Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury

Summary

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 primates^9,10,11, 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 model^13,16, 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 device^18,19.

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

1. Select at random 30 adult male rats, and divide them into two groups: TBI and sham.
2. 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.
3. Perform baseline assessments of the neurological severity score before injury in both groups of rats, as detailed in step 3 and Table 1.
4. 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.
5. 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 reports^18,20.

1. Infiltrate the scalp with 0.5% bupivacaine (see the Table of Materials), perform a 10 mm incision, and retract the tissues laterally.
2. Perform craniotomy^18,20 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. 
3. Induce TBI^18,19 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.
4. Perform craniotomy on the group of sham-operated rats (Figure 1). Do not induce TBI for the sham-operated group.
5. 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.
6. 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 dysfunction^21,22,23 (Table 1).

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

1. Randomly divide the rats into cages 1 week before the test.
   NOTE: Each cage should contain one sham-operated rat and one TBI rat.
2. 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).
3. 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 tunnel^15,19,25.
4. 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.
5. Place the apparatus on a table with a height of 80 cm above the floor.
6. Place each rat in the apparatus for 15 min for habituation on the first 2 days. Start the task after 2 habituation days.
7. 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.
8. 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.
9. 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

1. 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.
2. 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.
3. Perform data analysis²³ manually by two analysts blinded to the groups.

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 system^22,23; a score of 24 indicated a severe neurological dysfunction, and a score of 0 represented intact neurologi...

Discussion

Clinical studies indicate that brain injury may increase the risk of psychiatric disorders^26,27. Moreover, TBI affects the development of social behavior^28,29. 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 ...

Materials

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
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