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* These authors contributed equally
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.
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.
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.
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
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.
3. Preparing for the metric task
4. Performing the metric task
NOTE: Performing the metric task consists of two periods: 1) habituation (15 min) and 2) test (5 min) period.
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.
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...
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...
The authors have nothing to disclose.
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.
Name | Company | Catalog Number | Comments |
2% chlorhexidine in 70% alcohol solution | SIGMA - ALDRICH | 500 cc | For 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 suture | 4-00 | ||
Bottles | Techniplast | ACBT0262SU | 150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution |
Bottlses (four) for topological an metric tasks | For 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 diameter | Glass Hole Saw Kit | Optional. | |
Digital Weighing Scale | SIGMA - ALDRICH | Rs 4,000 | |
Dissecting scissors | SIGMA - ALDRICH | Z265969 | |
Ethanol 99.9 % | Pharmacy | 5%-10% solution used to clean equipment and remove odors | |
EthoVision XT (Video software) | Noldus, Wageningen, Netherlands | Optional | |
Fluid-percussion device | custom-made at the university workshop | No specific brand is recommended. | |
Gauze Sponges | Fisher | 22-362-178 | |
Gloves (thin laboratory gloves) | Optional. | ||
Heater with thermometer | Heatingpad-1 | Model: HEATINGPAD-1/2 | No specific brand is recommended. |
Horizon-XL | Mennen Medical Ltd | ||
Isofluran, USP 100% | Piramamal Critical Care, Inc | NDC 66794-017 | Anesthetic liquid for inhalation |
Office 365 ProPlus | Microsoft | - | Microsoft Office Excel |
Olympus BX 40 microscope | Olympus | ||
Operating forceps | SIGMA - ALDRICH | ||
Operating Scissors | SIGMA - ALDRICH | ||
PC Computer for USV recording and data analyses | Intel | Intel® core i5-6500 CPU @ 3.2GHz, 16 GB RAM, 64-bit operating system | |
Plexiglass boxes linked by a narrow passage | Two transparent 30 cm × 20 cm × 20 cm plexiglass boxes linked by a narrow 15 cm × 15 cm × 60 cm passage | ||
Purina Chow | Purina | 5001 | Rodent 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) | Techniplast | 2000P | No specific brand is recommended |
Scalpel blades 11 | SIGMA - ALDRICH | S2771 | |
SPSS | SPSS Inc., Chicago, IL, USA | 20 package | |
Stereotaxic Instrument | custom-made at the university workshop | No specific brand is recommended | |
Timing device | Interval Timer:Timing for recording USV's | Optional. Any timer will do, although it is convenient to use an interval timer if you are tickling multiple rats | |
Topological and metric tasks device | Self made in Ben Gurion University of Negev | White circular platform 200 cm in diameter and 1 cm thick on table | |
Video camera | Logitech | C920 HD PRO WEBCAM | Digital video camera for high definition recording of rat behavior under plus maze test |
Windows 10 | Microsoft |
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