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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Abstract

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Introduction

The prevalence of neurological deficits such as attention, executive function, and certain memory deficits after moderate traumatic brain injury (TBI) is more than 50 percent1,2,3,4,5,6,7,8. TBI can lead to severe impairments in spatial short-term, long-term, and working memory9. These memory impairments have been observed in rodent models of TBI. Rodent models have enabled the development of techniques to test memory, allowing for deeper examinations into the effect of TBI on memory processing in neural memory systems.

Two tests, related to topological and metric spatial information processing respectively, assist with measuring spatial working memory (SWM). The topological test depends on changing the size of environmental space or related spaces of connection or enclosure around an object, while the metric test assesses changes in angles or distance between objects10,11. Goodrich-Hunsaker et al. first adapted the human topological test for rats10 and applied the metric task to dissociate the roles of the parietal cortex (PC) and dorsal hippocampus in spatial information processing11. Similarly, Gurkoff and colleagues evaluated metric, topological, and temporal ordering memory tasks after lateral fluid percussion injury9. There is a correlation between damage to certain regions of the brain and impairment of metric or topological memory. It has been suggested that metric memory impairment is related to lesions in bilateral dorsal dentate gyrus and cornu ammonis (CA) sub-region CA3 of the hippocampus, and that topological memory impairment is related to bilateral parietal cortex lesions10,12.

The purpose of this protocol is to assess spatial memory deficit in a rat population via a metric task. This method is a suitable alternative to investigate mechanisms of SWM after brain injury, and its advantages include the relative ease of implementation, high sensitivity, low cost of reproducibility, the possibility of dynamic observation, and a low stress environment. Compared to other behavioral tasks such as the Barnes maze13,14, Morris water navigation task15,16,17, or spatial maze tasks18,19, this metric test is less complicated. Due to its ease of implementation, the metric test requires a shorter and less stressful training period and takes place over only 2 days9: 1 day for habituation and 1 day for the task. Moreover, our proposed test is easier to perform than other low stress tests, such as the novel object recognition (NOR) task, and does not require the extra day of habituation20.

This paper provides a straightforward model for evaluating SWM after brain injury. This assessment of post-TBI SWM may assist in a more comprehensive investigation of its pathophysiology.

Protocol

The experiments were performed following the recommendations of the Declarations of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev. A protocol timeline is illustrated in Figure 1.

1. Surgical procedures and fluid percussion TBI

  1. Select male and female adult Sprague-Dawley rats, housed at a room temperature of 22 ± 1 °C, and humidity of 40%-60%, with 12-12 h light-dark cycles.
  2. Provide food as chow and water ad libitum. Perform experiments between morning hours, i.e., 6:00 a.m. and 12:00 p.m.
  3. Perform a baseline neurological assessment for both the control and TBI groups prior to the start of the experiment (see section 2 below).
  4. Anesthetize the rats with inhaled 4% isoflurane for induction and 1.5% for maintenance of anesthesia. Ensure that rat is immobilized by testing pedal reflex or movement in response to an irritant.
    ​NOTE: Use a continuous isoflurane administration system for anesthesia. Perform all procedures in aseptic conditions.
  5. Perform parasagittal fluid-percussion injury as previously described21,22.
  6. Subcutaneously inject 0.2 mL of 0.5% bupivacaine along the prospective incision site, prior to incision. ransfer the rat to the recovery room and continue monitoring the neurological (e.g., paralysis), respiratory (e.g., respiratory arrest) and cardiovascular state (e.g., decreases in soft tissue perfusion, changes in color of pupils, and bradycardia) for 24 h. Prior to emergence from anesthesia, administer 0.01 - 0.05 mg/kg intramuscular buprenorphine as postoperative analgesia. Repeat doses every 6 - 12 h for at least 48 h.

2. Evaluation of Neurological Severity Score (NSS)

NOTE: Assessment of neurological deficit was performed and graded using an NSS, as previously described23,24. The maximum score of alteration in motor function and behavior is 24 points. A score of 0 indicates an intact neurological status and 24 indicates severe neurological dysfunction, as previously described24.

  1. Test the rat's inability to leave a circle (50 cm in diameter) when placed in its center. Perform this task three times, with each session lasting 30 min, 60 min, and more than 60 min each.
    NOTE: If picking up rats by the tail, hold the base of the tail.
  2. Test the rat for a loss of righting reflex.
    1. Place the animal on its back in the palm of the researcher's hand. Give a score of 1 if the animal is able to right itself25 (standing on all four paws).
  3. Test the rat for hemiplegia, the inability of the rat to resist forced positioning.
  4. Raise the rat by its tail to test the reflexive bending of the hindlimb.
  5. Put the rat on the floor to test its ability to walk straight.
  6. Perform testing for three reflexive behaviors: the pinna reflex, the corneal reflex, and the startle reflex.
    1. For the pinna reflex, perform light tactile stimulation to test ear retraction as previously described25.
    2. To test the corneal reflex, monitor blink response when applying a soft stick lightly to the eye and measure on a scale of 0 (no response) to triple eye blink (3), as previously described25.
    3. For the startle reflex, drag a pen across the top of the wire cage and record response with a scale from 0 (no response) to 3 (1 cm jump or more), as previously described25.
  7. Grade the rat based on loss of seeking behavior and prostration (not moving their whiskers, sniffing, or running after being transferred to a new environment)24.
  8. Test limb reflexes for the placement on the left and right forelimbs, and then the left and right hindlimbs.
  9. Analyze functionality via the beam balancing task with a beam that is 1.5 cm wide. Perform the test for sessions lasting 20 seconds, 40 seconds, and more than 60 seconds.
  10. Run the beam walking test with three different beams: 8.5 cm wide, 5 cm wide, and 2.5 cm wide.

3. Preparing for the metric task

  1. Equipment
    1. Place a black circular platform 200 cm in diameter and 1 cm thick on a table. The height of the table should be 80 cm above the floor.
    2. Establish two different objects in the center of circular platform 68 cm away from each other.
      NOTE: In this experiment, two glass bottles were used for objects, one round bottle with a height of 13.5 cm and another faceted bottle with a height of 20 cm. Fill bottles with water to ensure stability.
    3. Prepare a camera and install the required computer software for capturing, saving, and processing data. Install the camera at a height of 290 cm from the floor.
      NOTE: The distance between the platform and camera depends on the camera specifications. The camera frame should cover the entire area of the arena in which the test is being conducted. The distance for our experiment between the platform and the camera was 210 cm.
  2. Habituation
    1. On the day before the task, habituate the rat to the new environment for 10 min by placing on the arena without video recording.
      NOTE: Do not perform the neurological tasks and the metric task on the same day. 
      NOTE: Perform metric tests in a red light area.

4. Performing the metric task

NOTE: Performing the metric task consists of two periods: 1) habituation (15 min) and 2) test (5 min) period.

  1. Habituation period
    1. Establish two different objects in the center of the circular platform 68 cm away from each other.
    2. Place the rat on the end of the platform equidistant from the objects for a 15 min period, and record the video.
    3. Remove the rat from the platform and place in an individual cage for 5 min.
    4. Clean the platform with 5%-10% alcohol.
      ​NOTE: Up to 70% alcohol may be used to clean the platform in well-ventilated areas.
  2. Test period
    1. Reduce the distance between objects to 34 cm.
    2. Place the rat on the platform for 5 min and record the rat's exploration activity on video.
    3. Clean the platform with 5%-10% alcohol.

5. Data analysis

NOTE: Data analysis is performed by video tracking software specifically designed for animal behavior studies that automatically records animal activity and movement (see Table of Materials). This software automates a range of behavioral variables, including mobility, activity, and explorative behavior.

  1. Prior to analyzing the video files, insert the software hardware key. Start the video tracking software and open preset Template.
  2. In the Setup section, verify settings as follows: Arena, Trial Control, and Detecting Settings (see Figure 2a) .
    NOTE: For this experiment, parameters for the exploration area are defined as 6 cm around the object of interest. The time the rat entered into this area was measured.
  3. After verifying the settings, duplicate and rename them.
  4. On the general screen of the program, Grab Background by right clicking on the mouse.
  5. Select a video file for the background image. In the Browse menu, select the location of the video file.
  6. Capture the image and mark the investigated areas and zones, calibrating the image for analysis. Perform the same steps for Trial Control and Detecting Settings.
  7. In the general menu, select Trial List and download the list of video files for analysis.
  8. Add the videos and indicate the location with the required settings.
  9. Select acquisition and Start Trial (see Figure 2b,c). Export all data as Excel files (see Figure 2d).
    NOTE: Perform all calculations for the habituation and test periods. Metric task assessment is prepared with an advanced template.

Results

The significance of comparisons between groups was determined using the Mann-Whitney test. Statistical significance of results was considered at P < 0.05, while statistically high relevance was measured at P < 0.01.

The results showed no differences in NSS between all groups before intervention and 28 days after TBI. Each group consisted of 12 female or 12 male rats. The NSS scores obtained 48 h after TBI are presented in Table 1. Rats from the TBI group that showed si...

Discussion

By specifically targeting the metric spatial information process, this metric test provides a necessary tool toward understanding memory deficiency after TBI. The protocol presented in this paper is a modification of previously described behavioral tasks11. One previously described metric task used two different paradigms, each consisting of three habituation sessions and one testing session. The first paradigm consisted of moving the familiar objects closer together after habituation and the seco...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Professor Olena Severynovska; Maryna Kuscheriava M.Sc; Maksym Kryvonosov M.Sc; Daryna Yakumenko M.Sc; Evgenia Goncharyk M.Sc; and Olha Shapoval, PhD candidate at the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro University, Dnipro, Ukraine for their supportive and useful contributions. The data was obtained as part of Dmitry Frank's PhD dissertation.

Materials

NameCompanyCatalog NumberComments
2% chlorhexidine in 70% alcohol solutionSIGMA - ALDRICH500 ccFor general antisepsis of the skin in the operatory field
 Bupivacaine 0.1 %
4 boards of different thicknesses (1.5cm, 2.5cm, 5cm and 8.5cm)This is to evaluate neurological defect
4-0 Nylon suture4-00
BottlesTechniplastACBT0262SU150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution
Bottlses (four) for topological an metric tasksFor objects used two little bottles, first round (height 13.5 cm) and second faceted (height 20 cm) shape and two big faceted bottles, first 9x6 cm (height 21 cm) and second 7x7 cm (height 21 cm).
Diamond Hole Saw Drill 3mm diameterGlass Hole Saw KitOptional. 
Digital Weighing ScaleSIGMA - ALDRICHRs 4,000
Dissecting scissorsSIGMA - ALDRICHZ265969
Ethanol 99.9 % Pharmacy5%-10% solution used to clean equipment and remove odors
EthoVision XT (Video software)Noldus, Wageningen, NetherlandsOptional
Fluid-percussion devicecustom-made at the university workshop   No specific brand is recommended.
Gauze SpongesFisher22-362-178
Gloves (thin laboratory gloves)Optional.
Heater with thermometerHeatingpad-1Model: HEATINGPAD-1/2   No specific brand is recommended.
Horizon-XLMennen Medical Ltd
Isofluran, USP 100%Piramamal Critical Care, IncNDC 66794-017Anesthetic liquid for inhalation
Office 365 ProPlusMicrosoft-Microsoft Office Excel
Olympus BX 40 microscopeOlympus
Operating  forcepsSIGMA - ALDRICH
Operating  ScissorsSIGMA - ALDRICH
PC Computer for USV recording and data analysesIntelIntel® core i5-6500 CPU @ 3.2GHz, 16 GB RAM, 64-bit operating system
Plexiglass boxes linked by a narrow passageTwo transparent 30 cm × 20 cm × 20 cm plexiglass boxes linked by a narrow 15 cm × 15 cm × 60 cm passage
Purina ChowPurina5001Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical researc for over 5
Rat cages  (rat home cage or another enclosure)Techniplast2000PNo specific brand is recommended
Scalpel blades 11SIGMA - ALDRICHS2771
SPSSSPSS Inc., Chicago, IL, USA 20 package
Stereotaxic Instrumentcustom-made at the university workshop   No specific brand is recommended
Timing deviceInterval Timer:Timing for recording USV'sOptional. Any timer will do, although it is convenient to use an interval timer if you are tickling multiple rats
Topological and metric tasks deviceSelf made in Ben Gurion University of NegevWhite circular platform 200 cm in diameter and 1 cm thick on table
Video cameraLogitechC920 HD PRO WEBCAMDigital video camera for high definition recording of rat behavior under plus maze test
Windows 10Microsoft

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