This work was supported by R01 GM088156 and T32 HL007713-18. We would like to thank Bill Pennie and Michael Carman from the University of Pennsylvania Research Instrumentation Shop for their help in assembling our righting reflex apparatus.

All procedures involving animals outlined herein have been approved by the University of Pennsylvania&#39;s Institutional Animal Care and Use Committee.
1. Overview of the Testing Apparatus
<ol>
	<li>The testing apparatus consists of 24 clear acrylic cylindrical chambers 10 cm in length and 5 cm in diameter (total volume of 200 ml). This size is appropriate for a typical 25 g adult mouse. Chambers have ports at each end for gas inlet and outlet. The outlet end is removable so that animals ma...

<ol>
	<li>Meyer, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+theorie+der+alkoholnarkose.+I.+Mittheilung.+Welche+Eigenschaft+der+An#228;sthetica+bedingt+ihre+narkotische+Wirkung?.">Zur theorie der alkoholnarkose. I. Mittheilung. Welche Eigenschaft der An#228;sthetica bedingt ihre narkotische Wirkung?.</a> Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmakol. 42, 109-137 .</li><li>Overton, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studien &#252;ber die Narkose: Zugleich ein Beitrag zur allgemeinen Pharmakologie. , (1901).</li><li>Bignall, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+levels+of+neural+organization:+the+righting+reflex+as+a+model.">Ontogeny of levels of neural organization: the righting reflex as a model.</a> Exp. Neurol. 3 (3), 566-573 (1974).</li><li>Franks, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anaesthesia:+from+molecular+targets+to+neuronal+pathways+of+sleep+and+arousal.">General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal.</a> Nat. Rev. Neurosci. 9 (5), 370-386 (2008).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab. Anim. 27 (1), 30-39 (1993).</li><li>Schernthaner, A., Lendl, C., Busch, R., Henke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+three+medetomidine--midazolam--ketamine+combinations+for+neutering+of+ferrets+(Mustela+putorius+furo)].">Clinical evaluation of three medetomidine--midazolam--ketamine combinations for neutering of ferrets (Mustela putorius furo)].</a> Berliner und M&#252;nchener tier&#228;rztliche Wochenschrift. 121 (1-2), 1-10 (2008).</li><li>Mohammad, F. K., Zangana, I. K., Abdul-Latif, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medetomidine+sedation+in+sheep.">Medetomidine sedation in sheep.</a> Zentralblatt f&#252;r Veterin&#228;rmedizin. Reihe A. 40 (5), 328-331 (1993).</li><li>Nicholls, E. A., Louie, G. L., Prokocimer, P. G., Maze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halothane+anesthetic+requirements+are+not+affected+by+aminophylline+treatment+in+rats+and+dogs.">Halothane anesthetic requirements are not affected by aminophylline treatment in rats and dogs.</a> Anesthesiology. 65 (6), 637-641 (1986).</li><li>Tung, A., Szafran, M. J., Bluhm, B., Mendelson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+Deprivation+Potentiates+the+Onset+and+Duration+of+Loss+of+Righting+Reflex+Induced+by+Propofol+and+Isoflurane.">Sleep Deprivation Potentiates the Onset and Duration of Loss of Righting Reflex Induced by Propofol and Isoflurane.</a> Anesthesiology. 97 (4), 906-911 (2002).</li><li>Pal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Minimum+Alveolar+Concentration+for+Isoflurane+and+Sevoflurane+in+a+Rodent+Model+of+Human+Metabolic+Syndrome.">Determination of Minimum Alveolar Concentration for Isoflurane and Sevoflurane in a Rodent Model of Human Metabolic Syndrome.</a> Anesth. 2 (2), 297-302 (2012).</li><li>Eger, E. I., Saidman 2nd, ., J, L., Brandstater, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+halothane+and+cyclopropane+anesthesia+in+dogs:+correlation+with+some+theories+of+anesthetic+action.">Temperature dependence of halothane and cyclopropane anesthesia in dogs: correlation with some theories of anesthetic action.</a> Anesthesiology. 26 (6), 764-770 (1965).</li><li>Vitez, T. S., White, P. F., Eger, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2nd+Effects+of+hypothermia+on+halothane+MAC+and+isoflurane+MAC+in+the+rat.">2nd Effects of hypothermia on halothane MAC and isoflurane MAC in the rat.</a> Anesthesiology. 41 (1), 80-81 (1974).</li><li>Antognini, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+eliminates+isoflurane+requirements+at+20+degrees+C.">Hypothermia eliminates isoflurane requirements at 20 degrees C.</a> Anesthesiology. 78 (6), 1152-1156 (1993).</li><li>McKenzie, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+on+the+anaesthetic+potency+of+halothane,+enflurane+and+ethanol+in+Daphnia+magna+(Cladocera:+Crustacea).">Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea).</a> Comp. Biochem. Physiol. C. 101 (1), 15-19 (1992).</li><li>Icaza, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane-Induced+Changes+in+Righting+Response+and+Breathing+are+Modulated+by+RGS+Proteins.">Isoflurane-Induced Changes in Righting Response and Breathing are Modulated by RGS Proteins.</a> Anesth. Analg. 109 (5), 1500-1505 (2009).</li><li>Drexler, B., Antkowiak, B., Engin, E., Rudolph, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+anesthetic+targets+by+mouse+molecular+genetics+approaches.">Identification and characterization of anesthetic targets by mouse molecular genetics approaches.</a> Can. 2 (2), 178-190 (2011).</li><li>Wafford, K. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+Occurring+Variability+in+Anesthetic+Potency+Among+Inbred+Mouse+Strains.">Naturally Occurring Variability in Anesthetic Potency Among Inbred Mouse Strains.</a> Anesth. 91 (3), 720-726 (2000).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+modular+chambers+for+rapid+evaluation+of+anesthetic+sensitivity.">High throughput modular chambers for rapid evaluation of anesthetic sensitivity.</a> BMC Anesthesiol. 6 (1), 13 (2006).</li><li>Hu, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypnotic+Hypersensitivity+to+Volatile+Anesthetics+and+Dexmedetomidine+in+Dopamine+&#946;-Hydroxylase+Knockout+Mice.">Hypnotic Hypersensitivity to Volatile Anesthetics and Dexmedetomidine in Dopamine &#946;-Hydroxylase Knockout Mice.</a> Anesthesiology. , (2012).</li><li>Kelz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+orexins+in+emergence+from+general+anesthesia.">An essential role for orexins in emergence from general anesthesia.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (4), 1309-1314 (2008).</li><li>Moore, J. 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L., McGinty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-waking+discharge+patterns+of+ventrolateral+preoptic/anterior+hypothalamic+neurons+in+rats.">Sleep-waking discharge patterns of ventrolateral preoptic/anterior hypothalamic neurons in rats.</a> Brain Res. (1-2), 178-188 (1998).</li><li>Nelson, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sedative+component+of+anesthesia+is+mediated+by+GABAA+receptors+in+an+endogenous+sleep+pathway.">The sedative component of anesthesia is mediated by GABAA receptors in an endogenous sleep pathway.</a> Nat. Neurosci. 5 (10), 979-984 (2002).</li><li>Li, K. Y., Guan, Y., Krnjevi&#263;, K., Ye, J. 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Though assessment of LORR in a single mouse is a seemingly straightforward task, it is nevertheless essential to maintain identical physiological conditions between subjects in order to collect reliable data from a group of animals. The tightly regulated, high-capacity LORR apparatus presented here offers a way to standardize experiments and maximize efficiency. By following the basic tenets of thermoregulation and equal flow distribution, this system can be easily recreated and customized to fit individual experimenters...

General anesthetics are defined by their ability to cause a reversible state of hypnosis in a wide variety of species, yet an explanation as 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 potency and lipid solubility, which suggested general membrane disruptions as the basis for hypnosis1,2. More recent evidence 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 anesthetic responsiveness. Though a mouse cannot be asked about its subjective awareness under 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 position3. The dose of anesthesia at which a mouse loses its righting reflex correlates well with human hypnotic doses4.
Assessment of loss of righting reflex (LORR) has become a widely used laboratory standard for testing anesthetic sensitivity in mice as well as a variety of other species including rat, guinea pig, rabbit, ferret, sheep, and dog5-8. The dose of a given anesthetic at which LORR will occur for members of a species is extremely consistent, but it can be shifted significantly by environmental factors. For example, sleep-deprived rats are more sensitive to both volatile and intravenous anesthetics9&#160;and rats with high aerobic capacity are less sensitive to isoflurane10. Hypothermia has also been shown to decrease the dose of numerous anesthetics required for hypnosis in a large spectrum of species11-14. In order to reliably identify the anesthetic dose at which LORR occurs in a group of experimental animals, it is critical that the assessment environment be carefully controlled to minimize stress, maintain euthermia, and deliver equal amounts of drug to all subjects. Not surprisingly, genetic factors are also known to alter anesthetic sensitivity15-18. Consequently, careful consideration should also be given to controlling for genetic background19.
We have developed an apparatus that ensures identical gaseous anesthetic delivery to each of 24 mice while maintaining a constant 37&#160;oC environment. The transparent cylindrical design of our exposure chambers allows for fast LORR assessment and easy integration of telemetric physiological measurements. This system has been shown to accurately measure isoflurane, halothane, and sevoflurane induction EC50 and time to emergence in wild-type mice20. We have also used this system to observe changes in anesthetic sensitivity in mice with genetic mutations and targeted hypothalamic lesions21-23. Here we describe two ways in which anesthetic sensitivity may be assessed after a pharmacological intervention using our controlled environment apparatus. Steady-state phenotyping of volatile anesthetic induction and emergence sensitivity requires 8-10 hours and is consequently best tailored for studies in which experimental conditions do not change, such as in chronic or long-acting pharmacological interventions. However, for short-acting treatments whose effects dissipate significantly over time we also present a simple procedure to evaluate changes in righting reflex following stereotactically-targeted microinjections or intravenous drug treatments that significantly impact anesthetic emergence. These tests represent a small subset of the potential applications for this controlled environment system, which could be adapted for any number of subjects of a variety of species to receive any type of inhaled therapeutic.

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			<td height="42" style="height:42px;width:115px;">Name of the Reagent</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Oxygen</td>
			<td style="width:88px;">Airgas</td>
			<td style="width:88px;">OX300</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Isoflurane</td>
			<td style="width:88px;">Butler Schein</td>
			<td style="width:88px;"></td>
			<td style="width:171px;">Any volatile anesthetic of interest may be substituted</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="17" style="height:17px;"></td>
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			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Name of Material</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Mass flow meter- 10 SLPM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2309</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Mass flow meter- 500 SCCM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2305</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Anesthetic agent analyzer/gas indicator</td>
			<td style="width:88px;">AM Bickford</td>
			<td style="width:88px;">FI-21 Riken</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Heating water pump</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">13-874-175</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Temperature transponders</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">IPTT-300</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">RF temperature reader</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">DAS-6007</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

assessing changes in volatile general anesthetic sensitivity of mice after local or systemic pharmacological intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state 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 animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 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.

Figure 1 demonstrates the utility of the step-wise LORR assay for determining long-term effects of a pharmacological intervention. Ibotenic acid (IBA) is an agonist of the glutamatergic N-methyl-D-asparate (NMDA) receptor that is often used as an excitotoxin to cause permanent neuronal lesions. Here we injected 10 nl of 1% IBA bilaterally into the ventrolateral preoptic area (VLPO) of C57BL/6J mice one week prior to testing. The majority of neurons in this nucleus exhibit low rates of firing during wakef...

Watch this Scientific Journal Video about Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention at JoVE.com

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

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

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Unit 1

Local Anesthetics

Drugs for Pain Management: Opioid Analgesics and General Anesthetics

SCIENTISTS IN ACTION

13.4K ビュー.  Perelman School of Medicine, University of Pennsylvania. 立ち直り反射の消失は、長い実験動物でも催眠呼ばれる、意識不明のための標準的な行動の代理を務めています。薬理学的介入によって引き起こされる揮発性麻酔薬感受性における変化は、任意の吸入治療剤の送達のために適合させることができる注意深く制御されたハイスループット評価システムを用いて検出することができる。 

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

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals.  Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled...

time-lapse 3d imaging of phagocytosis by mouse macrophages

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic...

local anesthetic thoracoscopy for undiagnosed pleural effusion

Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe...

Anesthetic Thoracoscopy for Diagnosis of Pleural Effusions

characteristics of pain changes in rats with nerve injury within 24 hours after one-time tuina intervention

Author Spotlight: Exploring the Initiation Mechanism of &#039;Three-Method and Three-Acupoint&#039; Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent...

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

battery of behavioral tests assessing general locomotion, muscular strength, and coordination in mice

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these...

dynamic assessments of coronary flow reserve after myocardial ischemia reperfusion in mice

Author Spotlight: Improved Localization and Monitoring of Coronary Flow Reserve Using Modified PLAX View in Mice

The present protocol describes a modified parasternal long-axis view for swift and precise localization of the left anterior descending artery. This approach is designed to be simpler and more user-friendly while facilitating the examination of dynamic changes in coronary flow reserve following myocardial ischemia-reperfusion in...

CFR Changes Post-Ischemia: Implications for Microvascular Dysfunction

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measuring changes in tactile sensitivity in the hind paw of mice using an electronic von frey apparatus

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory...

monitoring of systemic and hepatic hemodynamic parameters in mice

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in...

measuring local anaphylaxis in mice

Measuring Local Anaphylaxis in Mice

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen...

wide-field, real-time imaging of local and systemic wound signals in arabidopsis

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

assessing the dynamic changes in coronary flow reserve (cfr) before and after ischemia-reperfusion

Assessing the Dynamic Changes in Coronary Flow Reserve (CFR) Before and After Ischemia-Reperfusion

systemic and local drug delivery for treating diseases of the central nervous system in rodent models

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration.  Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in...

local and global methods of assessing thermal nociception in drosophila larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

a mouse model for pathogen-induced chronic inflammation at local and systemic sites

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic...

systemic injection of neural stem/progenitor cells in mice with chronic eae

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous...

systematic assessment of well-being in mice for procedures using general anesthesia

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered...

assessing teratogenic changes in a zebrafish model of fetal alcohol exposure

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1.  We then seek to find potential interventions and rapidly test them in this animal...

Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Salidroside&#039;s Mechanism in Suppressing MCF-7 Cell Migration and Proliferation

Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer mechanisms have been only incompletely elucidated. Hence, this protocol intended to decode the potential of Sal in regulating the PI3K-AKT-HIF-1&#945;-FoxO1 pathway in the malignant proliferation of human breast cancer MCF-7 cells. First, the pharmacological activity of Sal against MCF-7 was evaluated by CCK-8 and cell scratch assays. Moreover, the resistance of MCF-7 cells was measured by migration and Matrigel invasion assays. For cell apoptosis and cycle assays, MCF-7 cells were processed in steps with annexin V-FITC/PI and cell cycle-staining detection kits for flow cytometry analyses, respectively. The levels of reactive oxygen species (ROS) and Ca2+ were examined by DCFH-DA and Fluo-4 AM immunofluorescence staining. The activities of Na+-K+-ATPase and Ca2+-ATPase were determined using the corresponding commercial kits. The protein and gene expression levels in apoptosis and the PI3K-AKT-HIF-1&#945;-FoxO1 pathway were further determined using western blot and qRT-PCR analyses, respectively. We found that Sal treatment significantly restricted the proliferation, migration, and invasion of MCF-7 cells with dose-dependent effects. Meanwhile, Sal administration also dramatically forced MCF-7 cells to undergo apoptosis and cell cycle arrest. The immunofluorescence tests showed that Sal observably stimulated ROS and Ca2+ production in MCF-7 cells. Further data confirmed that Sal promoted the expression levels of pro-apoptotic proteins, Bax, Bim, cleaved caspase-9/7/3, and their corresponding genes. Consistently, Sal intervention prominently reduced the expression of the Bcl-2, p-PI3K/PI3K, p-AKT/AKT, mTOR, HIF-1&#945;, and FoxO1 proteins and their corresponding genes. In conclusion, Sal can be used as a potential herb-derived compound for treating breast cancer, as it may reduce the malignant proliferation, migration, and invasion of MCF-7 cells by inhibiting the PI3K-AKT-HIF-1&#945;-FoxO1 pathway.

Author Spotlight: Exploring Salidroside&#039;s Molecular Mechanisms in Breast Cancer Treatment 

The present protocol describesa comprehensive strategy for evaluating the pharmacological action and mechanism of salidroside in inhibiting MCF-7 cell proliferation and migration.

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer ...

Developing an Exercise Program for Rheumatoid Arthritis Patients

Evaluation of Changes in Hydration and Body Cell Mass with Bioelectrical Impedance Analysis after Exercise Program for Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown benefits for RA patients, its impact on hydration and body cell mass remains uncertain. The presence of pain, inflammation, and joint changes often restrict activity and make traditional body composition assessments unreliable due to altered hydration levels. Bioelectrical impedance is a commonly used method for estimating body composition, but it has limitations since it was primarily developed for the general population and does not consider changes in body composition. On the other hand, bioelectrical impedance vectorial analysis (BIVA) offers a more comprehensive approach. BIVA involves graphically interpreting resistance (R) and reactance (Xc), adjusted for height, to provide valuable information about hydration status and the integrity of the cell mass.
Twelve women with RA were included in this study. At the beginning of the study, hydration and body cell mass measurements were obtained using the BIVA method. Subsequently, the patients participated in a six-month dynamic exercise program encompassing cardiovascular capacity, strength, and coordination training. To evaluate changes in hydration and body cell mass, the differences in the R and Xc parameters, adjusted for height, were compared using BIVA confidence software. The results showed notable changes: resistance decreased after the exercise program, while reactance increased. BIVA, as a classification method, can effectively categorize patients into dehydration, overhydration, normal, athlete, thin, cachectic, and obese categories. This makes it a valuable tool for assessing RA patients, as it provides information independent of body weight or prediction equations. Overall, the implementation of BIVA in this study shed light on the effects of the exercise program on hydration and body cell mass in RA patients. Its advantages lie in its ability to provide comprehensive information and overcome the limitations of traditional body composition assessment methods.

Author Spotlight: Implementation of BIVA for Analyzing Disease Risk Factors in Patients with Low Body Cell Mass 

This protocol assesses alterations in hydration and body cell mass status using bioelectrical impedance vectorial analysis following a dynamic exercise program designed for patients with rheumatoid arthritis. The dynamic exercise program itself is detailed, highlighting its components focused on cardiovascular capacity, strength, and coordination. The protocol details steps, instruments, and limitations.

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown ...

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed pleural effusions. This single-port procedure is conducted with the patient under mild sedation and involves a contralateral decubitus position. It is performed in a sterile setting, typically a bronchoscopy suite or surgical theater, by a single operator with support from a procedure-focused nurse and a patient-focused nurse.
The procedure begins with a thoracic ultrasound to determine the optimal entry point, usually in the IV-V intercostal space along the midaxillary line. Lidocaine/mepivacaine, with or without adrenaline, is used to anesthetize the skin, thoracic wall layers, and parietal pleura. A designated trocar and cannula are inserted through a 10 mm incision, reaching the pleural cavity with gentle rotation. The thoracoscope is introduced through the cannula for systematic inspection of the pleural cavity from the apex to the diaphragm. Biopsies (typically six to ten) of suspicious parietal pleura lesions are obtained for histopathological evaluation and, when necessary, microbiological analysis. Biopsies of the visceral pleura are generally avoided due to the risk of bleeding or air leaks. Talc poudrage may be performed before inserting a chest tube or indwelling pleural catheter through the cannula. The skin incision is sutured, and intrapleural air is removed using a three-compartment or digital chest drainage system. The chest tube is removed once there is no airflow, and the lung has satisfactorily re-expanded. Patients are usually discharged after 2-4 h of observation and followed up on an outpatient basis. Successful LAT relies on careful patient selection, preparation, and management, as well as operator education, to ensure safety and a high diagnostic yield.

Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure.

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed ...

Exploring Obliterative Airway Disease in Lung Transplants

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions.
In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. 
Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months.
In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

Author Spotlight: Investigating the Key Factors of Obliterative Bronchiolitis After Lung Transplantation

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...