This work was supported by the research unit FOR 2688 - Wa1336/12 of the German Research Foundation and by the Marie Sk&#322;odowska-Curie grant agreement No. 860436-EVIDENCE. T. J. and C. W. acknowledge funding from French German University (DFH/UFA). A. D. acknowledges funding by the Young Investigator Grant of Saarland University.

Blood sample collection and experiments were approved by the &#34;&#196;rztekammer des Saarlandes&#34;, ethics votum 51/18, and performed after informed consent was obtained according to the Declaration of Helsinki. Standard measurements should be performed with ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood (standard EDTA concentration of 1.6 mg/mL blood, European norm NF EN ISO 6710), in Westergren tubes. The volume required to fill the Westergren tube depends on the manufacturer (as lower parts sometimes contain a wider reservoir); The volume should be about 1 mL of full blood, and 800 &#181;L for the tubes indicated in the Table of Materials. The method described below is however valid no matter the specific suspension and container shape, as long as the hematocrit of the probed samples is higher than 25%16. Volumes, containers, suspending medium, and additives should therefore be selected according to the specific objectives of the performed research.
1. Experiments and measurements
NOTE: Record the sedimentation rate of the sample every minute.
<ol>
	<li>Sample preparation (if required): If a control hematocrit or suspending liquid is required, start by washing the cells and preparing the samples (as an example, we show how to prepare samples with various fibrinogen levels, by mixing autologous serum and plasma). Serum can indeed be approximated as fibrinogen-free plasma26,27, and can be used in order to decrease the aggregation of RBCs, in otherwise physiological conditions. In order to prepare several samples, collect the blood in standard EDTA tubes of 9 mL and the serum in standard serum tubes (with silica beads as clotting activators) of 9 mL.
	<ol>
		<li>Centrifuge the blood samples (i.e., 9 mL of standard EDTA and serum tubes in the chosen example) at 3,000 x g for at least 7 min, for optimal compaction of the packed erythrocytes. Replace the supernatant with PBS or the desired suspending liquid, if a sufficient quantity is available. If it is simply required to control the hematocrit in autologous plasma, proceed directly to step 1.1.3. Otherwise, gently mix after inclusion of the supernatant for washing.</li>
		<li>Repeat three times. Perform the last wash with the desired suspending liquid in any case.
		<ol>
			<li>In the chosen example, prepare mixtures of autologous plasma and serum with determined proportions (e.g., 25%/75%, 50%/50%, or 75%/25% of plasma-serum volume fraction). For example, when preparing 2.5 mL of the 25%/75% plasma-serum mixture, add 0.625 mL of plasma to 1.875 mL of serum.</li>
		</ol>
		</li>
		<li>Extract the required volume of packed cells and suspend it in the desired liquid. Process the packed cells as a highly viscous liquid (using standard pre-pipetting and/or reverse-pipetting or a positive displacement pipette28). In the chosen example, for a 4 mL sample at a hematocrit of 45%, suspend 1.8 mL of packed cells within 2.2 mL of the plasma-serum mixture.</li>
	</ol>
	</li>
	<li>If the hematocrit of the sample is not controlled, determine it by high-speed microcentrifugation (other standard methods are also suitable).
	<ol>
		<li>Extract the required amount of sample for hematocrit determination: fill the micro-hematocrit capillaries by immersing its lower tip in the liquid. Stop it by covering the top opening when the required amount of sample rises in the tube by capillary suction.</li>
		<li>Seal the capillaries with sealing wax. Place them in the micro-hematocrit centrifuge and run it at 15,000 x g(12,000 rpm) for 5 min, or according to the manufacturer&#39;s instructions.</li>
		<li>Read the hematocrit level on the capillary and write it down for reference.</li>
	</ol>
	</li>
	<li>Set up a camera for recording the samples&#39; sedimentation. To avoid overloading the memory or draining the battery, power the device from the mains and save the pictures directly to a computer or external hard drive.
	<ol>
		<li>Set up a steady camera in front of the holder where the ESR tubes will be left at rest. Use a white, illuminated background (white sheets of paper in the background work perfectly).</li>
		<li>Using empty Westergren tubes, preferably without markings, set the focus and the field of view of the camera to obtain the highest resolution where the samples will be placed. Preferably, ensure that the borders of the pictures are aligned in the vertical and horizontal direction.</li>
		<li>Take a picture of a scaled tube to extract the pixel resolution. The use of RGB pictures is recommended.</li>
		<li>Set the light and the exposure time of the camera to have a white background, but no saturation. The example in Figure 1 has been obtained using a Canon EOS M50, with an exposure time of 1/15s, an aperture of F8.0, Tungsten white balance, in single shot mode with manual focus, and an ISO speed of 1,000.</li>
	</ol>
	</li>
	<li>Prepare and place the ESR tubes.
	<ol>
		<li>Fill the bottom container of the Westergren tube with the volume corresponding to the manufacturer&#39;s instructions. Insert the Westergren tube in the bottom container as indicated by the manufacturer.</li>
		<li>As soon as the first tube is ready, place it in the holder and start the camera recording. Recording one picture every minute usually gives a good resolution of the ESR kinetic curve.</li>
		<li>Prepare and place the next samples. Make sure not to stand in front of any sample when a picture is taken.</li>
		<li>Let the measurements run for at least 2 h, in order to compare with standard measurements at 30 min, 1 h, and 2 h. However, it is also better to see the saturation and arrest of the sedimentation. For healthy samples, or in case of inflammation, recording the samples overnight, between 12 h and 24 h, is more than sufficient since the curvature of the fastest samples is visible within 3 h13,19. However, if a condition decreases the sedimentation velocity, as a decrease in fibrinogen concentration does (i.e., in the chosen example, high serum volume fraction), recordings of 50 h or more might be required to obtain the most accurate information16,19.</li>
	</ol>
	</li>
</ol>
2. Image analysis
NOTE: Once the images are recorded, extract the ESR curve. An example of Matlab code is provided as Supplementary File 1 (MatlabCodeImageAnalysisSampled.m).
<ol>
	<li>Select or identify a region of interest (ROI) where only one tube is visible, the lower border being located under the lowest position of the erythrocyte cell-free plasma interface but within the sample (see&#160;Figure 1A, B). If required, rotate the picture so that the vertical direction is aligned along the first component of the picture.</li>
	<li>Convert the ROI of the RGB picture into a gray-scale picture or matrix Gr. Usually, combining the three-color channels (red [R], green [G] and blue [B]) as Gr = 2 * R - B - G&#160;is very efficient with a clear background (see Figure 1C and MatlabCodeImageAnalysisSampled.m lines 121-128).</li>
	<li>Binarize the picture. For a healthy sample, using the Otsu threshold29 usually gives a consistent result (see Figure 1D and line 133 of MatlabCodeImageAnalysisSampled.m). For samples with a high hematocrit or with some hemolysis, it might be better to adjust it manually or use another automatic method (as done in lines 129-131 of MatlabCodeImageAnalysisSampled.m), depending on the exact contrast obtained between the various phases inside the tube.</li>
	<li>Obtain the average of the pixel (or elements) values of Gr along the horizontal direction. This step minimizes the noise and efficiently averages the possible interface irregularities (see Figure 1E and MatlabCodeImageAnalysisSampled.m line 137).</li>
	<li>Before computing the variations, smooth the curve with a moving average, especially if the tubes contain some horizontal markings (see Figure 1F). For the provided examples, a moving window of ~2.5 mm (50 pixels) was used for this process (see line 138 of MatlabCodeImageAnalysisSampled.m). Then, identify the interface position as the point with the highest intensity variation (see Figure 1G).</li>
	<li>Repeat for each picture and each sample. For each sample, save the positions of the interface along time in an appropriate format to fit the physical model with any suitably fit software.</li>
</ol>
3. Fitting of the physical model
<ol>
	<li>Using any suitably fit software, knowing the hematocrit and the initial height of the blood column h0, find the values of the delay time t0, the dimensionless time &#947;, and the final packed volume fraction &#934;m of the erythrocytes that minimizes the sum of squared residual deviations for the physical model17. A Matlab code (ShapeAnalyzerIntegrated.m) is provided as Supplementary File 2 as an example of how to perform such a fit. See Supplementary File 2 for further instructions. As described elsewhere16,17, erythrocytes at physiological hematocrit sediment as a soft particle gel, where the important physical parameters are the difference in density between the plasma and RBCs of the donor &#916;&#961;, the RBC characteristic diameter rRBC, the plasma viscosity at room temperature &#951;, and the donor&#39;s hematocrit &#934;. Using these parameters, and assuming that the gravitational stress is the main driving process for the plasma to flow upward through the porous network formed by the erythrocytes after a delay time t0, one obtains the temporal evolution16,17: 
	<img alt="Equation 1" src="/files/ftp_upload/64502/64502eq1v6.png" style="margin: auto;" /> 
	with <img alt="Equation 2" src="/files/ftp_upload/64502/64502eq2v6.png" style="margin: auto;" />, <img alt="Equation 3" src="/files/ftp_upload/64502/64502eq3v6.png" style="opacity: 0.9; margin: auto;" />, and <img alt="Equation 4" src="/files/ftp_upload/64502/64502eq4v6.png" style="margin: auto;" />&#160;being the average disk radius of an erythrocyte. An example of a Matlab code performing this is provided as an attachment (ShapeAnalyzerIntegrated.m fits the function defined in SedimFit.m [Supplementary File 3]). Alternatively, G/&#947; can also be used directly as a fit parameter with units of 1/t.</li>
	<li>Once the quantitative curve is extracted from the picture, save the physical parameters of the sample. Traditional ESR values at 30 min, 1 h, or 2 h can still be extracted from the curve for reference (see ShapeAnalyzerIntegrated.m lines 123-132).</li>
</ol>

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The authors have no conflicts of interest to declare relevant to the content of this article.

For the automated protocol to work efficiently, it is important to have a clear background and proper illumination. A dark background might prevent the existence of an efficient binarization threshold. For samples with some hemolysis, which usually occurs (increases) over time, it is important to verify first that the chosen binarization threshold is relevant for both the initial and final pictures.
When it comes to the binarization process of the picture, the choice of the ROI and binarizatio...

The erythrocyte sedimentation rate (ESR) is a medical in vitro clinical tool, formally introduced in evidenced-based medicine during the twentieth century1,2,3,4. It is currently used worldwide as a nonspecific inflammatory test, or to monitor the evolution of some specific conditions5,6,7,8. This is mainly due to an increase in the fibrinogen concentration, but also in other plasma components such as IgM1,9,10,11. According to the current Westergren standard protocol, ESR values are reported as the measurement of the cell-free plasma layer at a given time point (30 min or 1 h) after leaving a vertical tube of a typical size of 20 cm vertically at rest12. However, this measurement method has been criticized since qualitatively different stages in the sedimentation process have been reported, including a delay before reaching the maximum settling velocity13. This delay lasts more than 1 h in approximately half of healthy samples14. The velocity during this phase obeys a different scaling than during the second, faster phase of the sedimentation15. Restricting the readout to the average settling velocity during the first hour then compares a different mix of various blood properties between different individuals.
Moreover, it has recently been demonstrated that the usual theoretical considerations behind this protocol were erroneous16,17,18. At physiological hematocrit (above approximately 25%), red blood cells (RBCs) do not sediment as separate aggregates, but rather as a continuous, so-called percolating, network of RBCs17,18, obeying a different set of physical equations than the usually mentioned Stokes sedimentation16,17. It has been shown that considering a physical description based on the time-resolved measurements of the sedimentation (whole curve) was more robust in some novel medical contexts19,20. Moreover, these measurements could be used to shed light on the physical mechanisms altering the ESR in pathologies in which cell shapes are altered19,20. Additionally, a slow ESR can have a useful medical interpretation, as indicated in the measurements of a cohort of neuroacanthocytosis syndrome patients19,20. This article reviews how to practically implement the measurement of physically-meaningful parameters, based on the whole ESR kinetics. More accurately, the method presented here extracts the maximum sedimentation speed Um, the value of which can be corrected to consider the effect of the hematocrit of the donor16,17. This parameter is more accurate and thus more reliable than the traditional measurement16,17,19,20.
In addition, in some fundamental research, instead of monitoring the inflammation state of a given patient, it is interesting to exclude the effect of hematocrit on the ESR21,22,23, or to investigate the role of RBCs in a modified ESR19,20,24,25 between different donors. It might be useful to compare samples which are not directly full blood samples from patients. Therefore, resuspending RBCs with a controlled hematocrit in the autologous plasma, or in a plasma-substituent, might be used as the first step of ESR measurement. For instance, solutions of Dextran 70 kDa with a concentration of 55 mg/mL in phosphate buffered saline (PBS) produces a sedimentation range within the control range for healthy cells19. This manuscript also shows how such steps should be conducted, and that the presented analysis is also relevant in these cases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anticoagulant (EDTA or Heparin) tube (for blood sample)</td>
			<td>SARSTEDT</td>
			<td>267001 or 265</td>
			<td>Anticoagulated blood sample to characterize</td>
		</tr>
		<tr>
			<td>Camera EOS M50</td>
			<td>Canon</td>
			<td>Kit EF-M18-150 IS STM</td>
			<td>Any camera should work, provided that sector alimentation, connection to computer for automated shooting and adapted objective are available</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HERMLE</td>
			<td>302.00 V03 - Z 36 HK</td>
			<td>Requirements: at least 3000 x g ofr 7 min.</td>
		</tr>
		<tr>
			<td>Micro-centrifuge</td>
			<td>MLW</td>
			<td>TH21</td>
			<td>or any other way to determine the hematocrit</td>
		</tr>
		<tr>
			<td>Micro-hematocrit capilaries</td>
			<td>Fisher scientific</td>
			<td>11884040</td>
			<td>or other capillaries/containers for hematocrit determination</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td>1x PBS, pH 7.4, 298 Osm</td>
		</tr>
		<tr>
			<td>Pipettes (e.g. positive displacement pipette)</td>
			<td>Gilson</td>
			<td>FD10006</td>
			<td>Pipette required to manipulate blood and/or packed cells.Other models are of course suitable, but be careful to treat blood and pakced cells as highly viscous fluids.</td>
		</tr>
		<tr>
			<td>Wax sealing plate</td>
			<td>Hirschmann</td>
			<td>9120101</td>
			<td>Sealing wax for the micro-hematocrit capillaries</td>
		</tr>
		<tr>
			<td>Westergren tubes</td>
			<td>Praxindo</td>
			<td>A9244560</td>
			<td>Any other standard Wetsergren tube should work too</td>
		</tr>
		<tr>
			<td>White background with illumination</td>
			<td>/</td>
			<td>/</td>
			<td>White sheet(s) of paper behind the samples, with usual room light is perfcetly sufficient.</td>
		</tr>
	</tbody>
</table>

erythrocyte sedimentation rate: a physics-driven characterization in a medical context

Erythrocyte (or red blood cell) sedimentation rate (ESR) is a physical derived parameter of blood which is often used in routine health checks and medical diagnosis. For instance, in the case of inflammation, a higher ESR is observed due to the associated increase in fibrinogen and other plasma proteins. It was believed that this increase was due to the formation of larger aggregates of red blood cells (RBCs) caused by the increase in fibrinogen. Indeed, fibrinogen is an agent-fostering aggregation of RBCs and in the Stokes regime-assumed to be observed in blood-larger aggregates sediment faster. However, all models of ESR measurements based on this hypothesis require further specific physical assumptions, not required in any other system. Besides, modern studies in the field of colloidal suspensions have established that attractive particles form percolating aggregates (i.e. aggregates as wide as the container). The sedimentation of these colloids then follows a so-called &#34;colloidal gel collapse&#34;. Recently, it has been shown that RBCs actually follow the same behavior. This hypothesis also allows to efficiently and analytically model the sedimentation curve of RBCs, from which robust and physically-meaningful descriptors can be extracted. This manuscript describes how to perform such an analysis, and discusses the benefits of this approach.

An example of an image sequence correctly acquired is provided as Supplementary Movie 1 (MovieS1.avi). A series of characteristic fits of the model is shown for various conditions in Figure 2. Fibrinogen concentration was determined from the fibrinogen concentration in the plasma Fib0, assuming that the serum does not have any fibrinogen at all. Hence, Fib = C Fib0, where C is the plasma volume fraction in the plasma-...

Watch this Scientific Journal Video about Erythrocyte Sedimentation Rate: A Physics-Driven Characterization in a Medical Context at JoVE.com

Erythrocyte Sedimentation Rate: A Physics-Driven Characterization in a Medical Context

Erythrocyte sedimentation rate (ESR) is a physical parameter, often used in routine health checks and medical diagnosis. A theoretical model that allows to extract physically-meaningful parameters from the whole sedimentation curve, based on modern colloidal knowledge, has recently been developed. Here, we present a protocol to automatically collect the ESR over time, and extract the parameters of this recent model from this automated data collection. These refined parameters are also likely to improve the medical testimony.

Erythrocyte (or red blood cell) sedimentation rate (ESR) is a physical derived parameter of blood which is often used in routine health checks and medical ...

erythrocyte-sedimentation-rate-physics-driven-characterization

Research

JoVE Journal

Medicine

3.1K Views.  Saarland University. Erythrocyte sedimentation rate (ESR) is a physical parameter, often used in routine health checks and medical diagnosis. A theoretical model that allows to extract physically-meaningful parameters from the whole sedimentation curve, based on modern colloidal knowledge, has recently been developed. Here, we present a protocol to automatically collect the ESR over time, and extract the parameters of this recent model from this automated data collection. These refined parame...

Video: Erythrocyte Sedimentation Rate: A Physics-Driven Characterization in a Medical Context

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Stereo-Electro-Encephalo-Graphy (SEEG) With Robotic Assistance in the Presurgical Evaluation of Medical Refractory Epilepsy: A Technical Note

SEEG is a method and technique which is used for accurate, invasive recording of seizure activity via three dimensional recordings. In epilepsy patients who are deemed appropriate candidates for invasive recordings, the decision to monitor is made between the subdural grids versus SEEG. Invasive neuromonitoring for epilepsy is pursued in patients with complex, medically refractory epilepsy. The goal of invasive monitoring is to offer resective surgery with the hope of allowing seizure freedom. SEEG&#39;s advantages include access to deep cortical structures, an ability to localize the epileptogenic zone (EZ) when subdural grids have failed to do so, and in patients with non-lesional extra-temporal epilepsies. In this manuscript, we present a succinct historical overview of the SEEG and report on our experience with frameless stereotaxy under robotic. An imperative step of SEEG insertion is planning the electrode trajectories. In order to most effectively record ictal activity via SEEG trajectories should be planned based upon a hypothesis of where the seizure activity originates the presumed epileptogenic zone (EZ). The EZ hypothesis is based on a standardized preoperative workup including video-EEG monitoring, MRI (magnetic resonance imaging), PET (positron emission tomography), ictal SPECT (Single-photon emission computed tomography), and neuropsychological assessment. Using a suspected EZ, SEEG electrodes can be placed minimally invasively yet maintain accuracy and precision. Clinical results showed the ability to localize the EZ in 78% of difficult to localize epileptic patients.1

Stereo-Electro-Encephalo-Graphy SEEG With Robotic Assistance in the Presurgical Evaluation of Medical Refractory Epilepsy: A Technical Note

Stereo-electroencephalography (SEEG) aids in localization of epileptogenic zones, however, remains relatively underutilized in the United States. The goal of this abstract is to provide a brief introduction to the technique of SEEG and further a detailed technique of using robotic assistance in the placement of SEEG electrodes.

SEEG is a method and technique which is used for accurate, invasive recording of seizure activity via three dimensional recordings. In epilepsy patients ...

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 ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...