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 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' 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).
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'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'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).
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' 2006 model31.
2. Asphyxial Cardiac Arrest (ACA)
3. T-maze
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 ± 4.96%) was significantly lower as compared to the control group (62.96 ± 6.07%) (*p0.05)35 due to the fact that rats submitted to ACA developed a side bias as compared to control (82.14 ± 4.57% v. 62.89 ± 2.86%, *p0.05) (Figure 3a and 3b)35, thus presented with lower spontaneous alternation rate. Results were expressed as means ± S.E.M. Data analyzed using one-way ANOVA followed by Turkey's post-hoc test37. p < 0.05 (95% confidence level) was considered statistically significant.
Figure 1. The T-maze design.
The T-shaped platform of the maze (for rats) was built with a 600 mm x 165 mm start arm and 400 mm x 100 mm goal arms at the upper apex of the "T". The thickness of the walls was 5.5 mm x 8 mm (floor thickness). A central partition was at the junction of the start arm to the goal arm, where this partition extended from the back wall of the T-maze and 100 mm into the start arm dividing the goal arms. The dotted squares represent the location of the "T" junction block that blocked either arm. Please click here to view a larger version of this figure.
Figure 2. Experimental timeline for ACA/sham surgery and T-maze.
Before the day of the ACA surgery, rats were handled 4 times (5 min/each time, at day 0) to acclimatize the rat to human touch. After ACA surgery, the rats were permitted to heal and stabilize over the following 3 days. After recovery, the rats performed the spontaneous alternation tests for 3 consecutive days (12 runs, 4 runs/day). Each rat performs 4 runs per day. The experimental timeline is the same for sham surgery. Please click here to view a larger version of this figure.
Figure 3. Short-term memory deficits after ACA.
An increase in side preference rate can be observed in rats subjected to ACA (a). Spontaneous alternation rate was decreased in rats with ACA as compared with control (b). Numbers in parentheses indicate the animals used per group. Results were expressed as means ± S.E.M. *P < 0.05 indicates significantly different from control. Statistical analysis was evaluated by one-way ANOVA with Tukey's post hoc test. This figure has been modified from Lee et al., 201735. Please click here to view a larger version of this figure.
Modifications were made in the present study as compared to Deacon and Rawlins' 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' 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 rats' tail while lowering the guillotine door. Any unnecessary stress will inhibit the rat's performance on subsequent runs. Thus, we utilized a "T" junction block as the gate in the present study to eliminate any grinding noise from sliding plastic guillotine doors during test. This step can significantly reduce rats' anxiety during the T-maze experiment.
Habituation is a critical step for successful T-maze studies since anxious rats are not motivated to run in the maze. Thus, it is imperative to ensure that the rats acclimatize to human touch, height, and moving from the cage to the maze before the experiments. Anxious rats make squeaking or hissing sound when operators try to touch them. Additionally, anxious rats either present with long freezing responses or refuse to run in the maze during the entire T-maze experiments. They usually spend most of the time in the start arm without exploring the maze. If the rats do not run in the maze, gently touch their tail and they will run. The other possible reason is that the rats received insufficient habituation. Gently put the rat back in the cage and wait for 10 min to decrease their anxiety. If the rat fails to complete the run again, that rat is excluded from the study. If the rats cannot complete a single run in 3 min, put it back to its cage and wait for 10 min. If the rat still fails to complete the run, the rat must be excluded from the study. The rats should not perform the test for an extended duration or frequency or stay in the testing room for more than an hour, otherwise they will stop running due to lack of novelty.
Previous studies have shown that 70% of rats exhibit left or right bias38,39. This lateral bias worsens T-maze performance in the rat39. Thus, we specified the inclusion and exclusion criteria for rats in the present study. We have tested a total of 15 rats received ACA surgery in our T-maze study. The average alternation rate in ACA-treated rats is 29 ± 4 %. Since control or sham rats do not have any hippocampal injury or learning/memory deficits, we hypothesized that control or sham rats should have better T-maze performance (alternation rate ≥ 29 ± 4 %) as compared to ACA-treated animals. Therefore, control or sham rats with alternation rate < 29 % (the total number of alternation ≤ 1), which suggest the rats either have side preference or failed to learn in the maze should be excluded from the study. We have applied this exclusion criteria to 25 control rats. 6 of them (24%) performed with high side preference rate (85 ± 3%) and low spontaneous alternation rate (lower than 29%) were excluded from the studies.
The T-maze spontaneous alternation experiment is highly dependent on the rats' natural tendency to explore novelty. Thus, the major limitation of the T-maze spontaneous alternation protocol is that the rat will eventually cease to run in the maze. Based on our past experiences, the rat is only willing to run in the maze for three consecutive days. Since asphyxial cardiac arrest mainly results in short-term memory deficits, we can extrapolate our current findings to other brain injury/diseases (i.e. Alzheimer's disease, Parkinson's disease, and transient ischemic attack) that are also related to short-memory deficits.
The authors have nothing to disclose.
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.
3D Printer | MakerBot | Replicator | Fifth generation |
3D Printer Filament | Hatchbox | PLA, 1.75 mm filament diameter | |
200 Proof Pure Ethanol | Koptec | V1005SG | |
Sani-Chips | PJ Murphy-Forest Products | Size: 8 to 20 mesh; 2.2 cubic foot/package; autoclavable bags | |
Rat | Charles River Laboratories | Sprague-Dawley | |
Vecuronium bromide | Sun Pharmaceutical | 47335-931-40 | 10 mg |
Epinephrine | Par Pharmaceutical | 42023-103-01 | Adrenalin Chloride Solution 1 mg/mL, 1:1000 |
Buprenorphine Hydrochloride Injection | Pfizer | 00409-2012-32 | 0.3mg/mL |
SketchUp | Trimble Inc. | 3D modeling software | |
VentElite Small Animal Ventilator | Harvard Apparatus | 55-7040 | Animals raging in size from mouse to guinea pig (10g to 1kg) |
PowerLab 8/35 | Adinstruments | PL3508 | 8 analog input channels – 4 of which can be used in differential mode. |
Bio Amps | Adinstruments | FE132 | 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. |
Quad Bridge Amp | Adinstruments | FE224 | A four-channel, non-isolated bridge amplifier designed to allow the PowerLab to connect to most DC bridge transducers. |
LabChart 8 | Adinstruments | ||
ABL80 FLEX CO-OX blood gas analyzer | Radiometer | pH / p CO2 / p O2 | |
SURFLO Teflon I.V. Catheter | Terumo | sc-361556 | Only use the flexible thin wall catheter (49-mm long) |
Pipet/Infusion Needle | Hamilton | 7748-03 | 17-gauge; 93-mm long; 10-degree angle |
Classic T3 Vaporizer | SurgiVet | VCT302 | Classic T3 Isoflurane Funnel Fill |
ENVIRO-PURE Charcoal Canister | SurgiVet | 32373B10 | Designed to absorb waste anesthetic gas |
O2 single flowmeter | SurgiVet | 32375B1 | 0-1000 mL |
N2O Flowmeter | VetEquip | 401721 | 0-4LPM |
Clay Adams Intramedic Luer-Stub Adapter (Sterile) | Becton Dickinson | 427565 | 23 gauge |
Micro Forceps | Black and Black surgical | B3FRC-18 RM-8 | 7 1/4" (18 cm), 8mm RH, counterweight w/ guide pin 2mm, platform 6 x .3 mm, curved. |
Halstead Mosquito Forceps | Roboz | RS-7111 | Curved; 5" Length, 1.3 mm tip diameter, 2.1 mm jaw width |
Mixter Forceps | Roboz | RS-7291 | 5.25" Curved Extra Delicate, 1.1 mm tips |
Castroviejo Micro Dissecting Spring Scissors | Roboz | RS-5650 | Straight, Sharp Points; 9 mm Cutting Edge; 0.15 mm Tip Width; 3 1/2" Overall Length |
Mayo-Stille Scissors | Roboz | RS-6891 | 5.5" Round Curved |
Dumont #5 Forceps | Roboz | RS-5058 | 45 Deg Dumoxel Tip Size .10 x .06 mm |
Olsen-Hegar Combination Scissor And Needle Holder | Roboz | RS-7884 | Cross Serration Tip; 5.5" Length |
Moloney Forceps | Roboz | RS-8254 | Serrated; Slight Curve; 4.5" Length |