The authors would like to thank Drs Silvia B Campos-Bilderback and George J Rhodes for completing surgical procedures involving placement of venous access lines. They would also like to thank Sara E Wean for maintaining the Munich-Wistar colonies consisting of both Simonsen and Fr&ouml;mter strains. This work was supported by funding provided to the Indiana Center for Biological Microscopy, and the National Institutes of Health grants P30-DK079312, and 5RO1-DK091623 awarded to Bruce A Molitoris.

1. Conjugation of Rat Serum Albumin to Sulfo-Rhodamine 101 Sulfonyl Chloride (Texas Red)
<ol>
	<li>Dissolve 100 mg of Rat Serum Albumin (RSA) in 6.667 ml of 100 mM Sodium Bicarbonate pH 9.0; final concentration 15 mg/ml in a 50 ml conical tube.</li>
	<li>Place solution in ice/water beaker and cool to between 0 to 4 &deg;C.</li>
	<li>Add 200 &mu;l of high quality anhydrous Dimethyl Formamide (DMF) to a 10 mg vial of Texas Red Sulfonyl Chloride (TRSC); vortex on medium for 15 sec.</li>
	<li>Vortex RSA solution on medium and add the dissolved TRSC.</li>
	<li>Wrap 50 ml conical in foil (Parafilm can be used to assure a tight seal is formed), place horizontally in ice bucket w/ ice only and place on rocker to agitate solution slowly (avoid formation of bubbles), allow reaction to continue at 0 to 4 &deg;C for 1 hr.</li>
	<li>Make 5 L of 0.9% saline solution, and wet a 50 kDa molecular weight cut off chamber which can be either a) dialysis membrane with clips, b) dialysis tubing as in a float-a-lyzer, or c) slide-a-lyzer cassette (all suitable for removing unconjugated TRSC).</li>
	<li>Place reaction mixture in the dialysis chamber and place in the 5 L container w/ the 0.9% saline solution, dialyze overnight at 4 &deg;C with a gentle agitation using a stir bar.</li>
	<li>Change the 5 L dialysis solution in morning and in late afternoon until an overnight incubation results in virtually no color change (This typically takes ~ 48 hr with at least 4 exchanges). Additionally, with the MWCO of 50 kDa being so close to the MW of RSA, 66kDa; it is possible to never produce a clear solution; this will be dependent on the distribution in the pore size of the membrane; 60 hr and 5 exchanges is more than sufficient time.</li>
	<li>Measure volume and divide the initial weight of 100 mg by the volume to give approximate concentration of TR-RSA; typically concentrations range from 10-13 mg/ml. The final dye:albumin ratio should be ~4:1, 1 fluorophore per 15,000 Daltons of protein MW. Store at 4 &deg;C; NEVER FREEZE the TR-RSA solution, as resulting fragmentation that may occur will alter permeability values.</li>
</ol>
2. Preparing the Inverted Microscope Stage for Imaging/ Microscope Settings
<ol>
	<li>Place ~ 4-7 pieces of autoclave tape (approximately &frac34;" long) perfectly stacked over each other inside a 50 mm dish with a 40 mm coverslip bottom (coverslip bottom dish). These should be placed closer to one of the edges so that the edge of the tape will make contact with the edge of curvature of the kidney but not block the objective light path (Figures 1A and 1B); larger rats will require more space between the tape and edge of dish.</li>
	<li>Place 2 Repti-therm pads next to the stage alongside the 50 mm dish (Figure 1A). Place a warming water jacket blanket over the stage.</li>
	<li>Maximize efficiency when collecting images by assuring the objective turret has a 10x air or 20x (air or water immersion) objective and a higher powered water immersion objective to collect images for quantitation.</li>
	<li>During imaging the most efficient way to add water to the objectives is to rotate them to the side and add water using a 1 cc syringe with a long piece of PE-200 tubing that can reach the top of the objectives.</li>
	<li>Set the excitation intensity of the 10watt laser to ~15-20% using the neutral density filters on the software. The gallium-arsenide phosphide non-descanned photodetectors are set to 750 to collect green emissions, and 625 to collect red emissions. Blue emission (such as the nuclear stain Hoechst 33342) are collected using a standard nondescanned multialkaline detector with a setting between 750-800.</li>
	<li>To assure the proper collection of the low intensity emissions within the Bowman's space, make sure the lower limits of the detectors are set so as not to exclude these values. Visual warning markers will indicate if the sensitivity setting is too low and these values are being given an intensity value of zero.</li>
	<li>Load a 1 cc syringe with ~ 5-8 mg of the fluorescent albumin solution diluted with 0.9% sterile saline to bring up the total volume to 1 cc.</li>
</ol>
3. Exposing the Kidney in a Munich Wistar Rat for Intravital 2-Photon Imaging
<ol>
	<li>Start with a pre-anesthetized rat using Pentabarbitol (50 mg/ml solution, 120 &mu;l/100 g body weight), Inactin (130 mg/ml solution, 120 &mu;/100 g body weight), or Isofluorane (5% induction, 1.5 to 2.5% maintenance), an indwelling venous access line (either jugular or femoral venous), and the left flank shaved from just below the ribcage to just above the left thigh.</li>
	<li>Place the rat flat on its right side so that the left, shaved side is facing up; make sure it is flat on the table, with its posture elongated and not crouched, with front paws touching each other and rear paws touching each other (Figure 2A).</li>
	<li>Very gently palpate to feel the kidney to determine where it naturally lays within the retroperitoneum and draw a straight line parallel to the body (from ribcage to thigh) using a Sharpie (Figure 2A).</li>
	<li>Pick up the skin with a pair of toothed forceps, and pinch the skin along the drawn line using a pair of hemostats to crush the tissue and prevent bleeding. Cut along the incision using a pair of surgical scissors. Crushing the outer skin and the muscle layers prior to cutting will dramatically reduce and typically eliminate bleeding.</li>
	<li>Repeat this procedure for the outer muscle layer, which is thin.</li>
	<li>For the incision into the inner muscle layer, which will expose the peritoneum, re-palpate the kidney to estimate size. Pinch an incision line smaller than the kidney; assuring the incision is just over the kidney. It is best to make this incision too small and make it larger if needed. If this is made too big, suturing will be required. This last incision is critical; too far over in any direction will affect stability on the stage and induce either motion artifacts from breathing (best case scenario) or will stretch the renal pedicle and adversely reduce renal perfusion (worst case scenario).</li>
	<li>Locate the kidney (as shown in Figure 2B) and grip the surrounding fat using forceps, working towards the bottom pole of the kidney in a hand over hand fashion.</li>
	<li>Once the lower pole of the kidney is reached, gently pull the kidney through the incision while very gently squeezing below the kidney to exteriorize. If the incision is too small, crush the muscle layer and cut to widen it; repeat the procedure to exteriorize kidney.</li>
</ol>
4. Placing the Munich Wistar Rat on the Stage for Imaging
<ol>
	<li>Place the kidney towards the edge of the coverslip dish with the kidney slightly rotated towards the dorsal (back side) of the rat so the ventral side of the kidney is making contact with the coverslip bottom dish (Figure 1A). Add a warmed, sterile 09.% saline solution to the dish.</li>
	<li>Look through the 10x or 20x objective and check for motion. If motion is detected, roll the rat over slightly so the thorax is farther away from the coverslip bottom dish; make sure the kidney is as close to the edge of the dish without stretching the renal pedicle (Figure 1B).</li>
</ol>
5. Acquiring Images to Quantify Renal Permeability of Albumin
<ol>
	<li>Calculating the glomerular permeability (Glomeruluar Sieving Coefficient; GSC) of any macromolecule will require taking reference background images of individual glomeruli prior to infusion of the fluorescent molecule. If your microscope is equipped with a motorized stage capable of marking locations, find and center individual glomeruli using the lower powered objective and mark each location. A dual pass Fluorescein/Rhodamine epifluorescence filter is ideal for this procedure although either emission filter will work (phase contrast or other non-fluorescence sources of illumination will not work). Glomeruli will appear as empty circular structures surrounded by proximal tubules having an inherent yellow-orange autoflourescence when viewed through the dual pass filter.</li>
	<li>If your microscope does not have a motorized stage, scan the kidney in a raster pattern with the low power objective and make a rudimentary map of where the individual glomeruli are located; relying on natural landmarks such as large superficial blood vessels located above the renal capsule or fat patches.</li>
	<li>Switch the turret to the higher power water immersion objective and take 3D data sets of each glomeruli making sure the capillary loops and Bowman's space are clearly visible. Using a pseudocolor palette (something other than B/W) will help to visualize these structures.</li>
	<li>Focusing in on a superficial blood vessel slowly infuse in the fluorescent albumin making sure time is given to allow for systemic distribution. For molecules with low GSC it is essential to maximize the intensity values in the plasma but not to reach levels that will saturate the photo-detectors in the microscope. This increases detectability of filtered molecules. Note: there is typically a 5-7 sec lapse between the time the fluorescent albumin is introduced to the time it appears on the screen (acquiring full frames at ~ 1 frame/second).</li>
	<li>Allow approximately 10 min to allow any potential small molecular weight fragments to clear before acquiring 3D volumes at 1 &mu;m intervals to be used in calculating albumin GSC. Typically, the Simonsen's strain of Munich Wistar rats has far fewer surface glomeruli, so all that can be visualized are imaged and quantified. The Fr&ouml;mter strain has a far greater number so we limit the number quantified to ~10.</li>
	<li>At the end of the study the rat is euthanized via an overdose of the anesthetic used in the study. A dual pneumothoracotamy is performed to insure euthanasia.</li>
</ol>
6. Calculating GSC for Fluorescent Albumin from 3D Volumes
<ol>
	<li>Using Metamorph image processing software load the 3D data sets for each glomeruli, both the background set and set taken after infusion of the fluorescent albumin.</li>
	<li>In the volume containing the fluorescent albumin locate a superficial capillary loop with enough space empty space between its defined margins and the edge of the Bowman's capsule.</li>
	<li>In the background volume locate the same focal plane which should contain all the visual cues of the albumin containing image. Select a region within the capillary loop of interest and note the average intensity reading. Next select a region within the Bowman's space and note the average intensity reading. These will be used as background values.</li>
	<li>For quantitation, select the similar region within the Bowman's space in the albumin containing image. Do this for at least two other regions to take an average value for the average intensity within Bowman's space.</li>
	<li>Select the capillary loop with the brightest plasma intensity and draw a region around it. Next using the threshold function, highlight the bright values within the circulating plasma, avoiding the dark streaks that are circulating RBC's. Note the average intensity values of the selected plasma space. It is important to preferentially select the bright areas of the plasma because factors within the blood will only serve to cause and underestimation of plasma fluorescence levels.</li>
	<li>Using a Microsoft Excel spreadsheet enter the values to calculate the GSC where:</li>
</ol>
<img alt="Figure 1" fo:content-width="2.95in" fo:src="/files/ftp_upload/50052/50052eq1highres.jpg" src="/files/ftp_upload/50052/50052eq1.jpg" />

<ol>
	<li>Russo, L. M., 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=The+normal+kidney+filters+nephritic+levels+of+albumin+retrieved+by+proximal+tubule+cells;+retrieval+is+disrupted+in+nephritic+states.">The normal kidney filters nephritic levels of albumin retrieved by proximal tubule cells; retrieval is disrupted in nephritic states.</a> Kidney International. 71, 504 (2007).</li><li>Russo, L. M., 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=Impaired+tubular+uptake+explains+albuminuria+in+early+diabetic+nephropathy.">Impaired tubular uptake explains albuminuria in early diabetic nephropathy.</a> Journal of the American Society of Nephrology. 20 (3), 489 (2009).</li><li>Sandoval, R. M., 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=Multiple+factors+influence+glomerular+albumin+permeability+in+rats.">Multiple factors influence glomerular albumin permeability in rats.</a> Journal of the American Society of Nephrology. 23 (3), 447 (2012).</li><li>Tojo, A., Endou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarenal+handling+of+proteins+in+rats+using+fractional+micropuncture+technique.">Intrarenal handling of proteins in rats using fractional micropuncture technique.</a> American Journal of Physiology. 263, 601 (1992).</li><li>Asgeirsson, D., 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=Glomerular+sieving+of+three+neutral+polysaccharides,+polyethylene+oxide+and+bikunin+in+rat:+Effects+of+molecular+size+and+conformation.">Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat: Effects of molecular size and conformation.</a> Acta Physiologica. 191 (3), 237 (2007).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+endocytosis+in+vivo+using+intravital+two-photon+microscopy.">Quantifying endocytosis in vivo using intravital two-photon microscopy.</a> Methods in Molecular Biology. 440, 389 (2008).</li><li>Dunn, K. W., 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=Live-animal+imaging+of+renal+function+by+multi-photon+microscopy.">Live-animal imaging of renal function by multi-photon microscopy.</a> Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).</li></ol>

The authors have nothing to disclose.

The steps highlighted here represent what we feel to be ones that will produce consistent and accurate permeability values because they circumvent the following pitfalls:
<ol>
 <li>Scattering: The use of a red emitting fluorophores allow for more efficient collection of light since longer wavelength photons are less prone to scatter. Using either green or blue emitting fluorophores will introduce greater variation in GSC's because of the increased variability in intensity values from the capillary loops and...

<table border="1">
 <tbody>
 <tr>
 <td>Olympus Floview 1000 confocal/Multi-photon microscope</td>
 <td>Olympus America</td>
 <td></td>
 <td>Filters for detectors: Blue 430/100, Green 525/50, Red 605/90</td>
 </tr>
 <tr>
 <td>Mode-Locked Ti:Sapphire Mai Tai Laser</td>
 <td>Spectra-Physics</td>
 <td></td>
 <td>Tunable excitation wavelengths: ~750-1150 nm</td>
 </tr>
 <tr>
 <td>Gallium arsenide phosphide photodetectors</td>
 <td>Hamamatsu Corp</td>
 <td></td>
 <td>Note: Front or Side mounted configurations available.</td>
 </tr>
 <tr>
 <td>Metamorph Image processing Software</td>
 <td>Molecular Dynamics</td>
 <td></td>
 <td>Note: Version 6.1r1</td>
 </tr>
 <tr>
 <td>Microsoft Excel</td>
 <td>Microsoft Corportation</td>
 <td></td>
 <td>2007 version</td>
 </tr>
 <tr>
 <td>Handling Forceps</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78266-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Mayo Dissecting Scissors</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72962</td>
 <td></td>
 </tr>
 <tr>
 <td>CA Micro scissors Model 1C300</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78180-1C3</td>
 <td></td>
 </tr>
 <tr>
 <td>Kelly Hemostatic Forceps (straight)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72930</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Jacket Blanket + Heating Pad</td>
 <td>Gaymar</td>
 <td></td>
 <td>T/Pump PN 11184-000 Blanket-66N111CC</td>
 </tr>
 <tr>
 <td>Repti-Therm Under Tank Heater</td>
 <td>ZooMed</td>
 <td></td>
 <td>RH-4</td>
 </tr>
 <tr>
 <td>Texas Red Sulfonyl Chloride</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>Cat# T-353</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat Serum Albumin</td>
 <td>Sigma Aldrich</td>
 <td>Cat# A-6272</td>
 <td></td>
 </tr>
 <tr>
 <td>High Quality Anhydrous DMF</td>
 <td>Sigma Aldrich</td>
 <td>Cat# 270547</td>
 <td></td>
 </tr>
 <tr>
 <td>Strate-Line Autoclave Tape</td>
 <td>Fisher Scientific</td>
 <td>Cat# 11-889-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 70665-07</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

Figure 3 shows an example of an image taken from a surface glomerulus of a Munich Wistar Fr&ouml;mter rat and the steps taken to determine the permeability of fluorescent albumin. The GSC value for albumin of 0.0111 derived for this individual glomerulus fall within the range seen in this strain of Munich Wistar rats when in the fed condition3. The stability seen in these images is due to the careful planning and execution of instructions depicted in Figures 1 and 2. As ...

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Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

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10.4K Views.  Indiana University School of Medicine. Техника использования высокого разрешения intavital 2-фотонной микроскопии непосредственно визуализировать и количественно gloemrular поверхность фильтрации в клубочках. Этот метод позволяет прямое определение характеристик проницаемости макромолекул в нормальн?...

Video: Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat 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 ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...