Name: dynamic light scattering analysis for the determination of the particle size of iron-carbohydrate complexes
Uploaded: 2023-07-07T12:30:00
Description: Watch this Scientific Journal Video about Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes at JoVE.com

1. Operating the machine
<ol>
	<li>Starting up the machine and software 
	NOTE: Supplemental Figure S1A-D describes the steps for starting up the machine and software.
	<ol>
		<li>Switch on the instrument at least 30 min before starting the measurements, and then start the PC.</li>
		<li>Double-click on the instrument software icon to start the program.</li>
		<li>Enter the username and password in the login window. Ensure that each user has their own account.</li...

<ol>
	<li>Auerbach, M., Gafter-Gvili, A., Macdougall, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+iron:+A+framework+for+changing+the+management+of+iron+deficiency.">Intravenous iron: A framework for changing the management of iron deficiency.</a> Lancet Haematology. 7 (4), e342-e350 (2020).</li><li>Generic drugs: FDA should make public its plans to issue and revise guidance on nonbiological complex drugs. US Government Accountability Office Available from: <a target="_blank" href="https://www.gao.gov/products/gao-18-80">https://www.gao.gov/products/gao-18-80</a> (2017)</li><li>Klein, K., 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+EU+regulatory+landscape+of+non-biological+complex+drugs+(NBCDs)+follow-on+products:+Observations+and+recommendations.">The EU regulatory landscape of non-biological complex drugs (NBCDs) follow-on products: Observations and recommendations.</a> European Journal of Pharmaceutical Sciences. 133, 228-235 (2019).</li><li>Astier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+select+a+nanosimilar.">How to select a nanosimilar.</a> Annals of the New York Academy of Sciences. 1407 (1), 50-62 (2017).</li><li>Rottembourg, J., Kadri, A., Leonard, E., Dansaert, A., Lafuma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+two+intravenous+iron+sucrose+preparations+have+the+same+efficacy.">Do two intravenous iron sucrose preparations have the same efficacy.</a> Nephrology Dialysis Transplantation. 26 (10), 3262-3267 (2011).</li><li>Aguera, M. L., 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=Efficiency+of+original+versus+generic+intravenous+iron+formulations+in+patients+on+haemodialysis.">Efficiency of original versus generic intravenous iron formulations in patients on haemodialysis.</a> PLoS One. 10 (8), e0135967 (2015).</li><li>Alphandery, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+oxide+nanoparticles+for+therapeutic+applications.">Iron oxide nanoparticles for therapeutic applications.</a> Drug Discovery Today. 25 (1), 141-149 (2020).</li><li>Arami, H., Khandhar, A., Liggitt, D., Krishnan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery,+pharmacokinetics,+biodistribution+and+toxicity+of+iron+oxide+nanoparticles.">In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles.</a> Chemical Society Reviews. 44 (23), 8576-8607 (2015).</li><li>Nikravesh, N., 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=Factors+influencing+safety+and+efficacy+of+intravenous+iron-carbohydrate+nanomedicines:+From+production+to+clinical+practice.">Factors influencing safety and efficacy of intravenous iron-carbohydrate nanomedicines: From production to clinical practice.</a> Nanomedicine. 26, 102178 (2020).</li><li>Pai, A. B., 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=In+vitro+and+in+vivo+DFO-chelatable+labile+iron+release+profiles+among+commercially+available+intravenous+iron+nanoparticle+formulations.">In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.</a> Regulatory Toxicology and Pharmacology. 97, 17-23 (2018).</li><li>Brandis, J. E. P., 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=Evaluation+of+the+physicochemical+properties+of+the+iron+nanoparticle+drug+products:+Brand+and+generic+sodium+ferric+gluconate.">Evaluation of the physicochemical properties of the iron nanoparticle drug products: Brand and generic sodium ferric gluconate.</a> Molecular Pharmaceutics. 18 (4), 1544-1557 (2021).</li><li>Di Francesco, T., Sublet, E., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicines+in+clinical+practice:+Are+colloidal+iron+sucrose+ready-to-use+intravenous+solutions+interchangeable.">Nanomedicines in clinical practice: Are colloidal iron sucrose ready-to-use intravenous solutions interchangeable.</a> European Journal of Pharmaceutical Sciences. 131, 69-74 (2019).</li><li>Di Francesco, T., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+and+easily+reproducible+protocol+for+the+determination+of+size+and+size+distribution+of+iron+sucrose+using+dynamic+light+scattering.">A robust and easily reproducible protocol for the determination of size and size distribution of iron sucrose using dynamic light scattering.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 89-93 (2018).</li><li>The Center for Research on Complex Generics. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/drugs/guidance-compliance-regulatory-information/center-research-complex-generics">https://www.fda.gov/drugs/guidance-compliance-regulatory-information/center-research-complex-generics</a> (2021)</li><li>Drug products, including biological products, that contain nanomaterials - Guidance for industry. Center for Drug Evaluation and Research. FDA-2017-D-0759. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/drug-products-including-biological-products-contain-nanomaterials-guidance-industry">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/drug-products-including-biological-products-contain-nanomaterials-guidance-industry</a> (2022)</li><li>US Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Draft+guidance+on+ferric+oxyhydroxide.">Draft guidance on ferric oxyhydroxide.</a> US Food and Drug Administration. , (2021).</li><li>Zou, P., Tyner, K., Raw, A., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+of+iron+carbohydrate+colloid+drug+products.">Physicochemical characterization of iron carbohydrate colloid drug products.</a> The AAPS Journal. 19 (5), 1359-1376 (2017).</li><li>D'Mello, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+landscape+of+drug+products+containing+nanomaterials+in+the+United+States.">The evolving landscape of drug products containing nanomaterials in the United States.</a> Nature Nanotechnology. 12 (6), 523-529 (2017).</li><li>Q2 (R1) Validation of analytical procedures: Text and methodology guidance for industry. FDA-2017-D-6821. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r1-validation-analytical-procedures-text-and-methodology-guidance-industry">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r1-validation-analytical-procedures-text-and-methodology-guidance-industry</a> (2005)</li><li>Reflection paper on the data requirements for intravenous iron-based nano-colloidal products developed with reference to an innovator medicinal product. European Medicines Agency Available from: <a target="_blank" href="https://www.ema.europe.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-iron-based-nano-colloidal-products-developed_en.pdf">https://www.ema.europe.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-iron-based-nano-colloidal-products-developed_en.pdf</a> (2015)</li><li>Fischer, K., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+and+novel+applications+of+particle+sizing+by+dynamic+light+scattering.">Pitfalls and novel applications of particle sizing by dynamic light scattering.</a> Biomaterials. 98, 79-91 (2016).</li><li>Caputo, F., 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=Asymmetric-flow+field-flow+fractionation+for+measuring+particle+size,+drug+loading+and+(in)stability+of+nanopharmaceuticals.+The+joint+view+of+European+Union+Nanomedicine+Characterization+Laboratory+and+National+Cancer+Institute+-+Nanotechnology+Characterization+Laboratory.">Asymmetric-flow field-flow fractionation for measuring particle size, drug loading and (in)stability of nanopharmaceuticals. The joint view of European Union Nanomedicine Characterization Laboratory and National Cancer Institute - Nanotechnology Characterization Laboratory.</a> Journal of Chromatography A. 1635, 461767 (2021).</li><li>Yusa, S., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6+-+Polymer+characterization.">Chapter 6 - Polymer characterization.</a> Polymer Science and Nanotechnology. , 105-124 (2020).</li></ol>

DLS has become a fundamental assay for the determination of the size and size distribution of nanoparticles for applications in drug development and regulatory evaluation. Despite advances in DLS techniques, methodologic challenges still exist regarding the diluent selection and sample preparation, which are especially relevant for iron-carbohydrate complexes in colloidal solutions. Importantly, DLS methods for iron-carbohydrate nanomedicines have not yet been studied extensively in the biological milieu (e.g., the plasm...

Iron sucrose (IS) is a colloidal solution comprised of nanoparticles consisting of a complex of a polynuclear iron-oxyhydroxide core and sucrose. IS is widely employed to treat iron deficiency among patients with a wide variety of underlying disease states who do not tolerate oral iron supplementation or for whom oral iron is not effective1. IS belongs to the drug class of complex drugs as defined by the Food and Drug Administration (FDA), which is a class of drugs with complex chemistry commensurate with biologicals2. The regulatory evaluation of complex drug products may require additional orthogonal physicochemical methods and/or preclinical or clinical studies to accurately compare follow-on complex drugs3,4. This is important because several studies have reported that the use of IS versus a follow-on IS product does not produce the same clinical outcomes. This underscores the criticality of the use of novel and orthogonal characterization techniques that are suitable for detecting differences in the physicochemical properties between IS products5,6. 
The accurate elucidation of the size and size distribution of IS is of clinical importance, as particle size is a major influential factor in the rate and extent of opsonization-the first critical step in the biodistribution of these complex drugs7,8. Even slight variations in the particle size and particle size distribution have been related to changes in the pharmacokinetic profile of iron-oxide nanoparticle complexes9,10. A recent study by Brandis et al. showed that particle size measured by DLS was significantly different (14.9 nm &plusmn; 0.1 nm vs. 10.1 nm &plusmn; 0.1 nm, p &lt; 0.001) when comparing a reference listed drug and a generic sodium ferric gluconate product, respectively11. The consistent batch-to-batch quality, safety, and efficacy of iron-carbohydrate products are entirely dependent on the manufacturing process scale-up, and potential manufacturing drift must be carefully considered9. The manufacturing process may result in residual sucrose, and this will vary based on the manufacturer12. Any modifications in the manufacturing process variables can lead to significant changes in the final complex drug product with regard to the structure, complex stability, and in vivo disposition9. 
To assess drug consistency and predict the drug's in vivo behavior, contemporary orthogonal analytical methodologies are required to determine the physicochemical properties of complex nanomedicines. However, there is a lack of standardization of methodologies, which can result in a high degree of interlaboratory variation in result reporting13. Despite the recognition of these challenges by global regulatory authorities and the scientific community14, most of the physicochemical characteristics of IS remain poorly defined, and the full complement of critical quality attributes in the context of available regulatory guidance documents have not been defined15. The draft product-specific guidance documents issued by the FDA for iron-carbohydrate complexes suggest DLS as a procedure to evaluate the size and size distribution of follow-on products16,17.
Several publications have detailed established DLS protocols to determine IS nanoparticle dimensions13,18. However, because the sample preparation, procedure conditions, instrumentation, and instrumentation setting parameters are different among the published methods, the DLS results cannot be directly compared in the absence of a standardized method to interpret the results13,18. The diversity in methodologies and data-reporting approaches limit the appropriate evaluation of these characteristics for comparative purposes19. Importantly, many of the DLS protocols previously published to evaluate IS do not account for the effect of the diffusion of sucrose in the suspension due to the presence of free sucrose, which has been shown to spuriously elevate the Z-average-calculated hydrodynamic radii of the nanoparticles in colloidal solutions13,18. The present protocol aims to standardize the methodology for the measurement of the particle size and distribution of IS. The method has been developed and validated for this purpose.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>NA</td>
			<td>equipped with Zetasizer software 7.12, Helium Neon laser (633 nm, max. 4 mW) and 173&#176; backscattering geometry</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic cuvettes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LLG-Disposable plastic cells</td>
			<td>LLG labware</td>
			<td>LLG-K&#252;vetten, Makro, PS; Order number 9.406011</td>
			<td></td>
		</tr>
		<tr>
			<td>low-particle water</td>
			<td></td>
			<td></td>
			<td>&#160;(The use of freshly deionized and filtered (pore size 0.2 &#956;m) water is recommended).</td>
		</tr>
		<tr>
			<td>Microlitre pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Venofer 100 mg/5 mL</td>
			<td>Vifor Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask 25 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosphere</td>
			<td>Thermo</td>
			<td>3020A</td>
			<td>Particle Standard</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Origin Pro v.8.5&#160;</td>
			<td>Origin Lab Corporation</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic light scattering analysis for the determination of the particle size of iron-carbohydrate complexes

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

The method described was validated according to ICH Q2(R1)20, which involved the measurement of test solutions under varying conditions. The precision was only 0.5% RSD for the Z-average size, while a maximum of 3.5% RSD was calculated for the PDI. The mean results from different analysts and days only differed by 0.4% for the Z-average size and 1.5% for the PDI. Statistics were calculated from 12 measurements performed by two analysts on varying days. Neither changes in the test concentration in ...

Watch this Scientific Journal Video about Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes at JoVE.com

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally ...

Dynamic light scattering, or DLS, is a fundamental method to evaluate intact particle size and distribution. However, analysis of iron carbohydrate nanoparticles does pose some challenges. An advantage of DLS include wide available instrumentation, easy to perform the analysis, and establish protocols for the analysis of nanomaterials.One of the goals of DLS is to evaluate the poly dispersity profile of the nanoparticles, which may ultimately affect the interaction with the biological milieu. For the demonstration of the procedure, will be Cintia Batista Marques, a PhD candidate from University of Geneva. After starting the machine in the instrument software, select the storage location in the opened window, name the measurement file, and confirm the details by clicking on save.Select the required standard operating procedure from the dropdown list in the instrument interface. If an older SOP is needed, select browse for SOP from the list and confirm the selection by clicking on the green arrow. To begin the measurement process, click on the green start button at the bottom of the instrument interface screen.Then, fill one milliliter of the undiluted particle standard into a polystyrene cuvette and close it with the lid. Once filled, ensure that there are no air bubbles. If any air bubbles are present, remove them by tapping lightly on the cuvette.Place the cuvette in the cell holder of the instrument with the arrow mark facing forward and close the measuring chamber cover. Load the unit parameter SOP and enter sample name SST 20 nanometer particle standard in the start window. Additionally, add a note that includes an identifier number and the expiry date of the standard.To measure iron sucrose solution, pipette 0.5 milliliters of an iron sucrose solution with 2%mass by volume iron content into a 25 milliliter volumetric flask and fill up to the mark with low particle water, resulting in a solution containing 0.4 milligrams iron per milliliter. Now place the plastic cuvette containing the measuring solution in the device with the arrow mark facing forward and close the lid. Load the parameter SOP again and enter sample name batch number in the start window.Start the measurement and when it finishes, indicated by an acoustic signal, close the measurement window. Calculate the mean value of six individual measurements. Mark the individual measurements in the records view of measurement file and right click on create average result.Add the name of the mean value under sample name. Then confirm by clicking on okay. Wait for the software to create a new record at the end of the list and look for a name entered as well as the average result in that record.The size distribution plots by intensity, volume, and number are shown. Size distribution by intensity impacted by a second peak is provided as an example of a bad result. Poor quality data showed an additional signal at 5, 000 nanometers.The size distribution by number differed by up to a factor of two from the proposed intensity based Z average. Only slightly lower values were calculated by the size distribution by volume. When using DLS to characterize iron carbohydrate nanoparticles, it is important to ensure correct SOP, sample dilution, and also cuvette filling.Asymmetrical flow field flow fractionation and size exclusion chromatography and small angle x-ray scattering can be performed as additional orthogonal physico-chemical methods. Validated protocols for DLS greatly improve the robustness of initial and comparative characterization of iron carbohydrate nanoparticles. However, the sample preparation and the bias of the technique toward large particle sizes needs to be considered in the context of orthogonal methodologies.

dynamic-light-scattering-analysis-for-determination-particle-size

Research

JoVE Journal

Medicine

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the so-called &#34;evaporative&#34; subtype, that is mainly caused by meibomian gland dysfunction (MGD). Intense pulsed light (IPL) devices employ high intensity pulses of polychromatic lights with a broad range of wavelength (515-1200 nm). IPL treatment has been utilized for years in the field of dermatology, and then its use was applied to ophthalmology for the treatment of MGD. Recently, a new device employing IPL was specifically designed for the periocular application. This procedure determines the thermal selective coagulation and ablation of superficial blood vessels and telangiectasias of the eyelids skin, reducing the release of inflammatory mediators and tear cytokines levels, and improving meibomian glands outflow. IPL treatment is noninvasive and easy to perform, lasts for only a few minutes and can be conducted in an office setting. In the present study, 19 patients underwent 3 sessions of IPL treatment. After treatment, both mean noninvasive break-up time and lipid layer thickness grade significantly increased, as a result of an improvement of tear film stability and quality, respectively. Conversely, no statistically significant changes were found for meibomian gland loss and tear osmolarity. Furthermore, the vast majority of the treated patients (17/19; 89.5% of the total) perceived an improvement of their ocular discomfort symptoms after IPL treatment. Although IPL treatment provides an improvement of both ocular surface parameters and ocular discomfort symptoms after one cycle of three sessions, regular repeated treatments are usually required to maintain the persistence over the time of its beneficial effects.

Dry eye disease is an increasingly common condition, which strongly impair patients&#39; life quality. Recently, a new device employing intense pulsed light, specifically designed for the periocular area, has been shown to improve tear film stability and ocular discomfort symptoms in dry eye disease owing to meibomian gland dysfunction.

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...