better understand the interactions between anesthetics and pharmacological challenges in brain functional imaging studies.

The ability of intravenous cocaine to induce Fos protein expression in anesthetized rats was tested. Two anesthetic regimens commonly used for in vivo FMRI of animals, i.v. alpha-chloralose and gaseous isoflurane, were studied in separate cohorts. The first experiment included three groups that received the following treatments: saline i.v. and no anesthetic; 2 mg/kg cocaine i.v. and no anesthetic; and 2mg/kg cocaine i.v. under 36 mg/kg/h alpha-chloralose anesthesia. The second experiment had a factorial design of four groups that were either nonanesthetized or isoflurane-treated and were either given saline or cocaine (2 mg/kg, i.v.). Anesthetized rats were maintained for 2 h under 2.5-3.5% isoflurane anesthesia, while nonanesthetized rats were kept in an alternative environment for the same time period. Rats were given 2 mg/kg cocaine or saline i.v., 30 min into the test session. Rats were perfused and their brains were processed for Fos immunohistochemistry 90 min after the i.v. treatment. In both experiments, the frontal cortex and striatum of the cocaine-treated nonanesthetized rats expressed Fos in greater amounts than the saline-treated nonanesthetized rats, as expected. The alpha-chloralose treatment prevented cocaine-induced Fos expression across all eight subregions of the striatum and frontal cortex that were examined. In contrast, isoflurane only partially attenuated Fos expression in the orbital and Cg2 subregions of frontal cortex. These results suggest a strong advantage for using isoflurane, as opposed to alpha-chloralose, when studying anesthetized rats for in vivo effects of psychostimulants.

Focal ventricular tachycardia (VT) in acute myocardial ischemia is closely related to triggered activity (TA), which may be blocked by scavenging reactive oxygen species (ROS).This study analyzed effects of acutely administered ROS scavenger-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) on VT in vivo and TA in vitro.Forty-three alpha chloralose anesthetized dogs with coronary artery occlusion were studied. Three-dimensional activation mapping helped to locate the origin of focal or reentrant VT. TEMPO (30 mg/kg intravenously) or vehicle was given. Endocardium excised from the site of origin of VT was studied using standard microelectrode techniques and measures of ROS.Reentry and focal VT induction were both highly reproducible. TEMPO blocked focal VT in 6 of 11 dogs (P.05), but 9 of 9 dogs with reentrant VT continued to have VT re-induced after TEMPO. TEMPO did not alter effective refractory period (168 +/- 3 to 171 +/- 3 ms), mean blood pressure (88 +/- 3 to 81 +/- 3 mm Hg), and size of ischemia (42% +/- 3% vs 40% +/- 4%). In vitro, TEMPO (10(-3) M, n = 14) produced no change in action potentials. Nevertheless, TA was reversibly attenuated from 5.3 +/- 1.1 to 0.4 +/- 0.4 complexes with TEMPO (n = 15, P.05). Lucigenin-enhanced chemiluminescence and dihydroethidium staining showed increased ROS in ischemic endocardium; TEMPO dramatically reduced ROS in ischemic sites.TEMPO, a scavenger of ROS, prevented triggered activity associated with focal VT during myocardial ischemia in areas of increased ROS. Antioxidant therapy may play an important role in blockade of focal VT under the conditions of myocardial ischemia.

The effect of thioridazine on sinusal automaticity, myocardial excitability and auriculoventricular conduction have been studied in dogs by measuring the spontaneous heart rate, the effective refractory period of the atrial contractile tissue, the time of auriculonodal and infrahisian conduction. Chloralose anaesthetized dogs were administered 10 mg/kg thioridazine hydrochloride via intravenous route. While sinusal automaticity was not altered, conduction times were prolonged in the suprahisian and mainly the infrahisian part and the atrial refractory period increased. These effects are related to quinidine-like properties and consistent with cardiotoxic effects previously reported in clinical practice.

Direct measurements of deep-brain and body-core temperature were performed on rats to determine the influence of cerebral blood flow (CBF) on brain temperature regulation under static and dynamic conditions. Static changes of CBF were achieved using different anesthetics (chloral hydrate, CH; alpha-chloralose, alphaCS; and isoflurane, IF) with alphaCS causing larger decreases in CBF than CH and IF; dynamic changes were achieved by inducing transient hypercapnia (5% CO(2) in 40% O(2) and 55% N(2)). Initial deep-brain/body-core temperature differentials were anesthetic-type dependent with the largest differential observed with rats under alphaCS anesthesia (ca. 2 degrees C). Hypercapnia induction raised rat brain temperature under all three anesthesia regimes, but by different anesthetic-dependent amounts correlated with the initial differentials--alphaCS anesthesia resulted in the largest brain temperature increase (0.32 +/- 0.08 degrees C), while CH and IF anesthesia lead to smaller increases (0.12 +/- 0.03 and 0.16 +/- 0.05 degrees C, respectively). The characteristic temperature transition time for the hypercapnia-induced temperature increase was 2-3 min under CH and IF anesthesia and approximately 4 min under alphaCS anesthesia. We conclude that both, the deep-brain/body-core temperature differential and the characteristic temperature transition time correlate with CBF: a lower CBF promotes higher deep-brain/body-core temperature differentials and, upon hypercapnia challenge, longer characteristic transition times to increased temperatures.

Summary. 1. Experiments have been performed with usnic acid on eats anaesthetized with nembutal or, in a few cases, with chloralose. 2. 10 mg sodium usnate/kg body weight caused a prompt and heavy rise in oxygen consumption accompanied by a pronounced increase in rate and amplitude of respiration and by a gradual rise in body temperature, lasting for about 5 hours and then dowly falling. 3. Larger doses are as a rule lethal. 4. Lethal doses cause an intense rigor mortis. This effect is opposite to the effect of vulpinic acid but similar to the Well known action of the dinitrophenols. 5. In normal anaesthetized cats there is a steep rise in arterial blood sugar to a level of 300–400 mg% lasting for about the same time as the increased respiratory volume. In fasted cats the rise is much slower and somewhat smaller. 6. It is concluded that the major part of the rise in arterial blood sugar is due to an increased breakdown of glycogen to glucose, but there may be increased gluconeogenesis. 7. The actions of usnic acid in the cat are, in the whole, very similar to those of the dinitrophenols.

Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura. It negatively impacts tissue injury during stroke, cerebral contusion and intracranial hemorrhage. Susceptibility to CSD has been assessed in several experimental animal models in vivo, such as after topical KCl application or cathodal stimulation. Various combinations of anesthetics and ambient conditions have been used by different laboratories making comparisons problematic and differences in data difficult to reconcile. We systematically studied CSD susceptibility comparing commonly used experimental anesthetics (isoflurane, alpha-chloralose, and urethane) with or without N(2)O or normobaric hyperoxia (100% O(2) inhalation). The frequency of evoked CSDs, and their propagation speed, duration, and amplitude were recorded during 2 h topical KCl (1 M) application. We found that N(2)O reduced CSD frequency when combined with isoflurane or urethane, but not alpha-chloralose; N(2)O also decreased CSD propagation speed and duration. Urethane anesthesia was associated with the highest CSD frequency that was comparable to pentobarbital. Inhalation of 100% O(2) did not alter CSD frequency, propagation speed or duration in combination with any of the anesthetics tested. Our data show anesthetic modulation of CSD susceptibility in an experimental model of human disease, underscoring the importance of proper study design for hypothesis testing as well as for comparing results between studies.

Understanding the link between the hemodynamic response and the underlying neuronal activity is important for interpreting functional magnetic resonance (fMRI) signals in human and animal studies. Simultaneous electrophysiological and functional imaging measurements provide a knowledge of information processing and communication in the brain with high spatial and temporal resolution. In this study, a range of neural and blood oxygenation level-dependent (BOLD) responses were elicited in the rat somatosensory cortex by changing the type of anesthesia (urethane or alpha-chloralose) and the electrical forepaw stimulus frequency (1–15 Hz). Duration of the stimulus was 30 s. Electrical local field potential and BOLD fMRI responses were recorded simultaneously. Under urethane anesthesia, integrated neural activity and BOLD responses increased with increasing stimulus frequency up to 11 Hz, after which both responses plateaued. In contrast, in alpha-chloralose-anesthetized rats both responses were measurable only at 1 and 3 Hz. Although neuronal and BOLD responses were nonlinear as a function of frequency over the 1 to 15 Hz stimulation range under both anesthetics, tight neural–hemodynamic coupling was observed independently of the anesthetic agent. Anesthetic agents influence neuronal activity in a different manner, but the relationship of neuronal activity and BOLD response remains the same.

Two adult and 1 juvenile free-flying greater sandhill cranes (Grus canadensis tabida) were diagnosed with capture myopathy after alpha-chloralose baiting and physical capture during a banding and feeding ecologic study. Blood samples were collected for serum biochemical analysis at the time of capture for the 2 adults, and at 24 hours postcapture, at various intervals during treatment, and at the time of release for all 3 birds. Concentrations of creatine kinase, aspartate transaminase, and lactate dehydrogenase were high within 1 hour of capture and peaked approximately 3 days after capture. By days 10-17 after capture, creatine kinase and lactate dehydrogenase concentrations both decreased to within the reference range measured for cranes at capture, but aspartate transaminase concentrations remained 2-5 times higher than the measured reference range. Treatment consisted of corticosteroids, selenium/vitamin E, parenteral fluids, and gavage feedings. Physical therapy consisted of assisting the cranes to stand and walk 2-8 times a day, massaging leg muscles, and moving limbs manually through the range of motion. The adults were released when they were able to stand up independently and were pacing in the pen. The juvenile was released 12 hours after it was able to stand independently but was returned to the pen when it fell and could not rise. It was treated supportively for an additional 3 days and then successfully released. Both adult cranes were observed on their territories with their original mates after release and returned to their territories for the subsequent 8 years, raising chicks most years. After release, the juvenile was observed in a flock of cranes near its natal territory for the next 2 days.

Antagonists of NMDA receptors can inhibit both the transmission of pain signals from the intestine and enteric reflexes. However, it is unknown whether doses of the NMDA antagonist, ketamine, that are used in anaesthetic mixtures suppress motility reflexes and visceromotor responses (VMRs). In fact, whether intestinal motility is affected by NMDA receptor blockers in vivo has been little investigated. We studied the effects of ketamine and memantine, administered intravenously or intrathecally. Rats were maintained under alpha-chloralose plus xylazine or pentobarbitone anaesthesia; VMR and jejunal motility were measured. Under alpha-chloralose/xylazine anaesthesia, i.v. ketamine inhibited VMRs at 6 mg kg h(-1), but not at 3 mg kg h(-1). It did not inhibit propulsive reflexes in the jejunum at 10 mg kg h(-1), but reduced them by 30% at 20 mg kg h(-1). Under alpha-chloralose/pentobarbitone anaesthesia, i.v. ketamine reduced propulsive reflexes at 40 mg kg h(-1) and VMR at 10 mg kg h(-1). Memantine inhibited VMRs at 20 mg kg h(-1) and propulsion at 2 mg kg h(-1). Ketamine and memantine, intrathecally, prevented VMRs, but not jejunal propulsion. We conclude that peripherally administered ketamine reduces both VMR and motility reflexes, but not at doses used in anaesthetic mixes (1.8-2.4 mg kg h(-1)). Effects on motility reflexes are likely to be due to non-NMDA receptor actions, possibly on nicotinic receptors.

Background: Granule cells occupy a strategic position in the transmission of afferent information to the cerebellar cortex. They are also the most abundant type of neurons in the cerebellum. The functions of the cerebellum are thought to be sensitive to acute alcohol intoxication. The effects of acute alcohol intoxication on the in vivo physiology of cerebellar granule cells are, however, not completely known. Methods: We studied chloralose-anesthetized cats at ethanol doses relevant to human drinking (0.3–1.2 g/kg). We recorded the electrophysiological responses of granule cell clusters to auditory and visual stimulation, and simultaneously monitored the concentration of ethanol in the cerebrospinal fluid (CSF). Results: At an intravenous ethanol dose of 0.3 g/kg, CSF ethanol concentration peaked in 10 minutes at 17 mM, equivalent to a blood alcohol concentration (BAC) of about 0.08 g/dL. Ethanol quickly and almost completely abolished both auditory and visual responses from granule cells. Complete or near-complete inhibition lasted 15 to 20 minutes; ∼50% recovery required an additional 15 minutes, and a full recovery yet another 15 minutes. A higher ethanol dose at 1.2 g/kg resulted in a more severe inhibition and required longer time for recovery. The relationship between ethanol dose, CSF ethanol concentration, and granule cell responses was dynamic and nonlinear, critically depending upon the elapsed time. Conclusions: Cerebellar granule cell sensory responses are highly sensitive to ethanol inhibition. A rapid development of acute tolerance appears to be a major factor contributing to the dynamic and nonlinear relationship among ethanol dosage, CSF ethanol concentration, and granule cell responses. It is likely that a generalized de-afferentation of the cerebellum from its mossy fiber afferents, followed by the subsequent development of acute tolerance may play major roles by which alcohol intoxication affects cerebellar functions.

Alpha-chloralose is commonly used during animal fMRI studies for anesthesia, however, recovery of animals is difficult, limiting experimental design. The use of a less invasive anesthetic would enable chronic experiments. The present study compares functional activity in the spinal cord of the alpha-chloralose and halothane-anesthetized rat. Functional MRI of the rat cervical spinal cord was performed on 6 alpha-chloralose and 6 halothane-anesthetized rats in a Bruker 7 T MR system during electrical stimulation of the right forepaw. Following imaging, four animals from each group were perfused and spinal cords removed for immunohistochemical analysis. Areas of c-fos expression were identified with immunofluorescent labeling to confirm the presence of neuronal activity. Functional activity and c-fos expression were observed predominantly between the fifth and seventh cervical spinal cord segments. Areas of fMRI activation in the spinal cord correspond well with spinal cord physiology. Areas of c-fos expression confirmed that neuronal activity was present in the regions of fMRI activity. The regions and amount of fMRI activity observed were similar for both anesthetics. Functional magnetic resonance imaging of the spinal cord can be achieved using both alpha-chloralose and halothane anesthesia in rats. We therefore suggest that halothane may be used as an anesthetic agent for chronic fMRI studies of the spinal cord.

The effects of pulse lung inflation (LI) on expiratory muscle activity and phase duration (Te) were determined in anesthetized, spontaneously breathing dogs ( n = 20). A volume syringe was used to inflate the lungs at various times during the expiratory phase. The magnitude of lung volume was assessed by the corresponding change in airway pressure (Paw; range 2–20 cmH2O). Electromyographic (EMG) activities were recorded from both thoracic and abdominal muscles. Parasternal muscle EMG was used to record inspiratory activity. Expiratory activity was assessed from the triangularis sterni (TS), internal intercostal (IIC), and transversus abdominis (TA) muscles. Lung inflations 2O consistently inhibited TS activity but had variable effects on TA and IIC activity and expiratory duration. Lung inflations resulting in Paw values >7 cmH2O, however, inhibited expiratory EMG activity of each of the expiratory muscles and lengthened Te in all animals. The responses of expiratory EMG and Te were directly related to the magnitude of the lung inflation. The inhibition of expiratory motor activity was independent of the timing of pulse lung inflation during the expiratory phase. The inhibitory effects of lung inflation were eliminated by bilateral vagotomy and could be reproduced by electrical stimulation of the vagus nerve. We conclude that pulse lung inflation resulting in Paw between 7 and 20 cmH2O produces a vagally mediated inhibition of expiratory muscle activity that is directly related to the magnitude of the inflation. Lower inflation pressures produce variable effects that are muscle specific.

The spontaneous cerebral hemodynamic fluctuations observed during the resting state have been frequently visualized using functional magnetic resonance imaging (rsfMRI). However, the neuronal populations and neuroelectric characteristics underlying the functional connectivity of cerebrohemodynamic activities are poorly understood. We investigated the characteristics of bi-hemispheric functional connectivity via electrophysiology and rsfMRI in the primary sensory cortex of rats anesthetized by α-chloralose. Unlike the evoked responses, the spontaneous electrophysiological activity was concentrated in the infragranular layers and could be classified into subtypes with distinctive current sources and sinks. Both neuroelectric and rsfMRI signals were interhemispherically correlated in a layer-specific manner, suggesting that there are independent neural inputs to infragranular and granular/supragranular layers. The majority of spontaneous electrophysiological activities were bilaterally paired with delays of up to ~50 ms between each pair. The variable interhemispheric delay implies the involvement of indirect, multi-neural pathways. Our findings demonstrated the diverse activity patterns of layer-specific electrophysiological substrates and suggest the recruitment of multiple, non-specific brain regions in construction of interhemispheric functional connectivity.

The relationship between peri-infarct depolarizations (PIDs) and infarction was investigated in a model of preconditioning by cortical freeze lesions (cryogenic lesions, CL) in the Spontaneously Hypertensive Rat. Small (< 5 mm3) lesions produced 24 hours before permanent focal ischemia were protective, without impacting baseline cerebral blood flow (CBF) and metabolism. Prior CL reduced infarct volume, associated with improved penumbral CBF as previously showed for ischemic preconditioning. The brief initial procedure avoided sham effects on infarct volume after subsequent occlusion under brief anesthesia. However, under prolonged isoflurane anesthesia for perfusion monitoring both sham and CL rats showed reduced PID incidence relative to naive animals. This anesthesia effect could be eliminated by using α-chloralose during perfusion imaging. As an additional methodological concern, blood glucose was frequently elevated at the time of the second surgery, reflecting buprenorphine-induced pica and other undefined mechanisms. Even modest hyperglycemia (>10 mmol/L) reduced PID incidence. In normoglycemic animals CL preconditioning reduced PID number by 50%, demonstrating associated effects on PID incidence, penumbral perfusion, and infarct progression. Hyperglycemia suppressed PIDs without affecting the relationship between CBF and infarction. This suggests that the primary effect of preconditioning is to improve penumbral perfusion, which in turn impacts PID incidence and infarct size.

Pulmonary embolism (PE) is a leading cause of sudden cardiac death, and a model is needed for testing potential treatments. In developing a model, we compared the hemodynamic effects of isoflurane and α-chloralose in an acute swine model of PE because the choice of anesthesia will likely affect the cardiovascular responses of an animal to PE. At baseline, swine that received α-chloralose (n = 6) had a lower heart rate and cardiac output and higher SpO2, end-tidal CO2, and mean arterial pressure than did those given isoflurane (n = 9). After PE induction, swine given α-chloralose compared with isoflurane exhibited a lower heart rate (63 ± 10 compared with 116 ± 15 bpm) and peripheral arterial pressure (52 ± 12 compared with 61 ± 12 mm Hg); higher SpO2 (98% ± 3% compared with 95% ± 1%), end-tidal CO2 (35 ± 4 compared with 32 ± 5), and systolic blood pressure (121 ± 8 compared with 104 ± 20 mm Hg); and equivalent right ventricular:left ventricular ratios (1.32 ± 0.50 compared with 1.23 ± 0.19) and troponin I mean values (0.09 ± 0.07 ng/mL compared with 0.09 ± 0.06 ng/mL). Isoflurane was associated with widely variable fibrinogen and activated partial thromboplastin time. Intraexperiment mortality was 0 of 6 animals for α-chloralose and 2 of 9 swine for isoflurane. All swine anesthetized with α-chloralose survived with sustained pulmonary hypertension, RV-dilation-associated cardiac injury without the confounding vasodilatory or coagulatory effects of isoflurane. These data demonstrate the physiologic advantages of α-chloralose over isoflurane for anesthesia in a swine model of severe submassive PE.

Sildenafil has been shown to be an effective treatment of pulmonary arterial hypertension and is believed to present with pulmonary selectivity. This study was designed to determine the site of action of sildenafil compared with inhaled nitric oxide (NO) and intravenous sodium nitroprusside (SNP), known as selective and nonselective pulmonary vasodilators, respectively. Inhaled NO (40 ppm), and maximum tolerated doses of intravenous SNP and sildenafil, (5 μg·kg−1·min−1and 0.1 mg·kg−1·h−1), respectively, were administered to eight dogs ventilated in hypoxia. Pulmonary vascular resistance (PVR) was evaluated by pulmonary arterial pressure (Ppa) minus left atrial pressure (Pla) vs. flow curves, and partitioned into arterial and venous segments by the occlusion method. Right ventricular hydraulic load was defined by pulmonary arterial characteristic impedance (Zc) and elastance (Ea) calculations. Right ventricular arterial coupling was estimated by the ratio of end-systolic elastance (Ees) to Ea. Decreasing the inspired oxygen fraction from 0.4 to 0.1 increased Ppa − Pla at a standardized flow of 3 l·min−1·m−2from 6 ± 1 to 18 ± 1 mmHg (mean ± SE). Ppa − Pla was decreased to 9 ± 1 by inhaled NO, 14 ± 1 by SNP, and 14 ± 1 mmHg by sildenafil. The partition of PVR, Zc, Ea, and Ees/Ea was not affected by the three interventions. Inhaled NO did not affect systemic arterial pressure, which was similarly decreased by sildenafil and SNP, from 115 ± 4 to 101 ± 4 and 98 ± 5 mmHg, respectively. We conclude that inhaled NO inhibits hypoxic pulmonary vasoconstriction more effectively than sildenafil or SNP, and sildenafil shows no more selectivity for the pulmonary circulation than SNP.

The significance of changes in cerebral oxygen consumption in focally activated brain tissue is still controversial. Since the rate of cerebral oxygen consumption is tightly coupled to that of tricarboxylic acid cycle which can be measured from the turnover kinetics of [4-(13)C]glutamate using in vivo (1)H{(13)C} magnetic resonance spectroscopy, changes in tricarboxylic acid cycle flux rate were assessed in primary somatosensory cortex of alpha-chloralose anesthetized rats during electrical forepaw stimulation. With markedly improved (1)H{(13)C} magnetic resonance spectroscopy technique and the use of high magnetic field strength of 11.7 T accessible to the current study, [4-(13)C]glutamate at 2.35 ppm was spectrally resolved from overlapping resonances of [4-(13)C]glutamine at 2.46 ppm and [2-(13)C]GABA at 2.28 ppm as well as the more distal [3-(13)C]glutamate and [3-(13)C]glutamine. The results showed a significantly increased V(TCA) in focally activated primary somatosensory cortex during forepaw stimulation, corresponding to approximately 51 +/- 27% (n = 6, mean +/- SD) increase in cerebral oxygen consumption rate. Considering the high efficiency in producing adenosine triphosphate by oxidative metabolism of glucose, the results demonstrate that aerobic oxidative metabolism provides the majority of energy required for cerebral focal activation in alpha-chloralose anesthetized rats subjected to forepaw stimulation.

Functional magnetic resonance imaging (fMRI) in anesthetized rodents has been commonly performed with alpha-chloralose, which can be used only for terminal experiments. To develop a survival fMRI protocol, an isoflurane (ISO) -anesthetized rat model was systematically evaluated by simultaneous measurements of field potential (FP) and cerebral blood flow (CBF) in the somatosensory cortex. A conventional forepaw stimulation paradigm with 0.3 ms pulse width, 1.2 mA current, and 3 Hz frequency induced 54% less evoked FP and 84% less CBF response under ISO than alpha-chloralose. To improve stimulation-induced responses under ISO, 10-pulse stimulations were performed with variations of width, current, and frequency. For widths of 0.1-5.0 ms and currents of 0.4-2.0 mA, evoked FP and CBF increased similarly and reached a plateau. The evoked FP increased monotonically for intervals from 50 to 500 ms, but the CBF peaked at an interval of 83 ms (approximately 12 Hz frequency). These data suggest that different anesthetics profoundly affect FP and CBF responses in different ways, which requires optimizing stimulation parameters for each anesthetic. With the refined stimulation parameters, fMRI consistently detected a well-localized activation focus at the primary somatosensory cortex in ISO-anesthetized rats. Thus, the ISO-anesthetized rat model can be used for cerebrovascular activation studies, allowing repeated noninvasive survival experiments.

Microgravity and simulated microgravity may cause cardiovascular deconditioning, but mechanisms of instantaneous responses to inverse-orthostasis are not studied. Hence, we investigated transient and steady state cardiovascular changes by combining the tilt technique with cardiovascular telemetry. Normotensive and NO-deprived hypertensive Wistar rats were used to analyze responses of mean arterial blood pressure, heart rate, contractility, spontaneous baroreflex sensitivity (sBRS), and autonomic balance. Inverse-orthostasis tests were carried out by 45 degrees head-down tilting (repeated 3 x 5 mins "R", or sustained for 120 mins "S"). In normotensive rats, horizontal control blood pressure was R111.3 +/- 1.7/S110.4 +/- 2.3 mm Hg and heart rate was R385.2 +/- 5.9/S371.1 +/- 6.1 BPM. Head-down tilt induced an increase in blood pressure by R5.9/S10.6 mm Hg, while heart rate, contractility, sBRS, and autonomic balance did not change. The hypertensive response was sustained, could be prevented by prazosin (10 mg/kgbw), and augmented by subanesthetic doses of chloralose (26 and 43 mg/kgbw). In NO-suppressed hypertension, control blood pressure and heart rate were R132.4 +/- 2.9/S130.0 +/- 4.1 mm Hg and R339.2 +/- 7.9/S307.2 +/- 23.6 BPM, respectively. Head-down tilt further increased blood pressure by R5.1/S10.5 mm Hg. These data demonstrate that conscious rats respond to inverse-orthostasis by sustained elevation of blood pressure independent of NO synthesis. This response is neither due to increased contractility and altered sBRS, nor due to non-specific stress, but probably due to sympathetic activation elicited by gravity-related reflexes, which increase peripheral resistance.

Administration of ABT-594, a potent agonist for nicotinic acetylcholine receptors with selectivity for the alpha4beta2 receptor subtype, is known to modulate a diverse array of behaviors including those associated with nociception, anxiety and motor function. In this study, we sought to gain insight into the neural actions of ABT-594, in vivo, by conducting functional magnetic resonance imaging in awake and anesthetized rats. Using T(2)*-weighted gradient echo imaging and an ultrasmall superparamagnetic iron oxide contrast agent, functional imaging was conducted on a 4.7 T magnet to measure changes in relative cerebral blood volume. In awake, restrained, male Sprague-Dawley rats that were acclimated to the imaging environment, injection of ABT-594 (0.03-0.3 micromol/kg, i.v.) evoked changes to relative cerebral blood volume in several neural regions including the cingulate, somatosensory, motor, auditory, and pre-frontal cortices as well as the thalamus and the periaqueductal gray/dorsal raphe. These effects were typically bimodal with significant decreases in relative cerebral blood volume at the 0.03 micromol/kg dose and increases at the higher doses (0.1 and 0.3 micromol/kg). The decreases and increases in relative cerebral blood volume were often observed within the same region, but triggered by different doses. Both increases and decreases in relative cerebral blood volume were blocked by pretreatment with the noncompetitive nicotinic acetylcholine receptor antagonist, mecamylamine (5 micromol/kg, i.p.) in awake rats. Administration of ABT-594 (0.1 micromol/kg, i.v.) to alpha-chloralose-anesthetized rats did not significantly alter relative cerebral blood volume in any brain region suggesting an anesthetic-related interference with the effects of ABT-594. The neural regions affected by administration of ABT-594 corresponded well to the known pre-clinical behavioral profile for this compound, and demonstrate the utility of using functional magnetic resonance imaging in awake animals to study pharmacological action.

Previously, magnetic resonance spectroscopy studies of alterations in cerebral metabolite concentration during functional activation have been focused on phosphocreatine using 31P MRS and lactate using 1H MRS with controversial results. Recently, significant improvements on the spectral resolution and sensitivity of in vivo spectroscopy have been made at ultrahigh magnetic field strength. Using highly resolved localized short-TE 1H MRS at 11.7 T, we report metabolic responses of rat somatosensory cortex to forepaw stimulation in alpha-chloralose-anesthetized rats. The phosphocreatine/creatine ratio was found to be significantly decreased by 15.1 +/- 4.6% (mean +/- SEM, P < 0.01). Lactate remained very low (approximately

The cholinergic agonist pilocarpine injected intraperitoneally (ip) increases mean arterial pressure (MAP) and superior mesenteric (SM) vascular resistance and reduces submandibular/sublingual gland (SSG) vascular resistance. In the present study, we investigated the effects of electrolytic lesions of the anteroventral third ventricle (AV3V) region on the changes in MAP, SM, and SSG vascular resistances induced by ip pilocarpine. Male Holtzman rats anesthetized with urethane (1.0 g/kg) and chloralose (60 mg/kg) were submitted to sham or electrolytic AV3V lesions and had pulsed Doppler flow probes implanted around the arteries. Contrary to sham rats, in 1-h and 2-day AV3V-lesioned rats, pilocarpine (4 micromol/kg) ip decreased MAP (-41 +/- 4 and -26 +/- 4 mm Hg, respectively, vs. sham: 19 +/- 4 mm Hg) and SM (-48 +/- 11 and -45 +/- 10%, respectively, vs. sham: 41 +/- 10%) and hindlimb vascular resistances (-65 +/- 32 and -113 +/- 29%, respectively, vs. sham: 19 +/- 29%). In 7-day AV3V-lesioned rats, pilocarpine produced no changes on MAP and SM and hindlimb vascular resistances. Similar to sham rats, pilocarpine reduced SSG vascular resistance 1 h after AV3V lesions (-46 +/- 6%, vs. sham: -40 +/- 6%), but it produced no effect 2 days after AV3V lesions and increased SSG vascular resistance (37 +/- 6%) in 7-day AV3V-lesioned rats. The responses to ip pilocarpine were similar in 15-day sham and AV3V-lesioned rats. The cholinergic antagonist atropine methyl bromide (10 nmol) iv slightly increased the pressor response to ip pilocarpine in sham rats and abolished for 40 min the fall in MAP induced by ip pilocarpine in 1-h AV3V-lesioned rats. The results suggest that central mechanisms dependent on the AV3V region are involved in the pressor responses to ip pilocarpine. Although it was impaired 2 and 7 days after AV3V lesions, pilocarpine-induced salivary gland vasodilation was not altered 1 h after AV3V lesions which suggests that this vasodilation is not directly dependent on the AV3V region.

The aim of this study was to characterize the properties of somatosensory neurons in the first 2 cervical spinal dorsal horns (C1 and C2 DHs) and compare them with those previously described for the rostral subnucleus caudalis (rVc). A total of 74 nociceptive neurons classified as wide-dynamic-range (WDR) or nociceptive-specific (NS), as well as 72 low-threshold mechanoreceptive (LTM) neurons, was studied in urethane/chloralose-anesthetized rats. The majority of LTM neurons were located in laminae III/IV and had a small mechanoreceptive field (RF) that included the posterior face and cervical tissues. In contrast, the nociceptive neurons were located in laminae I/II or V/VI, and the RF of each C1 and C2 DH nociceptive neuron included a part of the face and in 47% of them the RF included a region supplied by upper cervical afferents. There was a gradual caudal shift in the neuronal RF from nasal/intraoral tissues towards the neck as recording sites progressed from rVc to C1 and C2 DHs. In contrast to LTM neurons, many C1 and C2 DH nociceptive neurons received mechanosensitive convergent afferent inputs from cervical and craniofacial deep tissues (e.g., tongue muscles or temporomandibular joint), and over 50% could be activated by hypoglossal (XII) nerve electrical stimulation. We propose that C1 and C2 DHs represent part of the caudal extension of the Vc, and that Vc and C1 and C2 DHs may act together as one functional unit to process nociceptive information from craniofacial and cervical tissues, including that from deep craniofacial tissues.

Incidence of orthostatic hypertension is estimated at 5% but is even more prevalent in borderline hypertension and autonomic neuropathies. The aim of this study was to develop a potential model to investigate orthostatic hypertension. We used normotensive and hypertensive Wistar rats to analyze responses and diurnal variations of arterial blood pressure, heart rate, temperature, and locomotor activity by telemetry. Orthostatic tests were carried out during 45 degrees head-up tilt (R, repeated 3 times for 5 minutes; or S, sustained for 120 minutes). Hypertension was induced by blockade of nitric oxide synthesis. In normotensives, horizontal control blood pressure was R115.4 +/- 1.4/S113.7 +/- 1.6 mm Hg and heart rate R386.4 +/- 7.0/S377.9 +/- 8.8 bpm. Head-up tilt increased blood pressure by R4.5/S8.4 mm Hg, including a 3.8 mm Hg hydrostatic component. The sustained hypertensive response was prevented by prazosin (10 mg/kgbw) and augmented by a subanesthetic dose of chloralose (26 mg/kgbw). In NO-deprived hypertension, horizontal control blood pressure and heart rate were R138.4 +/- 2.6/S140.3 +/- 2.7 mm Hg and R342.1 +/- 12.0/S346.0 +/- 8.3 bpm, respectively. Tilt increased blood pressure further by R4.2/S9.4 mm Hg. In both normo- and hypertensives, variables exhibited similar diurnal rhythms except for nighttime locomotor activity, reduced from 3.7 +/- 0.4 to 2.8 +/- 0.3 counts/s. These data demonstrate that conscious rats respond to sustained orthostasis with hypertension, probably as a result of increased sympathetic output. Decreasing stress using a subanesthetic dose of chloralose increased this response, reducing the inhibitory effect on hypertensive responses.

The mechanisms by which α-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that α-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and α-chloralose-urethane. With α-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants ( P < 0.05). Avertin was associated with reduced contractility compared with α-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type ( P < 0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (α-chloralose-urethane) demonstrate that α-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. β-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

Functional magnetic resonance imaging (fMRI) in animal models provides a platform for more extensive investigation of drug effects and underlying physiological mechanisms than is possible in humans. However, it is usually necessary for the animal to be anesthetized. In this study, we have used a rat model of direct cortical stimulation to investigate the effects of anesthesia in rodent fMRI. Specifically, we have sought to answer two questions (i) what is the relationship between baseline neuronal activity and the BOLD response to stimulation under halothane anesthesia? And (ii) how does the BOLD response change after transferring from halothane to the commonly used anesthetic alpha-chloralose? In the first set of experiments, we found no significant differences in the amplitude of the BOLD response at the different halothane doses studied, despite electroencephalography (EEG) recordings indicating a dose-dependent reduction in baseline neuronal activity with increasing halothane levels. In the second set of experiments, a reduction in the spatial extent of the BOLD response was apparent immediately after transfer from halothane to alpha-chloralose anesthesia, although no change in the peak signal change was evident. However, several hours after transfer to alpha-chloralose, a significant increase in both the spatial extent and peak height of the BOLD response was observed, as well as an increased sensitivity to secondary cortical and subcortical activation. These findings suggest that, although alpha-chloralose anesthesia is associated with a greater BOLD response for a fixed stimulus relative to halothane, there is substantial variation in the extent and magnitude of the response over time that could introduce considerable variability in studies using this anesthetic.

Brain metabolism of glucose and lactate was analyzed by ex vivo NMR spectroscopy in rats presenting different cerebral activities induced after the administration of pentobarbital, alpha-chloralose, or morphine. The animals were infused with a solution of either [1-(13)C]glucose plus lactate or glucose plus [3-(13)C]lactate for 20 min. Brain metabolite contents and enrichments were determined from analyses of brain tissue perchloric acid extracts according to their post-mortem evolution kinetics. When amino acid enrichments were compared, both the brain metabolic activity and the contribution of blood glucose relative to that of blood lactate to brain metabolism were linked with cerebral activity. The data also indicated the production in the brain of lactate from glycolysis in a compartment other than the neurons, presumably the astrocytes, and its subsequent oxidative metabolism in neurons. Therefore, a brain electrical activity-dependent increase in the relative contribution of blood glucose to brain metabolism occurred via the increase in the metabolism of lactate generated from brain glycolysis at the expense of that of blood lactate. This result strengthens the hypothesis that brain lactate is involved in the coupling between neuronal activation and metabolism.