Name: motor and hippocampal dependent spatial learning and reference memory assessment in a transgenic rat model of alzheimer's disease with stroke
Uploaded: 2016-03-22T13:00:00
Description: Watch this Scientific Journal Video about Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke at JoVE.com

This work was made possible with funding from the Canadian Institutes of Health Research (CIHR) and the authors would like to thank CIHR for their funding support.

The appropriate institutional animal ethics committee should approve all of the behavioral procedures prior to starting experimentation. All animal work described here was approved by Western University Animal Use Subcommittee and follows the Canadian Council on Animal Care guidelines.&#160;These animal experiments were performed during the light phase.
1. Cylinder Task for Gross Forelimb Motor Assessment
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	<li>Equipment Setup
	<ol>
		<li>Acquire a Plexiglas cylinder with a perforated lid that will accommoda...

<ol>
	<li>Auld, D., Kornecook, T., Bastianetto, S., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+the+basal+forebrain+cholinergic+system:+relations+to+&#946;-amyloid+peptides,+cognition,+and+treatment+strategies.">Alzheimer's disease and the basal forebrain cholinergic system: relations to &#946;-amyloid peptides, cognition, and treatment strategies.</a> Prog Neurobiol. 68 (3), 209245 (2002).</li><li>Huang, Y., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer+Mechanisms+and+Therapeutic+Strategies.">Alzheimer Mechanisms and Therapeutic Strategies.</a> Cell. 148 (6), (2012).</li><li>Querfurth, H., LaFerla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> New Engl J Med. 362 (4), 329-344 (2010).</li><li>Karran, E., Mercken, M., Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li><li>Akiyama, H., 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=Inflammation+and+Alzheimer's+disease.">Inflammation and Alzheimer's disease.</a> Neurobiol Aging. 21 (3), (2000).</li><li>Butterfield, D., Drake, J., Pocernich, C., Castegna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+oxidative+damage+in+Alzheimer's+disease+brain:+central+role+for+amyloid+&#946;-peptide.">Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid &#946;-peptide.</a> Trends Mol Med. 7 (12), 548554 (2001).</li><li>Kalaria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cerebral+ischemia+in+Alzheimer's+disease.">The role of cerebral ischemia in Alzheimer's disease.</a> Neurobiol Aging. 21 (2), 321-330 (2000).</li><li>Selkoe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+genes,+proteins,+and+therapy.">Alzheimer's disease: genes, proteins, and therapy.</a> Physiol Rev. 81 (2), 741-766 (2001).</li><li>Snowdon, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+infarction+and+the+clinical+expression+of+alzheimer+disease:+The+nun+study.">Brain infarction and the clinical expression of alzheimer disease: The nun study.</a> JAMA. 277 (10), 813 (1997).</li><li>Clarke, J., 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=Overexpression+of+APP+provides+neuroprotection+in+the+absence+of+functional+benefit+following+middle+cerebral+artery+occlusion+in+rats.">Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in rats.</a> Eur J Neurosci. 26 (7), 1845-1852 (2007).</li><li>Whitehead, S., Hachinski, V., Cechetto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+Between+a+Rat+Model+of+Cerebral+Ischemia+and+&#946;-Amyloid+Toxicity+Inflammatory+Responses.">Interaction Between a Rat Model of Cerebral Ischemia and &#946;-Amyloid Toxicity Inflammatory Responses.</a> Stroke. 36 (1), 107-112 (2005).</li><li>Agca, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transgenic+rats+producing+human+&#946;-amyloid+precursor+protein+as+a+model+for+Alzheimer's+disease:+Transgene+and+endogenous+APP+genes+are+regulated+tissue-specifically.">Development of transgenic rats producing human &#946;-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically.</a> BMC Neuroscience. 9 (1), 28 (2008).</li><li>Bayona, N. A., Gelb, A. W., Jiang, Z., Wilson, J. X., Urquhart, B. L., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+neuroprotection+in+cerebral+ischemia+and+its+effects+on+low-molecular-weight+antioxidants+and+skilled+motor+tasks.">Propofol neuroprotection in cerebral ischemia and its effects on low-molecular-weight antioxidants and skilled motor tasks.</a> Anesthesiology. 100 (5), 1151-1159 (2004).</li><li>Langdon, K., Clarke, J., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+to+high+fat+diet+is+bad+for+your+brain:+exacerbation+of+focal+ischemic+brain+injury.">Long-term exposure to high fat diet is bad for your brain: exacerbation of focal ischemic brain injury.</a> Neuroscience. 182, 82-87 (2011).</li><li>Brownlow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+rescue+of+memory+deficits+induced+by+calorie+restriction+in+a+mouse+model+of+tau+deposition.">Partial rescue of memory deficits induced by calorie restriction in a mouse model of tau deposition.</a> Behav Brain Res. 271, 7988 (2014).</li><li>Halagappa, V., 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=Intermittent+fasting+and+caloric+restriction+ameliorate+age-related+behavioral+deficits+in+the+triple-transgenic+mouse+model+of+Alzheimer's+disease.">Intermittent fasting and caloric restriction ameliorate age-related behavioral deficits in the triple-transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 26 (1), (2007).</li><li>Maalouf, M., Rho, J., Mattson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroprotective+properties+of+calorie+restriction,+the+ketogenic+diet,+and+ketone+bodies.">The neuroprotective properties of calorie restriction, the ketogenic diet, and ketone bodies.</a> Behav Brain Res. 59 (2), (2008).</li><li>Tillerson, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+limb-use+effects+on+the+behavioral+and+neurochemical+effects+of+6-hydroxydopamine.">Forced limb-use effects on the behavioral and neurochemical effects of 6-hydroxydopamine.</a> Neuroscience. 21 (12), 4427-4435 (2001).</li><li>Sierksma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+spatial+memory+function+in+APPswe/PS1dE9+mice+after+chronic+inhibition+of+phosphodiesterase+type+4D.">Improvement of spatial memory function in APPswe/PS1dE9 mice after chronic inhibition of phosphodiesterase type 4D.</a> Neuropharmacology. 77, (2013).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Behav Brain Res. 36 (1), 60-90 (2001).</li><li>Vorhees, C., Williams, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: procedures for assessing spatial and related forms of learning and memory.</a> Nat Protoc. 1 (2), 848-858 (2006).</li></ol>

The combination of stroke and Alzheimer&#39;s disease results in very distinct behavioral pathologies that can affect both motor and cognitive function depending on the severity of each condition. Thus, it is necessary to make use of a variety of behavioral tasks to determine the individual contributions of these conditions, as well as give some insight into the combined and potentially interactive effects in the co-morbid condition. The data presented demonstrate three cost-efficient, time-efficient and sensitive behavi...

Alzheimer&#39;s Disease (AD) is the most prevalent form of dementia in the elderly population and a debilitating neurodegenerative disease. Histopathologically, AD presents itself as amyloid plaques, neurofibrillary tangles and neuronal loss. Amyloid plaques consist primarily of beta-amyloid peptide (A&#946;) that has been produced through an altered proteolytic cleavage of amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase enzymes1,2,3. The cleavage product, A&#946;, deposits in the brain creating pathological amyloid plaques and has toxic effects on the brain that can lead to the characteristic learning impairments and memory loss. All of these steps together are referred to as the &#34;amyloid cascade hypothesis&#34;3,4. While this hypothesis is important when investigating AD, other cellular changes have been found to precede these plaque formations that strays from the original amyloid cascade pathway. These other cellular changes are thought to contribute to early memory loss, learning impairments and other cognitive dysfunctions involved in AD prior to plaque formation3,5,6.
With AD becoming increasingly prevalent, risk factors for developing AD are becoming an extremely important focus of research. Although age is the principal risk factor for sporadic forms of AD, other risk factors have been identified, including stroke7,8. Stroke is not only a risk factor, but it can also exacerbate already present dementias. For example, clinically, the progression of AD has been shown to be worse in patients who had previously experienced stroke9. Moreover, increased APP expression and A&#946; accumulation has been found in experimental animal models of A&#946; toxicity combined with induced stroke10,11. Since there is this important interaction between stroke and AD, it is essential that these two pathologies be further researched together in co-morbid models to better understand pathophysiology and behaviors implicated in both conditions.
To investigate co-morbid conditions, an appropriate model had to be developed in which a stroke could interact with A&#946; to produce AD-like pathology. For the first time, an APP21 transgenic rat that has a mutated human APP gene incorporated into its DNA was used to achieve an appropriate model of AD. The mutations are the Swedish double missense and Indiana single missense mutations, which have both been implicated in familial forms of AD4,8,12. In the absence of an additional insult, this rat model ages without developing the characteristic A&#946; plaques or neurofibrillary tangles12. Therefore, in an effort to induce AD-like behavioral pathology, a small stroke is introduced into the right striatum to mimic the small subcortical strokes often present in dementia patients9. The stroke in the APP21 transgenic rat embodies the co-morbid condition and allows investigation of various types of behavioral changes implicated in both disease conditions. In particular, this induction of AD-like pathology and cognitive deficits in the adult rat allows us investigate the earliest molecular and cognitive changes preceding AD.
Since the goal is to determine the first signs of behavioral changes and since both stroke and AD have very distinct behavioral pathologies, when studying the co-morbid model, behavior tasks need to assess a variety of behavioral phenotypes. There are a battery of relatively sensitive tests that can be done to analyze motor and cognitive behavior in rodent models that involve a variety of paradigms and equipment. To specifically analyze forelimb and hindlimb motor function, the cylinder task and beam-walk task have been implemented to detect motor deficits and monitor locomotion in this model. Other sensitive tasks designed to specifically assess fine forelimb motor skill (i.e. the staircase task and single pellet reaching task) require food deprivation11,13,14. To avoid any of the known effects of food deprivation on disease pathologies15,16,17, these tests have been deemed unsuitable for this study. The cylinder task assesses the spontaneous use of the rat&#39;s forelimbs during rearing in a novel environment and can detect asymmetry between forelimbs in rats with unilateral stroke10,18. A major benefit to this task is that the apparatus can be utilized for other behavior tasks, such as the Porsolt forced swim task19. Contrary to the cylinder task, the beam-walk task also allows analysis of hindlimb and forelimb motor control, in addition to locmotion10,14. Beam walking includes a locomotor component, a balance component and skilled foot placement. Both of these tests are cost-efficient, straightforward, and time-efficient and elucidate the effects of stroke and AD on differences in limb functioning.
Aside from changes in motor function, AD involves memory deficits that can present in early stages of the disease progression. When addressing AD-like pathologies in a rodent model, it is crucial that hippocampal dependent learning and memory is assessed because the hippocampus is an important brain structure largely affected in AD2. The hippocampus is an essential brain region for spatial learning and memory and its function can be tested using various maze paradigms in rodents. One of the mostly widely used maze tasks for rodent models of different diseases is the Morris water maze20. The Morris water maze utilizes spatial cues to assist the rat in locating a stationary hidden platform and tests spatial reference memory when the platform is removed. A valuable advantage of the water maze setup is that it is highly adaptable depending on the proposed research question20.
For the first time, the techniques described have been used to assess motor and cognitive function in a novel co-morbid rat model of stroke and AD. involving small strokes in an APP21 transgenic rat model.&#160;Co-morbidity was achieved by inducing vasoconstriction of the blood vessels in the striatum to produce a small stroke in APP21 transgenic rats. This stroke model has been well established as a co-morbid condition in an alternative rat model of AD11. Advancement into this novel APP21 transgenic rat model was intended to produce a more translationally valuable model. While the behavioural tasks are described using a co-morbid stroke and AD rat model, these tasks can be further applied to other models of stroke or models of other neurological diseases (i.e. Parkinson&#8217;s disease). The general methodology described will be widely applicable to these other disease states, but behavior timelines and paradigms may require alteration based on the proposed research question and model. In addition to being adaptable, the tasks described are effective in demonstrating minor deficits, while also being cost and time-efficient.&#160;

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			<td height="125" style="height:125px;width:216px;">Cylinder</td>
			<td style="width:115px;">Western University</td>
			<td style="width:167px;">Plexiglas Cylinder 
			Cylinder Diameter: 23 cm 
			Cylinder Height: 40 cm 
			Platform Height: 30 cm 
			Mirror Length: 35 cm 
			Mirror Width: 26.5 cm</td>
			<td style="width:320px;">This was made specifically by Western University Machine Services for our lab. Please contact your own chosen manufacturer to design this product.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Handheld Video Camera</td>
			<td style="width:115px;">JVC</td>
			<td style="width:167px;">GZ-E200</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Video Camera Tripod</td>
			<td style="width:115px;">Slik</td>
			<td style="width:167px;">F163</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AM/FM Clock Radio</td>
			<td style="width:115px;">Sylvania</td>
			<td style="width:167px;">SCR1388</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">White Board</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:167px;">Width: 71.12 cm 
			Height: 55.88 cm</td>
			<td style="width:320px;">These can be purchased at any store (i.e. Walmart, University bookstores)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dry-erase Marker</td>
			<td style="width:115px;">Expo</td>
			<td style="width:167px;">Expo Dry-erase Original Marker</td>
			<td style="width:320px;">These can be purchased at any store (i.e. Walmart, University bookstores)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Wooden Beam</td>
			<td style="width:115px;">Rona</td>
			<td style="width:167px;">Plywood 
			Width: 2 cm 
			Length: 100cm</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VWR General Purpose Laboratory Tape</td>
			<td style="width:115px;">VWR Intl.</td>
			<td style="width:167px;">89097-920</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">iMovie &#39;11</td>
			<td style="width:115px;">Apple Inc.</td>
			<td style="width:167px;">Version 9.0.9 (1795)</td>
			<td style="width:320px;">This is the version used in this manuscript, but any other iMovie version or video editing software could be used.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Morris Water Maze Pool with Platform</td>
			<td style="width:115px;">Stoelting Co.</td>
			<td style="width:167px;">60136/60035</td>
			<td style="width:320px;">These are not the exact products used in the video, but these are essentially identical.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Platform Cue</td>
			<td style="width:115px;">-</td>
			<td style="width:167px;">-</td>
			<td style="width:320px;">The platform cue used was created using a small metal stand and white spherical foam ball. These can likely be purchased at any store with home improvement materials (i.e. Walmart, Rona etc.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mainstays 71&#34; Floor Lamp</td>
			<td style="width:115px;">Walmart</td>
			<td>HW-F0377SLV</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ANY-Maze Behavioural Tracking Software</td>
			<td style="width:115px;">Stoelting Co.</td>
			<td style="width:167px;">60000</td>
			<td style="width:320px;">There are ANY-maze&#174; bundles that include the camera with or without a computer and accessories.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Compact Video Camera</td>
			<td style="width:115px;">Logitech</td>
			<td style="width:167px;">V-U0023</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laptop</td>
			<td style="width:115px;">Hewlett-Packard</td>
			<td style="width:167px;">HP&#174; Pavilion dv6 Notebook PC</td>
			<td style="width:320px;">Laptop Specifics: AMD A6-3420M APU with Radeon&#8482; HD Graphics 1.50 GHz, 6.00 GB RAM, 64-bit operating system.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Americana Non-toxic Acrylic Paint</td>
			<td style="width:115px;">DecoArt</td>
			<td>DAO67-9</td>
			<td style="width:320px;">This can be ordered on the DecoArt site or purchased in store at DecoArt&#174; retailers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poster Board</td>
			<td style="width:115px;">Walmart</td>
			<td>PA-1961</td>
			<td style="width:320px;"></td>
		</tr>
	</tbody>
</table>

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.

The behavioral tasks described were used to demonstrate the effects of stroke in an APP21 transgenic rat model of Alzheimer&#39;s disease. The combination of stroke and the APP21 transgene is expected to result in greater motor deficit in the affected limbs, as well as increased memory deficits.
The cylinder task assessed gross forelimb motor function and is represented as the use of the affected forelimb. Furthermore, the beam-...

Watch this Scientific Journal Video about Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke at JoVE.com

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

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 ...

The overall goal of these behavioral tests is the assess motor function, as well as spacial learning and memory performance in a novel comorbid rat model of Alzheimer's disease and stroke. This method can help answer key questions in the dementia field, such as how stroke and Alzheimer's disease pathology interact, producing a phenotype of potential enhanced motor deficit and accelerated cognitive decline. The main advantage of these techniques is that they test for a range of brain functions while being easy to conduct, cost efficient, and suitable for repeated testing at later time points.The implications of this technique extend toward prevention of dementia after stroke because novel preventative compounds can be tested for their potential to ameliorate the comorid behavioral phenotype. To begin, place a mirror at a 45 degree angle beneath a previous mounted cylinder apparatus. Then affix a video camera to a tripod and position it at a distance of approximately 50 centimeters from the apparatus.Check that the entire diameter of the cylindar in the mirror can be visualized on the camera. Next, choose an acclimatized rat and on a white board write down the animal's number and additional trial information. Place this board in front of the mirror and then press record on the video camera.Continue by picking up the rat by the base of the tail and placing it in the cylinder. Then secure the lid of the apparatus and remove the white board so that it no longer blocks the mirror. Once the white board has been removed, proceed to film the animal for five minutes, a period that constitutes a single trial.At the end of this time, stop the recording, return the rat to it's home cage and clean the cylinder with paper towels and water. Then repeat the previously described steps twice more to achieve a total of three trials per rat. Once data has been collected for all rats, import the camera files into a video editing program and proceed to quantify the number of four limb contacts as described in the text protocol.First, position two shelving units approximately 100 centimeters apart from one another in front of a black wall. Then, use tape to anchor the ends of a previously acquired wooden beam to the shelves so that there is approximately one meter of unsupported wood between them. Next place a video camera on a stand and position it so that the camera captures the entire length of the beam.Proceed to choose a rat that has undergone a beam walking training session and record it's number and additional trial information on a white board. When finished, tape the white board to the wall behind the beam. Continue by placing the animal's home cage and an environmental enrichment tube which serve as a motivating queue which the rat can immediately enter after crossing plank, at one end of the beam.Then press record on the video camera. Immediately afterwards, pick up the rat by the base of the tail and place it on the opposite end of the beam without the cage and tube. Record the animal until it sucessfully traverses the full length of the elevated wood, which marks the end of the trial.If the rat pauses midway across the beam, gently touch it's tail to promote movement. Once the animal completes the task, move it's home cage to the other end of the beam. The change the trial number on the white board and repeat the procedure as previously described.When all animal's trials have been recorded, import the camera files into a video editing program and analyze the relevant step, limb slips, and fall data as describe in the text protocol. Start by securing a video camera above the center of an empty, circular pool. Then, designate the four quadrants of the tank by placing tape along the rim of the pool and align them properly with the outline of the tank in the tracking software program.Next, fill the tank with water to a depth of 36 centimeters and then add black, non-toxic paint until the liquid if opaque in order to ensure the platform is not visible below the surface of the water and to better visualize white rats. Once the pool has been prepared, surround it with blank wall surfaces. Then prepare spatial cues by cutting four shapes from different colors of poster board.Afterwards, attach one shape to each of the walls associated with the north, south, east, and west pool locations. To perform a spatial learning experiment, continue by placing a circular platform in the southwest quadrant of the pool. In the accompanying computer program, check that this structure is aligned with a designated platform area in the quadrant.Proceed by grasping a rat by the base of it's tail and gently placing it at a designated start site in the water along the pool wall. Once the animal is in the tank, quickly move our of it's sight and signal another experimenter to start the tracking software. Allow the rat to swim until it locates and climbs onto the platform.At which point, the other researcher should stop the trial on the computer. Let the animal remain on the platform for 30 seconds. If the rat fails to find the platform within the allotted trial time, here 90 seconds, guide the animal to the structure and let it stay there for 30 seconds.Afterwards, lift the rat by the base of the tail and remove it from the maze. Placing it on a towel draped over an arm. Repeat this procedure three additional times per animal with an inter-trial interval of 20 minutes, so that each rat completes of total of four trials.Once the spatial learning experiment has been completed, remove the platform from the pool to commence the probe trials. In the software, check that the previous platform position, in the southwest quadrant remains defined. Then introduce a rat into the pool.Placing it in the water along the wall of the tank. As before, immediately move out of the animal's sight and instruct another experimenter to start the tracking software. At the end of the 30 second probe trial, grasp the animal by the base of the tail, remove it from the tank and then place it one a towel covered arm.Repeat this procedure one week later. Once spatial learning and probe trials have been performed for all animals, analyze the data as described in the text protocol. Four limb use data from the cylinder apparatus was collected from APP21 transgenic rats three days before and seven and 20 days after stroke induction.When standardized and compared to the value representing equal four limb use, the red line here, these results indicate that comorbid rats do not statistically demonstrate a four limb deficit. Similar results were obtained for wild type animals with or without stroke and APP21 transgenic rats alone. However, comorbid animals showed differences in locomotion associated motor function.Beam task data collected for these rats seven days prior to and 21 days after stroke induction reveals a significant increase in the steps needed to traverse the plank, which was not observed in other groups. Morris water maze results also reveal memory deficits in comorbid animals, as evidenced by this graph showing the percentage change in latency, the time animals take to reach the quadrant of the pool where the platform once was between the first and second probe trials. Here APP21 rats with stroke take significantly longer in the second trial to enter the target quadrant.A difference that was not demonstrated by the transgenic rats alone or wild type animals with stroke. This indicates that comorbid animals have a long term memory deficit. While attempting this procedure, it's important to keep all variables as consistent as possible throughout and across testing sessions and create a stress free environment for the animals.After watching this video, you should have a good understanding of how to assess motor and cognitive performance in rat models of post-stroke dementia.

motor-hippocampal-dependent-spatial-learning-reference-memory

Research

JoVE Journal

Medicine

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 ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...