Name: utilizing the modified t-maze to assess functional memory outcomes after cardiac arrest
Uploaded: 2018-01-05T14:00:00
Description: Watch this Scientific Journal Video about Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest at JoVE.com

This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke grant 1R01NS096225-01A1, the American Heart Association grants AHA-13SDG1395001413, AHA-17GRNT33660336, AHA-17POST33660174, the Louisiana State University Grant in Aid research council, The Malcolm Feist Cardiovascular Research Fellowship, and the Evelyn F. McKnight Brain Institute.

All experimental procedures were conducted in accordance with the guidelines of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (LSU Health Sciences Center-Shreveport) for the usage of male Sprague Dawley rats (300-350 g, 9-10 weeks old). Rats were fasted overnight before the ACA surgery.
1. T-maze apparatus design and setting
NOTE: Base the T-maze design on the Deacon and Rawlins&#39; 2006 model31</s...

<ol>
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Modifications were made in the present study as compared to Deacon and Rawlins&#39; protocol31. The 3D printer was used to build the T-maze. The 3D-printing provides affordable and cost-effective alternatives to commercialized T-maze. To reduce rats&#39; anxiety during the test, the T-maze was performed in the dark room with minimum illumination. Once the rat entered one of the goal arms, we gently blocked the opposing arm. This avoids possible stress from the test, as well as possible damage to r...

According to the American Heart Association (2017), cardiac arrest (CA)-induced mortality occurs every four minutes, and affects over 400,000 people per year in the United States1. It is well-documented that CA can cause neuronal brain injury as a result of insufficient blood perfusion2,3,4. CA-induced brain injury occurs in the ischemia-sensitive CA1 region of the hippocampus5,6,7, affecting neurons that are critical to learning and memory8,9,10,11,12. Moreover, the loss of dendritic spine density, under ischemic conditions in the hippocampus (i.e. CA1 neurons), plays a critical role in spatial memory impairment13,14,15. Due to these pathological changes after CA, behavioral disorders such as: anxiety, depression, post-traumatic stress disorder, and memory loss are more prevalent. Although there have been advances in medical technology (i.e. efficient ambulatory service) that correlate with improved CA survival rates, most of the neuroprotective treatments (except for hypothermia) fail to improve functional outcomes after CA16,17. CA survivors typically have a poor quality of life, and are burdened with incremental medical spending16.
Cognitive status assessments for cerebral ischemia via behavioral tests are important to determine both drug efficacy and ultimately develop a successful clinical trial. In the 1940s, Edward Tolman designed the first behavior trial to study hippocampus-based spatial memory18. Subsequently, different mazes (i.e. Morris water maze, radial maze, T- or Y-maze, and Barnes maze) were developed to evaluate hippocampal-based spatial learning and memory in rats19,20,21,22,23. One of the more widely used behavioral test is the Morris water maze, which examines spatial learning and memory in rat models24. However, the Morris water maze requires the rat to swim and exert full motor function and control. For ischemia experiments such as the asphyxial cardiac arrest (ACA, a rat model of CA) model, cannulation of the femoral artery/vein are required to obtain vital blood pressure, blood gases and introduction of various drugs. Since femoral artery/vein cannulation can inhibit leg mobility rendering the rat&#39;s ability to swim properly, the Morris water maze may not be the most appropriate to test cognitive impairments under ACA.
The Barnes maze is the other widely used behavioral test to examine spatial learning and memory in rodent models. The Barnes maze does not require the exertion of full motor function and control, and thus less stressful than the Morris water maze. In the past, we performed experiments using the Barnes maze to determine if functional learning/memory differences occur between control or sham versus ACA-induced rats. The data obtained for the Barnes maze did not have the resolution to test cognitive impairments following mild to moderate ACA due to the fact that the Barnes maze is widely used to measure reference (long term) memory25,26, while ACA-induced neurocognitive deficits more closely related to working (short-term) memory27,28,29,30 suggesting that the Barnes maze is less viable for assessing memory function in our ACA model.
We thus developed a modified T-maze using spontaneous alternation test to evaluate working (short-term) memory after ACA. The modified T-maze spontaneous alternation test&#39;s major advantage is its simplicity and minimal stress on the rats as compared to other behavioral tests due to the fact that the modified T-maze does not require prior animal training, as well as heavy computational analysis or sub-routines (i.e. video imaging of the rat) as required by the Morris water maze and Barnes maze. Here we show that the modified T-maze spontaneous alternation test is a simple and yet highly efficient behavioral trial paradigm that can offer enough resolution to accurately detect and evaluate hippocampal function in diseases that cause short-term memory loss (i.e. ACA).

<table>
	<tbody>
		<tr>
			<td>3D Printer</td>
			<td>MakerBot</td>
			<td>Replicator</td>
			<td>Fifth generation</td>
		</tr>
		<tr>
			<td>3D Printer Filament</td>
			<td>Hatchbox</td>
			<td></td>
			<td>PLA, 1.75 mm filament diameter</td>
		</tr>
		<tr>
			<td>200 Proof Pure Ethanol</td>
			<td>Koptec</td>
			<td>V1005SG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sani-Chips</td>
			<td>PJ Murphy-Forest Products</td>
			<td></td>
			<td>Size: 8 to 20 mesh; 2.2 cubic foot/package; autoclavable bags</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Charles River Laboratories</td>
			<td>Sprague-Dawley</td>
			<td></td>
		</tr>
		<tr>
			<td>Vecuronium bromide</td>
			<td>Sun Pharmaceutical</td>
			<td>47335-931-40</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>Epinephrine</td>
			<td>Par Pharmaceutical</td>
			<td>42023-103-01</td>
			<td>Adrenalin Chloride Solution 1 mg/mL, 1:1000</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Pfizer</td>
			<td>00409-2012-32</td>
			<td>0.3mg/mL</td>
		</tr>
		<tr>
			<td>SketchUp</td>
			<td>Trimble Inc.</td>
			<td></td>
			<td>3D modeling software</td>
		</tr>
		<tr>
			<td>VentElite Small Animal Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-7040</td>
			<td>Animals raging in size from mouse to guinea pig (10g to 1kg)</td>
		</tr>
		<tr>
			<td>PowerLab 8/35</td>
			<td>Adinstruments</td>
			<td>PL3508</td>
			<td>8 analog input channels &#8211; 4 of which can be used in differential mode.</td>
		</tr>
		<tr>
			<td>Bio Amps</td>
			<td>Adinstruments</td>
			<td>FE132</td>
			<td>The Bio Amp is a galvanically isolated, high-performance differential bio amplifier optimized for the measurement of a wide variety of biological signals such as ECG, EMG and EEG recordings.</td>
		</tr>
		<tr>
			<td>Quad Bridge Amp</td>
			<td>Adinstruments</td>
			<td>FE224</td>
			<td>A four-channel, non-isolated bridge amplifier designed to allow the PowerLab to connect to most DC bridge transducers.</td>
		</tr>
		<tr>
			<td>LabChart 8</td>
			<td>Adinstruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABL80 FLEX CO-OX blood gas analyzer</td>
			<td>Radiometer</td>
			<td></td>
			<td>pH / p CO2 / p O2</td>
		</tr>
		<tr>
			<td>SURFLO Teflon I.V. Catheter</td>
			<td>Terumo</td>
			<td>sc-361556</td>
			<td>Only use the flexible thin wall catheter (49-mm long)</td>
		</tr>
		<tr>
			<td>Pipet/Infusion Needle</td>
			<td>Hamilton</td>
			<td>7748-03</td>
			<td>17-gauge; 93-mm long; 10-degree angle</td>
		</tr>
		<tr>
			<td>Classic T3 Vaporizer</td>
			<td>SurgiVet</td>
			<td>VCT302</td>
			<td>Classic T3 Isoflurane Funnel Fill</td>
		</tr>
		<tr>
			<td>ENVIRO-PURE Charcoal Canister</td>
			<td>SurgiVet</td>
			<td>32373B10</td>
			<td>Designed to absorb waste anesthetic gas</td>
		</tr>
		<tr>
			<td>O2 single flowmeter</td>
			<td>SurgiVet</td>
			<td>32375B1</td>
			<td>0-1000 mL</td>
		</tr>
		<tr>
			<td>N2O Flowmeter</td>
			<td>VetEquip</td>
			<td>401721</td>
			<td>0-4LPM</td>
		</tr>
		<tr>
			<td>Clay Adams Intramedic Luer-Stub Adapter (Sterile)</td>
			<td>Becton Dickinson</td>
			<td>427565</td>
			<td>23 gauge</td>
		</tr>
		<tr>
			<td>Micro Forceps</td>
			<td>Black and Black surgical</td>
			<td>B3FRC-18 RM-8</td>
			<td>7 1/4&#34; (18 cm), 8mm RH, counterweight w/ guide pin 2mm, platform 6 x .3 mm, curved.</td>
		</tr>
		<tr>
			<td>Halstead Mosquito Forceps</td>
			<td>Roboz</td>
			<td>RS-7111</td>
			<td>Curved; 5&#34; Length, 1.3 mm tip diameter, 2.1 mm jaw width</td>
		</tr>
		<tr>
			<td>Mixter Forceps</td>
			<td>Roboz</td>
			<td>RS-7291</td>
			<td>5.25&#34; Curved Extra Delicate, 1.1 mm tips</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Dissecting Spring Scissors</td>
			<td>Roboz</td>
			<td>RS-5650</td>
			<td>Straight, Sharp Points; 9 mm Cutting Edge; 0.15 mm Tip Width; 3 1/2&#34; Overall Length</td>
		</tr>
		<tr>
			<td>Mayo-Stille Scissors</td>
			<td>Roboz</td>
			<td>RS-6891</td>
			<td>5.5&#34; Round Curved</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>45 Deg Dumoxel Tip Size .10 x .06 mm</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Combination Scissor And Needle Holder</td>
			<td>Roboz</td>
			<td>RS-7884</td>
			<td>Cross Serration Tip; 5.5&#34; Length</td>
		</tr>
		<tr>
			<td>Moloney Forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
	</tbody>
</table>

utilizing the modified t-maze to assess functional memory outcomes after cardiac arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

ACA (global cerebral ischemia) mainly causes working (short-term) memory deficits28,29. To assess the function of learning and memory after ACA, we used the modified spontaneous alternation test to evaluate working (short-term) memory30. Results from spontaneous alternation test suggest that the alternation rate from three consecutive days in the ACA group (26.19 &#177; 4.96%) was significantly lower as com...

Watch this Scientific Journal Video about Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest at JoVE.com

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

The overall goal of this modified T-maze is to evaluate functional learning and memory after cardiac arrest induced cerebral ischemia. These modified team mates can help answer hippocampus function in disease that cause short-term memory deficits, such as cardiac arrest. The major advantage of the modified T-maze is it's relatively simple design.That is powerful enough to measure functional learning memory after ischemia. Additionally the data analysis is simple and straight forward. Since asphyxial cardiac arrest mainly results in short-term memory deficits, the modified T-maze can also be applied to other central nervous system diseases, that cause short-term memory deficits.Such as Alzheimers disease, Parkinson disease and transient ischemic attack. After femoral and arterial and venous catheterization, adjust physiological parameters as needed by modulating stroke volume, oxygen, or nitrous oxide levels. Then use a one milliliter syringe connected with a 23 gauge lure stub adaptor, to administer vecuronium bromide via femoral vein and wait for two minutes.Ensure that the blood pressure is at or around 100 millimeters of mercury. Induce apnea for six minutes by disconnecting the endotracheal tube from the ventilator. Further block the endotracheal tube by a one milliliter syringe to ensure complete apnea.During the last minute of apnea, adjust the respiratory rate of the ventilator to 80 breaths per minute and increase oxygen to two liters per minute with zero percent nitrous oxide to blow out any remaining isoflurane or nitrous oxide remaining in the ventilator. After the six minutes of apnea, remove the one milliliter syringe from the endotracheal tube. Reconnect the endotracheal tube to the ventilator.Next, use a one milliliter syringe connected with a 23 gauge lure stub adaptor to administer epinephrine via femoral vein. Administer manual chest compressions by the thumb, index and middle finger on the animals chest, in a light circular motion on the X and Z axis, until the return of spontaneous circulation. Then use another one milliliter syringe connected with a 23 gauge lure stub adapter to administer sodium bicarbonate via femoral vein, immediately after return to spontaneous circulation, to alleviate respiratory acidosis.10 minutes after resuscitation, measure blood gases again to determine the acid base status. Use a hemostat to clamp the femoral artery and vein. Slowly and gently remove arterial and venous catheters using blunt tip forceps.Then legate femoral artery and vein with a 5 O silk suture to prevent bleeding. Close the skin overlying the surgical site with a 3 O silk suture using the interrupted suturing technique, to minimize the chance of wound reopening. After the rat starts breathing on its own, 30 to 60 minutes after resuscitation, disconnect from the ventilator and gently remove the endotracheal tube.Place the rat in the baby incubator overnight. Finally transfer the rat to the individual cage and return the rat to the animal facility with regular chow and water. On the day before surgery, handle each rat for five minutes and never elevate the rat from the cage when handling.Then gently pick it up by it's tail with one hand, while the other hand supports it's legs, and let it jump from the hand to the cage five times. Separate each rat into individual cages so they will not dominate for food and or fight. Then three days after sham or ACA surgery, transfer the rat with the cage into a quiet and dark room before the start of the first run.Only turn on a low power desk lamp, and place it in the corner of the testing room to maintain minimum illumination. Allow the rat to adapt to darkness for 10 minutes. Next spread a thin layer of bedding to cover the entire floor of the maze and place the rat at the start arm.Allow each rat three minutes to explore the right or left goal arm. After the rat commits to one of the goal arms, when all four paws of the rat have entered the goal arm, lock the T junction between the start arm and the opposing goal arm to prevent the rat from entering the opposing goal arm. Leave the rat in the maze for 30 seconds.Then pick up the rat and place it back in it's cage for approximately 30 seconds. Then remove the T junction block from the T-maze. Place the rat at the start arm again and repeat the test to determine spontaneous alternation.Have rats perform four runs per day with a 10 minute break after the first two runs. Finally, change the bedding during the 10 minute break and between animals to eliminate scent bias. Clean the T-maze with 75%ethanol followed by distilled water at the end of each experimental day.Results from the spontaneous alternation test suggest that the alternation rate from three consecutive days in the ACA group was significantly lower compared to the control group. This is due to the fact that rats subjected to ACA developed an increase in side preference compared to controls. After watching this video, you should have a good understanding of how to utilize this modified T-maze to evaluate short-term memory deficit after cerebral ischemia in a rodent.While attempting this modified T-maze, it is important to gently block the opposite arm after the rat enters one of the goal arms. Additionally, T-maze should be performed in dark room with minimal illumination, which reduces rat anxiety.

Research

JoVE Journal

Behavior

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