Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Summary

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Abstract

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Introduction

Mild traumatic brain injury (mTBI), or concussion, is the most prevalent type of injury and can lead to various short-term and chronic symptoms¹. These symptoms may include dizziness, headaches, depression, and anhedonia, among others, leading to significant suffering for individuals affected by mTBI². Since most mTBIs are caused by blunt force trauma³, it becomes imperative to develop animal models that accurately mimic such injuries. These models are essential for gaining a better understanding of the injury and its underlying mechanisms, offering a controlled environment with reduced variability and heterogeneity compared to human studies.

Numerous well-established rodent models have been developed for traumatic brain injury (TBI), including fluid percussion injury (FPI)⁴, controlled cortical impact (CCI)⁵, weight-drop injury⁶, blast traumatic brain injury⁷, and others. However, these models primarily focus on replicating moderate to severe TBI scenarios. In contrast, the experimental models specifically designed to simulate mTBI have received relatively less attention and remain underexplored⁸. Therefore, there is a critical need to establish a stable and reproducible animal model that accurately represents mTBI. Such a model would significantly enhance our understanding of the neurobiological and behavioral consequences associated with mTBI.

One cannot distinguish the functional deficits in mTBI rats compared to normal rats via casual observation after the effects of anesthesia have worn off. Therefore, it is necessary to administer specific tests. In humans, a wide range of clinical assessments are used to evaluate patients^9,10,11. Similarly, establishing a successful model in the rat model also requires using rapid assessment tools to determine its validity.

In this study, we present a closed-head mTBI rat model, enabling the investigation of mTBI in a manner that closely resembles the human condition. The detailed description of the model and its validation procedure provides a comprehensive understanding of the experimental approach utilized in studying mTBI.

Protocol

Animal experiments were approved by the Central South University Animal Care and Use Committee. All studies were conducted in line with the welfare and ethical principles of laboratory animals.

1. Animal feeding and anesthetization procedure

1. Group house 280-320 g Sprague-Dawley male rats and maintain them on a 12 h/12 h light/dark cycle with access to food and water ad libitum. Perform the study after the rats acclimatize for 6 days.
2. Anesthetize the rat with 3% isoflurane at 0.6 L/min air flow in an induction box until it is non-responsive to paw or tail pinch. Maintain the flow rate for 30 s.
   NOTE: Pain medications were not used as they would interfere with the rat's response in the neurobehavioral assessments.

2. Preoperative setup

1. Position a sponge with a hardness value of 35D (weight of 35 kg/m3 of sponge), with identical length and width but a thickness of 12 cm, within an acrylic box (15 cm x 22 cm x 43 cm) lacking a top cover.
2. Trim a tin foil (a thickness of 20 µm) and affix it onto the acrylic box using adhesive tape to form a destructible surface capable of supporting the weight of a rat. Additionally, mark a cut line measuring approximately 10 cm to be the designated location for positioning the rat's head.
3. With the aid of an iron stand, firmly secure the PVC tube in place. Prepare a perforated weight, weighing 550 grams with an 18-millimeter diameter. Attach the weight to a fishing line at a height of 1 meter inside a polyvinyl chloride or PVC tube. and adjust the position of the guide tube 3 centimeters above the tin foil.
4. Prepare a helmet and a pillow. Make a helmet using a stainless-steel disk measuring 10 mm in diameter and 3 mm in thickness. Prepare a wedge-shaped sponge pillow to place beneath the rat's head, ensuring that it is perpendicular to the direction of gravity.
   NOTE: A schematic diagram of the impact apparatus is presented in Figure 1. The helmet serves the purpose of identifying the impact location and enhancing the distribution of external force. The pillow is utilized to guarantee uniform and stable damage.

3. mTBI induction

1. Quickly place the anesthetized rat on its chest on the tin foil.
   NOTE: Two operators are required for mTBI induction-one for the preparation and the other for verification.
2. Preparation: Place the pillow beneath the rat, ensuring its head is parallel to the foil paper. Align the helmet with the rat's ears and secure it in place.
3. Verification: Verify that the PVC pipe is positioned directly above the helmet. Once both operators confirm the correct setup, proceed to the next step.
4. Induction of head rotation: Release the weight, allowing it to fall and strike the rat's head, inducing a fall onto the sponge and a 180° rotation.
5. Place the rat on its back in a clean cage.

4. Sham induction

1. Treat the rat the same way as the previous mTBI induction description, but do not subject it to the head impact.

5. Validation procedure: Acute neurobehavioral assessments

NOTE: The following assessments were modified based on the Neurological Severity Scores⁹ and the protocol by Flierl et al.¹⁰. All these assessments were performed 10 min after the rat recovered the righting reflex.

1. Time of loss of consciousness: Record the duration from when the rat is anesthetized to when it recovers the righting reflex.
   NOTE: The righting reflex is the process in which the rat turns over when placed on its back. Loss of righting reflex is to be considered as a humane endpoint, and the animal must be euthanized as per institutional guidelines.
2. First seeking-behavior time: Record the duration from when the rat is anesthetized to when it shows the seeking behavior for the first time.
   NOTE: Seeking behavior is a sign of interest in the environment, a physiological response.
3. Ability to escape
   1. Place the rat in the middle of a circular apparatus (0.5 m diameter and 0.3 m height) with an exit (12.5 cm long and 9 cm wide).
   2. Record the time the rat takes to exit the circle.
      NOTE: If the rat does not exit the circle within 180 s, record the time as 180 s.
4. Beam balance ability test
   1. Place the rat on a 3 cm, 2 cm, and 1.5 cm wide beam for 1 min accordingly.
   2. If the rat maintains a balance with a steady posture on the beam, score it as 0.
   3. If the rat grasps the side of the beam, give a score of 1. If the rat hugs the beam and one limb falls off it, score it as 2.
   4. If the rat hugs the beam and the two limbs fall off it or spin on it (>60 s), score it as 3.
   5. If the rat attempts to balance on the beam but falls off (> 40 s), score it as 4.
   6. If the rat attempts to balance on the beam but falls off (>20 s), score it as 5.
   7. If the rat does not attempt to balance or hang on the beam and falls off within 20 s, score it as 6.
      NOTE: The beam balance test does not require a pre-trial.

6. Validation procedure: Neurobehavior assessment

NOTE: Prior to the behavioral experiments, the rats were handled for 2 min daily for 3 consecutive days to minimize stress and novelty disruption. All behavioral experiments were performed by placing the animals in the test environment for 60 min prior to the start of the experiment.

1. Motor coordination ability (Beam task)
   1. Experimental setup
      1. Place the rats on one end of the balance beam (1.5 m long and 75 cm above the floor). Place an escape box (a slanted bedding home cage) on the other end.
      2. Position a foam padding below the beam to mitigate the potential risk of injury to rats in case of falls during the test.
      3. Turn on the video camera.
      4. Schedule testing days at specific time points post-injury or post-sham treatment (e.g., day 1, day 3, and day 7).
   2. Training phase (2 days)
      1. Train the rats to cross the 4 cm wide beam 3 consecutive times, followed by two trials on the 2 cm wide beam.
      2. During the training, gently guide the rats across the beam until they can cross them easily without interference.
   3. Balance beam experiment
      1. Place the rats on the 2 cm wide beam for 5 consecutive trials.
      2. Record the start and end of each trial when the rat's nose crosses the starting and finishing lines, respectively.
      3. Return the rats to their cages at the end of the experiment.
   4. Baseline testing
      1. Conduct the balance beam experiment before the injury or treatment.
      2. Calculate the average values from these 5 consecutive trials to establish the baseline for each rat.
   5. Data analysis
      1. Analyze the time to cross the beam and the total number of hindfoot slips using video analysis by researchers blinded to the experimental conditions.
2. Anxiety (Open field test)
   1. Experimental setup
      1. Prepare the open field arena, ensuring it is clean and free from any previous odor cues. Divide the arena into three zones: a central inner zone (33 cm x 33 cm), a middle zone (66 cm x 66 cm), and an outer zone.
   2. Testing phase
      1. Place a rat in the center of the open field arena and start the timer. Allow the rat to explore the arena for 5 min freely. After 5 min, carefully and gently return the rat to its home cage.
   3. Data collection
      1. Measure the total distance traveled by the rat during the 5-minute exploration period. Determine the time the rat spends in the central inner, middle, and outer zones.
   4. Data analysis
      1. Use the total distance traveled as a measure of overall exploratory behavior and locomotor ability. Calculate the time spent in the central inner zone as an indicator of anxiety-like responses.
3. Learning and memory abilities (Morris water maze test)
   1. Ensure the water maze apparatus is in proper condition. Dye the water black and place cues in the four cardinal directions. Position the platform 2.5 cm below the water surface.
   2. Set up a monitoring system to record and observe the behavior of the rats.
   3. Trail day
      1. Quickly place the rat into the water maze. If the rat fails to reach the platform within 2 min, gently guide it using the wooden stick.
      2. Allow the rat to familiarize itself with the maze environment while standing on the platform for 20 s, then remove it. Once the rat is on the platform, let it stay for 20 s, then remove it.
   4. Daily repetition
      1. Repeat the training day procedure, placing the rat in the water from different quadrants. Repeat step 6.3.3. Continue the training for 5 consecutive days.
   5. Probe test day: On the 6^th day, remove the platform and place the rat in the same quadrant for 2 min.
   6. Observation and recording: Utilize the monitoring system to monitor the rat's behavior on trial and probe test days.
   7. Cleaning: After removing the rat from the water maze, use a towel to dry it thoroughly.

Representative Results

The apparatus used in this work was a modified version of the Kane model and Richelle Mychasiuk's pediatric model^11,12. In this study, SD rats were assigned to sham and mTBI groups. To demonstrate the reproducibility of this model, we conducted three independent replicates of this model along with the acute neurobehavioral assessment, with each experiment involving 8-12 rats. In this study, we used more than 30 mTBI rats, with 2 rats experiencing mortality due to anesthetization. However, no rats succumbed to brain injury during the experiment. The results of these experiments are presented in Figure 2. Additionally, neurobehavioral assessments were carried out during the acute and subacute stages (Figure 3, Figure 4, and Figure 5).

Acute neurobehavioral assessment results

All these assessments were performed after anesthesia/impact 0 min (time of loss of consciousness and first seeking-behavior time) or 10 min (circle exiting and beam balance), respectively.

As shown in Figure 2A, mTBI rats spent significantly more time recovering from unconsciousness, which aligns with results obtained in previous studies^12,13. The seeking behavior in rats considered a normal physiological activity, exhibited a statistically significant increase in the recovery period within the mTBI group (Figure 2B). This finding suggests that the mTBI rats required a longer duration to regain their locomotion, olfaction, tactile probing, and environmental scanning abilities.

The circle existing test has replaced the original sensory tests in the neurological severity score, which previously relied on examiners' subjective observations such as the placing and proprioceptive tests. The mTBI rats spent a significantly longer time exiting from the circle compared to the sham rats (Figure 2C). The statistical analysis using a two-way ANOVA for circle exiting time showed a significant main effect of injury (F [1, 36] = 21.29, p < 0.0001), indicating a difference between the mTBI and sham groups. However, different trials had no significant effect (F [2, 36] = 0.1396, p = 0.87).

The results of the beam-balance test were analyzed using a two-way ANOVA, followed by Bonferroni's multiple comparisons for differences between group means (Figure 2D). There was a significant overall effect of injury in all wide beam tasks (3 cm: F = 13.89, p < 0.001; 2 cm: F = 42.7, p < 0.001; 1.5 cm: F = 27.25, p < 0.001), indicating that the mTBI rats exhibited balance impairment compared to the sham rats after 10 min following the impact. According to three independent repeated experiments, the 2 cm and 1.5 cm wide balance beam showed better discrimination between the sham and the mTBI groups than the 3 cm wide beam.

Neurobehavior assessment results

Motor coordination ability was assessed using the beam task at 1 day pre-anesthesia/ injury and 1 day, 3 days, and 7 days post-anesthesia/injury (Figure 3). The total number of hindlimb slips (Figure 3A) was analyzed by repeated measured two-way ANOVA, and Bonferroni's multiple comparisons found that mTBI rats displayed significantly more hindlimb slips at day 1 post-injury compared with sham rats (Figure 3A; p < 0.01). However, after a 2-day recovery, no alterations were seen in the hind-mistakes, with a total number of slips resolving back to sham levels after 7 days. Notably, all 6 mTBI rats had more post-impact hindlimb slips than their baseline performance. The slightly increased hindlimb slips in sham rats might associated with the lack of practice balance beam. At post-injury 1 day and 3 days, the mTBI rats spent more time traversing the 150 cm beam (39.8 s ± 3.79 s vs. 28.68 s ± 0.82 s, 37.06 s ± 4.06 s vs. 29.28 s ± 3.42 s), although there were no differences between mTBI rats and sham rats in the time taken to traverse the beam at all time-points (Figure 3B).

There were no significant differences in the distance traveled between the sham and mTBI groups (Figure 4A). Anxiety-like behavior was evaluated by measuring the time spent in the center zone during the open-field test. At both 3 days and 7 days post-injury, the mTBI rats exhibited a significant reduction in the time spent in the center zone compared to the sham rats. This finding indicates that the mTBI rats displayed higher levels of anxiety-like behavior following the impact within 7 days (Figure 4B,C).

The Morris water maze learning days results revealed that the mTBI rats required more time to locate the hidden platform than the sham rats, indicating impaired spatial learning and memory in the mTBI group (Figure 5). Subsequently, during the probe trial, the mTBI rats exhibited deficits in retaining spatial memory, as evidenced by spending less time searching for the removed platform. Notably, no significant difference was observed in swimming speed between the sham and mTBI groups, supporting the consistent findings observed in the distance traveled analysis conducted in the open field test. These results suggest that the impact did not have a discernible effect on spontaneous locomotor function.

Figure 1: Impact apparatus for mTBI in rats. (A) The top view and side view of the pillow and helmet in the relative position of the rat's head. The red dotted line shows the helmet position. (B) An image of the entire assembly showing a vertical guide tube for the dropped weight positioned above the rat stage and collecting sponge. (C) A still captured from an impact video depicting the 180° rotation of the rat following head impact and the subsequent acceleration/rotation. Please click here to view a larger version of this figure.

Figure 2: Acute neurobehavioral assessment outcomes following sham, mTBI rats, repeated three times independently. (A) Significantly increased time of the loss of consciousness after discontinuation of anesthesia in rats received a mTBI versus sham rats. There was a significant group (P < 0.0001, two-way ANOVA) effect but no significant time (P = 0.6226) effect or a group x time (P = 0.5803) interaction. (B) mTBI rats showed their first seeking behavior after anesthesia. (C) Sham rats spent less time escaping the 60 cm circle (*p < 0.01, **p < 0.001, unpaired t-test). (D) The performance in beam balance score of 3 cm, 2 cm, and 1.5 cm wide beam. The results of Bonferroni's multiple comparisons for each group are shown in the figures. Data presented as mean ± standard error of the mean. N = 8-12 rats were used per experiment. Please click here to view a larger version of this figure.

Figure 3: The beam task performance before impact and following impact on day 1, day 3, and day 7. (A) The mTBI rats made more hindlimb slips at post-injury day 1 (*p < 0.001, repeated measured 2-way ANOVA). (B) The average traverse time of sham rats is less than that of mTBI rats. Data presented as mean ± standard error of the mean (N = 6/group). Please click here to view a larger version of this figure.

Figure 4: The performance of the open field test in the pre-injury day 1 and post-injury day 1, day 3, day 7, and day 14. (A) There was no difference between sham and mTBI rats in the distance traveled. (B) mTBI rats spent less time in the center than sham rats at day 3 and day 7 (*p < 0.01, **p < 0.001, repeated measured 2-way ANOVA), with no apparent differences at pre-injury day 1 and post-injury day 1 and day 14. (C) Track map of mTBI rats in post-mTBI day 1, day 3, day 7, and day 14. Data presented as mean ± standard error of the mean (N = 6-10/group). Please click here to view a larger version of this figure.

Figure 5: The performance in Morris water maze. (A) There was no difference in speed in the swimming ability test among the sham and mTBI rats. (B) Latency to the hidden platform of the reference memory task on the trial day. (C) The rats crossed the platform more times in the 2-min probe test trial after 5 trial days. Sham (5.14 ± 0.65) vs. mTBI (3.56 ± 0.6), (*p < 0.01, Unpaired t-test). Data presented as mean ± standard error of the mean (N = 9/group). Please click here to view a larger version of this figure.

Discussion

This model successfully simulates a closed-head mTBI without the need for scalp incision or skull opening, providing a more accurate representation of the impact scenario observed in human cases. The avoidance of scalp incision helps prevent inflammatory responses that may not align with the actual situation. Compared to Richelle Mychasiuk's pediatric model¹², the model used in this study is specifically tailored for adult rats weighing between 280-320 g, enabling us to obtain valuable insights into the effects of mTBI on adult individuals. Additionally, incorporating key components such as a pillow and helmet facilitates the delivery of more uniform impacting force and assists the operator in precisely identifying the target area for impact.

It is important to emphasize that the impact procedure in rats was conducted without anesthesia maintenance, so the depth of anesthesia was confirmed prior to initiating the impact. We ensured that the rats had no response by gently shaking the anesthesia induction box and extending the anesthesia time by an additional 30 s to ensure an adequate level of anesthesia. It is recommended to complete the entire impact process within 1 min.

The impact apparatus described in this study is relatively easy to construct and can be replicated in almost any laboratory using the provided specifications. This promotes greater standardization and comparability of experimental data across different research settings. Furthermore, the validation data obtained from this study serve as a valuable resource for researchers in addressing specific scientific questions. By analyzing the neurobehavioral outcomes observed in this study, researchers can make informed decisions and tailor the experimental approach to align with their specific research objectives. This enhances the overall quality and relevance of future studies on mTBI and facilitates advancements in our understanding of its underlying mechanisms and associated outcomes.

This study exclusively involved male rats for or implementing the closed-head mTBI model. Given prior research indicating sex-specific variations in anxiety-like behavior and persistent cognitive and somatic symptoms related to mTBI^14,15, future studies should be performed on female rodents.

Materials

Name	Company	Catalog Number	Comments
Acrylic box	In-house	N/A	15 cm x 22 cm x 43 cm
Anesthesia Machine	RWD Life Science Co.	R540 Mice & Rat Animal Anesthesia Machine
Helmet	In-house	N/A	Stainless-steel disk measuring 10 mm in diameter and 3 mm in thickness
Morris water maze	RWD Life Science Co.		Diameter 150 cm, height 50 cm,platform diameter 35 cm
Open field	RWD Life Science Co.	63007	Width100 cm, height 40 cm
Panlab SMART V3.0	RWD Life Science Co.	SMART v3.0
Perforated weight	In-house	N/A	Weight of 550 g and diameter of 18 mm
Pillow	In-house	N/A	Wedge-shaped sponge to place beneath the rat's head