M-V.G-S. is supported by a BrightFocus Foundation Alzheimer's Disease Research Fellowship Award (A2015309F) and an Alzheimer's Association, California Southland Chapter Young Investigator Award. T.M.W. is supported by an ARCS Foundation and John Douglas French Alzheimer's Foundation Maggie McKnight Russell-JDFAF Memorial Postdoctoral Fellowship. This work was supported by the National Institute on Neurologic Disorders and Stroke (1R01NS076794-01, to T.T.), an Alzheimer's Association Zenith Fellows Award (ZEN-10-174633, to T.T.), and an American Federation of Aging Research/Ellison Medical Foundation Julie Martin Mid-Career Award in Aging Research (M11472, to T.T.). We are grateful for startup funds from the Zilkha Neurogenetic Institute, which helped to make this work possible. &#8195;

Statement of research ethics: All experiments involving animals detailed herein were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and performed in strict accordance with National Institutes of Health guidelines and recommendations from the Association for Assessment and Accreditation of Laboratory Animal Care International.
1. Rodent Brain Isolation and Preparation for Immunostaining
DAY 1:
<ol>
	<li>Place aged TgF344-AD rats (14-month-old) or APP/PS1 mice (12-month-old) under continuous deep isoflurane anesthesia (4%). Assess the depth of anesthesia by toe pinch and the absence of withdrawal reflex.</li>
	<li>Cut through both sides of the rib cage and lift to expose the heart. Insert a 23 G needle into the left ventricle of the heart and make a small incision into the right atrium. Proceed to exsanguination by transcardial perfusion with ice-cold phosphate-buffered saline (PBS) using a peristaltic pump (30 mL&#160;for mice;&#160;150 - 200 mL&#160;for rats).</li>
	<li>Make a caudal midline incision into the skin and move the skin and muscle aside. Cut through the top of the skull along the midline and between the eyes. Remove the bone plates and isolate the whole brain from the skull.</li>
	<li>Place the brain into a coronal rodent brain matrix and slice it into quarters. Incubate posterior quarters O/N (16 h) in paraformaldehyde fixative (4% PFA in PBS) at 4 &#176;C. Wash 3x in PBS, and then transfer to 70% ethanol. Caution: PFA is toxic and should be handled under a chemical hood with appropriate personal protection equipment.</li>
</ol>
DAY 2:
<ol start="5">
	<li>Place brain quarters into embedding cassettes and progressively dehydrate tissue in successively more concentrated 1 h ethanol baths (70%, 80%, 95%, and 100% x3).</li>
	<li>Clear ethanol from the tissue with three successive 100% xylene baths (1 h each). Caution: Xylene is toxic and should be handled under a chemical hood with appropriate personal protection equipment.</li>
	<li>Embed tissue in paraffin blocks after two molten paraffin wax baths (56 - 58 &#176;C, 90 min each).</li>
</ol>
DAY 3:
<ol start="8">
	<li>Cut 10 &#956;m-thick sections of paraffin-embedded brains using a microtome. Dip sections into a water bath (50 &#176;C for 1 min) and apply to microscope slides. Leave the slides to dry O/N, ensuring tissue adhesion to the slide.</li>
</ol>
2. Immunostaining
Note: Different combinations of antibodies can be utilized for the staining procedure described below. Antibody cocktails compatible with brain tissue from rats and mice are listed in Table 1.
DAY 4:
<ol>
	<li>Deparaffinize brain sections using 2x 100% xylene baths (12 min each).</li>
	<li>Rehydrate brain sections in successive ethanol baths &#8212; 100% for 10 min, 95% for 5 min, 80% for 10 min, and finally 70% for 15 min &#8212; followed by 3x PBS washes [5 min at RT, with light agitation]. Meanwhile, heat antigen retrieval solution to 95 - 97 &#176;C on a hot plate with magnetic bar stirring.</li>
	<li>Incubate brain sections in antigen retrieval solution at 95 - 97 &#176;C for 30 min. Then, wash 3x in PBS (5 min at RT, with light agitation).</li>
	<li>Quickly dry slides using delicate task wipers to avoid tissue drying, and draw a hydrophobic barrier around the tissue area with a hydrophobic barrier pen. Fill the encircled tissue region with blocking buffer [PBS containing 0.3% Triton X-100 and 10% Normal Donkey Serum (NDS)], and incubate at RT for 1 h in a humidified chamber.</li>
	<li>Replace blocking buffer with Iba1 primary antibody (diluted in blocking buffer) to label mononuclear phagocytes, and incubate O/N at 4 &#176;C in a humidified chamber. For antibody hosts and working dilutions, see Table 1 and the Table of Materials.</li>
</ol>
DAY 5:
<ol start="6">
	<li>Rinse primary antibody with 3x PBS baths (5 min at RT with light agitation). Incubate with fluorescent secondary antibody (conjugated with a 594 nm emission fluorophore) for 1 h (in blocking buffer at RT in the dark) followed by 3x PBS baths (5 min at RT with light agitation). At this time, maintain sections in the dark to avoid fluorescent signal bleaching.</li>
</ol>
DAY 6 - 7:
<ol start="7">
	<li>Repeat steps 2.5 &#38; 2.6 with CD68 (rat brains) or LAMP1 (mouse tissue) antibodies and appropriate secondary antibodies (coupled with a 488 nm emission fluorophore) to label phagolysosomes.</li>
</ol>
DAY 7 - 8:
<ol start="8">
	<li>Repeat steps 2.5 &#38; 2.6 with OC (rat tissue) or 4G8 (mouse brains) antibodies and appropriate secondary antibodies (coupled to a 647 nm fluorophore) to label A&#946; deposits. 
	NOTE: Alternatively, 6E10 antibody can be used successfully both on mouse and rat tissue. For appropriate antibody combinations, see the Table of Materials.</li>
	<li>Allow sections to completely dry O/N at RT in the dark. Then, cover specimens with a cover slip sealed by fluorescence mounting media containing DAPI.</li>
</ol>
3. Acquisition of Large Z-stack Confocal Datasets
Note: This protocol requires a fully automated laser scanning confocal microscope equipped with a 60X objective and 405 nm, 488 nm, 594 nm, and 647 nm lasers. All equipment is computer controlled by imaging and laser control software. Prior to beginning the imaging protocol, power on the computer, epifluorescent lamp, microscope, lasers and camera.
DAY 9:
<ol>
	<li>Select the 60X microscope objective. Add immersion oil to the lens, and place the sample onto the microscope stage slide holder. Raise the objective until the oil makes contact with the slide. Adjust the focal plane to locate amyloid plaques in the hippocampus or cerebral cortex using epifluorescent illumination through the oculars.</li>
	<li>Acquire confocal images of activated mononuclear phagocytes surrounding amyloid deposits in the hippocampus or cortex of rodents by confocal microscopy (60X magnification, z-stack steps: 0.25 &#956;m &#60; z &#60; 0.40 &#956;m, number of steps 25 &#60; n &#60; 35).</li>
</ol>
4. q3DISM
Note: In order to yield significant results, we suggest analyzing a minimum of 3 images per animal/region of interest. For each image, the abundance of cells to analyze may vary depending on experimental paradigms. In the representative results shown in this report, we analyzed 3 cells/condition (e.g., mononuclear phagocytes distant from or associated with plaques; see Figures 1C - D and 2C - D).
DAY 10:
<ol>
	<li>Analyze confocal datasets with scientific 3D image processing and analysis software colocalization (coloc) module for spatial proximity of Iba1/CD68 (rat tissue) or Iba1/LAMP1 (mouse tissue) staining in all z-planes simultaneously. Create Iba+/CD68+ or Iba+/LAMP1+ colocalization channels that correspond to phagolysosomes within activated mononuclear phagocytes.
	<ol>
		<li>Select TRITC for channel A (corresponding to Iba1 staining coupled with 594 nm fluorophore) and FITC for channel B (corresponding to CD68 or LAMP1 staining coupled with 488 nm fluorophore). On the right-hand side of the software window for &#39;mode check,&#39; select &#39;threshold,&#39; and for &#39;coloc intensities,&#39; select &#39;source channels&#39;.</li>
		<li>Click &#39;Edit&#39; to select &#39;coloc color&#39; on the right-hand side of the software window.</li>
		<li>For each channel independently, adjust thresholds to include specific staining and exclude background/non-specific signals. Once adjusted, do not change thresholds between images to ensure an unbiased analysis. The colocalized voxels (pixels from all z-stacks) will appear in the color selected in step 4.1.2 in all z-stacks simultaneously.</li>
		<li>Click &#39;build coloc channel&#39;. The colocalization channel created will appear in the display adjustment window.</li>
		<li>Click on the coloc channel to open channel statistics. The &#39;% of volume/material A above threshold colocalized&#39; represents the % of Iba1 signal (voxels corresponding to the 594 nm fluorophore) colocalized with LAMP1 or CD68 signal (voxels corresponding to the 488 nm fluorophore). More simply, this is the monocyte volume occupied by phagolysosomes (see Figures 1C and 2C). 
		NOTE: &#39;% of volume/material B above threshold colocalized&#39; corresponds to % of LAMP1 or CD68 signal colocalized with Iba1. This should be close to 100%, as phagolysosomes are intracellular structures. Values are based on ratio of signal from all z-stacks above threshold colocalized.</li>
	</ol>
	</li>
	<li>Using the coloc module, analyze the coloc channel created in step 4.1 for spatial proximity with OC (rat tissue) or 4G8 (mouse tissue) A&#946; signals. This allows for quantitation of A&#946; encapsulated within phagolysosomes.
	<ol>
		<li>Select the channel A coloc dataset (corresponding to the Iba1/CD68 or Iba1/LAMP1 coloc channel created in step 4.1) and Cy5 for channel B (corresponding to OC or 4G8 staining coupled with 647 nm fluorophore).</li>
		<li>Build a coloc channel as described in steps 4.1.2 to 4.1.5.</li>
		<li>Click on the coloc channel to open channel statistics. The &#39;% of volume/material A above threshold colocalized&#39; represents the % of Iba1/LAMP1 or Iba1/CD68 (voxels corresponding to the coloc channel built in step 4.1.5.) colocalized with OC or 4G8 signal (voxels corresponding to the 647 nm fluorophore). This is the phagolysosomal volume occupied by A&#946; (see Figures 1D and 2D). 
		NOTE: &#39;% of volume/material B above threshold colocalized&#39; corresponds with % of total A&#946; signal colocalized with phagolysosomes. This can be used to evaluate the fraction of total A&#946; deposits encapsulated within phagolysosomes (not shown in the present representative results).</li>
	</ol>
	</li>
	<li>Use the surpass module to reconstruct the confocal image stacks and generate 3D models of A&#946; encapsulated within monocyte phagolysosomes.
	<ol>
		<li>In the &#39;display adjustment&#39; window, select TRITC (to show only Iba1 staining). In the &#39;volume properties&#39; window, click &#39;add new surface&#39;.</li>
		<li>In step &#39;1/5 algorithm&#39;, in &#39;settings,&#39; select &#39;surface.&#39; Also, in &#39;color,&#39; select color type in palette or RGB and adjust transparency (red is set at 60% transparency in Figures 1B and 2B). Check box &#39;select region of interest&#39;. Click next.</li>
		<li>In Step &#39;2/5 region of interest,&#39; draw a window around the cell of interest by adjusting x, y and z coordinates (see white boxes in Figures 1A and 2A). Click next.</li>
		<li>In Step &#39;3/5 source channel&#39;, select the TRITC source channel. Check the box &#39;smooth,&#39; and set surface area detail level to 0.4 &#956;m. For &#39;thresholding,&#39; select absolute intensity. Click next.</li>
		<li>In Step &#39;4/5 threshold,&#39; adjust threshold so that the volume created overlaps perfectly with the TRITC channel signal. Click next.</li>
		<li>In Step &#39;5/5 classify surfaces,&#39; in section &#39;filter type,&#39; select objects depending on their size to be included or excluded from the volumes to be created. Click finish, execute all creation steps, and exit the wizard.</li>
		<li>Repeat steps 4.3.1 to 4.3.6 to create a 3D surface for the FITC channel (LAMP1+ or CD68+ phagolysosomes).</li>
		<li>Repeat steps 4.3.1 to 4.3.6 to create a 3D surface for the Cy5 channel (OC+ or 4G8+ A&#946; deposits).</li>
	</ol>
	</li>
</ol>

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L., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Microglia+Cross+Talk+through+Complement+Activation+Modulates+Amyloid+Pathology+in+Mouse+Models+of+Alzheimer's+Disease.">Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.</a> J Neurosci. 36 (2), 577-589 (2016).</li><li>Novotny, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+Synthetic+A&#946;+to+In+Vivo+Active+Seeds+and+Amyloid+Plaque+Formation+in+a+Hippocampal+Slice+Culture+Model.">Conversion of Synthetic A&#946; to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.</a> J Neurosci. 36 (18), 5084-5093 (2016).</li><li>Tartaro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fluorescence-based+in+vivo+phagocytosis+assay+to+measure+mononuclear+phagocyte+system+function+in+the+rat.">Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.</a> J Immunotoxicol. 12 (3), 239-246 (2015).</li></ol>

The authors have nothing to disclose.

The protocol that we describe in this report for true 3D quantitation of A&#946; phagocytosis in vivo by mononuclear phagocytes relies on specific labeling of cellular and subcellular compartments as well as A&#946; deposits. Specifically, we use Iba1 (Ionized-calcium Binding Adaptor molecule 1), a protein that is involved in membrane ruffling and phagocytosis upon cell activation18,19, to stain cerebral mononuclear phagocytes. While Iba1+ cells are generally regarded as brain-resident microglia, it remains possible that peripherally infiltrating mononuclear phagocytes comprise a subset of immunolabeled cells. Intracellular phagolysosomes are revealed with antibodies recognizing members of the Lysosomal/endosomal-associated membrane glycoprotein (LAMP) family: LAMP120 or CD6821. The latter is primarily localized to lysosomes and endosomes, with a smaller fraction circulating to the cell surface. LAMP1 and CD68 antibodies have both been successfully used in rat and mouse tissue, and the decision on which to use is primarily dependent on the host of other antibodies used for co-staining (see Table 1). It is important to be aware that various antibodies can be used for detection of A&#946;. In mouse tissue, 4G8 (recognizing human and mouse amino acid residues 17 - 24 of A&#946; peptide22, Figure 1) and 6E10 (recognizing human amino acid residues 1 - 1723, data not shown) have been used successfully, allowing detection and quantitation of A&#946; content in LAMP1+ or CD68+ phagolysosomes present in Iba1+ mononuclear phagocytes. Using our q3DISM methodology, we observed increased A&#946; load in Iba1+ cells associated with global reduction of A&#946; load in brains of APPPS1/IL-10-/- mice16, whereas others showed decreased A&#946; uptake into Iba1+ cells accompanied by increased A&#946;&#160;load and plaque volume in brains of Rag-5xfAD mice24. These results suggest that the phenomenon observed is a &#34;snapshot&#34; of active A&#946; phagocytosis, although our methodology cannot be used to predict if the phagocytosed material is efficiently digested and cleared.
In rat tissue, we detected A&#946; deposits using OC (recognizing soluble A&#946;42 oligomers/fibrils25,26, Figure 2) or 6E1017 (not shown). Interestingly, very little OC+ fibrils were present in monocyte phagolysosomes in TgF344-AD rat brains. This phenomenon has previously been reported in APP23 transgenic mice using immunogold staining and 3D reconstruction27. Note: a variety of antibodies for detection of different A&#946; species/conformers are available and can potentially be tested and employed at the user&#39;s discretion.
Our q3DISM technique has been adopted by us and others to quantify A&#946; phagocytosis in the context of AD16,24,28. However, the method can be adapted to evaluate virtually any form of phagocytosis. It may also be applied to tissues other than the brain, and to other animal species (including humans). The procedures described in this protocol rely on standard immunostaining, detected with fluorescence and imaged with a confocal microscope. In this report, we have used paraffin-embedded tissue according to guidelines provided by the antibody manufacturers. If desired, the present protocol could also be optimized for fresh tissue (embedded in 2% agarose in PBS). The protocol can also be optimized to detect phagocytosis of coaggregating proteins. This is made possible by staining and acquiring images composed of more than 4 fluorescent colors29. In this case, the creation of the first colocalization channel (monocyte/phagolysosome) would be followed by creation of a second colocalization channel (aggregated protein 1/co-aggregated protein 2). Then, the two channels would be used for 3D analysis and modeling.
The q3DISM technique is limited principally by the specificity and sensitivity of immunostaining, quality of antibodies, and colocalization approach. This multistep procedure has a few potential pitfalls that can affect quality of the staining and, therefore, 3D quantitation of A&#946; uptake. The first critical step of the procedure is the isolation of rodent brains. Indeed, careful tissue handling is fundamental to avoid damage to the brain and to guarantee that brain structures remain intact when sectioned. Second, tissue fixation time is quite important since over-fixation (more than 16 h in 4% PFA) limits detection of subcellular phagolysosomes and A&#946; content. Importantly, the successive staining of 1) monocytes, 2) phagolysosomes and 3) A&#946; is critical for the success of the method. It is essential to use the antibodies consecutively- not concomitantly. Additionally, acquisition of the z-stack confocal images is of utmost importance, since 3D modeling of cells, phagolysosomes and A&#946; peptide as well as colocalization analyses depend on the quality of imaging of cellular and subcellular structures through the thickness of the tissue section. Finally, adjustment of thresholds in the software is crucial for 3D modeling and for colocalization analysis. Indeed, thresholds for the different channels need to be carefully chosen, since they will determine which signal is included (specific) or excluded (background) from the analysis. It is also fundamental that the thresholds chosen remain consistent between samples in the case of the comparison of different animals/specimens. So far, classical immunohistostaining, magnetic resonance imaging and positron emission tomography have been the imaging methods of choice for in vivo visualization and quantitation of A&#946;30. Although highly useful for large-scale quantitation of amyloid burden in brains of rodents or AD patients, these methods are inefficient for visualization of intracellular A&#946;. Other techniques using confocal microscopy, fluorescence spectroscopy and flow cytometry exist and have been used by us and others to discriminate and quantify intracellular A&#946; content in vitro16,31,32. Immunogold staining coupled with 3D reconstruction was previously used to highlight the absence of amyloid fibrils within microglia in vivo in APP23 transgenic mice27, and another study successfully used confocal imaging and 3D surface reconstruction of microglia-associated with &#946;-amyloid plaques to show limited A&#946; interaction and uptake33. However, these techniques do not allow for quantitation of intra-phagolysosomal A&#946; species. Others have recently developed a fluorescence-based in vivo assay allowing the measurement of phagocytic activity by peripheral macrophages in the rat34. This method ingeniously allows for quantitation of fluorescent bioprobes in phagolysosomes in vivo, however, data available so far are limited to the periphery.
To our knowledge, q3DISM is the first method that affords 3D visualization and quantitation of A&#946; phagocytosis in brains of rodent AD models. This formidable tool will undoubtedly pave the way to a deeper understanding of cerebral A&#946; phagocytosis, and may also prove useful in other contexts.

Alzheimer&#39;s Disease (AD), the most common age-related dementia1, is characterized by cerebral amyloid-&#946; (A&#946;) accumulation as &#34;senile&#34; &#946;-amyloid plaques, chronic low-level neuroinflammation, tauopathy, neuronal loss, and cognitive disturbance2. In AD patient brains, neuroinflammation is earmarked by reactive astrocytes and mononuclear phagocytes (referred to as microglia, although their central vs. peripheral origin remains unclear) surrounding A&#946; deposits3. As the innate immune sentinels of the CNS, microglia are centrally positioned to clear brain A&#946;. However, microglial recruitment to A&#946; plaques is accompanied by very little, if any, A&#946; phagocytosis4,5. One hypothesis is that microglia are initially neuroprotective by phagocytozing small assemblies of A&#946;. However, eventually these cells become neurotoxic as overwhelming A&#946; burden and/or age-related functional decline, provokes microglia into a dysfunctional proinflammatory phenotype, contributing to neurotoxicity and cognitive decline6.
Recent Genome-wide Association Studies (GWAS) have identified a cluster of AD risk alleles belonging to core innate immune pathways7 that modulate phagocytosis8-11. Consequently, the immune response to cerebral amyloid deposition has become a major area of interest, both in terms of understanding AD etiology and for developing new therapeutic approaches12-14. Yet, there is a vital need for methodology to evaluate A&#946; phagocytosis in vivo. To address this unmet need, we have developed quantitative 3D in silico modeling (q3DISM) to enable true 3D quantitation of cerebral A&#946; phagocytosis by mononuclear phagocytes in rodent models of Alzheimer-like disease.
Limited only by the extent to which they recapitulate disease, animal models have proven invaluable for understanding AD pathoetiology and for evaluating experimental therapeutics. Owing to the fact that mutations in the Presenilin&#160;(PS) and Amyloid Precursor Protein (APP) genes independently cause autosomal dominant AD, these mutant transgenes have been extensively used to generate transgenic rodent models. Transgenic APP/PS1 mice simultaneously coexpressing &#34;Swedish&#34; mutant human APP (APPswe) and &#916; exon 9 mutant human presenilin 1 (PS1&#916;E9) present with accelerated cerebral amyloidosis and neuroinflammation15,16. Further, we have generated bi-transgenic rats coinjected with APPswe and PS1&#916;E9 constructs (line TgF344-AD, on a Fischer 344 background). Unlike transgenic mouse models of cerebral amyloidosis, TgF344-AD rats develop cerebral amyloid that precedes tauopathy, apoptotic loss of neurons, and behavioral impairment17.
In this report, we describe a protocol for immunostaining microglia, phagolysosomes and A&#946; deposits in brain sections from APP/PS1 mice and TgF344-AD rats, and acquisition of large z-dimensional confocal images. We detail in silico generation and analysis of true 3D reconstructions from confocal datasets allowing quantitation of A&#946; uptake into microglial phagolysosomes. More broadly, the methodology that we detail here can be used to quantify virtually any form of phagocytosis in vivo.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:384px;">Isoflurane</td>
			<td style="width:152px;">Abbott</td>
			<td style="width:127px;">NDC 0044-5260-05</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Dissecting scissors</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-582</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Dissecting scissors Blunt tip</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-588</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tweezers</td>
			<td style="width:152px;">VWR</td>
			<td>94024-408</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">23G needle</td>
			<td style="width:152px;">VWR</td>
			<td>BD305145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">peristaltic pump FH10</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td>72-310-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">PBS 10X</td>
			<td style="width:152px;">Bioland Scientific</td>
			<td style="width:127px;">PBS01-02</td>
			<td>Working concentration 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-200C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-305C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:152px;">EMS</td>
			<td style="width:127px;">15714-S</td>
			<td>Caution: Toxic. Working concentration 4% in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Ethanol</td>
			<td style="width:152px;">VWR</td>
			<td>89125-188</td>
			<td>Various concentrations, see protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tissue-Tek Uni-cassettes Sakura</td>
			<td style="width:152px;">VWR</td>
			<td>25608-774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Embedding and Infiltration Paraffin</td>
			<td style="width:152px;">VWR</td>
			<td>15147-839</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Disposable Microtome Blades&#160;</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">25608-964</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Water bath Leica HI 1210</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Micro slide Superfrost plus</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Xylene</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:127px;">534056-4X4L</td>
			<td style="width:249px;">Caution: Toxic&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Target Retrieval Solution 10X</td>
			<td style="width:152px;">DAKO</td>
			<td style="width:127px;">S1699</td>
			<td>Working concentration 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KimWipes</td>
			<td style="width:152px;">VWR</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Hydrophobic PAP pen</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">95025-252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Triton X-100</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">97062-208</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Normal Donkey Serum</td>
			<td style="width:152px;">Jackson Immuno</td>
			<td style="width:127px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Coverslips</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48393081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Prolong Gold antifade reagent with DAPI</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:127px;">P36935</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Glass Slide Rack</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">100492-942</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, rabbit)</td>
			<td style="width:152px;">Wako</td>
			<td style="width:127px;">019-19741&#160;</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, goat)</td>
			<td style="width:152px;">LifeSpan Bioscience</td>
			<td style="width:127px;">LS-B2645</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">rat CD68 [KP1] antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab955</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD68 [FA-11] antibody (monoclonal, rat)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab53444</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD107a (LAMP1) antibody (monoclonal, rat)</td>
			<td style="width:152px;">Affymetrix</td>
			<td style="width:127px;">14-1071</td>
			<td>Working concentration 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 17-24 (4G8) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39220</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 1-16 (6E10) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39320</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">OC antibody (polyclonal, rabbit)</td>
			<td colspan="2">Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:127px;">A-11001</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; rat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11006</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11037</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 goat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11080</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21235</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21443</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Immersion oil</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">A1 Confocal microscope</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">NIS Elements Advanced Research software</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Imaris:Bitplane software version 7.6</td>
			<td style="width:152px;">Bitplane</td>
			<td style="width:127px;"></td>
			<td>&#34;coloc&#34; and &#34;supass&#34; modules are used.&#160;Alternatively, the open-source freeware&#160;ImageJ can be used for colocalization&#160;analysis of confocal z-stacks datasets.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Using the multi-stage methodology for q3DISM detailed above, we are able to quantify A&#946; uptake into monocyte phagolysosomes in the brains of APP/PS1 mice (Figure 1) and TgF344-AD rats (Figure 2). Therefore, the q3DISM methodology has enabled analysis of mononuclear phagocytes in mouse and rat models of AD. Interestingly, the volume occupied by CD68+ phagolysosomes is significantly increased in Iba1+ mononuclear phagocytes associated with compared to distant from plaques both in APP/PS1 mice (Figure 1B,&#160;C) and TgF344-AD rats (Figure 2B,&#160;C). These data show that mononuclear phagocytes near plaques are poised for phagocytosis compared to cells located away from the plaque. In order to illustrate the range of data from staining different A&#946; conformers, we used various antibodies in mouse and rat tissue (detailed in Table 1 and discussion)- we are not seeking to directly compare mouse and rat models of AD with regards to A&#946; phagocytosis. Nonetheless, it was noteworthy that plaque associated mononuclear phagocytes had increased A&#946; uptake in APP/PS1 mice compared to cells distant from plaques (Figure 1D). TgF344-AD rats had very modest uptake of A&#946; fibrils overall, and no change in A&#946; fibril phagocytosis as a function of distance from plaques (Figure 2D).
<img alt="Table 1" src="/files/ftp_upload/54868/54868table1.jpg" /> 
Table 1: Immunostaining Mononuclear Phagocytes, Phagolysosomes and Amyloid-&#946; in Brain Tissue from APP/PS1 Mice or TgF344-AD Rats. Rat or mouse specific antibody cocktails are listed with their respective host in parentheses. Colors indicate the fluorophore used for the secondary antibody. Red: Alexa Fluor 594; green: Alexa Fluor 488; magenta: Alexa Fluor 647.
<img alt="Figure 1" src="/files/ftp_upload/54868/54868fig1.jpg" /> 
Figure 1: Visualization and Quantitation of Amyloid-&#946; Phagocytosis in APP/PS1 Mouse Brains. (A) Confocal images of brain sections from an APP/PS1 mouse showing Iba1+ cells (red), LAMP1+ phagolysosomes (green) and 4G8+ amyloid deposits (magenta). Inset 1 highlights a mononuclear phagocyte distant from the plaque, while inset 2 shows a mononuclear phagocyte associated with the plaque. Scale bar denotes 50 &#956;m. Smaller panels on the right are enlargements of Iba1, LAMP1 and 4G8 signals within insets 1 and 2. (B) 3D reconstruction of Iba1, LAMP1 and 4G8 signals for insets 1 and 2. Scale bar denotes 3 &#956;m. (C) Quantitation of Iba1+ mononuclear phagocyte volume occupied by LAMP1+ phagolysosomes. (D) Quantitation of intracellular LAMP1+ phagolysosomal volume occupied by 4G8+ A&#946;. Values are based on ratio of signal (voxels) above threshold that are colocalized. Data are shown as mean &#177;standard error of the mean (n = 6 cells) for mononuclear phagocytes distant from plaques (1), or associated with plaques (2). ** p &#60;0.01 by student&#39;s t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54868/54868fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54868/54868fig2.jpg" /> 
Figure 2: Visualization and Quantitation of Amyloid-&#946; Phagocytosis in TgF344-AD Rat Brains. (A) Confocal images of brain sections from a TgF344-AD rat showing Iba1+ cells (red), CD68+ phagolysosomes (green), and OC+ soluble fibrillar A&#946; (magenta). Inset 1 highlights a mononuclear phagocyte distant from the plaque, while inset 2 shows a mononuclear phagocyte associated with the plaque. Scale bar denotes 50 &#956;m. Smaller panels on the right are enlargements of Iba1, CD68 and OC signals within insets 1 and 2. (B) 3D reconstruction of Iba1, CD68 and OC signals for insets 1 and 2. Scale bar denotes 3 &#956;m. (C) Quantitation of Iba1+ mononuclear phagocyte volume occupied by CD68+ phagolysosomes. (D) Quantitation of intracellular CD68+ phagolysosomal volume occupied by OC+ A&#946;. Values are based on ratio of signal (voxels) above threshold that are colocalized. Data are shown as mean &#177;standard error of the mean (n = 6 cells) for mononuclear phagocytes distant from plaques (1), or associated with plaques (2). ** p &#60;0.01 by student&#39;s t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54868/54868fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

quantitative-3d-silico-modeling-q3dism-cerebral-amyloid-beta

Nanyang Technological University

Research

JoVE Journal

Medicine

7.9K Views.  University of Southern California (USC). We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-β (Aβ) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer's disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Video: Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

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

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

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