This work was supported by NIH R15 1R15DK106666-01A1 (to J. Liu) and NIH RO1 HL071556 (to J.I. Shapiro).

All animal care and experiments were approved by the Marshall University Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice (10-12 weeks old) were housed in a pathogen free animal facility in designated rooms equipped with cages that supply purified air under a 12 h light/dark cycle. Food and water were supplied ad libitum.
1. Surgery Preparation
NOTE: The surgical instruments and materials are obtained from different sources that are not specific to surgery operations. Instruments and materials from other sources can also be used for the same purpose. See the Table of Materials for a list of surgical instruments.
<ol>
	<li>Preparation for the surgery
	<ol>
		<li>Place the following within reach of the operating table: warming pad, halogen lamp, oxygen tank and isoflurane tank, isoflurane vaporizer, hair clipper, hair removal cream, scales, sterilized operating set, sterilized surgical linen, cotton swabs, 70% alcohol, polyhydroxydine solution (containing 1% iodine), 0.9% NaCl solution, 1 mL syringes and needles (30 G), 3-0 and 4-0 silk sutures, buprenorphine and penicillin, antibiotic ointment, and lubricant eye ointment.</li>
		<li>Clean a cage and place a heat pad under the bedding for mice housing after surgery.</li>
	</ol>
	</li>
</ol>
2. Step-one Surgery: Pole Ligation of Left Kidney
NOTE: Maintain sterile conditions during the surgery. Cover the autoclaved surgical instruments with a sterile pad. Keep more than one set of surgical instruments in hand for more than one surgery to prevent cross contamination during the surgery. If the instruments need to be used again, in the case of multiple surgeries, disinfect the instruments with betadine solution and 70% ethanol, and then sterilize in a germinator glass bead sterilizer for 5 min. Disinfect the operating area with 70% ethanol. Wear a gown, mask (to cover nose and mouth), cap (to cover head), and a pair of sterile gloves. Change gloves after each surgery.
<ol>
	<li>Wash and disinfect hands and wear sterile surgical gloves. Wear a head cap and a mask.</li>
	<li>Disinfect the table with 70% alcohol. Apply lubricant eye ointment to both eyes of the mouse to prevent eye dryness while under anesthesia.</li>
	<li>Place the mouse in an induction chamber of an isoflurane vaporizer system and induce anesthesia with a mixture of 2.5% isoflurane with 100% oxygen (0.8 - 1.2 L/min) for 1 - 2 min. 
	NOTE: The efficacy of anesthesia was determined by the absence of pain reflex elicited by pinching the tail, as well as by the absence of palpebral reflex by touching the eyelid.</li>
	<li>Inject penicillin (40,000 unit/kg body weight, IM) and buprenorphine (0.02 mg/kg body weight, SC) to prevent infection and pain during the surgery.</li>
	<li>Place the mouse on a warming pad (initially set at 42 &#176;C) with a rectal thermometer to maintain and monitor the body temperature around 37 &#176;C during the procedure. Place the mouse nose into the nose cone and ventilate with a mixture of 0.8-1.2% isoflurane with 100% oxygen (0.8-1.2 L/min) during the surgery.</li>
	<li>Place the mouse on the right side. Shave the hair of surgical area with a hair clipper and then remove with the hair removal cream. Disinfect the site with polyhydroxydine solution (containing 1% iodine) followed by 70% ethanol. Align autoclaved surgical linen on the surgical area.</li>
	<li>Make a ~1.0 cm lumbar incision and separate the muscle and fascia to expose the left kidney using the scalpel and forceps (Figure 1a).</li>
	<li>Pull out the left kidney very gently by pulling on the perirenal fat with small blunt forceps. In the process, in order not to disturb the adrenal gland, move the adrenal gland upwards from the kidney with forceps.</li>
	<li>Using blunt forceps, gently tease the connective tissue and adrenal gland from the superior end of the kidney towards the middle pylorus of the kidney where blood vessels enter and exit. Identify and isolate the ureter. 
	NOTE: The ureter lies around the inferior half of the kidney embedded in the connective tissue and clearly visible under light.</li>
	<li>Gently grab the connective tissue and separate from the kidney tissue to ensure that the ureter is not ligated.</li>
	<li>Before polar ligations, use a sterile ruler to identify the superior and inferior 1/3 part of kidney. The mouse kidney usually is around 1.25 - 1.5 cm long.</li>
	<li>Ligate using a 3-0 silk suture string and with proper force, approximately 0.4 cm from the superior portion towards the middle of pylorus and similarly, 0.4 cm from the inferior portion towards the pylorus (Figure 1a, dotted lines).</li>
	<li>After ligating both poles (Figure 1b), allow a 2-min observation time to make sure that there is no bleeding. 
	NOTE: In the process, it is very important to avoid ligation of the adrenal gland, renal artery, and ureter. If the left ureter is ligated, the mouse will die after removal of the right kidney in the step-two surgery. The ligation force can restrict blood flow to the poles without causing intra-renal bleeding. Within 1 - 2 min after ligation, the poles are discolored due to the limit of blood flow to the poles.</li>
	<li>Push the left kidney back to the original place. Close the muscle and skin with 4-0 and 3-0 silk suture strings, respectively. Apply antibiotic ointment to the surgical area for better recovery and disinfection.</li>
	<li>After ligation, house the mouse separately in a cage with a heat lamp over the cage so that the bedding area is close to 37 &#176;C (monitored with a thermometer), before placing the animals in individual cages. 
	NOTE: Monitor the mouse until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Allow easy access to water and food during recovery. Administer penicillin and buprenorphine every 12 h for the first 3 days post-surgery to prevent infection and pain. 
	NOTE: The mouse should recover from the trauma of the surgery within 2 - 3 days and should be back to normal eating, drinking and moving. At this time, house the mouse individually for full recovery. The following must be monitored: drinking, eating, walking patterns, awkward gait, hunched back, ocular and/or nasal discharge, and aggressive behavior. Euthanize the mouse if it shows any sign of seizure, coma, untreatable infection, difficulty to walk, loss of gait, unable to eat and drink, and loss of more than 15% pre-operation weight.</li>
</ol>
3. Step-two Surgery: Removal of Right Kidney
<ol>
	<li>Seven days later, expose the right kidney as described for the left kidney in steps 2.1 to 2.10, except place the mouse on its left side.</li>
	<li>Place the right kidney on the linen and clear surrounding fat and connective tissue with blunt forceps.</li>
	<li>Identify the renal artery and vein, and then place two ligatures (3-0 silk) each with a single loose knot around the vessels. Gently tease the connective tissue and adrenal gland from the superior end of the kidney towards the middle pylorus of the kidney using blunt forceps. Gently displace the ureter from the inferior end and tie all the vessels together (3-0 silk) without causing bleeding.</li>
	<li>Move the two loose ligature knots along the vessels. One towards the abdominal aorta side, and the other one towards the kidney side.</li>
	<li>First tie off the ligature knot (towards the abdominal aorta side) with double knots; a solid knot will change the color of the right kidney. Tie off the ligature knot (towards the kidney side) with double knots. Leave enough space between these two double knots so as to cut the vessels between the two knots without cutting the ligature knots.</li>
	<li>Cut the renal vessels between the two knots and remove the right kidney. Check for possible bleeding of the renal vessels.</li>
	<li>Dry the right kidney using sterile gauze and weigh it.</li>
	<li>Close the muscle incision with 4-0 silk suture string and then close the skin incision with 3-0 silk suture string.</li>
	<li>House the mouse separately in a clean solitary cage with a heat lamp over the cage (step 2.15) with easy access to water and food for at least 24 h to recovery. Administer penicillin and buprenorphine every 12 h for 3 days to prevent infection and pain. 
	NOTE: The mouse should recover from the trauma of the surgery within 2 - 3 days, and should be back to normal eating, drinking and moving.</li>
	<li>After surgery, monitor the mice twice daily for three days, and then once a day for the duration of the experiment. Follow the post-surgical monitoring and treatment procedures described in step 2.16.</li>
</ol>
4. Sham Surgery
<ol>
	<li>For the sham surgery, perform the same surgery procedures, including exposure of the kidney, dissection of tissue, and wound closure, but without the pole ligation of left kidney or removal of right kidney.</li>
</ol>
5. Evaluation of Experimental Uremic Cardiomyopathy
<ol>
	<li>One day before sacrifice, perform a transthoracic echocardiography and capture images18. 
	NOTE: Here, the transthoracic echocardiography was performed with a mouse handling table and rectal thermometer. The echocardiographic images were captured using an 18-38 MHz operating frequency transducer attached to an imaging system. Relative wall thickness (RWT), myocardial performance index (MPI), and left ventricle mass index (LVMI) were calculated as we described18.</li>
	<li>At the time of sacrifice, measure the body weight. Measure hematocrit (HCT) with a HCT centrifuge as per manufacturer&#39;s instructions. Prepare the plasma samples using heparin-coated tubes by centrifuging the blood samples for 10 min at 1,500 x g at 4 &#176;C in a tabletop refrigerated centrifuge. Measure cystatin C, creatinine and BUN using kits following the manufacturer&#39;s instructions. Perform the measurements in duplicate.</li>
	<li>At the end of the experiment, anesthetize the mice with ketamine (90 mg/kg, IP) and xylaxine (10 mg/kg, IP) and sacrifice by thoracotomy. Measure the heart weight after removing it from the chest. Prepare cardiac (LV) and kidney homogenates, as well as determination of Type-1 collagen (COL-1), phosphorylation of c-Src and protein carbonylation, as described before18,19.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The rat 5/6th PNx model has been widely used to study CKD. Because of the much smaller kidney size in mouse, the classical artery ligation and pole resection are very challenging in mouse models with possible high mortality rates and unexpected bleeding/blood loss.
We adopted a mouse PNx model with pole ligation to overcome the bleeding/blood loss. This PNx model takes less time with improved survival rate and high reproducibility. This pole ligation model develops the phenotypic ch...

CKD, also known as chronic renal failure, is a progressive loss of kidney function over time that eventually develops into permanent kidney failure. CKD, from early stage renal disease states to end-stage renal disease (ESRD), is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called &#34;uremic cardiomyopathy&#34;1,2,3. The uremic cardiomyopathy in patients with CKD or ESRD is associated with cardiovascular abnormalities, mainly caused by overload of left ventricular (LV) pressure and/or volume, leading to LV hypertrophy (LVH), LV dilation, and LV systolic dysfunction4,5,6. Cardiac fibrosis is another common pathological process of uremic cardiomyopathy that reduces cardiac compliance resulting in LV diastolic dysfunction. Severe cardiac fibrosis can lead to sudden cardiac death even in those without cardiac symptoms7.
The 5/6th PNx is a commonly used CKD animal model for animal studies involving renal failure, uremic cardiomyopathy and hypertension. PNx is achieved by ablation of 5/6 renal parenchyma. The rat model was initially developed with the two most common protocols employed being surgical resection or infarction. The rat PNx model is an extremely useful model to study uremic cardiomyopathy with substantial elevations in blood pressure, cardiac hypertrophy and impaired diastolic function. Later, mouse PNx models, operated with the similar techniques as the rat model, were developed due to the wide availability and ease of making genetic manipulations in the mouse system.
It is well-documented that systemic oxidant stress is a constant feature of both clinical and experimental uremic cardiomyopathy8,9. Furthermore, oxidant stress contributes to the uremic syndrome10, and plays a critical role in the pathogenesis of the cardiac abnormalities seen in uremic cardiomyopathy11,12,13. To this point, we have demonstrated that the rodent 5/6th PNx model causes physiological, morphological, and biochemical features of uremic cardiomyopathy14,15,16,17. In the mouse PNx model described here, PNx-operated mice developed significant oxidative stress, at least partially mediated by Na/K-ATPase signaling function, which is critical in PNx-mediated experimental uremic cardiomyopathy. Attenuation of the Na/K-ATPase signaling not only reduces oxidative amplification, but also ameliorates the phenotypical changes in PNx-mediated experimental uremic cardiomyopathy18.

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			<td height="21" style="height:21px;width:299px;">Iris Scissors, 11.5 cm, Straight</td>
			<td style="width:235px;">World Precision Instruments</td>
			<td style="width:172px;">501758</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">WPI Swiss Tweezers #5 11 cm, 0.1x0.06 mm Tips</td>
			<td>World Precision Instruments</td>
			<td>504506</td>
			<td></td>
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			<td height="42" style="height:42px;width:299px;">Jewelers #5 Forceps, 11cm, Straight, Titanium</td>
			<td>World Precision Instruments</td>
			<td>555227F</td>
			<td></td>
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			<td height="21" style="height:21px;">Iris Forceps, 10cm, Straight, Serrated</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
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			<td height="21" style="height:21px;">Tweezers #3, 11cm, 0.2x0.4mm Tips</td>
			<td>World Precision Instruments</td>
			<td>501976</td>
			<td></td>
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			<td height="20" style="height:20px;">Medesy Iris 4.5&#34; Curved Scissors, Stainless Steel</td>
			<td>Net23</td>
			<td>3512</td>
			<td>www.Net32.com</td>
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			<td height="20" style="height:20px;">Dr. Slick Iris Scissors; 3.5&#34;</td>
			<td>Avid Max</td>
			<td style="width:172px;">220-1-965-IrisScsrs-P&#160;</td>
			<td>www.Avid Max.com</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Miltex Iris Scissors 4 1/8&#34; Curved</td>
			<td>4mdmedical</td>
			<td style="width:172px;">V95-306</td>
			<td>www.4mdmedical.com</td>
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			<td height="20" style="height:20px;">5&#34; Hemostat clap, curved jaw</td>
			<td>PJTool</td>
			<td>4355</td>
			<td>www.pjtool.com</td>
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			<td height="20" style="height:20px;">sklar Knapp Iris scissors, straight and sharp/blunt 4&#34;</td>
			<td>Medical Device depot</td>
			<td>64-3430</td>
			<td>www.medicaldevicedepot.com</td>
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			<td height="20" style="height:20px;">Kelly Hemostatic Forceps straight 5.5&#34;</td>
			<td>Pilgtimmedical</td>
			<td>FA710-50</td>
			<td>www.pilgtimmedical.com</td>
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			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Hilltop Lab Animals Inc.</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;Mouse Handling Table and rectal thermometer</td>
			<td>Visualsonics</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">MicroScan transducer&#160;</td>
			<td>Vevo 1100</td>
			<td>MS400</td>
			<td></td>
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			<td height="21" style="height:21px;">Vevo 1100 Imaging System&#160;</td>
			<td>FUJIFILM VisualSonics Inc.</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cystatin C ELISA kit and mouse creatinine kit&#160;</td>
			<td>Crystal Chem. Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse BUN ELISA kit&#160;</td>
			<td>MyBioSource Inc&#160;</td>
			<td></td>
			<td></td>
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a mouse 5/6th nephrectomy model that induces experimental uremic cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

The data indicated that this modified 5/6th PNx model by pole ligation is a simple and effective model to investigate uremic cardiomyopathy. At four weeks post-surgery, this PNx model presents impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function. The results are summarized below.
At four weeks post-surgery, the PNx mice developed i...

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A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

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20.2K Views.  Marshall University. This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

Video: A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...