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 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.</li>
	<li>Each chamber is mounted on a rack that sits inside a water bath. The rack is fitted so that only the lower portion of the chambers (below the gas inlets) is submerged. For stability, the back end of the chamber rests on a support so that the entire chamber sits horizontally. This ensures even contact of the entire chamber with the bath.</li>
	<li>Polyethylene tubing connects an oxygen tank to an anesthetic vaporizer, and then passes through a 10 L/min flow meter. The tubing splits into 25 small-diameter resistors of equal length to ensure equal flow is delivered to each of the 24 chambers and to an agent analyzer.</li>
	<li>Vacuum lines exit each chamber at the opposite end 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 main vacuum line ensures atmospheric pressure conditions within each chamber.</li>
	<li>The bath is filled with enough water to fully contact the bottom of each chamber. The water is circulated through the bath and maintained at a constant 37&#160;oC by a pump.</li>
</ol>
2. Check the System Prior to Exposure
<ol>
	<li>Check that the temperature of the water bath is 37&#160;oC throughout the bath.</li>
	<li>Flow oxygen at a rate of 5 L/min (200 ml/min per chamber + agent analyzer). Submerge each chamber under water and look for bubbles or entry of water into the chamber, both of which are indicative of leaks. Seal any leaks before beginning the experiment.</li>
	<li>For each chamber, connect a 500 ml/min flow 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 5 L/min flows will be distributed evenly so that each chamber receives 200 ml/min flow. Any chamber not receiving the expected flow should have its inflow and outflow tubing checked for obstructions.</li>
	<li>Calibrate the agent analyzer to ensure a reading of 0.00% isoflurane when 100% oxygen is flowing.</li>
</ol>
3. Implant Temperature Transponder
<ol>
	<li>One week prior to habituation, anesthetize each mouse with 2% isoflurane.</li>
	<li>Sterilize the dorsal neck area with betadine.</li>
	<li>Inject a temperature transponder subcutaneously between the shoulder blades using the sterile, prepackaged injector needle.</li>
	<li>Monitor the injection site daily for infection and migration of the transponder.</li>
</ol>
4. Habituate Animals to Testing Chambers
<ol>
	<li>Four days before the first assessment, place all mice into individual chambers for 2 hr&#160;with 100% oxygen flowing.</li>
	<li>Repeat step 4.1 daily for the four days prior to assessment to avoid the confounding effects of stress due to a new environment.</li>
</ol>
5. Perform the Pharmacological Intervention that You Wish to Test for Effects on Anesthetic Sensitivity
<ol>
	<li>This intervention may be a stereotaxic injection into a specific part of the brain24, an intravenous or intraperitoneal injection25, or delivery of a drug to a specific brain area via cannula26.</li>
	<li>Because these procedures themselves may change anesthetic sensitivity compared to a na&#239;ve animal, a proper control group should undergo the same procedure with vehicle injections.</li>
	<li>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 6 below; otherwise, skip to step 7.</li>
</ol>
6. Assess Anesthetic Sensitivity using the Stepwise EC50 Determination for Induction and Emergence
<ol>
	<li>Place each animal into individual chambers with 100% oxygen flowing.</li>
	<li>Set the isoflurane concentration to 0.4%* for 15 min. During the last 2 min&#160;of this period, assess each animal&#39;s righting reflex by gently rolling the chamber until the mouse is placed on its back. The righting reflex is considered to be intact if and only if the mouse is able to restore all of its paws to the floor of the chamber within 2 min.
	<ol>
		<li>*Note that 0.4% isoflurane is a subhypnotic dose in C57BL/6J mice. If any mice lose their righting reflex at the first step, the initial dose was too large and should be reduced on subsequent days.</li>
	</ol>
	</li>
	<li>Record the state of righting reflex for each mouse and scan each mouse for temperature data. A template record is shown in Table 1.</li>
	<li>Increase the isoflurane concentration by ~0.05% for 15 min&#160;and repeat step 7.2. Continue to do this until all animals have lost their righting reflex.</li>
	<li>Optional: repeat the same procedure for decreasing step-wise isoflurane doses until all animals have regained their righting reflex (see step 6.3).</li>
	<li>To end the experiment, turn off the isoflurane and flush the entire system with 100% oxygen for 15 min. This will help to prevent hypoxia as the mice recover before being returned to their home cages and will protect the experimenter from any anesthetic exposure.</li>
	<li>Optional: if the number of animals or the number of anesthetic concentrations are limited due to resource or time constraints, the curve-fit parameter estimates-particularly the Hill slope-may have underappreciated, falsely low error estimate. In such cases, it may be necessary to repeat the anesthetic sensitivity measurement described in steps 6.1-6.6 on up to two additional experimental days to fully obtain the true Hill slope&#39;s parameter and its corresponding error estimation.</li>
</ol>
7. Assess Short-term Changes in Anesthetic Sensitivity with Time to Emergence
<ol>
	<li>Place each animal into individual chambers with 100% oxygen flowing.</li>
	<li>Set the isoflurane concentration to 1.2%, which corresponds to the induction ED99 for wild-type C57BL/6J mice20. Maintain for 30-60 min&#160;depending upon the expected duration of action of the acute intervention.</li>
	<li>Confirm LORR in all animals by gently rolling each chamber until mice are placed on their backs.</li>
	<li>Turn off isoflurane and flow 100% oxygen. Measure the time until each animal regains its righting reflex. This is defined by placement of all four paws on the floor of the chamber and confirmed by the presence of three consecutive tests with an intact righting reflex.</li>
</ol>

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The authors have nothing to disclose.

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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		<tr height="42">
			<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>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<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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13.5K Views.  Perelman School of Medicine, University of Pennsylvania. 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.

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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

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, ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

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 ...

Anesthetic Thoracoscopy for Diagnosis of Pleural Effusions

Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

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 ...