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, ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

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 ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...