Research in my laboratory is supported by the WKU Department of Biology startup funds, WKU Research Foundation RCAP-I grant # 11-8032 and by a KBRIN-AREA grant funded through a parent grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number 5P20GM103436-13. I would also like to acknowledge Dr. Tian Xu in whose laboratory I was introduced to this technique and Dr. Raymond Pagliarini who first established this technique in the Xu laboratory.

1. Induction of Benign Non-invasive Tumors
<ol>
	<li>Use stocks listed in Table 2 for the induction of benign tumors.</li>
	<li>Prepare a starter culture for the "tester" stock of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C&gt;y+&gt;Gal4, UAS-GFP</li>
	<li>Prepare a starter culture for the "tested" stock of the following genotype: w; FRT40A, UAS-RasV12/CyO</li>
	<li>Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.</li>
	<li>Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.</li>
	<li>Place the cross in a 25 &deg;C incubator and allow the males and females to mate and lay eggs for 24-48 hr.</li>
	<li>Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
	<ol>
		<li>Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).</li>
		<li>Continue to monitor the culture and repeat step 1.7.1 as needed.</li>
	</ol>
	</li>
	<li>Observe wandering third instar larvae under a fluorescence stereomicroscope as outlined in section 3.</li>
</ol>
2. Induction of Invasive Tumors
<ol>
	<li>Use stocks listed in Table 2 for the induction of invasive tumors.</li>
	<li>Use the starter culture from step 1.2 above of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C&gt;y+&gt;Gal4, UAS-GFP</li>
	<li>Prepare a starter culture for the "tested" stock of the following genotype: w; lgl4, FRT40A, UAS-RasV12/CyO</li>
	<li>Collect ten female virgins from the "tester" stock and ten males from the "tested" stock.</li>
	<li>Cross the males and females from the "tester" and the "tested" stock by placing them both in a vial with fly food.</li>
	<li>Place the cross in a 25 &deg;C incubator and allow the males and females to mate and lay eggs for 24-48 hr.</li>
	<li>Check the vials for sufficient egg deposition and for the presence of first instar larvae making sure that the culture is not drying out.
	<ol>
		<li>Add a few drops of autoclaved distilled water to the culture to keep it moist (if the culture is drying out).</li>
		<li>Continue to monitor the culture for dryness and repeat step 2.7.1 as needed.</li>
	</ol>
	</li>
	<li>Observe third instar larvae under a fluorescence stereomicroscope as outlined in section 3.</li>
</ol>
3. Observation of Benign and Invasive Tumors
<ol>
	<li>Use third instar larvae for benign tumor isolation and visualization.
	<ol>
		<li>Wet a paint brush in distilled water, and using this paint brush scrape a few third instar larvae from the wall of the culture vial.</li>
		<li>Place larvae in a depression slide with 1X PBS and then using a wet paint brush scrape off any food material from the larval epidermis.</li>
		<li>Place the larvae in a Petri plate and leave at -20 &deg;C freezer for 30 min to immobilize the larva.
		<ol>
			<li>Alternative method to immobilize larvae using FLYNAP.</li>
			<li>Place a FLYNAP wand dipped in FLYNAP to the vial from 3.1.3.2 and after plugging the vial let plugged vial with larvae stand for 30 to 40 min.</li>
		</ol>
		</li>
		<li>Place the immobilized larva on a glass slide, add a drop of light halocarbon oil, cover with a cover glass and observe under a fluorescence stereomicroscope with the capability to visualize Green Fluorescent Protein (GFP).</li>
		<li>The benign tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.</li>
	</ol>
	</li>
	<li>Select day 10 larvae (after induction) from the culture set in step #2 above for observation of invasive tumors. Day 10 larvae are selected because invasive tumor bearing larvae have an extended larval stage and generally fail to pupariate.</li>
	<li>Follow steps 3.1.1 to 3.1.4 except instead of third instar larvae use day 10 larvae.
	<ol>
		<li>The day 10 invasive tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.</li>
	</ol>
	</li>
	<li>If the fluorescent microscope is equipped with a digital camera then take pictures of the fluorescing benign and invasive tumor bearing larvae.</li>
</ol>
4. Dissection of the Cephalic Complex and Further Observation of Benign and Invasive Tumors
The translucent epidermis of the larva makes it difficult to observe the extent of invasion of tumors. Thus the extent of invasion is better visualized by dissecting the cephalic complex out of the larva. The following steps should be utilized to dissect the cephalic complex.
<ol>
	<li>Using a wet paint brush select third instar larvae (for benign tumors) and day 10 larvae (for invasive tumors) from the respective culture vials.</li>
	<li>Place larvae in a well of a dissection dish containing cold 1X PBS. Use paint brush to scrape off any food particles from the larval epidermis.
	<ol>
		<li>Transfer clean larvae to a fresh well of the dissection dish containing cold 1X PBS.</li>
		<li>Check for presence of GFP fluorescence using a stereomicroscope capable of detecting fluorescence. Discard non-GFP bearing larvae.</li>
	</ol>
	</li>
	<li>Add 1.0ml of cold 1X PBS to a fresh well on the dissection dish and using a pair of forceps transfer a larva bearing either the benign or invasive tumors (the dissection protocol remains the same for these two types of tumors).</li>
	<li>Hold the larva down using a pair of forceps about 2/3rds from the anterior end.
	<ol>
		<li>Use the other pair of forceps and smartly separate the posterior 1/3rd of the larva and discard it.</li>
		<li>Let go of the anterior 2/3rds of the larva. As the pressure inside the larva is released the contents of the larva will ooze out of the body cavity.</li>
		<li>Use a pair of forceps to remove the gut, fat body and other inner contents of the larva that have oozed out of the body cavity.</li>
		<li>Use a pair of forceps to hold the mouth hook of the larva and push it into the larval epidermis and using the other pair of forceps invert the larva completely.</li>
		<li>Use the forceps to gently and carefully remove the fat body, salivary glands, gut, the wing disc complex and any tracheal tubes. The cephalic complex should now be visible attached to the mouth hook of the inverted larva. The brain hemispheres and the ventral nerve cord (VNC) would still be connected to the larval epidermis through the nerves emanating from the VNC.</li>
		<li>Use a pair of forceps to gently break the connections between the cephalic nerves and the larval epidermis.</li>
		<li>As the nerve connections between the VNC and the larval epidermis are broken and excess fat body and other tissue removed, the cephalic complex would become clearly visible and will be attached to the larval epidermis only at the mouth hooks.</li>
		<li>The cephalic complex can be left attached to the larval epidermis if the complex needs to be fixed for downstream applications like antibody staining or for later visualization.</li>
		<li>If no further downstream applications would be performed then the cephalic complex can be detached from the larval epidermis and placed in a drop of a glycerol based mounting media for further observation and analysis. Use VECTASHIELD as the mounting media.</li>
	</ol>
	</li>
</ol>

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I have nothing to disclose.

Cancer is a complex disease with a much better understanding today than in the past. However, much still needs to be learnt and explained before we have a complete picture of the underlying mechanisms. The simple protocol presented here makes it possible to genetically induce benign and invasive tumors in a whole organism and then study the biology associated with the progression of tumors in this model. Most of the existing techniques in Drosophila and other organisms either utilize a cell culture based system ...

Progression of tumors from a benign to a metastatic state is a step wise process that is characterized by evasion of protective mechanisms present in the body 8. For example tumor cells in the body must be able to evade apoptosis and the immune system, breakthrough the specialized extracellular matrix (ECM) called Basement Membrane, and overcome any social controls imposed by the surrounding cells 8. It is through a step wise progression that the cancer cells acquire the ability to migrate and colonize distant sites in a process called metastasis. Our understanding of how the tumor cell overcomes the barriers imposed by the body is still in its infancy, however, the emerging picture from research done thus far points to a repeated use of normal developmental processes and signaling pathways by the cancer cells 11-13.
The fruit fly Drosophila melanogaster has contributed tremendously to our understanding of normal development and disease through use of sophisticated genetic techniques developed over the past several decades 14-17. Using mutagenesis and overexpression tools we have arrived at a better understanding of various oncogenes and tumor suppressor genes 18-22. However, tumor metastasis is a result of cooperation between several genetic lesions that has been studied primarily in cell culture models 23,24 as well as various xenograft models 25-27. These models though powerful have their limitations as they do not mimic entirely the conditions found in a living organism. Furthermore, transgenic models available in mice are cumbersome and not conducive to genetic analysis of invasive behavior 28,29. Several studies have attempted to understand invasion of tumor cells in Drosophila 30,31. These techniques primarily utilize transplantation of primary tumors to hosts and then rely on tracking the transplanted tumors for invasion of neighboring tissues 32,33. A powerful technique called MARCM 10 was adapted by Pagliarini and Xu to model tumor invasion in Drosophila 9. This elegant genetic modeling of tumor invasion exploited the cooperation between an activated oncogene and the loss of cell polarity. The power of this modeling lies in the fact that the invasive tumors are created in an intact organism thus circumventing the need for transplantation of tissues. To bring about the oncogenic cooperation, an activated oncogene like RasV12 is expressed in clones of cells in the larval eye-antennal disc. As a result of the MARCM technique these clones are also marked with green fluorescent protein (GFP) for easy visualization and are made homozygous for cell polarity mutants like lethal giant larvae, scribbled, and discs large. The result is GFP tagged invasive tumors in the cephalic complex. In this report I demonstrate how to induce, and visualize these invasive tumors both in the context of an intact larvae and in dissected out cephalic complex. The tumor induction presented here utilizes reagents on the second chromosome of Drosophila. In Table 2, I provide a listing of stocks on X and 3rd chromosomes that can be utilized for the same purpose. I believe that this simplified protocol will make this technique readily accessible to researchers interested in understanding the molecular basis of tumor progression.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

As a result of the protocol presented here the user will be able to induce benign tumors by overexpressing an activated oncogene in the larval eye antennal imaginal disc. The user will also be able to induce invasive tumors in the eye antennal disc by overexpressing an activated oncogene in clones of cells also mutant for a cell polarity gene. The tumors could be easily visualized with the help of a fluorescent stereomicroscope as "green fluorescent" tissue in the whole larvae or in cephalic complexes that have been diss...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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9.4K Views.  Western Kentucky University. Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Video: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

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 ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...