Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury
Özet
The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.
Abstract
Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social hierarchies that are often based on dominant-submissive relationships. The dominant-submissive relationship is a normal behavioral pattern among the individuals of a species. Traumatic brain injury is a frequent cause of social interaction impairment and the reorganization of dominant-submissive relationships in animal pairs. This protocol describes submissive behavior in adult male Sprague-Dawley rats after the induction of traumatic brain injury using a fluid-percussion model compared to naive rats through a series of dominant-submissive tests performed between 29 days and 33 days after induction. The dominant-submissive behavior test shows how brain injury can induce submissive behavior in animals competing for food. After traumatic brain injury, the rodents were more submissive, as indicated by them spending less time at the feeder and being less likely to arrive first at the trough compared to the control animals. According to this protocol, submissive behavior develops after traumatic brain injury in adult male rats.
Introduction
Intraspecies competition occurs when members of the same species compete for a limited resource at the same time1. In contrast, interspecies competition occurs between members of two different species2. Intraspecies competition is divided into two types, including interference (adapted) and exploitation (contest), and arises depending on the type of resource in contention, such as food and territory3.
The existence of social hierarchies is impossible without dominant-submissive relationships (DSRs). Dominance presents as "winning" and subordination as "losing" within pairs of animals4. However, DSRs appear not only in pairs but also in groups of three or more. In 1922, Thorleif Schjelderup-Ebbe described the dominance hierarchy in domestic chickens. The principal distinguishing signs between the dominant and subordinate animals were time spent at the feeder and aggressive behavior. The dominance hierarchy is divided into two forms: linear and nonlinear5. Linear dominance involves two groups, A and B. In this paradigm of transitive relationships6, group A dominates group B, or group B dominates group A. Nonlinear dominance occurs when there is at least one circular relationship: A dominates B, B dominates C, and C dominates A7.
Models for assessing dominant-submissive behavior exist for different species, including rodents, birds8, non-human primates9,10,11, and humans12. The dominant-submissive method is well represented in the literature and has been applied as a model to assess mania and depression13, as well as antidepressant drug activity14. This model has been used to investigate early life stress after maternal separation in adult rats15. The DSR paradigms can be divided into three models: the reduction of dominant behavior model13,16, the reduction of submissive behavior model14, and the clonidine-reversal of dominance model17.
This study demonstrates an investigation of DSR through tasks based on food competition. The advantages of this method are its easy reproducibility and the ability to observe and accurately analyze dominant-submissive behavior. In addition, the dominant-submissive behavioral task relies on food rather than territory, unlike comparable behavioral tasks, which makes this behavioral task lower cost and simpler and researchers do not need to undergo complicated training to perform the task and process the data.
The overall goal of the current study is to demonstrate the development of DSR after traumatic brain injury (TBI). TBI is associated with social impairments, depression, and anxiety. The model of inducing TBI is a simple and effective standard model that involves inducing traumatic brain injury with a fluid percussion device18,19.
Protocol
The experiments were approved by the Animal Care Committee of the Ben-Gurion University of the Negev.The experiments were performed following the recommendations of the Declarations of Helsinki and Tokyo and the Guidelines for the Care and Use of Laboratory Animals of the European Community. Adult male Sprague-Dawley rats, weighing 300-350 g, were used in the present study. The animals were housed at a room temperature of 22 °C ± 1 °C and a humidity of 40%-60% with light-dark cycles.
1. Animal preparation
- Select at random 30 adult male rats, and divide them into two groups: TBI and sham.
- Provide chow (see the Table of Materials) and water ad libitum.
NOTE: Perform all the steps of the test at the same time to control for the effect of time of day on behavioral performance. It is best to conduct the behavior tests in the morning (between 6:00 a.m. and 12:00 p.m.) to avoid disturbance from general activity. - Perform baseline assessments of the neurological severity score before injury in both groups of rats, as detailed in step 3 and Table 1.
- Anesthetize the rats with 4% (for induction) and 1.5% (for maintenance) isoflurane. Inject Buprenorphine (0.05-0.1 mg/kg; SC) for preemptive analgesia.
- Check for the immobilization of the rat by testing for a lack of movement or pedal reflex in response to a stimulant.
NOTE: For anesthesia administration, a continuous flow of isoflurane is recommended.
2. Surgical procedure
NOTE: All procedures are to be performed in aseptic conditions. Use sterile gloves. Change the gloves if any non-sterile surface is touched. Apply ophthalmic lubricant to both eyes to prevent drying. The parasagittal fluid-percussion injury was performed following previously published reports18,20.
- Infiltrate the scalp with 0.5% bupivacaine (see the Table of Materials), perform a 10 mm incision, and retract the tissues laterally.
- Perform craniotomy18,20 4 mm posterior and 4 mm lateral of bregma.
NOTE: The surgical area must be disinfected several times in a circular motion with both an iodine-based or chlorhexidine-based scrub and alcohol. - Induce TBI18,19 by a fluid-percussion device (see the Table of Materials) over 21-23 ms through the three-way stopcock.
NOTE: Perform moderate TBI with an amplitude of 2.5 atm. - Perform craniotomy on the group of sham-operated rats (Figure 1). Do not induce TBI for the sham-operated group.
- Perform 0.1% bupivacaine infiltration before closing the wound. Administer intramuscular buprenorphine (0.01-0.05 mg/kg) as postoperative analgesia before withdrawing the isoflurane.
NOTE: Repeat doses of buprenorphine every 12 h for at least 48 h. - Transfer the rat to the recovery room, and monitor its respiratory (e.g., respiratory arrest), neurological (e.g., paralysis), and cardiovascular state (e.g., changes in the color of pupils, decreases in soft tissue perfusion, and bradycardia) for 24 h.
3. Neurological severity score evaluation
NOTE: The highest possible score for behavioral alterations and motor function is 24 points. A score of 0 represents intact neurological status, and a score of 24 represents severe neurological dysfunction21,22,23 (Table 1).
- Evaluate the neurological severity score (NSS) as previously described24 on the TBI and sham rats before surgery, at 48 h after surgery (Figure 2A), and on day 28 after surgery (Figure 2B).
4. Studying the dominant- submissive behavior
- Randomly divide the rats into cages 1 week before the test.
NOTE: Each cage should contain one sham-operated rat and one TBI rat. - Perform one 15 min session every day for 2 days before testing so that rats can acclimate to the protocol.
NOTE: The dominant-submissive task was initiated on day 29 after injury (Figure 1). - Use an apparatus (see the Table of Materials) made from two transparent acrylic glass boxes (30 cm x 20 cm x 20 cm, Box A and Box B, Figure 3) connected by a slender 15 cm x 15 cm x 60 cm tunnel15,19,25.
- Fill a feeder (Figure 3) with sweetened milk, and place it at the tunnel's center. Use milk consisting of 10% sugar and 3% fat.
- Place the apparatus on a table with a height of 80 cm above the floor.
- Place each rat in the apparatus for 15 min for habituation on the first 2 days. Start the task after 2 habituation days.
- Randomly select one rat from the control group and one from the traumatic brain injury (TBI) group, and set them at equal distances from the feeder, allowing them to explore for 5 min.
- Allow the rats access to water ad libitum.
NOTE: The task lasted for 5 days. Food restriction was performed for the entire task period. The food was given every day for 1 h after the test period. - Clean the equipment with 5% alcohol before performing the subsequent tests with other rats.
NOTE: Cleaning the apparatus will eliminate the smell of the previous rats. Perform the test in a room with proper air circulation.
5. Recording the video and data analysis
- Position a camera, and install the recommended computer software (see the Table of Materials) to capture, save, and process the data.
NOTE: The camera needs to be installed at a height of 290 cm from the floor. - Record the video while the rats are in the arena.
NOTE: The camera and the apparatus were positioned 210 cm apart. The part of the arena where the test is conducted must be visible in the camera frame. - Perform data analysis23 manually by two analysts blinded to the groups.
Representative Results
Neurological severity score assessment
Neurological deficits were assessed in male rats after TBI using the NSS. The rats were divided into two groups: one TBI group and one control group. The control group was subjected to sham surgery. The NSS allowed for the assessment of motor function and behavior alteration by a points system22,23; a score of 24 indicated a severe neurological dysfunction, and a score of 0 represented intact neurological status. There were no significant differences in neurological deficits at 1 h before surgery between the TBI and sham-operated groups. The neurological deficits at 48 h after surgery were sufficiently greater for the TBI rats compared to the sham-operated rats (5-7, average: 6 vs. 0-0, average: 0; U = 0, p < 0.01, r = 0.89) (Figure 2A). At 28 days after surgery, the differences between the TBI and sham-operated groups were insignificant (Mann-Whitney U test19) (Figure 2B).
The dominant-submissive behavior assessment
The dominant-submissive behavior of male adult rats was assessed 30 days after surgery. This was done after the NSS assessment to ensure that there was no locomotor dysfunction. The dominant-submissive task was based on food competition and was assessed in terms of two main parameters: time spent on the feeder and who came first to the feeder. Time spent at the feeder was significantly lower for the TBI rats compared to the sham-operated rats (33.1 s ± 8.7 s vs. 55.9 s ± 21 s, t(28) = 3.14, p < 0.01, d = 1.15) (Figure 4A). Fewer TBI than sham-operated rats came first to the feeder (3 out of 15 vs. 12 out of 15, p < 0.01, according to a chi-square test and Fisher's exact test19) (Figure 4B).
Figure 1: Demonstration of the protocol timeline. The rats were divided into two groups: sham-operated and TBI. The TBI and craniotomy were performed when the rats reached 3 months old. The NSS scores were measured for the TBI and sham rats before the start of the experiment, at 48 h after surgery, and on day 28 after surgery. The assessment of dominant-submissive behavior was performed between day 29 and day 33 (for a total of 5 days) following the surgery. Please click here to view a larger version of this figure.
Figure 2: Neurological severity score assessment. Assessment of the neurological severity score at (A) 48 h and (B) 28 days following surgery, comparing the TBI group to the control group. P < 0.01 for (A), determined by a Mann-Whitney U test. Please click here to view a larger version of this figure.
Figure 3: Apparatus for the DSR behavior assessment. An apparatus made from two transparent acrylic glass boxes (30 cm x 20 cm x 20 cm, Box A and Box B) connected by a slender 15 cm x 15 cm x 60 cm tunnel, with a feeder in the center of the tunnel. Please click here to view a larger version of this figure.
Figure 4: The dominant-submissive behavior assessment. The assessment of dominant-submissive behavior was performed on day 33 following the surgery, comparing the TBI rats to the sham-operated control rats. Time spent on (A) the feeder and (B) the rat that came first at the feeder are shown. P < 0.01 for (A), determined by a t-test. P < 0.01 for (B), determined by the chi-square test and Fisher's exact test. Please click here to view a larger version of this figure.
Table 1: Scoring and grading system for the neurological severity score assessment. Please click here to download this Table.
Discussion
Clinical studies indicate that brain injury may increase the risk of psychiatric disorders26,27. Moreover, TBI affects the development of social behavior28,29. In this protocol, the TBI model had an effect on the presentation of dominant-submissive behavior. Dominant-submissive behavior manifested itself in terms of time spent on the feeder and who came first to the feeder.
In addition to the behavioral task performed here, other tasks for the assessment of dominant-submissive relations exist, such as the resident-intruder paradigm30,31 or the complex diving for food situation32,33,34. Each of these tasks targets a different aspect of social behavior. The resident-intruder paradigm is appropriate for measuring offensive aggression, defensive behavior, and social stress, and the complex diving for food situation is more appropriate for studying social hierarchies. The dominant-submissive task is the most suitable for assessing DSR.
The dimensions of the apparatus depend on the size of the rodents. The apparatus must have two Plexiglas chambers and one tunnel connecting them. In the center is a feeder with sweetened milk. For rats35, the dimensions of the chambers and tunnel are 24 cm x 17 cm x 14 cm and 4.5 cm x 4.5 cm x 52 cm, respectively. For the assessment of DSR after early life stress32, the dimensions of the apparatus are 30 cm x 20 cm x 20 cm for the chambers and 15 cm x 15 cm x 60 cm for the tunnel. The dimensions of the apparatus for mice36 are 12 cm x 8.5 cm x 7 cm and 2.5 cm x 2.5 cm x 27 cm for the chambers and tunnel, respectively.
This protocol has some critical steps. For the dominant-submissive task, it is necessary to clean the equipment after each subsequent trial with the alcohol solution. At the same time, the arena's surface must be dry and clean because any residual smell from previous animals can impact the behavior of the experimental animals. Constant ventilation and the absence of noise are necessary conditions in the room to avoid unnecessary stress factors that can influence behavioral patterns. The milk in the feeder should be replaced after each behavioral session. The behavioral tests are to be performed during the dark phase, and filming using a camera with high-resolution quality will enable images to be captured in the dark.
The limitations of this study include the small sizes of the groups, the assessment of locomotor activity only by the NSS, and not including weight in the data. Future studies could also incorporate locomotor function assessment by open field and/or elevated plus maze tests.
The neurological deficits at 48 h after the surgery were remarkably greater for the TBI rats than for the sham-operated rats. At 48 h after the injury, there were significant neurological deficits, indicating significant damage. When a neurological assessment was carried out on the rats on day 28 after injury, there were no significant differences between the sham rats and TBI rats; therefore, the submissive behavior of the injured group was not due to impaired neurological status. The locomotor activity was not impacted and did not impact the dominant-submissive behavior. Time spent at the feeder was significantly shorter for the TBI rats compared to the sham-operated rats. Fewer TBI rats than sham-operated rats came first to the feeder (Figure 4A). The principal findings of the present study indicated submissive behavior in rats after TBI and dominant behavior in the sham-operated rats. The TBI rats demonstrated submissive behavior on two parameters: time spent on the feeder and who came first to the feeder.
In summary, the main finding of this study was that TBI in adult rats leads to submissive behavior after 1 month. It is anticipated that this research will expand our ability to understand and assess social behavior after TBI. Future studies are expected to investigate the trait of submissive behavior as a predictor of the presence of previous brain injury.
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
The work done are part of Dmitry Frank's PhD thesis.
Materials
| 2% chlorhexidine in 70% alcohol solution | SIGMA – ALDRICH | 500 cc | For general antisepsis of the skin in the operatory field |
| 4 boards of different thicknesses (1.5 cm, 2.5 cm, 5 cm and 8.5 cm) | This is to evaluate neurological defect | ||
| 4-0 Nylon suture | 4-00 | ||
| Bottles | Techniplast | ACBT0262SU | |
| Bupivacaine 0.1 % | |||
| Diamond Hole Saw Drill 3 mm 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 | |
| Fluid-percussion device | custom-made at the university workshop | No specific brand is recommended. | |
| Gauze Sponges | Fisher | ||
| 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 |
| Logitech Webcam Software | Logitech | 2.51 | Software for video camera |
| 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, is a lifecycle nutrition that has been used in biomedical research |
| 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 | A 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 | |
| Video camera | Logitech | C920 HD PRO WEBCAM | Digital video camera for high definition recording of rat behavior under dominant submissive test |
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