Name: labeling and imaging of amyloid plaques in brain tissue using the natural polyphenol curcumin
Uploaded: 2019-11-01T14:30:00
Description: Watch this Scientific Journal Video about Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin at JoVE.com

Support for this study came from the Field Neurosciences Institute at Ascension of St. Mary&#39;s.

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Saginaw Valley State University. The human tissue was obtained from an established brain bank at the Banner Sun Health Institute in Arizona15,16.
1. Perfusion of the animals
<ol>
	<li>Prepare the fixative and perfusion buffers.
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		<li>Prepare 0.1 M sodium phosphate buffer by adding 80 g of sodium chloride (NaCl), 2 g of potassium chlorid...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+curcumin+forms+selectively+bind+fibrillar+amyloid+beta+in+post+mortem+Alzheimer's+disease+brains:+Implications+for+in-vivo+diagnostics.">Different curcumin forms selectively bind fibrillar amyloid beta in post mortem Alzheimer's disease brains: Implications for in-vivo diagnostics.</a> Acta Neuropathologica Communications. 6 (1), 75 (2018).</li><li>Koronyo, Y., 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=Retinal+amyloid+pathology+and+proof-of-concept+imaging+trial+in+Alzheimer's+disease.">Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease.</a> JCI Insight. 2 (16), (2017).</li><li>Koronyo, Y., Salumbides, B. C., Black, K. L., Koronyo-Hamaoui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+in+the+retina:+imaging+retinal+abeta+plaques+for+early+diagnosis+and+therapy+assessment.">Alzheimer's disease in the retina: imaging retinal abeta plaques for early diagnosis and therapy assessment.</a> Neurodegenerative Diseases. 10 (1-4), 285-293 (2012).</li><li>Koronyo-Hamaoui, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+amyloid+plaques+in+retinas+from+Alzheimer's+patients+and+noninvasive+in+vivo+optical+imaging+of+retinal+plaques+in+a+mouse+model.">Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model.</a> NeuroImage. 54 (Suppl 1), S204-S217 (2011).</li><li>Ran, C., 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=Design,+synthesis,+and+testing+of+difluoroboron-derivatized+curcumins+as+near-infrared+probes+for+in+vivo+detection+of+amyloid-beta+deposits.">Design, synthesis, and testing of difluoroboron-derivatized curcumins as near-infrared probes for in vivo detection of amyloid-beta deposits.</a> Journal of the American Chemical Society. 131 (42), 15257-15261 (2009).</li><li>Beach, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sun+Health+Research+Institute+Brain+Donation+Program:+Description+and+Experience,+1987-2007.">The Sun Health Research Institute Brain Donation Program: Description and Experience, 1987-2007.</a> Cell Tissue Bank. 9 (3), 229-245 (2008).</li><li>Green, S. J., Killiany, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subregions+of+the+inferior+parietal+lobule+are+affected+in+the+progression+to+AD.">Subregions of the inferior parietal lobule are affected in the progression to AD.</a> Neurobiology of Aging. 31 (8), 1304-1311 (2010).</li><li>Ono, K., Hasegawa, K., Naiki, H., Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+has+potent+anti-amyloidogenic+effects+for+Alzheimer's+beta-amyloid+fibrils+in+vitro.">Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.</a> Journal of Neuroscience Research. 75 (6), 742-750 (2004).</li><li>Garcia-Alloza, M., Borrelli, L. A., Rozkalne, A., Hyman, B. T., Bacskai, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+labels+amyloid+pathology+in+vivo,+disrupts+existing+plaques,+and+partially+restores+distorted+neurites+in+an+Alzheimer+mouse+model.">Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model.</a> Journal of Neurochemistry. 102 (4), 1095-1104 (2007).</li><li>Mutsuga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+curcumin+to+senile+plaques+and+cerebral+amyloid+angiopathy+in+the+aged+brain+of+various+animals+and+to+neurofibrillary+tangles+in+Alzheimer's+brain.">Binding of curcumin to senile plaques and cerebral amyloid angiopathy in the aged brain of various animals and to neurofibrillary tangles in Alzheimer's brain.</a> Journal of Veterinary Medical Science. 74 (1), 51-57 (2012).</li><li>Tei, M., Uchida, K., Mutsuga, M., Chambers, J. 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Our hypothesis was that Cur could be used as the quickest, easiest, and least expensive way to label and image A&#946; plaques in postmortem AD brain tissue when compared to other classical amyloid binding dyes, as well as A&#946;-specific antibodies. The aims of this study were to determine the minimum time required to label and image A&#946; plaques by Cur in postmortem AD brain tissue and determine whether Cur can be used as an alternative to A&#946; antibody for labeling A&#946; plaques. To this end, the A&#946;-labe...

Alzheimer&#39;s disease (AD) is one of the most common, age-related, progressive neurological disorders and one of the leading causes of death worldwide1,2. Learning, memory, and cognition impairment, along with neuropsychiatric disorders, are the common symptoms manifested in AD3. Although the etiology of AD has not been fully elucidated, the available genetic, biochemical, and experimental evidence indicates that gradual deposition of A&#946; is a definitive biomarker for AD4. This misfolded protein accumulates in intracellular and extracellular spaces and is thought to be involved in synaptic loss, increased neuroinflammation, and neurodegeneration in the cortical and hippocampal regions in brain affected by AD5. Therefore, histochemical detection of A&#946; in AD tissue is a crucial first step in developing non-toxic, anti-amyloid drugs to prevent AD progression.
During the past few decades, several dyes and antibodies have been used by many research laboratories to label and image A&#946; plaques in brain tissue, but some of these methods are time consuming and the dyes or antibodies used are expensive, requiring several accessory chemicals. Therefore, the development of an inexpensive means of detection of A&#946; plaques in the AD brain would be a welcome new tool. Many laboratories started using Cur, a promising anti-amyloid natural polyphenol, for labeling and imaging A&#946;, as well as a therapeutic agent for AD6,7,8,9. Its hydrophobicity and lypophilic nature, structural similarities with classical amyloid binding dyes, strong fluorescent activity, as well as strong affinity to bind with A&#946; makes it an ideal fluorophore for labeling and imaging of A&#946; plaques in AD tissue10. Cur binds with A&#946;-plaques and oligomers and its presence is also detected in intracellular spaces7,11,12,13. In addition, it has been shown that minimal amounts (1&#8722;10 nM) of Cur can label A&#946; plaques in 5x familial Alzheimer&#39;s disease (5xFAD) brain tissue7. Although the 1 nM concentration does not provide the optimal fluorescence intensity for counting of A&#946; plaques, a 10 nM or higher concentration of Cur does. Ran and colleagues14 reported that doses as low as 0.2 nM of difluoroboron-derivatized Cur can detect in vivo A&#946; deposits nearly as well as an infrared probe. Whether this dose is sufficient to label A&#946; plaques in tissue is still not clear. Most previous studies have used 20&#8722;30 min for staining A&#946; plaques using Cur, but optimal staining may require much less time.
The present study was designed to test the minimum time required by Cur to label A&#946; plaques in AD brain tissue and to compare the sensitivity for labeling and imaging of A&#946; plaques in brain tissue from the 5xFAD mice after staining with Cur with other conventional A&#946;-binding dyes, such as Thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC). The A&#946; labeling capability of these classical amyloid binding dyes was compared with Cur staining in paraffin-embedded and cryostat coronal brain sections from 5xFAD mice and from aged-matched human AD and control brain tissue. The findings suggest that Cur labels A&#946; plaques in a manner similar to A&#946;-specific antibodies (6E10) and moderately better than Thio-S, CR, or FJC. In addition, when intraperitoneal injections of Cur to 5xFAD mice were administered for 2&#8722;5 days, it crossed the blood-brain barrier and bound with A&#946; plaques7. Interestingly, nanomolar concentrations of Cur have been used to label and image A&#946; plaques in 5xFAD brain tissue7,14. Moreover, morphologically distinct A&#946; plaques, such as core, neuritic, diffuse, and burned-out plaques can be labeled by Cur more efficiently than with any of the other conventional amyloid binding dyes7. Overall, Cur can be applied to label and image A&#946; plaques in postmortem brain tissue from AD animal models and/or human AD tissue in an easy and inexpensive way, as a reliable alternative to A&#946;-specific antibodies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>IHC world, Woodstock, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aanimal model of Alzheimer&#39;s disease</td>
			<td>Jackson&#39;s laboratory, Bar Harbor, ME</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute alcohol</td>
			<td>VWR,Radnor, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 594</td>
			<td>Santacruz Biotech, Dallas, TX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody 6E10</td>
			<td>Biolegend, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody A11</td>
			<td>Millipore, Burlington, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound light microscope</td>
			<td>Olympus, Shinjuku, Japan</td>
			<td>Olympus BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Congo red</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>GMI, Ramsey, MN</td>
			<td>LeicaCM1800</td>
			<td></td>
		</tr>
		<tr>
			<td>Curcumin</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dystyrene plasticizer xylene</td>
			<td>BDH, Dawsonville, GA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter papers</td>
			<td>Fisher scientific, Pittsburgh, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Leica, Buffalo Grove, IL</td>
			<td>Leica DMI 6000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus, Shinjuku, Japan</td>
			<td>Olympus 1x70</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ploy-lysine coated charged glass slide</td>
			<td>Globe Scientific Inc, Mahwah, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>EMD Millipore, Burlington, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Vortex Pharmaceuticals limited, Dearborn, MI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin-S</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR,Radnor, PA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

labeling and imaging of amyloid plaques in brain tissue using the natural polyphenol curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin labels A&#946; plaques within a minute. When we stained 5xFAD tissue with Cur, we found that Cur label A&#946; plaques within 1 min. Although increased incubation time with Cur slightly increased the fluorescence intensity of A&#946; plaques, the number of observed A&#946; plaques was not significantly different between 1 min and 5 min staining time (Figure 1).
Cur can label A&#946; plaques...

Watch this Scientific Journal Video about Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin at JoVE.com

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

This protocol is the most efficient and cost-effective method for accurately labeling beta-amyloid plaques, as compared to amyloid-specific antibodies, which are currently the gold standard for labeling these plaques. This technique takes less time and is as equally effective as any of the commonly used amyloid labeling methods. Curcumin strongly binds to amyloid plaques and can be used as an imaging probe during PET scans of the brain and for relatively noninvasive screenings using retinal scanning.Curcumin also binds to neurofibrillary or tau tangle in Alzheimer's disease, alpha synuclein in Parkinson's disease, and mutant Huntington's proteins in Huntington's disease. Demonstrating the procedure with Panchanan Maiti will be Zackary Bowers, a graduate student, and Alexandra Plemmons, a research scientist from my lab. After confirming a lack of response to pain reflex in an anesthetized 12 month old Alzheimer's disease model mouse, place the mouse in the supine position on a surgery tray and make an incision into the abdomen and through the diaphragm.Insert a 21 gauge perfusion needle into the incision and make a small incision at the right auricle to allow the perfusion fluid to drain from the body. Use a gravity fed perfusion system to allow ice cold 0.1 molar PBS fluid to flow into the needle for five to six minutes at a 20 to 25 milliliters per minute flow rate. If the perfusion is performed correctly, a clear liver will be observed.A proper animal perfusion is essential because if the blood is not removed completely, curcumin may bind to the blood vessels and result in a green fluorescent background signal. When all of the PBS has been delivered, switch the buffer valve to an ice cold 4%paraformaldehyde solution for eight to 10 minutes before using scissors and forceps to free the brain from the skull. Then use a spatula to place the brain into a vial of 4%paraformaldehyde at least 10 times the tissue volume for four degrees celsius storage until processing.To obtain sections for cryostat sectioning, sequentially immerse the brain in graded sucrose solutions at four degrees celsius for 24 hours per concentration before using a cryostat at negative 22 degrees celsius to obtain 40 micrometer sections, placing 10 to 20 sections per well in a six well plate containing PBS supplemented with 0.02%azide as they are obtained. When all of the samples have been acquired, store the sections at four degrees celsius until further processing. To obtain sections for paraffin sectioning, dehydrate post-fixed perfused brain tissue with sequential graded alcohol solutions for two hours per concentration, followed by two one hour 100%alcohol immersions and two one hour xylene immersions at room temperature.After the last xylene immersion, penetrate the tissue with a one to one xylene to paraffin solution two times for one hour per incubation at 56 degrees celsius in a glass conical glass covered with aluminum foil before immersing the tissues in 56 degree celsius paraffin for four to six hours. At the end of the incubation, use a rotary microtome at room temperature to obtain five micrometer thick sections placing the sections in a 45 degree celsius tissue water bath as they are collected. For curcumin labeling of amyloid beta plaques in cryostat tissue sections, rinse the sections with PBS three times for five minutes per wash before immersing the sections in 70%ethanol for two minutes at room temperature.While the sections are incubating, dissolve one millimolar of stock curcumin in methanol and dilute the solution to a final working concentration of 10 micromolar with 70%ethanol. At the end of the incubation, immerse the sections in the working curcumin solution for 10 minutes at room temperature at 50 rotations per minute. At the end of the staining period, discard the curcumin solution and wash the sections with three two minute washes in 70%ethanol.After the last wash, transfer the sections to polylysine coated glass slides and mount a cover slip onto each slide with an appropriate organic mounting medium. Then view the sections on a florescence microscope using a 10 or 20x objective and the appropriate excitation and emission filters. For histochemical labeling of deep paraffinized tissue sections, immerse the five micrometer thick sections in xylene two times for five minutes at room temperature per immersion, followed by rehydration with graded one minute alcohol solution immersions.At the end of the incubation, wash the sections with graded alcohol solutions for two minutes per concentration followed by clearing with two five minute xylene immersions. After the second xylene immersion, mount each section on a microscope slide with a cover slip and an appropriate mounting medium for florescence microscopy imaging as demonstrated. For labeling of cryostat section specimens with anti-A-beta antibodies, wash the samples three times with fresh PBS per wash in individual wells of a 12 well plate before blocking any non-specific binding with 10%normal goat serum in PBS and 0.5%triton x-100 for one hour at room temperature.At the end of the incubation, discard the blocking solution and incubate the samples with a-beta specific antibody overnight at four degree celsius and 150 rotations per minute. The next morning, wash the sections with three 10 minute washes in fresh PBS per wash, followed by incubation with an appropriate secondary antibody conjugated with a red fluorophore for one hour at room temperature protected from light. At the end of the incubation, wash the sections three times with PBS followed by one wash with 70%alcohol.After the alcohol wash, incubate the sections with 10 micromolar curcumin for 10 minutes at room temperature followed by three one minute 70%alcohol washes. After the last wash, dehydrate the sections with 90%and 100%alcohol for one minute per concentration and clear the sections two times for five minutes per immersion with fresh xylene per incubation. Then mount and image the sections as demonstrated.For intracellular amyloid beta co-localization, stain the sections with anti amyloid beta antibody followed by staining with curcumin at room temperature and 50 rotations per minute protected from light as demonstrated. At the end of the incubation, counterstain with an appropriate nuclear dye for 10 minutes at room temperature and 50 rotations per minute protected from light followed by three washes in PBS. Then mount and image the sections by fluorescence microscopy as demonstrated.Although increasing the incubation time with curcumin slightly increases the fluorescence intensity of the A beta plaques, the number of observed plaques is not significantly different between one and five minutes of staining time. Curcumin can be used to stain A beta plaques and cryosectioned, paraffin embedded, and human Alzheimer's disease tissue samples. Labeling with A beta specific antibody before curcumin staining reveals that the curcumin is completely co-localized with A beta at the same plaques that bind the antibody.After staining with curcumin labeled A beta oligomers, curcumin co-localization with the oligomers can be observed in A beta plaques. Similarly, curcumin also co-localizes with the A beta specific antibody in intracellular spaces, confirming that the stain can label intracellular A beta. Curcumin labeling is also more prominent than conventional amyloid binding dyes.Curcumin derivatives found in tumeric extracts label A beta plaques comparatively to curcumin in human Alzheimer's disease brain tissue regardless of the type of mounting medium used. In addition, curcumin immunofluorescence signals and counterstaining intensity are maintained even after staining with typical nuclear stains and other cell markers. We met that constant sections should be thinner than 40 microns.This training procedure should be kept under 30 minutes and the optimal curcumin concentration is one to two micromolar. Double leveling retarded biomarkers can be performed on the same tissue and can provide a greater specificity of which types of brain cells are being leveled. Given that curcumin can reduce the amyloid plaque load, the precise mechanisms of how this process occurs is currently being investigated at both the cellular and molecular levels.

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Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Visualization of Amyloid &amp;#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...