The authors have no acknowledgements.

The methods detailed in this publication were reviewed by the institutional animal care and use committee (IACUC) at Hilltop Laboratory Animals to ensure proper care, use, and humane treatment of animals in compliance with applicable federal, state, and local laws and regulations, such as the federal Animal Welfare Regulations, or AWRs (CFR 1985), and Public Health Service Policy on Humane Care and Use of Laboratory Animals, or PHS Policy (PHS 1996).
1. General Guidelines for all Behavioral Assessment
<ol>
	<li>Before any animal handling, cover existing cage cards with a new cage card that indicates only the unique, blinded animal identifier. 
	NOTE: Investigators that handle mice during routine compound/placebo dosing are not permitted to handle mice for behavioral assessment.</li>
	<li>Dim or turn off the overhead lights and adjust the lighting such that illumination at the floor of the arena or maze is 30-35 lux.</li>
	<li>For studies that span several weeks, record body weights weekly as an indirect measure of overall health. 
	NOTE: Additional cage-side checks for coat quality, posture, gait and spontaneous locomotion can be included if more robust health checks are warranted.</li>
</ol>
2. Habituating Mice to Handling by Investigators
<ol>
	<li>Two days prior to any behavioral test, habituate the mice to handling. Remove the cage from the cage rack and place on a level surface.</li>
	<li>Remove the lid from the cage. Handle the mouse exactly as it would be handled during performance of the upcoming behavioral test. Place the mouse into a cupped hand above the home cage.</li>
	<li>Measure the latency to jump from the investigator&#39;s hand back into the home cage. Hold mice for a maximum of 5 s. 
	NOTE: Mice that exhibit latencies of &#8805;2 s are deemed &#34;habituated&#34;. Mice that exhibit latencies &#60;2 s during the first trial undergo 2 additional habituation sessions that day.</li>
	<li>Have the mice undergo 2 consecutive days of handling habituation. Note any mouse that is not habituated by the end of the 2nd day.</li>
</ol>
3. Assessing Spatial Working Memory by Measuring Spontaneous Alternation in a Y-maze8
<ol>
	<li>Prior to initial use, thoroughly clean the Y-maze with an unscented bleach germicidal wipe, 70% EtOH, followed by dH2O9. Clearly designate the arms of the maze as &#39;A&#39;, &#39;B&#39; &#38; &#39;C&#39; or other comparable unique identifiers.</li>
	<li>Prior to the start of a testing session, set the data acquisition system or video cameras and set up proper tracking of mice in the maze. Calibrate the distance in the maze using captured video images of a ruler or other object of known length. 
	NOTE: The behavioral methods in this procedure will work with a variety of data acquisition systems and the authors assume anyone performing this procedure is proficient in the use of their chosen data acquisition system. Power analyses indicate that sample sizes of 10-15 mice per group are required for a &#946; &#8804;0.2.</li>
	<li>Remove the cage from the rack and gently place it on a table in close proximity to the Y-maze. Remove the mouse from its home cage and gently place it into one arm of the Y-maze, facing the center. Have the investigator step far enough away from the maze so that the mouse cannot see the investigator.</li>
	<li>Activate the data/video acquisition system immediately after placement of the mouse into the maze.</li>
	<li>Press play and record the spontaneous behavior for each mouse for a period of 10 min. Once a session is complete, gently place the mouse back into its home cage and return the cage to the rack.</li>
	<li>Clean the maze thoroughly between each session with an unscented bleach germicidal wipe, 70% EtOH, followed by dH2O. Repeat from step 3.4 to assess all mice.</li>
	<li>Once all mice have completed exploration of the Y-maze, analyze the data from the acquisition system or manually score the videos of the sessions. An arm entry occurs when all 4 paws of the mouse cross the threshold of the central zone and into the arm and the animal&#39;s snout is oriented toward the end of the arm. 
	NOTE: Endpoints that will be analyzed include: total distance traveled in the maze, total distance traveled within each arm (including the central zone), total time spent in each arm (including the central zone), total number of arm entries, number of entries made into each arm, and a sequential list of arms entered to assess number of alternations made.</li>
	<li>A spontaneous alternation occurs when a mouse enters a different arm of the maze in each of 3 consecutive arm entries. Spontaneous alternation % is then calculated with the following formula. 
	<img alt="Equation 1" src="/files/ftp_upload/55523/55523eq1.jpg" /> 
	NOTE: For example, if the order of arm entry was: ABCCBABCABC, investigator would score a total of 6 spontaneous alternations (in order: ABC, CBA, ABC, BCA, CAB, ABC). With a total of 11 arm entries, the spontaneous alternation % would be 67%.</li>
	<li>Make the following quality control checks to ensure the data represent an unbiased assessment of spontaneous alternation.
	<ol>
		<li>Perform a Pearson&#39;s correlation of spontaneous alternation % to both total distance traveled and number of arm entries made. 
		NOTE: If there is a significant correlation of spontaneous alternation % to either parameter, then data should be further scrutinized due to potential influence of hyperdynamic locomotion on the apparent cognitive endpoint10.</li>
		<li>Analyze number of entries made into each arm with a 1-way ANOVA test. 
		NOTE: If this analysis is significant then this would indicate presence of cues in the environment that attracted mice to a particular region of the maze.</li>
	</ol>
	</li>
</ol>
4. Assessment of Intermediate-term Recognition Memory by Measuring Novel Object Recognition11,12,13
<ol>
	<li>For each phase of this test, thoroughly clean the open field arena with an unscented bleach germicidal wipe, 70% EtOH followed by dH2O prior to initial use.</li>
	<li>One day prior to object exposure, habituate the mice to the open field arena.
	<ol>
		<li>Prior to the start of the habituation session, set up the data acquisition system or video cameras and confirm proper tracking of mice in the maze. Calibrate the distance in the arena using captured video images of a ruler or other object of known length. Mark the corners of the arena in the software to permit scoring of positional biases. 
		NOTE: The behavioral methods in this procedure will work with a variety of data acquisition systems and the authors assume anyone performing this procedure is proficient in the use of their chosen data acquisition system. Power analyses indicate that sample sizes of 15-20 mice per group are required for a &#946; &#8804;0.2.</li>
		<li>Remove the cage from the rack and gently place on a table in close proximity to the arena.</li>
		<li>Remove the mouse from the home cage and gently place the mouse in the center of the arena. Turn on the tracking software and/or video recording system immediately after placing the mouse into the arena.</li>
		<li>Allow mice to freely explore the arena for 30 min. 
		NOTE: During this period, investigators will not disturb the mice.</li>
		<li>After the habituation session, place mice back into their home cage and clean the arena thoroughly with an unscented bleach germicidal wipe, 70% EtOH followed by dH2O.</li>
		<li>Repeat from step 4.2.2 until all mice have been habituated to the arena.</li>
		<li>After all mice have been habituated to the arena, analyze the video. 
		NOTE: Endpoints to analyze include total distance traveled in the arena and time spent near each corner. If relevant to the mouse model, stereotyped behaviors are included in these analyses (i.e., myoclonic corner jumping, circling, etc.). Mice exhibiting biases in time spent in particular regions of the arena are excluded from further experimentation as this will influence object exploration. 
		NOTE: The first phase of novel object recognition involves familiarizing mice to an object. Herein this portion of the novel object recognition procedure will be referred to as the Sample phase.</li>
		<li>Prior to the start of a sample phase session, place objects into the arena and fix them to the floor with a mounting putty so that animals cannot move the objects. Align two identical objects to a particular wall with enough distance between the walls and objects so that the mice can freely explore the objects from all angles.</li>
		<li>Set up the data acquisition system or video cameras and confirm proper tracking of mice and objects in the maze. Calibrate the distances in the arena using captured video images of a ruler or other object of known length.</li>
		<li>Mark the corners of the arena in the software to permit scoring of positional biases. Mark objects in software and track their exploratory behavior separately for each object (i.e., &#34;Object A&#34; and &#34;Object B&#34;).</li>
		<li>Remove the cage from the rack and gently place it on a table in close proximity to the arena.</li>
		<li>Remove the mouse from the home cage and gently place it into the center of the arena, facing the objects.</li>
		<li>Allow the mouse to freely explore the objects for 15 min. During this period do not disturb the mice.</li>
		<li>At the end of the session, gently place the mouse back into its home cage. Clean the arena and objects with 70% EtOH and dH2O. Place these objects back into the arena.</li>
		<li>Repeat step 4.2.11 until all mice are familiarized to an object.</li>
		<li>Once all mice have been familiarized to an object, analyze the videos. 
		NOTE: Object explorations are counted once the following criteria have been met: the mouse is oriented toward the object, the snout is within 2 cm of the object, the midpoint of the animal&#39;s body is beyond 2 cm from the object, and the previous criteria have been fulfilled for at least 1 s. Additionally, if an animal has satisfied the exploration criteria but exhibits immobility for &#62; 10 s then the exploratory bout is deemed finished.</li>
		<li>Calculate an object bias score for each mouse as follows. 
		<img alt="Equation 2" src="/files/ftp_upload/55523/55523eq2.jpg" /> 
		NOTE: Mice exhibiting an object bias score below 20% or above 80% are excluded from further experimentation.</li>
	</ol>
	</li>
	<li>The final phase of novel object recognition involves assessing exploratory behavior directed toward both a novel and familiar object in the environment, referred to here as the Test phase. This phase is performed 2-3 h after completion of sample phase.
	<ol>
		<li>Prior to the start of a test phase session, place objects into the arena and fix them to the floor so that the animals cannot move the objects.
		<ol>
			<li>Place the objects in the same position in the arena relative to the sample phase13.</li>
			<li>Balance the relative position of novel and familiar objects across genotypes and treatment groups.</li>
			<li>Ensure there is enough distance between the walls and objects so that the mice can freely explore the objects from all angles.</li>
		</ol>
		</li>
		<li>Setup the data acquisition system and/or video cameras. Confirm proper tracking of mice and objects in the maze. Calibrate distances in the arena using captured video images of a ruler or other object of known length.</li>
		<li>Mark corners of the arena in the software to permit scoring of positional biases. Mark objects in software and track exploratory behavior for each object individually (i.e., &#34;Novel&#34; and &#34;Familiar&#34;).</li>
		<li>Remove the cage from the rack and gently place it on a table in close proximity to the arena.</li>
		<li>Gently place animals into the center of the arena, facing the objects. Record mice freely exploring objects for 10 min.</li>
		<li>At the end of the test session, remove mice from the arena and place mice back into their home cage. Thoroughly clean arena and objects with an unscented bleach germicidal wipe, 70% EtOH and dH2O after each session.</li>
		<li>Repeat from step 4.4.3 until all animals have been assessed.</li>
		<li>Once object exploration is measured for all mice, videos are analyzed. 
		NOTE: Object explorations are counted once the following criteria have been met: the mouse is oriented toward the object, the snout is within 2 cm of the object, the midpoint of the animal&#39;s body is beyond 2 cm from the object, and the previous criteria have been fulfilled for at least 1 s. Additionally, if an animal has satisfied the exploration criteria but exhibits immobility for &#62; 10 s then the exploratory bout is deemed finished.</li>
		<li>Assess novel object recognition by comparing time spent exploring the novel to familiar object. Three methods are commonly reported in the literature.
		<ol>
			<li>Analyze raw time spent exploring both novel and familiar objects using a repeated measure test. This method is best used when genotype and/or treatment do not affect total exploration time.</li>
			<li>Calculate novelty preference, using the equation: 
			<img alt="Equation 3" src="/files/ftp_upload/55523/55523eq3.jpg" /> 
			NOTE: This provides the percentage of time spent exploring the novel object relative to the total time exploring objects. Values range from 0% (no exploration of novel object) to 100% (exploration only of the novel object), with a value of 50% indicating equal time spent exploring novel and familiar objects.</li>
			<li>Calculate discrimination index11, using the equation: 
			<img alt="Equation 4" src="/files/ftp_upload/55523/55523eq4.jpg" /> 
			NOTE: This yields the difference in time spent exploring the novel and familiar objects relative to the total time spent exploring objects. Values range from -1 (exploration only of the familiar object) to +1 (exploration only of the novel object, with a value of 0 indicating equal time spent exploring novel and familiar objects.</li>
		</ol>
		</li>
		<li>Remove animals that do not participate in the test session due to hyperdynamic locomotion or other stereotypies, from consideration11. 
		NOTE: Criteria used for removal must be objective and determined a priori for the mouse model (i.e., &#60;5th percentile for total exploration time and either &#62;100 average turn angle during test session or &#62;50th percentile time exhibiting myoclonic corner jumping).</li>
	</ol>
	</li>
</ol>
5. Assessment of Corticospinal Function in Mice with Limb Clasping14
<ol>
	<li>Video document the entire session. Record the video using a portable hand-held device (i.e., smartphone or equivalent). 
	NOTE: Power analyses indicate that sample sizes of 10-15 mice per group are required for a &#946; &#8804;0.2.</li>
	<li>Remove the home cage from the rack and place it on a table. Document the animal ID in the video prior to the next step.</li>
	<li>Gently remove the mouse from its cage and suspend by the tail for 5-10 s. The video must record the animal&#39;s hind and forepaws while suspended.</li>
	<li>After capturing at least 5 s of video, place the mouse back into its home cage, and return the cage to the rack.</li>
	<li>Clean the table. Repeat from step 5.2 until all mice have been recorded.</li>
	<li>Score limb clasping from videos of mice suspended by their tail on a scale from 0-4 (see Table 1 for description of scoring). Inspect videos of suspended mice and then assign a score based on the following criteria.
	<ol>
		<li>No limb clasping. Normal escape extension. One hind limb exhibits incomplete splay and loss of mobility. Toes exhibit normal splay.</li>
		<li>Both hind limbs exhibit incomplete splay and loss of mobility. Toes exhibit normal splay.</li>
		<li>Both hind limbs exhibit clasping with curled toes and immobility.</li>
		<li>Forelimbs and hind limbs exhibit clasping and are crossed, curled toes and immobility.</li>
	</ol>
	</li>
	<li>All mice are scored by 2 independent investigators. Any mouse where the 2 scores differ by more than 1 point is rescored once again.
	<ol>
		<li>Scores that differ are averaged.</li>
	</ol>
	</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/55523/55523table1.jpg" /> 
Table 1: Description of Limb Clasping Scores.

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J.M. Levenson is employed by Proclara Biosciences, Inc. C. Miedel, J. Patton, A. Miedel, and E. Miedel are employed by Hilltop Laboratory Animals.

Significance of the Technique with Respect to Existing Methods 
This procedure has been designed to screen for in vivo activity of compounds in transgenic mouse models of &#946;-amyloidosis and tauopathy. The staged approach employed here ensures detection of efficacious compounds in cognitive domains relevant to AD3. Moreover, the approach detailed here uses behavioral tests that have clearly defined endpoints, easily implementable quality control checks, can be run in a moderate throughput format, and require little intervention from the investigator. These characteristics result in assays that exhibit good reproducibility within animals and across cohorts, which results in low intra- and inter-assay variance and effect sizes (2 &#8804;f &#8804;6) that are robust enough to support behavioral profiling in a drug discovery environment.
Critical Steps within the Protocol 
Many mouse models in use for AD drug discovery exhibit behaviors consistent with heightened anxiety and aggression. This makes handling habituation essential for performing any of the behavioral tests described here. As these tests rely on unmotivated behaviors, rough handling by the investigator due to a hyperactive and anxious or aggressive mouse can significantly influence performance. Heightened anxiety could result in failure to perform the task, reducing the overall power of the test. Moreover, light levels in the arena are essential for facilitating the spontaneous locomotion needed for each test. Bright light tends to increase anxiety and suppress locomotion in rodents, therefore care should be taken to adjust ambient light levels to 30-35 lux in the arena.
Another critical aspect of the procedure is minimization of strong environmental cues that would interfere with an animal&#39;s ability to perform the tasks. Cleaning of the arena and objects in between runs is essential as mice are attracted to novel scents in the environment. Failure to thoroughly clean the arena and objects could result in skewing spontaneous activity of the mouse and masking true cognitive performance. Investigators should also minimize use of personal hygiene products and colognes/perfumes when performing these procedures. Lastly, rodents exhibit robust diurnal and circadian changes in many overt behaviors20 including learning and memory21. Therefore, to minimize variance due to diurnal rhythms in basal behaviors and cognitive performance, all tests should be done at the same time of day across cohorts and studies.
Further, specifically with regard to novel object recognition, the delay interval between sample and test phase, and the selection and placement of objects in the environment are critical parameters. Memory exists in 3 distinct forms: short term memory (STM), intermediate term memory (ITM) and long term memory (LTM)22,23. Changing the interval between sample and test phases from minutes (STM) to hours (ITM) or days (LTM) will change the type of memory tested by the procedure12. Moreover, prior to running the novel object recognition test, many objects should be screened in a test cohort of mice for potential biases in exploration. An object that is excessively attractive or repulsive to the test cohort cannot be used when assessing novel object recognition. Ideally all objects that will be employed in the test, when placed into an arena, will elicit equal exploration times from a na&#239;ve cohort of mice. Inadequate testing and optimization of objects can significantly reduce the power of novel object recognition.
Modifications and Troubleshooting 
There are several factors that could increase the apparent variability in the cognitive tests described here. Many mouse models of AD exhibit hyperdynamic locomotion3 which can mask or alter behaviors measured as the cognitive endpoint. Moreover, there is growing evidence that sex24,25,26 and even maternal genotype27 can influence development and progression of neuropathology and cognitive phenotypes in AD mouse models. Unexpected variability or failure to implement a behavioral task could be due to any of these factors. When first implementing a particular behavioral test, results should always be stratified by sex, age and if applicable, maternal genotype. Furthermore, the quality checks outlined in this procedure should always be performed to ensure that hyperactivity or other stereotyped behaviors are not interfering with quantification of cognitive endpoints.
Environment can also influence the spontaneous exploratory behavior of rodents. Scents or sounds that are undetectable to researchers could attract or repel mice, skewing results of cognitive tests that rely on spontaneous behavior. When initially establishing Y-maze or novel object recognition, performance of the control measures to ensure that there are no positional biases in exploration of objects and/or the environment is essential. If positional biases are observed then investigators must thoroughly scrutinize the environment and potentially adjust lighting, arena placement, location of testing room relative to other rooms in the facility (i.e., not near a high traffick area or heavy equipment) and arena cleaning procedures.
Habituation to the testing environment is key to achieving optimal performance in the novel object recognition test. For example, low total exploration times may be due to inadequate habituation. As an alternative to the procedures outlined here for handling (Section 2) and arena (Section 4.2) habituation, habituation to handling and the test environment can be performed as 3, 5 min sessions per day for 2 consecutive days.
Limitations of the Technique 
As with any procedure, these behavioral tests have limitations. These procedures have been employed because they test function of various cortical regions and hippocampus. If the mouse model does not exhibit functional deficits in brain regions probed by these tests, then these techniques will not be useful. Moreover, we have chosen cognitive tests that probe short-term memory. If the mechanism of action of the compound under preclinical assessment is not expected to affect short-term memory then these procedures should be modified accordingly (i.e., increasing the sample-test phase interval to test long-term memory). Lastly, these tests use unmotivated behaviors. Therefore, if a mouse model is excessively hyperactive or displays other stereotyped behaviors that prevent exploration of the environment then these procedures might not be optimal. As an alternative, one could use fear conditioning for Tg2576 or other &#946;-amyloidosis mouse models, or the spatial water maze for rTg4510 or other mouse models of tauopathy3.
Future Applications 
Once these procedures have been successfully adopted in the lab, several modifications or extensions can be made to assess additional cognitive and functional motor measures. For example, changing the novel object recognition task to determine if a mouse can recognize a change in placement of an object13. Alternatively, instead of using objects, one could use other mice and implement a test of social recognition. With respect to limb clasping and motor function, one could supplement that test with the wire hang and/or grip strength tests. The tests detailed in this method form a solid base to screen for compounds that have in vivo efficacy in translational mouse models for AD, and can be adapted or modified in many ways to best interrogate a particular mouse model or meet the needs of a unique drug discovery program.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative disorder resulting in debilitating cognitive decline that affects approximately 44 million people worldwide. Currently there are no available treatments for AD that are disease-modifying, emphasizing the urgent need for preclinical discovery of novel therapeutic strategies for this disease. A number of different transgenic mouse models have been created that recapitulate various aspects of AD1,2, including deficits in cognitive domains disrupted in patients3. These mouse models represent a useful tool for facilitating efficient screening in vivo.
When assessing a compound for potential in vivo efficacy, a staged approach must be taken that screens for efficacy in appropriate cognitive domains and also monitors behaviors that could influence the specific endpoints used to assess cognition. Many transgenic mouse models of AD exhibit hyperactivity and other behaviors that may interfere with a particular cognitive test, and prohibit its use in drug screening4. Moreover, for an approach to be implemented in a drug screening environment, the particular tests used should sustain at least a moderate throughput, have clearly defined endpoints, and a procedure that requires minimal intervention by the investigators. Using these criteria, behavioral screens can be implemented that exhibit the reproducibility, low intra- and inter-assay variance and effect sizes needed for compound screening. Detailed here are the methods we have employed to screen for compounds effective in mitigating the cognitive and motor phenotypes present in transgenic mouse models of &#946;-amyloidosis and tauopathy5,6. The methods described are adapted from commonly used behavioral paradigms reported in the literature7, with specific optimizations and quality control checks so that they may be used in transgenic mouse models relevant to AD. This protocol can be used with a variety of data acquisition and analysis systems and assumes that the investigator has a working knowledge of the associated software.

<table border="0" cellpadding="0" cellspacing="0" width="1375">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Topscan Lite-High Throughput</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;"></td>
			<td style="width:812px;">Automated behavioral analysis. Includes cameras and video acquisition system, laptop.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ObjectScan</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;"></td>
			<td>Software module for accurate object exploration quantification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Open field for mouse</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;">CSI-OF-M</td>
			<td>Arena for novel object recognition</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Y-maze for mouse</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;"></td>
			<td>Arms: 30cm long, 10 cm wide, 20cm high walls, placed 120deg apart.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Camera mount for open field</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;">Custom</td>
			<td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&#160; Two mounts, each covers two boxes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Camera mount for Y-maze</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;">Custom</td>
			<td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&#160; One mount covers two mazes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Marbles</td>
			<td style="width:211px;">Inperial Toy</td>
			<td>8565</td>
			<td>Standard (15.5mm Dia) glass marbles.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dice</td>
			<td style="width:211px;">Cardinal Industries</td>
			<td>770</td>
			<td>Standard (0.650 inch) white dice with black dots.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">LOCTITE Fun-Tak</td>
			<td style="width:211px;">Henkel</td>
			<td>B018A3AG0W</td>
			<td>Standard blue sticky tak</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">EtOH</td>
			<td style="width:211px;">Nexeo Solutions</td>
			<td style="width:136px;">82452</td>
			<td>100% Ethanol Diluted to 70% using distilled Water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">dH2O</td>
			<td style="width:211px;">Tulpenhocken Spring Water Co.</td>
			<td style="width:136px;">-</td>
			<td>PA D.E.P. #31, NJ D.O.H. #0049, NYSHD Cert. #320</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Paper towels</td>
			<td style="width:211px;">Procter &#38; Gamble</td>
			<td>B019DM86LA</td>
			<td>Bounty, White</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Handheld video camera</td>
			<td style="width:211px;">Apple, Inc.</td>
			<td style="width:136px;">MKV92LL/A</td>
			<td>Acquisition of Limb clasping video, Iphone 6S Plus (or functional equivalent).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gloves</td>
			<td style="width:211px;">SafePOINT, L.L.C.</td>
			<td style="width:136px;">GL640-2</td>
			<td>Standard, Powder free Latex Gloves, Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Light meter</td>
			<td style="width:211px;">Dr. Meter</td>
			<td style="width:136px;">LX1330B</td>
			<td>Lighting @ the bottom of Open Field= 35 LUX, Lighting @ bottom of Y-Maze= 32 LUX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach germicidal wipes</td>
			<td style="width:211px;">Clorox</td>
			<td style="width:136px;"></td>
			<td>Sterilization of equipment during &#38; after use</td>
		</tr>
	</tbody>
</table>

assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-&#946; and tau neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Aged Tg2576 mice exhibit robust deficits in spontaneous alternations made within a Y-maze15,16, a phenotype that can be replicated using the methods detailed here (Figure 1A). While a trend for increased arm entries is observed in these mice (Figure 1B), the hyperactivity observed in this line of mice did not affect the spontaneous alternation rate (Figure 1C). In contrast, aged rTg4510 mice appear to exhibit increased spontaneous alternation when placed into a Y-maze (Figure 1D). This is due to extreme hyperactivity (Figure 1E) and stereotypy10, which significantly interfere with measurement of spontaneous alternation (Figure 1F). When initially assessing mice in this task, it is critical to ensure that arm entries and/or distance traveled is not significantly correlated with the spontaneous alternation rate.
Prior to assessment of novel object recognition, mice are habituated to the arena where the test will be performed. During the habituation, hyperactivity (Figure 2A) and other stereotyped behaviors relevant to the mouse model can be assessed. During the sample phase, it is critical to measure exploration of each object separately so that mice that exhibit significant biases in exploratory behavior can be excluded from further assessment (Figure 2B, open circles). Novel object recognition is assessed by comparing exploration of a familiar and novel object, and is commonly analyzed in three different ways. If total exploratory time is comparable across genotypes and/or treatment groups, then raw time exploring each object and an appropriate repeated measures tests can be used to determine if there were differences in novel object recognition (Figure 2C). If a particular mouse strain exhibits differences in total exploratory time, novel object recognition can be assessed using either novelty preference (Figure 2D) or discrimination index (Figure 2E).
Limb clasping is a functional motor test that quantifies deficits in corticospinal function. Limb clasping, which is not a cognitive measure, is observed in several transgenic tau mouse models6,17,18,19 and recapitulates some of the functional motor deficits observed in late-stage AD patients. Suspension of mice by the tail elicits an escape response (Figure 3A, &#34;0&#34;). Deficits in the ability to splay the hind limbs and extend the toes are scored based on their severity on a scale from 0-4 (Figure 3A). Using the procedure outlined herein, one can observe significant limb clasping in rTg4510 mice (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/55523/55523fig1.jpg" /> 
Figure 1: Spontaneous Alternation in the Y-maze. (A) When placed into a Y-maze, mice adopt a lose-shift search strategy that results in a pattern of exploration whereby each arm is explored just once for every 3 arm entries. Aged Tg2576 mice exhibit a significant deficit in spontaneous alternation. Using the procedures outlined in this method, a significant restoration of spontaneous alternation was observed after treatment with a proprietary compound. Data was analyzed using 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (B) Number of arm entries was not significantly different across any of the groups monitored in this experiment. Data was analyzed using 1-way ANOVA test. Error bars indicate SEM. (C) There was no correlation between spontaneous alternation and the number of arm entries made, indicating that any differences in spontaneous locomotor activity did not impact quantification of spontaneous alternation. Correlation test was performed using Pearson&#39;s correlation analysis. (D) When placed into a Y-maze, rTg4510 mice (6 month) appear to exhibit significantly more spontaneous alternation relative to littermate WT mice. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (E) rTg4510 mice made significantly more arm entries due to their extreme hyperdynamic locomotion. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. ***p &#60;0.001. Error bars indicate SEM. (F) Spontaneous alternation behavior significantly correlated with arm entries, indicating the hyperdynamic locomotor phenotype obscured true spontaneous alternation. Correlation test was performed using Pearson&#39;s correlation analysis (r = 0.7, p &#60;0.0001). <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55523/55523fig2.jpg" /> 
Figure 2: Novel Object Recognition. (A) Arena habituation permits measurement of spontaneous locomotion and other stereotyped behaviors relevant to a particular mouse model. Here, aged Tg2576 mice (22 month) exhibit significantly more spontaneous locomotion relative to littermate WT mice. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-Veh were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (B) During sample phase, exploration of two identical objects was tracked separately. Mice that exhibit large biases toward exploration of one of the two objects (open circles) were excluded from test phase. (C-E) Novel object recognition was assessed by measuring the exploration of a novel and familiar object. Novel object recognition was assessed using (C) raw exploration time, (D) novelty preference or (E) discrimination index. Data in panel C were analyzed using a 2-way ANOVA with repeated measures and pairwise comparisons were made using Sidak&#39;s test. Data in panels D-E were analyzed with a 1-way ANOVA and post-hoc comparisons to Tg-Veh were made using Dunnett&#39;s test. *p &#60;0.05, **p &#60;0.01. Error bars indicate SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55523/55523fig3.jpg" /> 
Figure 3: Limb Clasping. (A) Representative images of mice exhibiting various degrees of limb clasping as described in Table 1. (B) rTg4510 mice (6 month) exhibit significant limb clasping relative to littermate WT mice as scored using these methods. Data were analyzed using a 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. ***p &#60;0.001, ****p &#60;0.0001. Error bars indicate SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-ÃŽÂ² and Tau Neuropathology at JoVE.co

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

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26.5K Views.  Hilltop Laboratory Animals. Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of β-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Video: Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

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 ...

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 ...