Which Animal Can Be Put Into A Hypnotic State When It Is Put Into Dorsal Recumbency

• Journal List
• J Vis Exp
• (80); 2013
• PMC3940706

J Vis Exp. 2013; (80): 51079.

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

Hilary S. McCarren

¹Department of Anesthesiology and Disquisitional Care, Perelman School of Medicine, University of Pennsylvania

^twoDepartment of Pharmacology, Perelman School of Medicine, Academy of Pennsylvania

⁴Center for Sleep and Circadian Neurobiology, Perelman Schoolhouse of Medicine, University of Pennsylvania

Jason T. Moore

¹Department of Anesthesiology and Critical Intendance, Perelman School of Medicine, Academy of Pennsylvania

ⁱⁱⁱDepartment of Neuroscience, Perelman School of Medicine, University of Pennsylvania

⁴Center for Sleep and Cyclic Neurobiology, Perelman School of Medicine, University of Pennsylvania

Max B. Kelz

^oneDepartment of Anesthesiology and Disquisitional Intendance, Perelman Schoolhouse of Medicine, University of Pennsylvania

²Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania

^threeSection of Neuroscience, Perelman School of Medicine, Academy of Pennsylvania

^ivHeart for Sleep and Cyclic Neurobiology, Perelman School of Medicine, University of Pennsylvania

Abstruse

1 desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic land in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the creature no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. Nosotros have developed a system to assess LORR in 24 mice simultaneously while carefully decision-making for potential confounds, including temperature fluctuations and varying gas flows. These chambers let reliable cess of anesthetic sensitivity as measured past latency to return of the righting reflex (RORR) post-obit a fixed coldhearted exposure. Alternatively, using stepwise increases (or decreases) in coldhearted concentration, the chambers also enable decision of a population's sensitivity to induction (or emergence) as measured by EC_l and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Keywords: Medicine, Result 80, Beefcake, Physiology, Pharmacology, Anesthesia, Inhalation, Behavioral Inquiry, General anesthesia, loss of righting reflex, isoflurane, anesthetic sensitivity, animal model

Introduction

Full general anesthetics are defined by their ability to cause a reversible state of hypnosis in a wide multifariousness of species, even so an explanation every bit to how such a diverse class of drugs can all elicit a singular endpoint remains elusive. A number of theories have been posited over the years, starting with the Meyer-Overton correlation between anesthetic dominance and lipid solubility, which suggested general membrane disruptions as the footing for hypnosis^1,2. More contempo show suggests that protein targets affecting neuronal signaling contribute to anesthetic effects. Mice have proven to be an indispensable model for exploring these theories because of the homology between murine and human being anesthetic responsiveness. Though a mouse cannot be asked nearly its subjective awareness nether full general anesthesia, certain primitive reflexes serve as useful surrogate measures of rodent hypnosis. In the first few days following birth, mice develop a reflexive righting response that prevents them from being passively placed in a supine positionⁱⁱⁱ. The dose of anesthesia at which a mouse loses its righting reflex correlates well with human being hypnotic doses⁴.

Assessment of loss of righting reflex (LORR) has become a widely used laboratory standard for testing coldhearted sensitivity in mice as well as a variety of other species including rat, republic of guinea grunter, rabbit, ferret, sheep, and dog^v-8. The dose of a given anesthetic at which LORR will occur for members of a species is extremely consequent, but it can exist shifted significantly by environmental factors. For example, sleep-deprived rats are more sensitive to both volatile and intravenous anesthetics^ix and rats with loftier aerobic capacity are less sensitive to isoflurane¹⁰. Hypothermia has too been shown to decrease the dose of numerous anesthetics required for hypnosis in a large spectrum of species^eleven-xiv. In order to reliably identify the anesthetic dose at which LORR occurs in a group of experimental animals, information technology is critical that the assessment environment be advisedly controlled to minimize stress, maintain euthermia, and evangelize equal amounts of drug to all subjects. Non surprisingly, genetic factors are also known to alter anesthetic sensitivity^15-eighteen. Consequently, careful consideration should also be given to decision-making for genetic groundwork^nineteen.

Nosotros have developed an apparatus that ensures identical gaseous anesthetic delivery to each of 24 mice while maintaining a abiding 37^oC environment. The transparent cylindrical design of our exposure chambers allows for fast LORR assessment and like shooting fish in a barrel integration of telemetric physiological measurements. This arrangement has been shown to accurately measure isoflurane, halothane, and sevoflurane induction EC_fifty and time to emergence in wild-type mice²⁰. We have too used this arrangement to discover changes in anesthetic sensitivity in mice with genetic mutations and targeted hypothalamic lesions^21-23. Here we describe ii ways in which anesthetic sensitivity may exist assessed after a pharmacological intervention using our controlled environment appliance. Steady-state phenotyping of volatile anesthetic consecration and emergence sensitivity requires eight-10 hours and is consequently best tailored for studies in which experimental weather do not alter, such as in chronic or long-acting pharmacological interventions. However, for short-acting treatments whose effects dissipate significantly over time nosotros as well present a elementary process to evaluate changes in righting reflex following stereotactically-targeted microinjections or intravenous drug treatments that significantly touch on anesthetic emergence. These tests correspond a small subset of the potential applications for this controlled surround organization, which could exist adjusted for whatsoever number of subjects of a variety of species to receive any type of inhaled therapeutic.

Protocol

All procedures involving animals outlined herein have been approved past the Academy of Pennsylvania's Institutional Brute Care and Employ Committee.

i. Overview of the Testing Apparatus

1. The testing apparatus consists of 24 clear acrylic cylindrical chambers 10 cm in length and 5 cm in diameter (total book of 200 ml). This size is appropriate for a typical 25 m adult mouse. Chambers have ports at each cease for gas inlet and outlet. The outlet end is removable then that animals may be easily loaded into the chamber. Gas port openings are carefully sealed with Teflon tape, while rubber o-ring gaskets are used to seal the removable end of the cylindrical chambers.

2. Each chamber is mounted on a rack that sits inside a water bath. The rack is fitted then that only the lower portion of the chambers (below the gas inlets) is submerged. For stability, the dorsum end of the bedchamber rests on a support so that the entire chamber sits horizontally. This ensures even contact of the entire bedroom with the bathroom.

3. Polyethylene tubing connects an oxygen tank to an anesthetic vaporizer, and so passes through a 10 L/min menstruation meter. The tubing splits into 25 small-scale-bore resistors of equal length to ensure equal period is delivered to each of the 24 chambers and to an amanuensis analyzer.

4. Vacuum lines exit each chamber at the reverse terminate of the gas inlet. This promotes unidirectional flow that eliminates rebreathing of exhaled carbon dioxide. The vacuum lines combine at a manifold to connect to an in-house suction line. A pop-off valve along the primary vacuum line ensures atmospheric pressure level conditions within each chamber.

5. The bath is filled with plenty water to fully contact the lesser of each bedroom. The water is circulated through the bath and maintained at a constant 37^oC by a pump.

2. Check the Arrangement Prior to Exposure

1. Check that the temperature of the h2o bath is 37^oC throughout the bath.

2. Flow oxygen at a rate of 5 L/min (200 ml/min per chamber + agent analyzer). Submerge each sleeping accommodation under water and look for bubbles or entry of water into the chamber, both of which are indicative of leaks. Seal any leaks before start the experiment.

3. For each chamber, connect a 500 ml/min menstruum meter in line after the chamber to make sure that flows are balanced across each of the 25 gas lines. This ensures that the input five L/min flows volition be distributed evenly and so that each chamber receives 200 ml/min period. Any chamber not receiving the expected menses should accept its inflow and outflow tubing checked for obstructions.

4. Calibrate the agent analyzer to ensure a reading of 0.00% isoflurane when 100% oxygen is flowing.

3. Implant Temperature Transponder

1. One week prior to habituation, anesthetize each mouse with 2% isoflurane.

2. Sterilize the dorsal neck surface area with betadine.

3. Inject a temperature transponder subcutaneously betwixt the shoulder blades using the sterile, prepackaged injector needle.

4. Monitor the injection site daily for infection and migration of the transponder.

4. Habituate Animals to Testing Chambers

1. Four days before the showtime cess, place all mice into individual chambers for 2 60 minutes with 100% oxygen flowing.

2. Repeat step 4.i daily for the four days prior to assessment to avert the confounding effects of stress due to a new surround.

5. Perform the Pharmacological Intervention that You Wish to Test for Effects on Anesthetic Sensitivity

1. This intervention may be a stereotaxic injection into a specific part of the encephalon²⁴, an intravenous or intraperitoneal injection²⁵, or commitment of a drug to a specific brain area via cannula²⁶.

2. Because these procedures themselves may alter anesthetic sensitivity compared to a naïve fauna, a proper control group should undergo the aforementioned process with vehicle injections.

3. Ensure that the pharmacological intervention has an appropriately long duration of action if you are planning to do a stepwise increasing and/or decreasing determination of anesthetic sensitivity as shown in step half-dozen below; otherwise, skip to footstep 7.

half-dozen. Assess Anesthetic Sensitivity using the Stepwise EC₅₀ Conclusion for Induction and Emergence

1. Place each animal into individual chambers with 100% oxygen flowing.

2. Gear up the isoflurane concentration to 0.4%* for 15 min. During the last ii min of this flow, appraise each creature'southward righting reflex past gently rolling the chamber until the mouse is placed on its back. The righting reflex is considered to exist intact if and but if the mouse is able to restore all of its paws to the floor of the chamber within 2 min.

   1. *Note that 0.iv% isoflurane is a subhypnotic dose in C57BL/6J mice. If whatever mice lose their righting reflex at the first step, the initial dose was too large and should be reduced on subsequent days.

3. Record the state of righting reflex for each mouse and scan each mouse for temperature information. A template tape is shown in Table 1.

4. Increment the isoflurane concentration by ~0.05% for 15 min and repeat step vii.ii. Continue to do this until all animals have lost their righting reflex.

5. Optional: repeat the same procedure for decreasing pace-wise isoflurane doses until all animals have regained their righting reflex (see step 6.3).

6. To cease the experiment, turn off the isoflurane and affluent the unabridged arrangement with 100% oxygen for xv min. This will help to prevent hypoxia as the mice recover earlier being returned to their home cages and volition protect the experimenter from whatever anesthetic exposure.

7. Optional: if the number of animals or the number of anesthetic concentrations are limited due to resources or time constraints, the curve-fit parameter estimates-particularly the Hill gradient-may have underappreciated, falsely low error estimate. In such cases, it may be necessary to echo the coldhearted sensitivity measurement described in steps 6.ane-half-dozen.half dozen on upwardly to two additional experimental days to fully obtain the true Hill slope'southward parameter and its corresponding fault estimation.

7. Appraise Brusk-term Changes in Coldhearted Sensitivity with Time to Emergence

1. Place each creature into individual chambers with 100% oxygen flowing.

2. Set the isoflurane concentration to 1.2%, which corresponds to the consecration ED₉₉ for wild-type C57BL/6J mice²⁰. Maintain for thirty-lx min depending upon the expected elapsing of action of the acute intervention.

3. Confirm LORR in all animals by gently rolling each chamber until mice are placed on their backs.

4. Turn off isoflurane and period 100% oxygen. Measure the time until each creature regains its righting reflex. This is defined by placement of all 4 paws on the floor of the chamber and confirmed by the presence of iii consecutive tests with an intact righting reflex.

Representative Results

Effigy 1 demonstrates the utility of the footstep-wise LORR analysis for determining long-term furnishings of a pharmacological intervention. Ibotenic acid (IBA) is an agonist of the glutamatergic N-methyl-D-asparate (NMDA) receptor that is often used equally an excitotoxin to cause permanent neuronal lesions. Hither we injected 10 nl of one% IBA bilaterally into the ventrolateral preoptic area (VLPO) of C57BL/6J mice one week prior to testing. The bulk of neurons in this nucleus exhibit low rates of firing during wakefulness and specifically increase their action during non-rapid middle movement sleep, rapid middle movement slumber, and with exposure to hypnotic doses of general anesthetics^23,27-29. Successful lesions in the VLPO should cause resistance to isoflurane-induced hypnosis. At each increasing level of isoflurane, the fraction of mice that had lost the righting reflex was plotted against anesthetic concentration on a log₁₀ calibration. Data for each group of mice (vehicle-injected and IBA-injected) was then fit with a sigmoidal dose-response curve. Considering this assay always starts with all animals upright and e'er ends with all animals having lost the righting reflex, bottom and summit constants were constrained to 0 and 1, respectively. The remaining complimentary parameters of the curves are the EC₅₀, or the concentration of anesthetic at which 50% of mice have lost their righting reflex, and the Hill slope, which reflects the population variance during their hypnotic country transition. An F-test was used to query whether a single consecration curve with shared EC₅₀ and Hill slope parameters best fit both vehicle and IBA groups or whether split induction curves with distinct parameters better fit the information. The degrees of freedom in this test arise from the raw data points underlying the curve fit and consequently depend upon the number of anesthetic concentrations tested and the number of parameters beingness fit-EC₅₀ and Hill slope in this case. Step-wise emergence data were analyzed and modeled identically to data for induction. Notation that the EC₅₀ for emergence is almost always lower than that of induction due to anesthetic hysteresis besides known as neural inertia³⁰. Reverse to anticipated results, animals that received IBA in the VLPO showed no significant differences in the EC₅₀ or Hill gradient for induction or emergence compared to vehicle- injected controls (F _2,80 = 1.73 and p = 0.184 for induction, F _2,88 = 2.89 and p = 0.061 for emergence). This indicates that mouse VLPO neurons are resistant to lesion with 1% IBA, a fact confirmed with post-mortem histology (non shown). Lu et al. accept previously demonstrated that a dose of 10% IBA is required to lesion the rat VLPO³¹, but histological exam of the mouse VLPO following injection of 10% IBA besides showed no significant prison cell loss (not shown). The rat VLPO is known to limited NMDA receptors³². Since x% IBA is able to exert an acute effect on coldhearted sensitivity when injected into the VLPO (run into Figure 2, discussion below), this argues that the mouse VLPO must too possess the NMDA receptors necessary for IBA's actions. Thus the reason for the discrepancy between species remains unclear. Successful mouse VLPO lesions have been achieved using a targeted galanin-saporin²³.

Though IBA does not have a long-term effect on isoflurane sensitivity when injected into the VLPO, the acute excitatory nature of this drug would exist expected to stimulate VLPO neurons and transiently increase anesthetic sensitivity. In Figure two, we have used the time to emergence test to demonstrate a large acute shift in isoflurane sensitivity immediately following bilateral IBA microinjection into the VLPO as evidenced by markedly prolonged hypnosis later cessation of anesthetic delivery (p < 0.001). Conversely, microinjection of IBA into the nearby medial septum caused no alter in time to emergence compared to vehicle-injected controls (p > 0.05). This finding adds an interesting facet to previous work showing that inactivation of this nucleus extends time to emergence^33,34. Data for experimental and command groups in the fourth dimension to emergence test was averaged and compared with a one-style ANOVA.

Fourth dimension	Isoflurane (% atm)	Mouse #i	Mouse #2	Mouse #iii	…
12:00 PM	0.4	-	-	-	-
12:15 PM	0.45	-	X	-	-
12:thirty PM	0.5	-	X	10	-
12:45 PM	0.55	-	X	X	-
…	0.6	-	X	X	Ten

Tabular array 1. Example of a Log Sheet for Long-term Anesthetic Sensitivity Assessment: Every 15 min the coldhearted dose was increased by 0.05% and righting reflex was assessed for each beast. "10" denotes animals that had lost their righting reflex for a given time bespeak and "-" denotes those that maintained their righting reflex.

Figure 1. Assessment of the Righting Reflex One Week Later Ibotenic Acrid Injection in the Ventrolateral Preoptic Nucleus: The long-term anesthetic sensitivity assay was conducted on mice with either vehicle or ibotenic acid (IBA) injected into the ventrolateral preoptic surface area (VLPO) one calendar week prior to testing. Induction and emergence data for each group was fit with a sigmoidal dose-response curve (induction in solid lines, emergence in dashed lines) along with the 95% conviction interval bracketing the best-fit curves (shaded bars). Anesthetic concentration was plotted on a log₁₀ scale. Overlapping 95% confidence intervals are shown in imperial. The sigmoidal dose-repsonse fits for both vehicle and IBA groups advise no evidence for distinct best-fit curves based on EC_l and Hill gradient. Click hither to view larger image.

Figure 2. Time to Emergence After Local Microinjection of Ibotenic Acid: Immediately prior to assessment, mice received a microinjection of the N-methyl-D-asparate (NMDA) receptor agonist ibotenic acid (IBA) into the ventrolateral preoptic nucleus (VLPO). This area is known to be activated during isoflurane-induced hypnosis. IBA injection led to an astute increase in the time to return of righting reflex compared to vehicle-injected controls (p < 0.001). Time to emergence for animals with IBA injected into medial septum did not differ from controls (p > 0.05). Click here to view larger image.

Discussion

Though assessment of LORR in a single mouse is a seemingly straightforward task, information technology is nevertheless essential to maintain identical physiological conditions between subjects in order to collect reliable data from a grouping of animals. The tightly regulated, high-chapters LORR apparatus presented here offers a way to standardize experiments and maximize efficiency. Past following the basic tenets of thermoregulation and equal catamenia distribution, this system can be easily recreated and customized to fit private experimenters' needs. Chamber size may exist scaled for other species, such every bit rats, and additional chambers may be accommodated by attaching more branch points to the inflow and vacuum. All subjects are easily visible through the clear acrylic chambers, which makes information technology possible to video tape experiments for secondary post-hoc confirmation of results. The acrylic is likewise uniform with radio frequency telemetry systems, which tin be used to monitor temperature, blood pressure, and biopotentials.

Nosotros present two dissimilar methods for assessing coldhearted sensitivity following a pharmacological intervention. Both the fourth dimension to emergence and the stepwise induction tests require the experimenter to score the presence or absenteeism of the righting reflex. Even with an explicit definition of LORR, such every bit "unable to place all four paws on the sleeping room flooring within 2 minutes of being rolled onto its back", assessment can be somewhat subjective. It is all-time to accept the same handling-blinded individual score each animal for the duration of the experiment to ensure consistency. When choosing which test to utilize for anesthetic sensitivity cess, the anticipated length of the outcome from the pharmacological intervention should exist the deciding cistron. Many drugs have a short elapsing of activeness, for which the acute time to emergence paradigm tin can provide useful information on anesthetic sensitivity in a limited period of fourth dimension. However, a drug may preferentially affect an beast's sensitivity to consecration of hypnosis rather than emergence; changes in time to induction are ofttimes difficult to observe because induction occurs quickly and thus requires continuous assessment. The longer stepwise test for EC_fifty of induction and emergence can give information on both archway to and exit from hypnosis. The total length of the experiment will depend on the size of the increment by which coldhearted concentration is altered at each footstep, with typical induction + emergence tests lasting about 8 hr. Decreasing the coldhearted pace size around the anticipated EC₅₀ and increasing the number of animals in each group will give a better fitted dose-response curve just would likewise lengthen the time required to complete the assay.

Some pharmacological interventions may differentially alter the infinitesimal ventilation of experimental animals when compared to their controls. This could cause 1 group to exhale the volatile anesthetic in the time to emergence test more apace than the other, thus confounding the results. Solt et al. depict a good alternative method for testing anesthetic sensitivity in this scenario³⁵. In their experiment, systemic methylphenidate is delivered during constant isoflurane exposure in animals that accept already equilibrated with anesthetic. Potential misreckoning effects on the minute ventilation are thus excluded during continuous coldhearted exposure equally anesthetic uptake and distribution during steady-state atmospheric condition are precisely balanced by metabolism and emptying. The chambers we describe could be easily modified with an additional gas-tight port to allow passage of tubing for intravenous or intracerebral drug delivery. It should as well be noted that the described 15 min of equilibration to each concentration of anesthetic in the step-wise assay might not exist sufficient in certain cases. Anesthetics with a higher solubility than isoflurane, such as halothane, will take longer to reach their full concentrations in the tissue. Larger animals and animals that undergo larger steps in anesthetic concentration may too require more than time to equilibrate. To determine if fifteen min is truly adequate for equilibration, anesthetic tissue levels at the aforementioned concentration of coldhearted on both the ascending and descending limbs of exposure should be measured.

In cases where an animal'due south ability to move is physically or pharmacologically hindered, LORR may non serve as a good surrogate measure of hypnosis. The most reliable and widely used alternative is cortical electroencephalographic (EEG) recordings. Though EEG may be better able to pick up more subtle changes in anesthetic sensitivity, it is significantly more expensive to set than the apparatus nosotros describe. Implanting EEG electrodes is an invasive and time-consuming procedure, and the ability to obtain data from multiple mice simultaneously is often limited by equipment availability. Moreover, analysis of EEG recordings is conceptually more abstract and difficult to interpret than the simple binary output of LORR assessment. For these reasons, behavioral tests like those described here are ofttimes more than feasible methods for rapidly screening anesthetic sensitivity. Notation that EEG patterns suggestive of arousal and hypnosis may not correlate well with behavior. LORR and EEG are distinct endpoints which both likely provide useful information regarding anesthetic sensitivity.

In addition to potential drug-induced changes in infinitesimal ventilation and mobility, there are several other limitations to the methods described herein. Though LORR is a standard surrogate for hypnosis across the field, the criteria and methodology used for its measurement differ across laboratories. Some advocate that mice should be rotated at a constant speed to assess the righting reflex. Continuous cess logically narrows the precise timing with which the righting reflex is lost and/or returns; however, the act of being turned supine may be more stimulating than simply remaining supine. In add-on, step-wise LORR cess is a time-consuming assay that may be further extended if xv minutes of equilibration at each step is constitute to be insufficient.

Despite these limitations, the potential applications for this protocol extend far beyond the specific instances we have presented. Clearly, pharmacological interventions are non the simply method by which coldhearted sensitivity might exist altered; targeted lesions, anatomic abnormalities, and genetic mutations may all be tested using the same stepwise EC₅₀ determination. The controlled environment system presented here can be used to deliver any kind of inhaled drug, such equally corticosteroids, antibiotics, or experimental therapeutics. The power to expose many mice to the same amount of drug at once makes this setup ideal for toxicology studies. Additionally, chambers serve as an ideal post-surgical recovery surround with regulated ambient temperature and fresh oxygen flow. This apparatus is useful for whatsoever instance in which basic animate being vital signs need to be monitored and controlled.

Disclosures

The authors take nothing to disembalm.

Acknowledgments

This work was supported by R01 GM088156 and T32 HL007713-eighteen. We would like to thank Neb Pennie and Michael Carman from the Academy of Pennsylvania Research Instrumentation Store for their aid in assembling our righting reflex apparatus.

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