Research to Prevent Blindness; Bartly J. Mondino M.D., Director of the Jules Stein Eye Institute, UCLA; and Ramiro Ramirez-Solis, Jacqui White, and Jeanne Estabel at the Sanger Institute, Wellcome Trust Genome Campus. This research complies with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

1. Blunt Dissection: Enucleation of the mouse eye in unfixed specimens
<ol>
 <li>Pull apart the eyelids to improve exposure and access to the posterior globe (eyeball) surface. </li>
 <li>Place a curved dressing forcep behind (under) the globe in the orbit (eye socket). The Mahajan Sharptip dressing forcep is a custom instrument with pointed tips to make this step easier (see materials and reagents table).</li>
 <li>Close the forcep and grasp the orbital connective tissue and optic nerve behind the globe while being careful to avoid squeezing the globe.</li>
 <li>Gently pull the forcep upward and pluck the eyeball from the orbit. The white thread-like tissue is the optic nerve.</li>
 <li>Place the eye in PBS. Using a 30-gauge needle, make a single puncture wound immediately posterior to the limbus by inserting the needle 1-2 mm into the eyeball. This allows fixatives, such as glutaraldehyde, that are otherwise unable to penetrate the eye tissue, to directly enter the eye. Creation of this puncture wound may not be necessary if a fixative, such as paraformaldehyde or alcohol, is capable of diffusing into ocular tissues of interest.</li>
 <li>Place the entire eye immediately into fixative for immersion fixation.</li>
</ol>
2. Sharp Dissection: Enucleation of the mouse eye following perfusion fixation
<ol>
 <li>Use a curved colibri forcep to hold the eyelid away from the globe.</li>
 <li>Orient curved Westcott dissection scissors parallel to the globe, while aiming towards the back of the orbit.</li>
 <li>Cut the fixed connective tissue that surrounds the globe from the inferior, medial, superior, and lateral sides.</li>
 <li>Place the curved forcep behind the globe, grasp the connective tissue without pressing on the globe, and pull forward to enucleate the eye. Since the orbital connective tissue is stiff from perfusion fixation, additional cutting of orbital tissues may be necessary to completely release the globe.</li>
</ol>
3. Fixation and Packaging
Shipping of fixed tissues must follow your institutional and postal service requirements including appropriate labels and packaging. The following protocol is an example of shipping a non-infectious biological sample at ambient temperature. This requires 3 layers of packaging but no class A/B biological substance or liquid nitrogen labels.
<ol>
 <li>Place the eye into a 5-mL glass scintillation vial with at least 3-mL fixative, or post-fixative buffer if fixation is short. Write the tracking number on the lid of the vial and seal it with Parafilm.</li>
 <li>Place scintillation vials into a biological sample approved shipping container. In our institute, for example, approved containers require three layers: a sealed container in which the sample is placed, an absorbent middle layer, and a protective outer layer. </li>
 <li>Place the container into a bubble-wrap package and then into an approved laboratory shipping container along with appropriate documentation.</li>
</ol>
4. Representative Results
Histological examination of eyes can be performed by a variety of methods including Hematoxylin and Eosin staining, enzymatic expression detection, transmission electron microscopy, and immunohistochemistry. Following immersion fixation in 4% paraformaldehyde, tissue was embedded in paraffin and sectioned on a microtome. In high-throughput phenotyping, we analyze pupil-optic nerve sections that sample all tissue types in the eye (Figure 1). Sections of tissue were also examined for lacZ expression (Figure 2), transmission electron microscopy of cellular organelles (Figure 3), and expression of specific molecules with antibodies (Figure 4).
In our phenotype screening method, it was not important to maintain orientation of the eye with respect to the nasal (medial canthus) and temporal (lateral canthus) sides. There are at least two options if eye orientation is necessary. After enucleation, we have applied light cautery to the temporal cornea at the three o&#x2019;clock position with a handheld cautery device. The lesion is obvious on histology and does not get lost during processing like histological inks. The downside is that cautery damages the cornea, which may be a critical tissue for examination. The other option requires a skilled anatomist that can identify the extraocular muscle insertions. Under a stereomicroscope, identification of the inferior and superior oblique muscle insertion marks the inferior and superior and aspect of the globe.3 With either orientation method, it is important to track the specimen as a right or left eye. Together, this will allow exact globe orientation prior to sectioning.
<img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Figure 1" /> 
Figure 1. Mouse eye histological images after remote acquisition. Mouse eyes were enucleated by sharp dissection following perfusion fixation with 4% paraformaldehyde. A. Pupil-optic nerve section stained with Hematoxylin and Eosin shows preservation of all tissue substructures. ON, optic nerve. (green scale bar = 500 microns) B. A higher magnification view of the normal retina shows its laminar structure: RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer (photoreceptor cell)(arrow); IS, photoreceptor inner segments (arrow head); OS, photoreceptor outer segments; RPE, retinal pigment epithelium; C, choroid. (green scale bar = 50 microns) C. Compared to the normal retina, this specimen is thinner due to photoreceptor degeneration. The ONL, IS, and OS are completely absent (arrow). (green scale bar = 50 microns) 
<img src="/files/ftp_upload/3184/3184fig2.jpg" alt="Figure 2" /> 
Figure 2. lacZ expression in transgenic mice. Eyes were enucleated by blunt dissection and immersed in 1 mg/ml X-gal solution (5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.02% NP40, and 0.1% sodium deoxycholate) overnight at 37&deg;C. Eyes were fixed in 2% formaldehyde/0.2% glutaraldehyde for 30 minutes and sectioned on a microtome. X-gal product (blue) labels: A. Ciliary epithelium, B. corneal epithelium, and C. the retinal ganglion cell (RGC) layer in the retina. INL, inner nuclear layer; ONL, outer nuclear layer.
<img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Figure 3" /> 
Figure 3. Transmission electron microscopy of retinal pigment epithelium (RPE). After blunt dissection and creation of a puncture wound, eyes were immersion fixed in 2.5% Paraformaldehyde/ 2.5% Glutaraldehyde in 0.1M sodium phosphate buffer. Eyes were secondarily fixed in 1% osmium textroxide, gradually dehydrated, and embedded in Spurr&#x2019;s resin. Tissue was sectioned at 90 nm and placed onto copper slot grids covered in Formvar and imaged using a transmission electron microscope. The micrograph shows, photoreceptor outer segments of the neurosensory retina, melanosomes (M) within the retinal pigment epithelium, and Bruch&#x2019;s membrane. 
<img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Figure 4" /> 
Figure 4. Immunohistochemical labeling of superoxide dismutase-3 (green) in the mouse vitreous. Eyes were enucleated by blunt dissection and underwent immersion fixation in 4% paraformaldehyde. They were embedded in paraffin and sectioned using a microtome. Tissue sections were incubated with rabbit polyclonal SOD3 antiserum diluted 1:50. SOD3 expression was detected using a goat anti-rabbit IgG (H+L)-Alexa Fluor 488 conjugated secondary antibody. Labeling of SOD3 is shown in green.

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

No conflicts of interest declared.

Most transgenic mice exist in laboratories that do not examine the eyes. Our video technique illustrates a simple, standardized method for remote surgical skill transfer to optimize tissue acquisition from laboratories with little experience with the eyes. This video technique helps overcome a major pitfall in high-throughput phenotyping, which is the use of a limited number of expert sites due to non-standardized tissue collection and fixation methods that prevent meaningful comparative phenotypic and molecular studies....

<table border="1">
 <tbody>
 <tr>
 <th>Name</th>
 <th>Company</th>
 <th>Catalog Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Curved Dressing Forcep</td>
 <td>Storz Ophthalmics</td>
 <td>E1408</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mahajan Sharptip dressing forcep</td>
 <td>Storz Ophthalmics</td>
 <td>E1406 (REF SP7-64520)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
	<td>Curved Westcott Scissors</td>
	<td>Storz Ophthalmics</td>
	<td>E3321 WH</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
 <td>15&deg; BD Beaver Microsurgical Blade</td>
 <td>Becton-Dickinson</td>
 <td>374881</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 Fine-Castroviejo Suturing Forceps</td>
 <td>Storz Ophthalmics</td>
 <td>E1805</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.12 Colibri forceps</td>
 <td>Storz Ophthalmics</td>
 <td>2/132</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30-gauge needle</td>
 <td>Becton-Dickinson</td>
 <td>305128</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biohazard Mailer</td>
 <td>Fisher</td>
 <td>03-523-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Fisher</td>
 <td>13-374-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass scintillation vials</td>
 <td>Wheaton</td>
 <td>4500413033</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS, pH 7.4</td>
 <td>Invitrogen</td>
 <td>70011-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>16% Paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>15700</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2.5% Paraformaldehyde/ 2.5% Glutaraldehyde in 0.1M sodium phosphate buffer</td>
 <td>Electron Microscopy Sciences</td>
 <td>15700 &amp; 16300</td>
 <td>Mixed in laboratory</td>
 </tr>
 <tr>
 <td>50% Glutaraldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>16300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Formvar</td>
 <td>Electron Microscopy Sciences</td>
 <td>15810</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Copper Slot Grid</td>
 <td>Electron Microscopy Sciences</td>
 <td>M2010-CR</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4% Osmium Tetroxide</td>
 <td>Electron Microscopy Sciences</td>
 <td>19140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-SOD3 antibody</td>
 <td>Abcam</td>
 <td>Ab21974</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat anti-rabbit Alexa Fluor 488</td>
 <td>Invitrogen</td>
 <td>A11070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spurr's embedding resin</td>
 <td>Electron Microscopy Sciences</td>
 <td>14300</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

mouse eye enucleation for remote high-throughput phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.1K Views.  University of Iowa. The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

Video: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...