Name: in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow
Uploaded: 2015-02-04T15:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

NOTE: The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments.
1. Preparation of Ultrasound-microinjection Instruments and Embryos
<ol>
	<li>Set up stage, mount, and probe (Figure 1) as well as surgical instruments (Figure 2). Place phosphate buffered saline (PBS) solution (pH7.4) in a 37 &#176;C warming bath. Fill microinjection needle entirely with mineral oil, using 1 ml syringe attached to a second flexible 25 G needle, through its base.</li>
	<li>Fix needle onto rotation mount arm, and empty needle of mineral oil solution. Re-fill with 2.5 &#956;l of TL solution. Ensure that no air bubbles are present within the injection needle. Rotate needle arm toward wall, away from the stage.</li>
	<li>Anesthetize the pregnant mother in anesthesia chamber via continuous infusion of isoflurane. When mother is rendered unconscious, transfer the anesthesia to nose tube positioned on the caudal side of the stage, and place mother in supine position with snout in nose tube to allow a continuation of a fully anesthetized state.</li>
	<li>Tape limbs in 45&#176; angles, or with hands and feet rested and positioned overtop ECG/Temperature monitor tabs. It is important that the pregnant dam is continually monitored to ensure that anesthesia is sufficient and that ointment in applied to the eyes to reduce drying.</li>
	<li>Apply hair removal product across lower abdomen of the mother, gently wipe off with dry cotton swabs, and then again with a 70% ethanol-saturated cotton swabs to remove any excess product and hair. Ensure to clean abdominal skin off of all hair at site of incision (Figure 3).</li>
	<li>Perform a laparotomy on the pregnant mother using fine surgical forceps and scissors. Take care to avoid cutting any major vessels or visceral organs. Make first, straight incision of epidermis 1.5-2 cm above vagina and continue cut towards ribs for approximately 2-3 cm. Expose the subcutaneous membrane, locate the linea alba (&#8220;white line&#8221;) (Figure 3), and cut parallel to initial incision, along the same length, to expose internal visceral organs and uterine saccules.</li>
</ol>
2. Extraction of Embryos
<ol>
	<li>Use 6-inch cotton-tipped applicators to maneuver mother and embryos. Gently push (do not force) on skin with applicator to manipulate first embryo out of incision opening. After extraction of the first embryo saccule, gently and slowly pull the remainder of the uterine horn through and onto the mother&#8217;s exterior. Avoid pulling intestine or other organs through incision at this stage.</li>
	<li>Quantify embryos and gently place left uterine horn back into mother. Then begin placing the right uterine horn back into mother starting with the embryo saccule furthest from the vagina on the horn and moving downward. Continue this until only two embryos remain exposed (Figure 4). Lastly, position embryos in a column above and parallel to incision line, being cognizant not to cut off uterine circulation.</li>
	<li>Place clay blocks and position between arms and body, as well as legs and tail. Ensure that the clay surfaces are approximately level with laparotomy incision. Wet the fenestrated Petri dish with 37 &#176;C PBS.</li>
	<li>Grasp extending forceps with right hand and fenestrated Petri dish with left hand (fenestration parallel to embryos) and bring closed forceps ~5-cm through fenestration, from the top of the Petri dish. Open forceps completely without tearing fenestration mesh.</li>
	<li>Maneuver hands above embryos and focus sight on the two embryo saccules. Without (or lightly) touching saccules with fenestration meshing or forceps, slide them through slit and set Petri dish onto mother&#8217;s abdomen. With forceps still extended, manipulate fenestrated mesh to seat it on either side and at base of each embryo (Figure 5) and pull forceps out of slit opening.</li>
	<li>Next, place blue rubber containing wall in Petri dish, without pinching or injuring embryos (Figure 6). Make sure clay blocks are firmly balancing Petri dish, embryo, and rubber-containing wall. Lastly, fill Petri dish with 37 &#176;C PBS until embryos are completely submerged (Figure 6), while controlling for leaks at fenestrated meshing.</li>
</ol>
3. Injection Procedure
<ol>
	<li>Lower injection stage with mother and embryos down using Z-level adjustment knob (Figure 1) and then rotate injection needle and mount arm and line up directly with ultrasound probe, with needle &#189; cm to the left and under the probe tip (Figure 7). Push needle-arm (not needle) 45&#176; away from the probe. Be careful not to hit the tip of the needle with the dish or ultrasound probe.</li>
	<li>Raise injection stage back to original height, with ultrasound probe directly above embryos Probe must be slightly submerged and within 3-4 mm of the embryonic tissue. Use X &#38; Y stage adjustment knobs (Figure 8) to amend embryo/mother/stage position to systematically locate the beating embryonic heart.</li>
	<li>Directly Center of ultrasound screen observe a black center target marker drawn (*). Locate left ventricle (preferable) or atrium using the X &#38; Y stage adjustment knobs (Figure 8) and then raise or lower the stage using revolving knob on the stage to position injection target precisely over the black center marker on the ultrasound screen.</li>
	<li>Use X stage adjustment knob to move embryo to the right, off of the ultrasound screen. Reposition microinjection needle and arm back into place, &#189; cm away from and 90&#176; to the ultrasound probe (Figure 7).</li>
	<li>Using microinjection mount adjustment knobs (Figure 9C-G), position needle with the tip clearly aligned with the center marker. Adjust injection angle (using knobs in Figure 9C &#38; E-G), and X, Y, and Z vectors of micro-needle to ensure needle tip is focused in the correct plane. Retract microinjection needle using solely the &#8220;injection&#8221; knob (Figure 9F), careful not to adjust other dimensions.</li>
	<li>Again, using solely the X fine-adjustment stage knob move embryo back under the probe, with target exactly on the center mark on the ultrasound screen. Ensure that the left ventricle is now exactly on the same X, Y, and Z plane.</li>
	<li>Next, with just the &#8220;injection&#8221; knob on the microinjection mount, slowly puncture embryo with the microinjection needle and gradually bring tip into the left ventricle. Inject the full 2.5 &#956;l of TL solution into left ventricle or until the empty warning beep sounds, by holding &#8220;empty&#8221; and tapping &#8220;fill&#8221; once (Figures 2A &#38; B). At this time of injection observe a shadow emanating from the tip of the needle which is the TL entering into the cardiac chamber (Figure 10). In reverse, quickly retract microinjection needle rotating the &#8220;injection&#8221; knob (Figure 9F) on the microinjection mount when injection is complete.</li>
	<li>Continue on to next embryo and repeat this procedure for remaining embryos following this series of steps and finally place the final embryos back into the dam&#8217;s abdomen. Switch anesthesia to chamber box and gently move mother here for 15 min from time of the last injection.</li>
</ol>
4. Harvesting Embryos and Analysis
<ol>
	<li>Sacrifice dam via cervical dislocation. Expand the laparotomy incision to easily remove uterine horns from the mother. Keep the embryos within uterine sac. Place embryos in chilled PBS.</li>
	<li>Dissect embryos from uterine sac (if necessary collect tissue for genotyping) and place the whole embryo in 4% Paraformaldehyde (PFA) overnight, then into 30% sucrose overnight, freeze in cryostat sectioning medium. Then cut the cryosection and place it onto slides for immunohistochemical analysis. Optionally, use tissues for whole-mount by dehydrating in 100% methanol, following fixation in 4% PFA.</li>
	<li>For immunohistochemical analysis place the cryosections into PBS to remove the OCT. Then block the sections with 10% normal donkey serum for 30 min. Then add PECAM primary antibody at 1:100 dilution overnight at 4 &#176;C. The next day, wash the slides in PBT 3 times for 10 min each and add donkey anti rat 594 secondary and incubate for 1 hr&#160;at RT. 
	NOTE: This is a highly demanding technique requiring the optimization and coordination of ultrasound and microinjection. There is a very steep learning curve related to this technique and requires four to six weeks of mentored injections before one becomes proficient.</li>
</ol>

<ol>
	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

The authors declare that they have no competing financial interests.

Microinjection anesthesia and time frame
With regards to anesthetization of the mother, it is essential to keep airflow constant (2-3 L/min) and at low PSI. The flow of the sedative must be held at approximately 1.75-2 L/min. Simultaneously, timeframes in which the injections take place must be closely monitored and controlled for with each litter. For each litter the injection procedure should be kept under 45 min. The importance of this time limit is paramount to the experiment, as each embr...

During embryonic development two discrete, yet simultaneous, vascular processes take place: angiogenesis, the process whereby a vessel grows from a major pre-existing vessel, and vasculogenesis, which is a de novo formation of vessels from residential endothelial progenitors1,2. Respectively, the former is synonymous with blood flow, while the latter is thought to largely take place in the absence of it.
Simultaneous to blood vessel formation, a cyclical and dynamic process of kidney progenitor cell synthesis, proliferation, and differentiation begins to unfold on embryonic day 9.5 (E9.5). At this point the ureteric bud (UB) invades dorsally into surrounding metanephric mesenchyme (MM), and continues until birth3. Repeated branching of the UB into rapidly condensing metanephric cap mesenchyme begins the formation of the functional units of the kidney, the nephron. With every new generation of UB and nephron, older generations are displaced into inner cortical and medullary regions, where they then undergo further maturation and differentiation within primarily vascular-dense environments. As evidenced by Dressler et al.3, this embryological process is precipitated by inductive signaling, such as crosstalk between UB and MM, and a myriad of extracellular factors 3-6. Two recently investigated extracellular factors within the developing pancreas and kidneys include oxygen tension and blood flow7,8. The latter will be discussed in further detail below with relation to kidney development.
In order to expose the inductive role that blood flow potentially plays in nephron progenitor cell differentiation, as well as in other organogenesis processes, precise and accurate methods of embryonic blood flow mapping is imperative.
Alternative methods of gauging blood flow include the prescription of ultrasound imaging and resin casts9,10. Conclusively, these modes have been shown to be inherently lacking in their capacity to contemporaneously unveil temporal and spatial juxtapositions between blood flow and stem cell differentiation. Resin casts, for example, provide a valid model of vessel patterning within adult tissues, however in immature vessels, such as with embryonic time points, vessels are grossly underdeveloped and leaky. Therefore, resin casts fail to hold within the tiny, oftentimes porous, vessels.
For these apparent obstacles, among others, we chose to incorporate ultrasound-guided in vivo intra-cardiac embryonic tomato lectin (TL) microinjections into our investigations of kidney development. In this procedure we utilize an ultrasound probe to synchronously guide a mounted micropipette needle filled with 2.5 &#956;l of TL solution into the left ventricle of mouse embryos at E11.5, E13.5, E15.5, and E17.5 time points. E17.5 is the latest developmental age as the needles are not strong enough to penetrate the more developed embryo.
The advantages of this microinjection method are abundant. Ultrasound-guided microinjection allows precise positioning of an injection needle within the embryonic left ventricle, passive and controlled expulsion of solution into the beating heart of the animal, minimal damage to heart and surrounding tissues, and the avoidance of sudden cardiac failure and death of the embryo prior to full-body perfusion. With the use of a FITC-labeled TL, any perfused vasculature will maintain the marker along its endothelial apical membrane. In combination with immunohistochemistry, utilizing Pecam (CD31, Platelet endothelial cell adhesion molecule) and various other vascular markers, we are able to clearly distinguish between perfused and un-perfused vessels, as well as characterize any aberrant staining of surrounding tissues.

<table><tbody><tr><td>DAPI</td><td>Sigma Aldrich</td><td>022M4004V</td><td>concentration 1:5,000</td></tr><tr><td>Pecam</td><td>BD Biosciences</td><td>553370</td><td>concentration 1:100</td></tr><tr><td>FITC-Tomato Lectin</td><td>Vector Laboratories</td><td>FL-1321</td><td>concentration 2.5 &amp;micro;l / embryo</td></tr><tr><td>Alexa Fluor-594 (Donkey Anti-Rat)</td><td>Jackson Immunoresearch</td><td>712-585-150</td><td>concentration 1:200</td></tr><tr><td>Microinjection Needle</td><td>Origio Mid Atlantic Devices</td><td>C060609</td><td></td></tr><tr><td>Mineral Oil</td><td>Fisher Scientific</td><td>BP26291</td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td>03-377-20</td><td></td></tr><tr><td>Clay Blocks</td><td>Fisher Scientific</td><td>HR4-326</td><td></td></tr><tr><td>Surgical Tape</td><td>Fisher Scientific</td><td>18-999-380</td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>NC9763655</td><td></td></tr><tr><td>Hair Removal Product</td><td>Fisher Scientific</td><td>NC0132811</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science tools</td><td>14084-08</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science tools</td><td>11064-07</td><td></td></tr><tr><td>Surgical Marking Pen</td><td>Fine Science tools</td><td>18000-30</td><td></td></tr><tr><td>Right angle forceps (for hysterectomy)</td><td>Fine Science tools</td><td>11151-10</td><td></td></tr></tbody></table>

in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

Vascular formation precedes flow in developing kidney
A majority of embryonic tissue (including the kidney) contains a dense vasculature (both unperfused and perfused), even at early embryonic time points. To better gauge and analyze blood flow within the developing kidney we utilized a method of in utero embryonic intracardiac microinjections. With the use of a high-resolution ultrasound to identify the embryonic heart at E11.5 through E17.5, and following the extraction and exposure of a sin...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.7K Views.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

Video: In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Cancer Research

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Medicine

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Biology

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Summary

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Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney