The authors wish to thank Malinda Spry for technical assistance. We also acknowledge current and past funding sources: the National Cancer Institute (R01 CA131075, R01 CA131075-02S1), the Wendy Will Case Cancer Research Fund, the Markey Cancer Foundation, the Children's Miracle Network and the Jennifer and David Dickens Melanoma Research Foundation.

1. Preparation of Forskolin for Topical Administration from a Crude Root Extract of the Plectranthus barbatus (Cohleus forskohlii) Plant
<ol>
	<li>Protocols for murine experiments followed the guidelines for ethical conduct in the care and use of animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky (Protocol # 00768M2004). The root extract is made up at 40% weight/volume in a standard dermatologic base of 70% ethanol, 30% propylene glycol.</li>
	<li>Weigh 200 g of crude forskolin root extract and transfer it to a beaker. To make 500 ml of a 40% (w/v) solution, resuspend 200 g of crude forskolin root extract by adding most but not all of the volume of the vehicle (70% ethanol, 30% propylene glycol) and bring the solution to roughly 450 ml.</li>
	<li>Stir for an hour at room temperature. The solution will be somewhat viscous and may require manual agitation to "lift" the extract into solution before the stir bar is able to take over.</li>
	<li>After an hour of stirring, pour the mixture into a graduated cylinder and bring the volume to 500 ml using vehicle that has been used to "rinse out" the beaker that was used to stir the slurry (to maximize recovery of forskolin from the beaker).</li>
	<li>Transfer the slurry to 50 ml polypropylene centrifuge tubes. Centrifuge (1,500 x g, room temperature, 15 min) using a table-top centrifuge. At this point, the insoluble material will be fairly compacted, allowing the supernatant to be easily poured off.</li>
	<li>Filter the solution through a 0.22 &mu;m cellulose acetate membrane to remove any residual insoluble material from the extract. We use a bottle-top system designed for cell culture, along with the use of pre-filters that come with the unit to prevent premature clogging of the membrane from insoluble components of the root extract. When making large volumes of the extract, filter roughly 100 ml at a time, changing the pre-filter between each added volume.</li>
	<li>When stored at room temperature, the extract maintains biologic activity up for up to one year.</li>
</ol>
2. Preparation of C57Bl/6 K14-Scf Mice for Topical Treatments
<ol>
	<li>Remove dorsal fur from the animals by electric shearing. Briefly anesthetize the animals with inhalational isoflurane to facilitate shearing of dorsal fur with electric shears outfitted with a 0.25 mm surgical preparatory head (Fisher Scientific). Preferably only use one type of anesthesia (e.g. ketamine/xylazine) to minimize risk of anesthetic overdose. The saturated inhalation chamber carries occupational risk when used outside a fume hood and delivers unknown amounts of anesthetic to animals. Ideally a precision vaporizer should be used. </li>
	<li>To remove residual hair stubble, treat the animals with a chemical depilatory. Administer anesthesia to the animal with an i.p. injection of ketamine 40 mg/kg and xylazine 4 mg/kg</li>
	<li>Once animals are adequately anesthetized (as judged by toe pinch), apply a fingertip-sized amount of depilatory cream to sheared dorsal skin using a gloved finger. Rub the cream into the skin for 30-60 sec or until hairs can clearly be seen in the cream as it is being moved around. Leave the cream on only for the minimum amount of time required for hair removal as prolonged exposure leads to chemical burning of the skin, epidermal breakdown and death from loss of epidermal integrity.</li>
	<li>Wipe the dorsal skin with water-soaked gauze pads repeatedly until all cream has been removed. Dry animals using soft paper towels, and allow them to recover in a warm secluded location (e.g. clean cages placed on a warming pad). Depilate the animals one-by-one and monitor closely throughout the procedure.</li>
</ol>
3. Topical Administration of Forskolin or Vehicle Control
<ol>
	<li>Animals should be treated one at a time. Briefly anesthetize with inhaled isoflurane by placing the mouse on top of a form-fitted nylon air-permeable filter under which has been placed an isoflurane-saturated paper towel in an isoflurane-saturated lidded clear glass jar in a fume hood. Expose the mouse to isoflurane for a sufficient time as to suppress voluntary muscular movements but to preserve spontaneous respiration (typically 10-20 sec). Leaving the animal in isoflurane too long will result in respiratory suppression and death. It is better to err on the side of "going light" and having to re-expose the mouse briefly to more isoflurane rather than to over-expose the animal to the drug and risk death.</li>
	<li>Remove the animal from the isoflurane chamber and place on a clean absorbent bench pad.</li>
	<li>Using a 1,000 &mu;l micropipette outfitted with a disposable polypropylene tip, draw up 400 &mu;l of 40% crude forskolin extract (vehicle control animals will receive 70% ethanol, 30% propylene glycol alone).</li>
	<li>Transfer the extract onto the back of the animal by dripping it onto the skin and then, using the side of the pipette tip, smear the extract over the dorsal skin until all the skin has been covered. There is no need to blot the skin after application.</li>
	<li>Return the mouse to its cage, and carefully observe until it recovers from anesthesia.</li>
	<li>In order that non-pigment cAMP effects should not confound UV sensitivity experiments, discontinue all topical treatments 2 days prior to UV exposure (pigment effect lasts several days beyond last topical treatment).</li>
</ol>
4. Skin Color Measurement by Reflective Colorimetry
<ol>
	<li>Briefly anesthetize the mouse with inhaled isoflurane (see above).</li>
	<li>Calibrate a Minolta colorimeter by placing the portable head on the standardized white surface provided with the colorimeter.</li>
	<li>Place the portable measuring head of the colorimeter flush with the dorsal skin of the animal ensuring that the 1 cm2 round aperture is completely pressed onto the skin. Take at least three separate measurements in different areas of dorsal skin.</li>
	<li>Calculate mean L* score &plusmn; SD per animal and per treatment group. Reflective colorimetry can be done at any point in the experiment.</li>
</ol>
5. Determination of UV Sensitivity by Calculation of "Minimal Erythematous Dose" (MED)
<ol>
	<li>Use animals that have been pre-treated with either vehicle or forskolin as described above. Anesthetize animals with intraperitoneal injection of a standard mixture of ketamine and xylazine (see above).</li>
	<li>Prepare a piece of UV-occlusive tape for MED testing. To generate holes in the tape, use a heavy-duty hole punch with a 1 cm2 circular cutout (Figures 2A and B). Having holes of a defined size and symmetric arrangement in the tape facilitates recognition of skin changes after irradiation. Over each hole in the tape, apply a small but easily detachable piece of tape that can be removed at defined times during UV exposure to allow administration of different UV doses.</li>
	<li>Once animals are adequately sedated, place the tape on the dorsal surface. Eye lubricant should always be utilized under anesthesia.</li>
	<li>Turn on the UV source consisting of two Westinghouse F15T8UV-B lamps with a peak output of 313 nm and a range of 280-370 nm. Allow the lamp to equilibrate to a constant UV output as measured by a UV photometer with a UVB sensor (generally takes a few minutes for the lamps to warm up).</li>
	<li>Based on the UV transmission rate as measured by the UV Photometer, calculate UV exposure time for each desired dose. For example, our lamp's UVB output measures 2.4 mW/cm2. Therefore, to administer 5 kJ/m2, the skin would need to be exposed to 208 sec (which is 3 min and 28 sec) of UVB radiation, as calculated below: 
	<img alt="Equation 1" fo:src="/files/ftp_upload/50670/50670eq1highres.jpg" src="/files/ftp_upload/50670/50670eq1.jpg" /></li>
	<li>Place sedated animals (each with occlusive tape in place) ventral surface down to ensure even UV exposure. To administer the chosen doses of UV radiation, sequentially remove the small occlusive tapes covering the holes to expose 1 cm2 areas of skin to the correct doses of radiation. Therefore, using the above example, if 40 kJ/m2 is the largest dose in the experiment, then the animal would be under the lamp for 27 min and 47 sec total and the skin in the 40 kJ/m2 condition would have no overlying tape the entire time. However, tape overlying the 5 kJ/m2 condition would be removed when there is 208 sec remaining in the exposure. Timing of tape removal should be done so that each condition ends simultaneously.</li>
	<li>After UV exposure, peel off the tape from the dorsal skin carefully, taking care not to rip the skin with sudden or overly-forceful movements. Place animals in a warm quiet place to allow recovery from anesthesia.</li>
	<li>Monitor mice for 24-48 hr to look for discreet areas of erythema (redness) or edema (swelling) corresponding to the anatomic sites exposed to the specific dose of UV irradiation. Document skin findings photographically.</li>
	<li>MED value corresponds to the minimum dose of UV that causes inflammation as defined by erythema and/or edema of the entire exposed circle of skin. Note that pigmentation of the skin can challenge determination of MED, however, erythema and edema can still generally be accurately assessed, thanks in part to the defined shape of the apertures in the tape during UV exposure.</li>
</ol>
6. Statistical Analysis
Analyze data between cohorts of mice by one way ANOVA with Bonferroni post test (Graph Pad PRISM). p values &lt;0.05 are considered statistically significant.

The authors declare that they have no competing financial interests.

Using an animal model of the fair-skinned human, we find that topical application of a forskolin-rich crude root extract robustly darkens the epidermis by stimulating melanin production in the skin. Epidermal melanization is dependent on the expression of stem cell factor in the basal epidermis, as occurs in human skin but not in genetically-unmodified mouse skin. The dorsal skin of genetically-unmodified mice lacks sufficient numbers of interfollicular melanocytes to impart pigment to the skin. Only in the setting of co...

The incidence of melanoma, the most deadly form of skin cancer, has increased dramatically over the last several decades in United States, particularly among fair-skinned individuals. Strong molecular and epidemiologic evidence implicates UV radiation as a major causative environmental factor 2-5. Increased UV exposure in the form of sun exposure and tanning bed use is likely to be responsible for much of increases in melanoma incidence 6-7. Melanoma risk seems particularly linked with sunburns 8, especially those early in life 9-10. Risk of sunburn is linked not only to dose and intensity of UV exposure, but also by inherited factors that influence cutaneous response to UV radiation. Skin pigmentation is one of the most important determinants of UV sensitivity, risk of sunburn and cancer risk. Melanoma occurs roughly twenty times more frequently in light-skinned persons compared to dark-skinned individuals 11-13.
	Melanin, a pigment produced by melanocytes in the epidermis, is the main determinant of skin complexion. Melanin comes in two major varieties: (1) eumelanin, a dark brown/black pigment effective at absorbing the energy of UV radiation, and (2) pheomelanin, a reddish/blonde pigment less effective at preventing penetration of UV photons into the skin. Skin color, UV sensitivity and melanoma risk are largely determined by epidermal eumelanin content 14-15. The more eumelanin in the epidermis, the less UV photons can penetrate into the skin. Because of low innate levels of eumelanin, fair-skinned individuals are much more prone to acute and chronic effects of UV radiation 16-18.	
	Skin pigmentation, melanoma risk and the ability to "tan" after UV exposure all correlate with the signaling ability of the melanocortin 1 receptor (Mc1r), a Gs-coupled seven transmembrane surface receptor on melanocytes 19-22. When Mc1r binds its cognate high-affinity ligand, &alpha;-melanocyte stimulating hormone (&alpha;-MSH), there is activation of adenylyl cyclase and production of the second messenger cAMP 23. The normal physiologic response of the skin after UV exposure includes epidermal production of &alpha;-MSH by keratinocytes 24-29. We and others hypothesize that keratinocyte-derived &alpha;-MSH binds to Mc1r on epidermal melanocytes, initiating downstream production of the cAMP second messenger through activation of adenylyl cyclase 30. cAMP levels control many aspects of melanocyte differentiation, including survival pathways, DNA repair and pigment synthesis. Mc1r signaling and cAMP clearly induce pigment enzyme levels and eumelanin production. When Mc1r signaling is intact and melanocytic cAMP levels are robust, eumelanin is produced and the skin darkens. However, if Mc1r signaling is defective and cytoplasmic cAMP levels remain low, pheomelanin is produced instead 1. Eumelanin synthesis can be stimulated pharmacologically by agents that raise cAMP levels 1,14,31-35.	
	Since the Mc1r protein is a major regulator of melanoma risk in humans 36-46, we are interested in mechanisms by which Mc1r protects melanocytes against UV-induced carcinogenesis. As a foundation for our studies, we generated a transgenic Mc1r-variant murine model on a pure C57BL/6 genetic background 1. In this model, stem cell factor (Scf) is constitutively expressed in the basal epidermis and epidermal interfollicular melanocytes are retained in the skin throughout life 47, in contrast to the non-transgenic mice in which melanocytes localize to the dermis in hair follicles. With the K14-Scf transgene incorporated, the epidermis becomes pigmented with the particular melanin pigments characteristic of the pigment strain of the animal 1. K14-Scf mice on the C57BL/6 genetic background with wild type Mc1r signaling have jet-black skin characterized by very high levels of eumelanin pigment. Not surprisingly, these animals are highly UV-resistant. In contrast, genetically matched K14-Scf C57BL/6 animals that harbor a mutant inactive Mc1r have almost no eumelanin in the epidermis. Instead, these "extension" animals (Mc1re/e) have a fair skin complexion caused by deposition of pheomelanin pigment (Figure 1A) and are much more UV-sensitive 48-49.	
	Pharmacologic compounds with chemical properties that allow penetration into the skin have been shown to potently induce eumelanin in the extension (Mc1re/e) K14-Scf animal model by directly manipulating cAMP levels in epidermal melanocytes in the skin. Melanin upregulation in this model has been reported by adenylyl cyclase activation 1 as well as phosphodiesterase 4 inhibition 35. In this article, we demonstrate the preparation and topical application of forskolin in extension (Mc1re/e) K14-Scf animals which model the fair-skinned UV-sensitive human. We show that twice daily application of the drug promotes accelerated melanization, that skin darkening is due to epidermal deposition of melanin pigment and that induced epidermal melanin protects against UV-induced sunburn through measurement of "minimal erythematous dose" (MED) 48.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Coleus Forskoli extract 20%</td>
			<td>Buckton Scott USA Inc. </td>
			<td>n/a</td>
			<td>Princeton, NJ</td>
		 </tr>
		 <tr>
			<td>Isothesia, Isoflurane , USP</td>
			<td>Butler Schein</td>
			<td>NCD 11695-6776-1</td>
			<td>Dublin, OH, USA</td>
		 </tr>
		 <tr>
			<td>Xylazine</td>
			<td>Anased Injection</td>
			<td>LA04612</td>
			<td>Shenandoah, Iowa, USA</td>
		 </tr>
		 <tr>
			<td>Ketamine HCl, USP</td>
			<td>Putney</td>
			<td>NDC 26637-411-01</td>
			<td>St. Joseph, MO, USA</td>
		 </tr>
		 <tr>
			<td>Ethanol</td>
			<td>Decon Labs.</td>
			<td>2705</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Propylene glycol</td>
			<td>Adesco</td>
			<td>05751L</td>
			<td>Solon, OH, USA</td>
		 </tr>
		 <tr>
			<td>Depilatory cream, Nair</td>
			<td>Church &amp; Dwight</td>
			<td>JF-11 4381322</td>
			<td>Priceton, NJ</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Germicidal Hg Lamp UV-B</td>
			<td>Westinghouse</td>
			<td>F15T8UV-B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Radiometer photometer</td>
			<td>International light</td>
			<td>1LT400A</td>
			<td>Peabody, MA,USA</td>
		 </tr>
		 <tr>
			<td>Chromameter</td>
			<td>Konica Minolta</td>
			<td>CR-400</td>
			<td>Ramsey, NJ, USA</td>
		 </tr>
		 <tr>
			<td>Data Processor for 	Chromameter CR-400</td>
			<td>Konica Monilta</td>
			<td>DR-400</td>
			<td>Ramsey, NJ, USA</td>
		 </tr>
		</tbody>
 </table>

pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

C57BL/6 mice were generated on eumelanotic, pheomelanotic or amelanotic backgrounds incorporating the K14-Scf transgene as described (Figure 1A). Cohorts of fair-skinned extension (Mc1re/e, Tyr+/+) mice were treated topically with twice daily doses of either vehicle (70% ethanol, 30% propylene glycol) or 40% crude Coleus forskohlii root extract (80 &mu;M per dose) for 5 days (Figure 2B). Effects of topical treatments on epidermal pigmentation were ...

Watch this Scientific Journal Video about Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model at JoVE.com

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

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18.1K Views.  University of Kentucky College of Medicine. Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Video: Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...