This research was supported by the Deutsche Forschungsgemeinschaft (CRC1039 A3) and the research funding program "Landesoffensive zur Entwicklung wissenschaftlich-&#246;konomischer Exzellenz" (LOEWE) of the State of Hessen, Research Center for Translational Medicine and Pharmacology TMP and the Else Kr&#246;ner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation - Pharma (TRIP).

1. EAE Induction in SJL/J Mice
<ol>
	<li>Mice
	<ol>
		<li>Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = 10 mice per group.</li>
		<li>For the assessment of medication effects, administer the drug and placebo for the control group continuously via the drinking water or via food pellets starting 3 or 5 days after immunization (n = 10 per group). During the peak of the disease, administer medication or placebo with milk or 3% sugar water-soaked corn flakes.</li>
	</ol>
	</li>
	<li>Immunization material
	<ol>
		<li>Use an EAE induction kit consisting of antigen (peptide of proteolipid protein, PLP139-151, 1 mg/mL emulsion) in an emulsion with complete Freund&#39;s adjuvant (CFA, heat-killed Mycobacterium tuberculosis H37 Ra) and 2 vials (5 &#181;g each) of lyophilized pertussis toxin (PTX).</li>
		<li>Dissolve the PTX (2 &#181;g/mL) in 1x phosphate-buffered saline (PBS; i.e., add 1.5 mL of PBS to each PTX tube, mix well, remove with the same pipet tip, and add to 1 mL of PBS in a 50-mL tube); mix well.</li>
	</ol>
	</li>
	<li>Immunization
	<ol>
		<li>Inject PLP/CFA subcutaneously in 2 portions, each 100 &#181;L, both at the base of the tail. Do not inject into the back of the neck, because any immune reactions in the skin in the upper back or neck will disturb imaging of the head and spinal cord.</li>
		<li>Inject 100 &#181;L of PTX intraperitoneally (i.p.), twice per mouse, the first 1 - 2 h after immunization and the second at 24 h.</li>
		<li>For the control mice, inject CFA without PLP (2 portions of 100 &#181;L) plus PTX without PLP.</li>
	</ol>
	</li>
	<li>Mouse handling after immunization
	<ol>
		<li>Weigh the mice every other day up to day 7, and then weigh them daily. 
		NOTE: Mice lose about 1 - 2 g of body weight during EAE. The decline marks the onset of EAE.</li>
		<li>Assess clinical symptoms daily from day 7 according to the standard scoring systems (i.e., Score 0: no obvious changes in motor functions; score 0.5: distal paralysis of the tail; score 1: complete tail paralysis; score 1.5: mild paresis of one or both hind legs; score 2: severe paresis of hind legs; score 2.5: complete paralysis of one hind leg; score 3: complete paralysis of both hind legs; score 3.5: complete paralysis of hind legs and paresis of one front leg. Euthanize mice with scores of 3.5 or higher for &#62; 12 h.</li>
	</ol>
	</li>
	<li>EAE course and time of imaging
	<ol>
		<li>Perform the first imaging at the onset of the disease, when the mice reach scores &#62; 1, which will occur around day 10 - 12 after immunization.</li>
		<li>Perform the second imaging at the peak, which will be reached 1 or 2 days after the initial symptoms develop and will last for 1 - 3 days. 
		NOTE: Subsequently, the mice will fully recover within 7 to 10 days. Imaging during the intervals may still show vascular leaks, but inflammation indicators should be negative.</li>
	</ol>
	</li>
</ol>
2. Bioluminescent and Near-infrared Imaging of Optic Neuritis and Brain Inflammation
<ol>
	<li>Setup of the imaging system
	<ol>
		<li>Perform in vivo imaging with any equipment that allows for the analysis of bioluminescence and near-infrared signals.</li>
		<li>Keep mice under 2 - 2.5% isoflurane anesthesia during all imaging procedures.</li>
		<li>Position one or two mice beside each other in the apparatus using the middle gas supplies. Position the upper spine in the center.</li>
		<li>Use two mice simultaneously, one per group, for evaluation of medication effects to compare pairs. This is important for bioluminescent imaging.</li>
		<li>Shield the site of immunization with black cloth and take a photograph and baseline image to assess the correct positioning of the mouse/mice. Use the B-focus with a 6.5-cm distance to the camera for all images.</li>
	</ol>
	</li>
	<li>Injection and imaging of bioluminescent inflammation probe
	<ol>
		<li>Use the in vivo imaging system settings: Epi-BLI, Em filter open, Ex filter block, fstop 1, binning 8, focus B = 6.5 cm, ad exposure 120 s; take a baseline image.</li>
		<li>Inject 100 &#181;L i.p. of the ready-to-use chemiluminescent reagent (40 mg/mL). Mix well before filling the syringe.</li>
		<li>Capture bioluminescence images 5, 10, and 15 min after injection. The time course of the bioluminescent peak differs between animals. 
		NOTE: The peak will occur 5 - 10 min after injection; a decline at 15 min indicates that no further images are required. Use mouse pairs of control and treatment groups to eliminate minor biases due to different time courses.</li>
		<li>Fill in descriptions relevant to the experiment. Observe a dialog box pop up automatically; it includes information, such as mouse strain, sex, time point, time of probe injection, group, etc. Save the files all in one folder; they will have time tags and all descriptions.</li>
	</ol>
	</li>
	<li>Injection and imaging of near-infrared fluorescent nanoparticles for BBB integrity
	<ol>
		<li>Use near-infrared epifluorescence imaging in the B-focus (distance: 6.5 cm). The excitation/emission maxima of the pegylated fluorescent nanoparticles are 675/690 nm. Capture two images at different wavelengths, at Ex640/Em700 and Ex675/Em720; use a 2-s exposure, binning 8, and fstop 2. Take a baseline image.</li>
		<li>Inject 70 &#181;L of pegylated fluorescent infrared nanoparticles i.v. through the tail vein and imagine the mice 3 h and 24 h after injection using the above settings. Mix the solution well before filling the syringe.</li>
		<li>Inject 0.9% sodium chloride in control mice, which will be needed to assess the specificity of the signal.</li>
	</ol>
	</li>
	<li>Injection and imaging of the near-infrared fluorescent probe for MMP activity
	<ol>
		<li>Shave or depilate the head and upper spine region cautiously one day before taking the baseline image. The skin must not be injured.</li>
		<li>Use near-infrared epifluorescence imaging in the B-focus (distance: 6.5 cm). The excitation/emission maxima of the MMP activatable probe are 680/700 nm. Capture two images at different wavelengths, at Ex640/Em700 and Ex675/Em720; use a 1-s exposure, binning 8, and fstop 2. Take a baseline image.</li>
		<li>Add 200 &#181;L of 1x PBS to the 1.5-mL tube of the ready-to-use solution (20 nmol/1.5 mL in 1x PBS) so that it will be sufficient for 10 mice; mix well before filling the syringe. 
		NOTE: The provided volume does not take into account that some volume is lost during syringe filling and injection.</li>
		<li>Inject 150 &#181;L of the probe i.v. through the tail vein 24 h before imaging. Inject PBS only in control animals to assess the specificity of the signal.</li>
		<li>At 24 h after injection, take Epi-FL images at least at two wavelengths, Ex640/Em700 and Ex 675/Em720, with the settings explained above (1-s exposure, focus B, binning 8, and fstop 2). 
		NOTE: The use of two wavelengths allows for spectral unmixing by subtracting the unspecific signal.</li>
	</ol>
	</li>
</ol>
3. Image Analyses
<ol>
	<li>Bioluminescence analysis (BLI)
	<ol>
		<li>Double-click the software to open it.</li>
		<li>In the upper menu bar, click on the file browser icon, go to the directory of the folder of the experiment, and select it; this will open all files in the folder in a table.</li>
		<li>Configure the columns showing the descriptions relevant to the experiment.</li>
		<li>For quality control, double-click on a file of a non-responder mouse without symptoms of EAE and/or a na&#239;ve mouse to check the specificity of the EAE signals. 
		NOTE: There should be no signal in na&#239;ve mice and minimal signal in non-responder mice.
		<ol>
			<li>Check the baseline image before the injection of the probe for each mouse as further control of the specificity of the signal; it should be negative.</li>
		</ol>
		</li>
		<li>To select an image, double-click on the first file of the first EAE mouse and check the bioluminescent intensity (LUT bar range) and localization. Check all images one by one. Close the file with the lowest intensity for each mouse (i.e., keep 2 out of 3 images for each mouse).</li>
		<li>Image adjustment and exportation
		<ol>
			<li>Double-click the first file to be quantified. Observe a new window pop up. Under &#34;options&#34; (upper menu), customize the labels to display in each image.</li>
			<li>In the right tool palette, go to &#34;image adjustment.&#34; By default, minimum and maximum intensities are set to &#34;auto&#34; and displayed in rainbow pseudocolor. Select &#34;manual&#34; to change the settings if necessary. 
			NOTE: For example, all exported images may have identical LUT bars (identical minima and maxima) to be easily comparable. The adjustment of the minima and maxima has no impact on the quantitative results.</li>
			<li>Click on image export, select png, the directory, and an image name</li>
		</ol>
		</li>
		<li>Quantification of Regions of Interest (ROI)
		<ol>
			<li>Go to the &#34;ROI&#34; tool in the tool palette. Select the ROI method (circle, rectangular, auto, or free-hand) and the number of ROIs. Observe the ROI window pop up in the image window.</li>
			<li>Using the mouse, adjust the size and position. Use identical ROI thresholds for all images if the auto-ROI tool is used. Use identical areas for all images if ROI sizes and positions are defined manually (e.g., circular ROIs).</li>
			<li>Click on &#34;measure RO.I. Observe a new window pop up. Customize the columns (e.g., file name, animal number, group, experiment, area, total counts, average counts, SD counts, min and max counts, area, time point, time of probe injection, etc.). Save the customized settings. When ready, select all (Ctrl + A), and copy and paste the table into a spreadsheet.</li>
			<li>Export the image as a png with the ROIs in place. Save and close the image file.</li>
		</ol>
		</li>
		<li>Repeat the procedure (steps 3.1.6 - 3.1.7) for all image files that need to be quantified. Copy all ROI quantifications into the spreadsheet. 
		NOTE: Here, results can be sorted by group, time point, etc. and statistically analyzed. Use the total counts of bioluminescence signals in the ROIs for statistical analyses.</li>
	</ol>
	</li>
	<li>Near-Infrared (NIR) analysis of fluorescent nanoparticles
	<ol>
		<li>Using the controls in the software, adjust the threshold of the image. 
		NOTE: This has no impact on the quantitative result.</li>
		<li>Visually compare the images captured at Ex/Em 675/720 and Ex/Em 640/700 to assess the specificity of the signal.</li>
		<li>Use the images captured at Ex/Em 675/720 for the quantitative analysis (excitation maximum: 680 nm). Define ROIs, for which the auto-ROI tool may be used. Adjust the auto-ROI threshold and use it for all images. Quantify the total radiant efficiency in ROIs (see step 3.1).</li>
	</ol>
	</li>
	<li>Near-Infrared (NIR) analysis of protease-sensitive probe
	<ol>
		<li>Using software controls, adjust the threshold of the image. 
		NOTE: This has no impact on the quantitative result. Perform spectral unmixing of the auto-fluorescence. The unmixing tool is implemented in Living Image. The auto-unmixing uses the Ex/Em 640/700 as the specific and 675/720 as the auto-fluorescent image.</li>
		<li>Select the unmixed image and define the ROIs, as described above. Use the total radiant efficiency in ROIs for quantitative and statistical analyses.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The present video shows techniques for bioluminescence and near-infrared fluorescence in vivo imaging of EAE in SJL/J mice. We show that bioluminescence imaging using an inflammation-sensitive probe mainly shows optic neuritis, and the quantification agrees with the clinical evaluation of EAE severity and the effects of medication. However, the bioluminescence imaging method was not able to detect inflammation of the lumbar spinal cord, which is a primary site of EAE manifestation17, like...

Multiple sclerosis is caused by the autoimmune-mediated attack and destruction of the myelin sheath in the brain and the spinal cord1. With an overall incidence of about 3.6 cases per 100,000 people a year in women and about 2.0 in men, MS is the second most common cause of neurological disability in young adults, after traumatic injuries2,3. The disease pathology is contributed to by genetic and environmental factors4 but is still not completely understood. Autoreactive T lymphocytes enter the central nervous system and trigger an inflammatory cascade that causes focal infiltrates in the white matter of the brain, spinal cord, and optic nerve. In most cases, these infiltrates are initially reversible, but persistence increases with the number of relapses. A number of rodent models have been developed to study the pathology of the disease. The relapsing-remitting EAE in SJL/J mice and the primary-progressive EAE in C57BL6 mice are the most popular models.
The clinical EAE scores, which describe the extent of the motor function deficits, and body weight are the gold standards to assess EAE severity. These clinical signs agree with the extent of immune cell infiltration and myelin destruction in the spinal cord and moderately predict drug treatment efficacy in humans5. However, these signs mainly reflect the destruction of the ventral fiber tracts in the spinal cord. Presently, there is no easy, non-invasive, reliable, and reproducible method to assess in vivo brain infiltration and optic neuritis in living mice. 
The in vivo imaging agrees with the 3 "R" principles of Russel and Burch (1959), which claim a Replacement, Reduction, and Refinement of animal experiments6, because imaging increases the readouts of one animal at several time points and allows for a reduction of the overall numbers. Presently, inflammation or myelin status is mainly assessed ex vivo via immunohistochemistry, FACS-analysis, or different molecular biological methods7, all requiring euthanized mice at specific time points. 
A number of in vivo imaging system probes have been developed to assess inflammation in the skin, joints, and vascular system. The techniques rely on the activation of bioluminescent or near-infrared fluorescent substrates by tissue peroxidases, including myeloperoxidase (MPO), matrix metalloproteinases (MMPs)8, and cathepsins9 or cyclooxygenase2. These probes have been mainly validated in models of arthritis or atherosclerosis9,10. A cathepsin-sensitive probe has also been used for fluorescence molecular tomographic imaging of EAE11. MMPs, particularly MMP2 and MMP9, contribute to the protease-mediated BBB disruption in EAE and are upregulated at sites of immune cell infiltration12, suggesting that these probes may be useful for EAE imaging. The same holds true for peroxidase or cathepsin-based probes. Technically, imaging of inflammation in the brain or spinal cord is substantially more challenging because the skull or spine absorb bioluminescent and near-infrared signals. 
In addition to inflammation indicators, fluorescent chemicals have been described, which specifically bind to myelin and may allow for quantification of myelination13. A near-infrared fluorescent probe, 3,3'-diethylthiatricarbocyanine iodide (DBT), was found to specifically bind to myelinated fibers and was validated as a quantitative tool in mouse models of primary myelination defects and in cuprizone-evoked demyelination14. In EAE, the DBT signal was rather increased, reflecting the inflammation of the myelin fibers5. 
An additional hallmark of EAE and MS is the BBB breakdown, resulting in increased vascular permeability and the extravasation of blood cells, extracellular fluid, and macromolecules into the CNS parenchyma. This can lead to edema, inflammation, oligodendrocyte damage, and, eventually, demyelination15,16. Hence, visualization of the BBB leak using fluorescent probes, such as fluorochrome-labeled bovine serum albumin5, which normally distribute very slowly from blood to tissue, may be useful to assess EAE.
In the present study, we have assessed the usefulness of different probes in EAE and show the procedure for the most reliable and robust bioluminescent technique. In addition, we discuss the pros and cons of near-infrared probes for MMP activity and BBB integrity.

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			<td height="21" style="height:21px;width:265px;">AngioSpark-680</td>
			<td style="width:267px;">Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;">NEV10149</td>
			<td style="width:636px;">Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MMP-sense 680</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td>NEV10126</td>
			<td>Imaging probe, activatable by matrix metalloproteinases, useful for imaging of inflammation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XenoLight RediJect Inflammation Probe</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td>760535</td>
			<td>Imaging probe, activatable by oxidases, useful for imaging of inflammation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLP139-151/CFA emulsion&#160;</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pertussis Toxin</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS Lumina Spectrum</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;"></td>
			<td>Bioluminescence and Infrared Imaging System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LivingImage 4.5 software&#160;</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td>CLS136334</td>
			<td>IVIS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Isoflurane</td>
			<td style="width:267px;">Abbott Labs, Illinois, USA</td>
			<td>26675-46-7</td>
			<td>Anaesthetic</td>
		</tr>
	</tbody>
</table>

bioluminescence and near-infrared imaging of optic neuritis and brain inflammation in the eae model of multiple sclerosis in mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

Time Course of Bioluminescence of Optic Neuritis
The bioluminescence signal of the inflammation probe was the strongest around the eyes and occurred exclusively in EAE mice with optic neuritis. A signal occurred in neither the non-EAE mice nor the mice not injected with the inflammation probe. The signal disappeared when the mice recovered. Hence, the signal is specific for optic neuritis, and the peak of the si...

Watch this Scientific Journal Video about Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice at JoVE.com

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

bioluminescence-near-infrared-imaging-optic-neuritis-brain

Research

JoVE Journal

Medicine

9.1K Views.  University Hospital Frankfurt. We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Video: Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

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 ...

The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability. The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed. The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing. The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient&#8217;s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing. This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research.

The Multiple Sclerosis Performance Test MSPT: An iPad-Based Disability Assessment Tool

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report methodologic details on a new test, the Multiple Sclerosis Performance Test (MSPT). This new approach to the objective of quantification of MS related disability provides a computer-based platform for precise, valid measurement of MS severity.

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical excitability. With well-established safety and tolerability, tDCS has been found to have the potential to ameliorate symptoms such as depression and pain in a range of conditions as well as to enhance outcomes of cognitive and physical training. However, effects are cumulative, requiring treatments that can span weeks or months and frequent, repeated visits to the clinic. The cost in terms of time and travel is often prohibitive for many participants, and ultimately limits real-world access. 
Following guidelines for remote tDCS application, we propose a protocol that would allow remote (in-home) participation that uses specially-designed devices for supervised use with materials modified for patient use, and real-time monitoring through a telemedicine video conferencing platform. We have developed structured training procedures and clear, detailed instructional materials to allow for self- or proxy-administration while supervised remotely in real-time. The protocol is designed to have a series of checkpoints, addressing attendance and tolerability of the session, to be met in order to continue to the next step. The feasibility of this protocol was then piloted for clinical use in an open label study of remotely-supervised tDCS in multiple sclerosis (MS). This protocol can be widely used for clinical study of tDCS.

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation tDCS in Multiple Sclerosis MS

The goal of this pilot study is to describe a protocol for the remotely-supervised delivery of transcranial direct current stimulation (tDCS) so that the procedure maintains standards of in-clinic practice, including safety, reproducibility, and tolerability. The feasibility of this protocol was tested in participants with multiple sclerosis (MS).

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...