Name: Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze (Video) | JoVE Journal
Uploaded: 2017-07-17T14:00:00.000+00:00
Duration: 6 min 9 s
Description: Learn to assess spatial memory in traumatic brain injury mouse models using the Radial Water Tread Maze, a non-swimming alternative to traditional tests. This method enables reliable cognitive evaluation in mice with motor deficits.

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5 minutes

This research was supported by the Institute for Translational Health Sciences pilot project grant opportunity (UL1TR000423), the University of Washington Center on Human Development and Disability, and the University of Washington Animal Behavior Core and Brain Imaging Core. We would like to acknowledge Dr. Warren Ladiges for his role in the development and dissemination of the original Radial Water Tread maze design and protocol presented here. We also thank Toby Cole for his assistance with this project.

All procedures and animal handling were conducted in accordance with the animal care guidelines issued by the National Institutes of Health and by the University of Washington Animal Care and Use Committee.
1. Surgery
<ol>
	<li>Anesthetize the mouse at 5% isoflurane in an induction box until unconscious. Confirm anesthesia by a reduction in the breathing rate and the absence of a withdrawal reflex following toe-pinch.</li>
	<li>Maintain anesthesia via nose cone at 2-2.5% throughout surgery. Monitor the breathing rate throughout surgery to ensure the mouse remains unconscious.</li>
	<li>Place the mouse prone on a heating pad and position the mouse in the stereotactic device using ear bars, ensuring that the head is secure and flat.</li>
	<li>Remove hair from the scalp using hair removal cream. Rinse the scalp thoroughly with saline.</li>
	<li>Clean the surgical site with an alternating iodine and 70% ethanol wash.</li>
	<li>Administer a subcutaneous injection of Lidocaine and Bupivacaine (1 mg/kg) at the scalp.</li>
	<li>With surgical scissors, make a longitudinal midline incision, and retract the skin to reveal the skull.</li>
	<li>Using a 5 mm trephine saw preform a craniotomy over the left frontoparietal cortex with center point at 2.5 mm behind bregma and 2.5 mm left of the midline. Carefully remove the circle of bone to expose the brain.</li>
	<li>Set the impactor device to a velocity of 6 m/s and 200 ms dwell time.</li>
	<li>Position the impactor device until the 3 mm convex impact tip is lightly touching the surface of the brain at 2.5 mm behind bregma and 2.55 mm left of the midline. Retract the impact tip, and lower by 1 mm (impact depth). When ready, fire the device, generating the desired impact.</li>
	<li>Cover the craniotomy with a sterile polypropylene disc cemented to derma with tissue adhesive, and suture the incision closed.</li>
	<li>Remove the mouse from anesthesia, and give IP injection of Buprenorphine (0.5 mg/kg).</li>
	<li>Allow the mouse to recover in a clean cage, warmed by a heating pad.&#160;The mouse should be monitored for signs of pain or distress over the next 24 h. 
	NOTE: Sham controls should receive identical treatment as above, with steps 1.8-1.9 omitted.</li>
</ol>
2. Radial Water Tread Maze Construction
<ol>
	<li>Bore 9 exit holes, large enough to accommodate 1-1/2 inch ABS DWV SPG x SJ trap adapters, at equal intervals around the circumference of a 32 inch diameter galvanized steel tub. Center these exit holes roughly 2-1/4 inches above the floor of the tub.</li>
	<li>Fit a 1-1/2 inch ABS DWV SPG x SJ trap adapter into each of the exit holes, and secure with the included ring nuts.</li>
	<li>With rubber stoppers, cap eight of the nine exits from the exterior of the apparatus. The final, uncapped exit will serve as the escape route. It does not matter which exit is designated as the escape route.</li>
	<li>Attach a 90&#176; ABS elbow to the exterior end of the remaining exit. The 90&#176; bend serves to prevent test subjects from visually determining the correct escape route from inside the maze.</li>
	<li>Construct the escape box from any opaque box capable of being sanitized and roughly 30 cm x 15 cm x 15 cm in size. Cut a hole on the side of the box, directly above the floor, large enough to accommodate a the 90&#176; ABS elbow.</li>
	<li>Attach the escape box to the terminal end of the 90&#176; ABS elbow.</li>
	<li>Slightly elevate the escape box (less than one inch) above the surface of the floor. This allows ample room for an electric heating pad, or other heating source, to be placed beneath the escape box.</li>
	<li>Print and laminate at least 5 unique, visual cues. Use simple, high contrast images that can be easily discerned from within the apparatus. Black and white clipart shapes (triangle, square, circle) are recommended.</li>
	<li>Using magnets, adhere the visual cues to the inner walls of the apparatus. Cues should be roughly equal distance apart, around the circumference of the apparatus.</li>
</ol>
3. Radial Water Tread Maze Protocol
NOTE: Water maze testing should begin only after the surgical site has healed (roughly one week post-surgery).
<ol>
	<li>Preparing for testing.
	<ol>
		<li>Allow mice to acclimate to the testing room for at least 30 min prior to commencing testing.</li>
		<li>Sanitize the apparatus using a 70% ethanol spray.</li>
		<li>Fill the apparatus with roughly 1 inch of cold (12-14 &#176;C) water.</li>
		<li>Place an electric heating pad, or other heating source, directly underneath the escape box. Keep the escape box dark and warm throughout the duration of testing.</li>
		<li>Position a bright light source over the apparatus. 
		NOTE: If using a lamp that might be visible to research animals from the apparatus itself, take care to ensure that the lamp is placed in the same position each day. The lamp itself might represent another visual cue for the mice to use to locate the escape box, and moving it drastically from day to day might complicate results.</li>
	</ol>
	</li>
	<li>Testing Protocol
	<ol>
		<li>Remove the mouse from its cage gently by the tail, and place in the center of the apparatus.</li>
		<li>As soon as the animal is in in the apparatus, begin timing.</li>
		<li>Once the animal has found the correct exit, and has located/entered the escape box, stop timing and record the number of seconds required to find the correct path.</li>
		<li>If the animal attempts to climb into a terminating hole and does not spontaneously reenter the maze after 10 s, guide the animal back into the center of the maze by hand.</li>
		<li>If the animal fails to find the correct path to the escape box within 3 min (180 s), score the trial as a failure and record as 180 s. Carefully guide the animal toward the correct path by hand.</li>
		<li>Allow the mouse to remain in the escape box for a 1 min, inter-trial rest.</li>
		<li>Once the 1 min rest has passed, remove the animal from the escape box and return to its home cage.</li>
		<li>Thoroughly sanitize the escape box and exits with a 70% ethanol spray to prevent the mouse from using olfactory cues to locate the correct escape route. This step should take no more than a few seconds.</li>
		<li>Return the mouse to the maze for the next trial.</li>
		<li>Repeat Steps 3.2.1-3.2.9 until the mouse has completed a total of three trials, and has either located or been led to the escape box three times.</li>
		<li>Following the final 1 min rest, return the mouse to its home cage.</li>
		<li>Drain and replace the water in the apparatus between animals to ensure consistent temperature throughout testing.</li>
		<li>Repeat steps 3.2.1-3.2.12 for each mouse to be tested.</li>
		<li>On the following day, repeat preparation steps 3.2.1-3.2.5. Take special care in ensuring that visual cues remain in consistent positions between testing days.</li>
		<li>Test animals using the above daily testing protocol for three trials a day for four days (training period), with a final three-trial test for memory retention on the fifth day. A sixth three-trial test (Long Term Memory retention) can be given on day twelve.</li>
		<li>Do not perform any tests between testing days five and twelve.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>If a mouse fails to complete the maze in under 180 s during any two-day period (i.e., a total of consecutive 6 trials all scored at 180 s), consider the mouse insufficiently motivated by the testing conditions, and remove from analysis.</li>
		<li>Calculate the mean latency to complete the maze for each subject by testing date by averaging their three daily trials for that day.</li>
		<li>Obtain group differences in memory retention using standard t-tests to compare groups at the fifth day test and long-term memory testing date. If more than two groups are being compared, one-way ANOVA followed by appropriate post-hoc analysis (such as Tukey&#39;s test) to follow up on any significance obtained should be employed instead.</li>
		<li>Obtain group differences in the acquisition period (days 1-4) via repeated measure analysis of variance.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The RWT maze protocol presented here successfully distinguishes between CCI-induced TBI mice and sham controls, and represents a feasible, mouse-centric, alternative to the MWM and Barnes circular maze. While the results reported here speak only to the use of the RWT maze in a TBI mouse model, this apparatus has been used successfully in aged and transgenic models where stress-induced noncompliance resulting from swim-based testing made using the MWM impractical15,17</...

Memory impairments are among the most common symptoms reported by patients following traumatic brain injury (TBI)1,2. Accurate identification and assessment of analogous memory deficits in animal models of TBI, therefore, are essential to our understanding of this condition and its management. Here, we present a protocol to test spatial memory in a mouse model of TBI using a Radial Water Tread (RWT) maze. This apparatus was previously shown to assess cognitive deficits in mouse models of controlled cortical impact (CCI)-induced TBI3, and represents a potential alternative to rat-validated, swim-based tests of cognition.
The growing diversity and availability of transgenic mouse models has led to a recent increase in the use of mice over rats in scientific research4. Despite this shift, researchers continue to rely on behavioral and cognitive tasks that were originally designed and validated for rat use. The most common tests currently used to assess cognition in mice, the Morris Water Maze (MWM) and the Barnes circular maze, were specifically designed to capitalize on instinctual behaviors found in rats5,6. Considering the genetic, neuroethological, and cognitive differences that exist between these two species4, it is unsurprising that mice consistently underperform on these tasks7,8.
Species-dependent differences in testing ability are particularly concerning in swimming-based cognitive tests, such as the MWM. While both rats and mice are proficient swimmers, researchers have identified several mouse strains that perform remarkably poorly on swimming-based cognitive tasks9,10,11,12,13. Even in wild-type animals, rats generally outperform mice7,8. While this could be interpreted as a species-specific difference in spatial memory, analogous follow-up testing using a dry-land maze revealed no species-dependent differences in cognitive performance8. A number of factors unrelated to cognition could account for this finding, including species-dependent differences in either swimming ability or search strategy. Indeed, factor analysis of mouse-specific search strategies in the MWM show that noncognitive factors (in particular, thigomotaxis and passivity [i.e., floating]) may play a more significant role in MWM performance than spatial learning14.
Here, we demonstrate the use of a cognitive test designed to capitalize on the instinctual behavior of mice, and which does not require swimming, to measure spatial memory impairment in a mouse model of CCI-induced TBI. While the RWT maze (Figure 1A-B) was conceived as a novel hybrid of the MWM and Barnes circular maze, it was specifically designed to take advantage of thigmotactic behavior instinctual to mice15,16. The apparatus consists of a 32 inch diameter galvanized steel tub in which nine evenly spaced exit holes have been bored. The holes are centered 2-1/4 inches above the floor of the tub and are sized to fit commonly available 1-1/2 inch ABS DWV SPG x SJ trap adapters. Eight of the exits are capped from the outside and blinded to a depth of 1 inch with rubber stoppers. The ninth is connected by a 90&#176; acrylonitrile butadiene styrene (ABS) elbow to an opaque plastic box from which the mouse can be easily removed after testing. Over the course of a brief acquisition period, the mouse is trained to use the unique visual cues lining the maze to locate this escape box. During testing, the maze is filled with an inch of cold water (12-14 &#176;C), sufficiently aversive enough to promote escape, but not deep enough that the mouse is required to swim.
The RWT maze represents a low-cost, low-maintenance alternative to the MWM, and has been used successfully in aged and transgenic mice15,17,18,19, and CCI-induced mouse models of TBI3. The protocol outlined here represents a simple and effective method for measuring spatial memory impairment requiring no pre-injury training, and could be easily modified to suit the particular needs of a research laboratory.

<table><tbody><tr><td>35 Gal. Hot Dipped Steel Round Tub</td><td>Home Depot</td><td>Internet #206638142</td><td>Needed: 1</td></tr><tr><td>1-1/2 in. ABS DWV SPG x SJ Trap Adapter</td><td>Home Depot</td><td>Internet #100344703, Store SKU #188956</td><td>Needed: 9</td></tr><tr><td>1-3/4 in. x 1-7/16 in. Black Rubber Stopper</td><td>Home Depot</td><td>Internet #100114974 Store SKU #755844</td><td>Needed: 8</td></tr><tr><td>1-1/2 in. ABS DWV 90 Degree Hub x Hub Elbow</td><td>Home Depot</td><td>Internet #100346663 Store SKU #188603</td><td>Needed: 1</td></tr><tr><td>HDX&lt;br/&gt; 10 Gal. Storage Tote</td><td>Home Depot</td><td>Internet #202523587 Store SKU #258804 Store SO SKU #258804</td><td>Needed: 1</td></tr><tr><td>Impact One Stereotaxic Impactor for CCI</td><td>Leica Biosystems</td><td>39463920</td><td>Needed: 1</td></tr><tr><td>Vernier Stereotaxic w/ Manual Fine Drive Stereotaxic Instrument for Small Animals</td><td>Leica Biosystems</td><td>39463001</td><td>Needed: 1</td></tr></tbody></table>

assessing spatial memory impairment in a mouse model of traumatic brain injury using a radial water tread maze

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

The RWT maze (Figure 1) was used to investigate injury-dependent spatial memory deficits in mice randomly assigned to receive either controlled cortical impact-induced TBI or sham surgery. The injury was generated using a solenoid-driven cortical impact with a 3 mm convex tip and the following injury parameters: 6 m/s strike velocity, 1 mm depth of penetration, and 200 ms contact time. Mice received cognitive testing starting at 35 days post-surgery, and were...

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

The overall goal of this Radial Water Tread Maze protocol is to test spatial memory in a mouse model of traumatic brain injury using a mouse centric cognitive task that does not require swimming. This method can be especially useful for testing cognition in mouse models, where motor deficits and or a lack of motivation makes swimming based cognitive testing protocols such as the Morris Water Maze impractical. Though this method could provide insight into group differences and cognition in a controlled cortical impact model of traumatic brain injury in mice, it can also be useful for measuring cognition in aged, and transgenic mouse models that are known to perform poorly in swimming based tests.On all testing days, perform the following preparatory steps. First, allow the mice to acclimate to the test room, while in their home cages for at least 30 minutes. The round maze features visual cues along the inner walls.The movable escape box positioned at one of nine different exit holes. While the mice acclimate, sanitize the exits and the box using a 70%ethanol spray. Next, fill the maze with roughly one inch of cold water.Keeping the water temperature consistent between trials is very important. So measure accurately, and adjust as needed. Now, position the escape box at one of the nine exit holes.Using a heating source to keep the box warm, ideally using a heating pad. Also, seal the box from light. The escape box should always be dark, warm and relatively odorless.The light source is also likely to be a cue so do not change its position between trials. Remove the test mouse from its cage gently by the tail, and place it in the center of the apparatus. Then immediately start timing how long it takes for the mouse to find the exit.If a mouse climbs into a terminating hole, and does not spontaneously reenter the maze after 20 seconds, do not stop the trial. Instead, guide the mouse back into the center of the maze by hand, and let the mouse complete the trial. If after three minutes, the mouse hasn't exited the maze, score the trial as a failure, and record the time as three minutes.Then by hand, carefully guide the animal along the straightest path to the exit. Once in the escape box, allow the mouse to remain there for one minute. Then transfer it back to its home cage while the maze gets reset.Between trials, thoroughly sanitize the escape box and exits with a 70%ethanol spray to prevent the mouse from using old factory cues to locate the correct escape route. This can be done pretty quickly. Now, repeat the trial on the same mouse twice more for a total of three consecutive trials.Every testing day consists of three trials per mouse. No more, and no less. In addition to sanitizing, drain the maze and refresh the water between testing different mice.Be sure to keep the water temperature between 12 and 14 degrees Celsius. Use containers of ice or colder hot water to adjust the tap water temperature into this range. Between testing days, it is important to make certain that the visual cues in the maze, and in the space surrounding the maze remain consistent.A complete testing regime includes five consecutive testing days all run in the same manner. The fifth day is considered the measure for memory retention. Provided no tests are performed between day six and 11, a test on day 12 can be used to measure long term memory.The radial water maze was used to investigate injury dependence spatial memory deficits in mice receiving either controlled cortical impact induced traumatic brain injury or a sham injury. The TBI surgery is described in detail in the text protocol and in a JoVE article by Romin, Gao and Chen from 2014. Briefly put, the injury was generated using a solenoid driven cortical impact with a three millimeter convex tip.The shams were operated on but impacted by the device. 35 days post surgery, the described testing regime was performed. All the mice passed the criteria test for a desire to escape the maze.So all were included in the analysis. The results show a clear difference between the TBI mice and sham controls. Escape latency was significantly longer for injured mice on the first day of training, and on both of the two memory test days.Thus, the described TBI affected the animal's capacity to learn. After watching this video, you should have a good understanding of how to properly perform cognition testing using a mouse specific test of spatial cognition. Once mastered, this technique can be done in less than 15 minutes per animal if it is performed properly.While attempting this procedure, it is important to remember that the positioning of visual cues and the water temperature must remain consistent across all trials.

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Research

JoVE Journal

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