Theses & Dissertations Boston University Theses & Dissertations
2014
The development of a repetitive
mild traumatic brain injury model
in adolescent mice
https://hdl.handle.net/2144/15053 Boston University
BOSTON UNIVERSITY SCHOOL OF MEDICINE
Thesis
THE DEVELOPMENT OF A REPETITIVE MILD TRAUMATIC BRAIN INJURY MODEL IN ADOLESCENT MICE
by
SHIVANI MARGUERITE SAITH B.S., University of Michigan, 2012
Submitted in partial fulfillment of the requirements for the degree of
© 2014 by
SHIVANI MARGUERITE SAITH All rights reserved
Approved by
First Reader
Vickery Trinkaus-Randall, Ph.D.
Professor of Biochemistry and Ophthalmology
Second Reader
Michael Whalen, M.D.
DEDICATION
I would like to dedicate this work to my supportive family and friends. I would not have had such a wonderful learning opportunity without the support of my mother and father,
ACKNOWLEDGMENTS
I would like to thank Dr. Michael Whalen for giving me this fantastic opportunity to write my thesis. I would also like to thank Julianne Golinski for taking time to edit my thesis and teach me various lab techniques, and to Lauren McAllister for guiding me through the behavioral testing. This thesis would not have been as enjoyable without their help.
THE DEVELOPMENT OF A REPETITIVE MILD TRAUMATIC BRAIN INJURY MODEL IN ADOLESCENT MICE
SHIVANI MARGUERITE SAITH ABSTRACT
While participation in youth sports bolster a myriad of health benefits, it can also pose a risk to the athlete’s health from the increasing prevalence of repetitive mild traumatic brain injuries (TBI), often referred to as concussions. The adverse effects from repeated traumatic blows give a combination of acute symptoms, which may potentially develop into long-term complications. There is little known about the epidemiology of concussions, and thus the development of an animal model would help enhance our understanding of this potentially debilitating injury. An appropriate animal model should mimic the conditions of how concussions occur, in that there is not an invasive method to induce the injury and follows the same biomechanics. In our adolescent repetitive mild TBI model, we utilized a free-falling weight to deliver the traumatic blow to anesthetized mice that allowed free head rotation after impact. The injured group received one hit daily over the course of three days. The mice then underwent several behavioral tests to analyze the cognitive deficits, and the pathology of the tissue was analyzed via silver, Hematoxylin and Eosin (H&E), and Fluoro Jade-B staining. The injured mice developed both short- and long-term memory and spatial learning deficits, symptoms commonly found in concussed athletes, but failed to show deficits in anxiety and depression tests. The Fluoro Jade-B, silver and H&E staining resulted in negative signals for cell death. This study properly demonstrates repetitive mild TBIs in an adolescent mice model.
TABLE OF CONTENTS
TITLE………...i
COPYRIGHT PAGE………...ii
READER APPROVAL PAGE………..iii
DEDICATION ... iv
ACKNOWLEDGMENTS ... v
ABSTRACT ... vi
TABLE OF CONTENTS ... vii
LIST OF IMAGES ... viii
LIST OF FIGURES ... ix
LIST OF ABBREVIATIONS ... x INTRODUCTION ... 1 METHODS ... 6 RESULTS ... 16 DISCUSSION ... 34 REFERENCES ... 41 CURRICULUM VITAE ... 46
LIST OF IMAGES
Image Title Page
1 Anesthetized mouse prior to dropping the weight 7
2 Anesthetized mouse after the weight has been dropped 7
3 CHI mouse immediately post-injury 8
4 PI-positive cells in the choroid plexus of a CHI mouse 27
5 PI-negative cells in the cortex of a CHI mouse 28
6 7 8 9 10 11 12 13
Fluoro Jade-B denate gyrus of a CCI mouse
Fluoro Jade-B close-up denate gyrus of a CCI mouse Fluoro Jade-B stained denate gyrus of a sham mouse Fluoro Jade-B stained denate gyrus of a CHI mouse Silver stained denate gyrus section of a sham brain Silver stained denate gyrus section of a CHI brain H&E stained denate gyrus section of a sham brain H&E stained denate gyrus section of a CHI brain
29 29 29 30 31 31 32 33
LIST OF FIGURES
Figure Title Page
1 Motor testing score descriptions 10
2 Short-term Morris water maze for 3HD male mice 17
3 Long-term Morris water maze for 3HD male mice 18
4 Short-term Morris water maze for single-hit male mice 19
5 Short-term Morris water maze for 3HD female mice 20
6 7 8 9 10 11 12 13 14 15 16
Short-term Morris water maze for single-hit female mice Motor testing score
Motor testing time
Elevated plus maze mean speed
Elevated plus maze percentage of time in open arms Overnight open field mean speed
Overnight open field distance traveled Y-maze score
Porsolt forced swim latency to first freeze Porsolt forced swim total freezing latency
Fear conditioning and extinction freezing latencies
21 22 22 23 23 24 24 24 25 25 26
LIST OF ABBREVIATIONS
ANOVA………..………… Analysis of Variance APOE……….….………...Apolipoprotein E CHI.………Closed Head Injury CT………Computed Tomography CCI………..Controlled Cortical Impact H&E……….Hematoxylin and Eosin HD………..Hit-Daily LOC………...Loss of Consciousness MWM……….Morris Water Maze PI………Propidium Iodide TBI………Traumatic Brain Injury
INTRODUCTION
Repetitive mild traumatic brain injuries (TBI), commonly referred to as multiple concussions, are an urgent concern in pediatric care. A concussion occurs after a traumatic blow transfers force to the brain, causing functional disturbances without discernable structural damage (Buzzini and Guskiewicz, 2006). Despite TBIs considered to be the most common injury in children, there is a lack of knowledge regarding the factors of how to treat it. The Center of Disease Control estimates that approximately half a million children are treated for a TBI in hospital emergency departments (Faul et al., 2010). Loss of consciousness is not necessary in order to have a concussion, but symptoms may include a combination of physical, cognitive, sleep, and emotional disturbances (Simma, Lütschg, and Callahan, 2013). One of the most detrimental cognitive symptoms is lack of concentration, which puts the child at risk of hindered performance in daily activities like focusing in school (McCrory et al., 2004). Excessive sleep and insomnia are both common symptoms that affect the adolescent’s recovery process and must be carefully monitored (Jamault and Duff, 2013). Whatever the combination of these short-term symptoms the adolescent may have, concussions have detrimental effects on daily functioning.
While symptoms may resolve in a matter of days to weeks, the brain is now more vulnerable to the next TBI and a second hit can prolong recovery compared to the first concussion (Guskiewicz et al., 2003). A more severe example is second impact syndrome, in which the adolescent receives another traumatic blow within days from the first hit (McCrory, Davis, and Makdissi, 2012). As demonstrated in the Fujita, Wei, and
Povlishock (2012) study, the timing of a second hit can dramatically affect axonal and vascular damage. The closer the second hit is to the first hit, the more damaging the results. However, multiple hits need not occur within this critical period in order to generate exponential effects. Damage from repetitive mild TBIs can accumulate and have long-term effects. Repetitive TBIs increase the risk of neurodegeneration and tau protein accumulation, which raise the risk of developing diseases such as Alzheimer’s and Parkinson’s (Daneshvar et al., 2011). Dementia pugilistica, also known as “punch drunk,” is a condition prevalent to boxers due to repetitive blows to the head that is characterized by motor deficits and mental confusion (Stern et al., 2011). This syndrome is now commonly referred to as chronic traumatic encephalopathy and is estimated to affect 15-20% of retired athletes (Simma, Lütschg, and Callahan, 2013).
Despite age-restrictive contact rules, sports have become a main contributor to repetitive mTBI (Simma, Lütschg, and Callahan, 2013). Sports not only provide opportunities for injuries during games, but traumatic hits can also occur in practices. While some athletes may brush off “a little bump” to the head, there is a critical rest period before a gradual increase in cognitively and physically demanding tasks should take place. The ideal rest period varies for each individual and should not involve any schoolwork or media devices (Shrey, Griesbach, and Giza, 2011). A five-step program has been developed before the athlete should return to play as recommended in the Zurich Consensus Statement (McCrory et al., 2013). The athlete should be asymptomatic for at least 24 hours before moving onto the next step. The first step after rest is light exercise that consists of low-intensity activities such as walking or riding a stationary bike. The
next step is exercise specific to the sport, such as running in soccer or skating in ice hockey. Eventually the athlete can join in non-contact drills, and then have no restrictions on activity level (Jamault and Duff, 2013).
Given the delicate recovery step-wise progression, detecting a concussion after it first occurs is vital for the health of the adolescent. It is rare to find an abnormal computed tomography (CT) or magnetic resonance imaging scan in order to diagnose a concussion, further complicating the already underreported injury (Khurana and Kaye, 2012). Instead, a CT scan should be used in case of a potential skull fracture or intra-cerebral lesion (McCrory et al., 2013). Other forms of imaging technology such as positron emission tomography or magnetic resonance spectroscopy have not been recommended for diagnosis purposes. Identifying overt symptoms is currently a more accurate method of evaluation than neuroimaging, and thus the development of an animal model can help discover and improve diagnosis techniques.
Currently, there are a few repetitive mild TBI translational models. In the past 40 years, several models have been proposed (Friess et al., 2009; Huh et al., 2007; Longhi et al., 2005; Prins et al., 2010; Raghupathi et al., 2004; Shitaka et al., 2011, Mychasiuk, Farran, & Esser, 2014; Sullivan et al., 2013, Browne et al., 2011; Alder et al., 2011; Namjoshi et al., 2013; Heldt et al. 2014; Dixon et al., 1987; Laurer et al., 2001; Longhi et al., 2005; Spain et al., 2010; Tang et al., 1997a, 1997b; Zohar et al., 2003; Dixon et al., 1991; Lighthall, 1988; Cernak et al., 1996; Leung et al., 2008; Meehan et al., 2012). Problems with these models include high mortality, no symptomatic behavioral deficits, and/or open head injuries (Prins et al., 2010). In the controlled cortical impact (CCI)
model, a piston delivers the impact to exposed brain tissue. This, however, induces considerable cell death and is only applicable to severe TBIs (Namjoshi et al., 2013; Dixon et al., 1991; Lighthall, 1988). Another example of an open head model is fluid percussion, where the injected fluid pressure creates the TBI (Alder et al., 2011; Dixon et al., 1987). Both of these models involve exposed dura due to the craniectomy performed, a characteristic not found in an average concussion. The third widely used model is an air blast pressure model, where a pressurized air gun induces a closed head injury (Heldt et al., 2014; Leung et al., 2008; Cernak et al., 1996). The animal is tightly restrained inside a tube with a hole cut out from the top for the pressure wave to hit. However, this model does not allow the animal’s head to have the free head rotation at impact like in sport concussions.
A proper model should mirror how sport concussions occur and generate closed head injuries (CHI) with low mortality and symptoms without gross pathology. In the Prins et al. (2010) rat model, they selected postnatal day 35 rats based on several developmental profiles that correspond to human adolescents. Using a mechanized piston, they targeted a precise location for two hits, 1 day apart and allowed free rotational acceleration of the head at impact based on the mechanics of a concussion. While this is a better model than an open head injury model, multiple concussions have more variability on location of impact. The weight-drop model uses a free falling weight on a mouse to deliver the blow in which the head is manually positioned to emulate the variability in real concussions (Mannix et al., 2013, Marmarou et al., 1994) In the Mannix et al. (2013) study, they used a free falling weight to produce repetitive TBIs on
adult mice. However, an adult animal model is not applicable to children as well. Since children are in the process of developing, they would have differing injury biomechanics and pathophysiological responses (Kirkwood et al., 2006). Thus in our 3-hit daily (HD) CHI model, we used the same weight drop method but selected adolescent mice to learn more on this particularly vulnerable age group. With such a high prevalence rate in adolescents, finding an adolescent animal model is imperative for the public health of future generations.
METHODS Animals
Male C57B1/6J mice (Jackson Labs, Bar Harbor, ME) between postnatal days 38 to 40 were used. The animals were housed in a 12-hour light/dark cycles at a Massachusetts General Hospital pathogen-free facility and were given food and water ad libitum. The mice were randomized into either the sham or CHI group, and four different cohorts of 3HD mice (n=8, n=4, n=6, n=4) were used to include for any variability on a given day. One cohort of single-hit male C57B1/6J mice (n=11) was included for comparison of injury effect. One cohort of 3HD (n=10) and single-hit female C57B1/6J mice (n=5) was included for comparison of injury effect with gender. Weights and loss of consciousness (LOC) times were taken on the first day of injury for the single-hit males and females, and 3HD females.
Closed Head Injury (CHI) Model
The CHI mice were anesthetized individually for 45 seconds in a chamber consisting of 4.5% isoflurane in a 70:30 nitric oxide and oxygen mixture. A CHI mouse was placed onto a delicate task wiper (Kimwipe) and held firmly by the tail with one hand while keeping the Kimwipe taut with the other hand (see image 1). The mouse’s head was positioned slightly off-midline under a vertical hollow 48 in PVC pipe. A 54 g bolt was released at the top of the pipe, allowing the head to freely penetrate the Kimwipe at impact (see image 2). The LOC time was recorded starting at the moment of impact and stopped when the mouse righted himself from being placed on its back (see image 3). The 3-hit CHI mice were hit once daily for three consecutive days, alternating the side of
each hit. The sham mice were anesthetized in the same procedure, but were placed directly back into their respective home cages after righting. Both male and female single-hit mice had an injury height of 48 in with a 54 g weight.
Image 1. An anesthetized mouse prior to dropping the weight. The mouse is placed on a taut Kimwipe with one hand holding the tail. The mouse’s head is positioned slightly off-centered, alternating the side of each hit.
Image 2. An anesthetized mouse after the weight has been dropped. The mouse’s head is free to move after impact, penetrating the Kimwipe. This method mimics the biomechanics of a concussion.
Image 3. A CHI mouse (lower right) placed on its back after a hit. The LOC time was recorded at the moment of impact until the mouse righted itself from this position (lower left).
Behavioral Testing
The mice underwent behavioral tests to determine various cognitive, somatic, and emotional effects of injury. Prior to each behavioral test, the mice were acclimatized to the respective room for 30 minutes. The mice were first tested in a short-term Morris Water Maze (MWM) 5 days post-injury of the third hit. Six weeks post-injury, the mice went through a long-term MWM for a second time and motor testing. Six weeks and one day post-injury, the mice went through the Y-maze, elevated plus maze, and overnight
open field. The forced swim test was conducted six weeks and two days post-injury. The fear conditioning and extinction tests were conducted six weeks and 3 days post-injury. The forced swim, and fear conditioning and extinction tests were conducted last to avoid any confounding anxiety-provoking side effects. The preliminary data of the short-term MWM for single-hit male, single-hit female, and the three-hit females were included for comparison of injury with gender effects. A cohort of female sham mice that were anesthetized once was used as the control for both groups of females.
Morris Water Maze
Each mouse was first tested in the MWM for short-term spatial learning and memory deficits (Washington et al., 2012). The MWM apparatus consists of a cylindrical tank (33 inch diameter x 24 inch height) filled approximately halfway with room-temperature water. The tank is divided up into four quadrants with symbols (star, square, circle, triangle) representing the four directions (North, South, East, West) in each trial. On day 1, the first five hidden trials were conducted. These trials consisted of a 4 in diameter cylindrical plexiglass platform hidden under 0.2 in of water and placed in the center of the southeast quadrant. The mouse was released in the water 2 in away from a direction and timed by hand (maximum 90 seconds) until the mouse reached and stayed on the hidden platform for three seconds. If the mouse was unable to find the platform within the 90-second time frame, the mouse was manually placed onto the platform for three seconds. A trial is completed once the mouse was placed at every direction, in which the direction order is randomized for each trial. On day 2, a probe and two visible trials were conducted. In the probe trial, the platform was removed, the mouse was placed
in the center of the northwest quadrant, and was recorded for location in the southeast quadrant where the original platform was located (maximum 60 seconds). The two visible trials consisted of a platform with a red tape border elevated 0.2 in from the water in the original position located in the southeast quadrant. The mice were reverse trained in the MWM with the platform located in the northwest quadrant for long-term spatial learning deficits and underwent 4 hidden trials and a probe trial on day 1 of testing, and a second probe and 2 visible trials on day 2. The second probe trial was recorded using the AnyMAZE program with overhead cameras.
Motor Testing
Motor testing was performed using an 18.5 in high wire trapeze to ensure the mouse was not hindered from motor deficits during the MWM, but from learning and memory deficits. The mouse was gently placed on the wire, and then scored for motor performance on a scale from 0 to 5 (see Figure 1) for a maximum time of 60 seconds. If the mouse lets go before the 30-mark, the mouse automatically gets a score of 0. The overall score is based on how the mouse performs for the majority of the time. The mouse automatically receives a time of 60 seconds with a score of 5 (climbs down a post and reaches the table).
Score Description
0 Falls from the wire before the 30-second mark
1 Holds onto the wire with only two feet
2 Holds onto the wire with all four feet
3 Holds onto the wire with all four feet and a complete tail wrap around
4 Moves towards a post with all four feet and a complete tail wrap around the wire
5 Climbs down a post and reaches the table
Figure 1. Descriptions for each motor test score. The overall score is based on the majority of the mouse’s performance. If the mouse falls from the wire before the 30-second mark, the score is automatically 0.
Elevated Plus Maze
The elevated plus maze tests anxiety-inducing behavior (Washington et al., 2012). The 24 in high apparatus consists of two 52 in by 3 in platforms intersecting at their midpoint, which is a 3 in by 3 in square. The closed arms of the platform have 4 in high walls to prevent falls whereas the open arms have no walls. It is considered anxious behavior if the mouse chooses to avoid the open arms. Each mouse was placed in the central square and recorded by the AnyMaze program for latency and distance in the center, closed, and open platforms for 5 minutes. During this time, it was noted if the mouse fell off the apparatus or had any bowel movements. After each trial is completed, the mouse was removed and the apparatus was cleaned with 70% ethanol to sterilize and eliminate scent. The mouse was assessed for mean speed and the percentage of time spent in the open arms.
Y-Maze
The Y-maze tests spatial working memory (Xu et al., 2009). The Y-shaped apparatus has three 13 in arms with 6 in walls, branching from a 3 in x 3 in x 3 in triangular intersection. Each arm is identified with a symbol (square, circle, star). There is one symbol at the end wall, and three near the intersection: one on the floor and two opposite from another on the walls. The mouse was placed behind a door on the square
maze for 5 minutes. The mouse’s activity was recorded by a Macbook laptop using the Photobooth program and was assessed for the number of times the mouse entered all three arms without re-entering the previous arm (i.e. ABC, ACB vs ABA, CBC) in a single triplet over the total number of arm entries. An arm entry consists of all four limbs in the designated areas. After each trial, the apparatus was wiped down with 70% ethanol to sterilize and eliminate scent.
Overnight Open Field
The overnight open field assay tests exploratory behavior and locomotor ability (Washington et al., 2012). The mice were placed in separate cages secured with metal wiring tops, each with an apple slice for nutrition and hydration. Overhead cameras using the AnyMAZE program recorded activity over the course of 8 hours overnight in minimal lighting. The mouse’s activity was assessed by mean speed and total distance traveled.
Porsolt Forced Swim
The forced swim tests depression-inducing behavior. The apparatus consists of a 4 L glass beaker filled with 2 L of room-temperature water. The mouse was placed in the water for five minutes and was recorded by the Photo Booth program on a Macbook Pro laptop computer. The mouse was scored based on latency to the first freeze and the total freezing time within the trial. Immobility criteria consist of the minimum amount of movement for the mouse to keep afloat.
Fear Conditioning and Extinction
Fear conditioning and extinction tests how the injury affects the mouse’s response to anxiety-provoking stimuli (Reger et al., 2012). The chamber consists of a 7 in x 7 in x
12 in apparatus with an electrical grid bottom inside a soundproof 17 in x 21 in x 21 in Stoelting box. The chamber was set with up with an overhead camera to record activity using the AnyMAZE program. On day 1 of testing (training), the apparatus was set up with grey panel walls and the bare electrical grid, scented with vanilla extract. Training consists of a 5-minute acclimation period followed by a 10 minute period that had five 1500 Hz, 30 second cue tones. The last three cue tones were accompanied with a two-second 6mA electrical shock prior to the cue tone ending. Mice that were on standby were placed outside the behavioral room to prevent them from hearing the cue tones. The apparatus was cleaned after each mouse with vanilla-scented 70% ethanol. On days 2 and 3 of testing (extinction), the apparatus was unscented and set up with striped black and white walls, and a grey plastic bottom on top of the electrical grid to prevent contextual association with cue tones. Extinction consists of a 2-minute acclimation period followed by a 25-minute period that has fifteen cue tones. These cue tones were the same as training day except none of them had an electrical shock. The apparatus was cleaned after each mouse with unscented 70% ethanol. Mice were scored based on freezing times of each cue tone.
Immunohistochemistry
Mice were deeply anesthetized, intraperitoneally injected with propidium iodide (PI), then transcardially perfused with 4% paraformaldehyde 4 hours post-injury of the third hit. After the brains were placed in 4% paraformaldehyde for 24 hours, they were placed in 30% sucrose for 2 days (as indicated by complete submergence to the bottom of
12 µm slices mounted on slides and 80 µm coronal sections placed in 4% paraformaldehyde free-floating for 5 days. CCI mice perfused 4 hours after injury were included for a positive control.
Silver Staining
Following the FD Neurosilver Kit II (FD NeuroTechnologies, Inc.) protocol for sliver staining, the free-floating slices went through a series of combinations of stock solutions. The blocking step (Solutions C and F) had two rinses of 15 minutes each to decrease the background stain. Permount was added to sections and slides were covered with coverslips and examined using a light microscope to detect dense silver precipitates, indicating neuronal degeneration.
Propidium Iodide
Cyrostat sections were examined for PI-positive cells, indicating cell death, in the cortex, hippocampus, sub-hippocampus, and thalamus through a bright field microscope. The choroid plexus is the positive control since the cells are permeable to PI and is indicated with the red staining.
Fluoro Jade-B
Cyrostat sections went through 10% paraformaldehyde, tap and distilled water, 70% and 100% ethanol, 0.06% potassium permanganate, and the staining solution made from 0.1% acetic acid and 0.01% stock solution of Fluoro Jade-B to detect degenerating neurons. The slides then had coverslips placed with DPX mounting medium.
Hematoxylin and Eosin (H&E)
Cryostat sections were dehydrated in a series of 100% and 60% ethanol. The slides were then transferred to hematoxylin, acid alcohol, lithium carbonate, and eosin with rinses of distilled water between each transfer. The slides were dehydrated in 100% ethanol before being cleared of substitutes in xylene. The slides then had coverslips placed with Permount.
RESULTS
Statistical Analysis
The averages, standard deviation, and standard error were assessed for each behavioral test. The results except for the fear conditioning are listed as mean ± standard deviation of the mean. Fear conditioning results are listed as mean ± standard error of the mean. The p-value for every behavioral test except for fear conditioning was found using a two-tailed t-test on Microsoft Excel with a statistical significance considered to be p < 0.05. The p-value for fear conditioning was calculated using a 2-way repeated measures ANOVA (analysis of variance) test using the program Prism with a statistical significance consider to be p < 0.05. In addition, a repeated measures ANOVA test was conducted for both MWM series.
Animals
Preliminary data of weights and LOC times taken on the first injury day were included in this study. There were significant differences between both groups of single-hit female and male mice compared to sham for LOC time (sham female 17 ± 1.6 seconds vs single hit female 91.2 ± 24.6 seconds, p < 0.001; sham male 34 seconds ± 4.9 seconds vs single-hit male 157.5 ± 86.2 seconds, p = 0.01). There were no significant differences in LOC time or weight for the sham and 3-hit females, or weights in both single-hit male and female mice compared to sham mice. One mouse in the first cohort (n = 8) of the 3-hit males died inbetween the two series of MWM testing due to an unknown reason.
Behavioral Testing Morris Water Maze
In the short-term (5 days post-injury of the third hit) MWM testing of the 3HD male mice (see figure 2), the CHI mice performed significantly worse than the sham mice (p < 0.03 for group repeated measures ANOVA). There were no significant differences between groups in the probe trial (sham 18.8 ± 5.8 seconds vs CHI 17.6 ± 11 seconds, p = 0.76). There were no significant differences in visible trial 1 (CHI 18.8 ± 15.3 seconds vs sham 12.7 ± 4.8 seconds, p=0.22) or trial 2 (CHI 11.8 ± 8.5 seconds vs sham 7.4 ± 2.3 seconds, p=0.12).
Figure 2. Short-term Morris water maze performance for CHI male mice that received one hit daily for 3 consecutive days and sham mice (p < 0.05 for group in hidden trials repeated measures ANOVA).
In the long-term (6 weeks post-injury of the third hit) MWM testing of the 3HD male mice (see figure 3), the CHI mice performed significantly worse than the sham mice in all hidden trials (p < 0.005 for group repeated measures ANOVA). There were no
significant differences in either probe trial 1 (CHI 16.1 ± 11.7 seconds vs sham 17.5 ± 6.3 seconds, p=0.725) or trial 2 (CHI 15.8 ± 7.7 seconds vs sham 15.3 ± 7.3 seconds, p=0.899). There were no significant differences in visible trial 1 (CHI 14.5 ± 11.8 seconds vs sham 7.1 ± 1.3 seconds, p = 0.054) and trial 2 (CHI 10.5 ± 8.1 seconds vs sham 6.3 ± 1 second, p = 0.101).
Figure 3. Long-term Morris water maze performance for CHI male mice that received one hit daily for 3 consecutive days and sham mice. There were statistically significant differences in all four hidden trials (H, p < 0.005). There were no significant differences in either visible trial (V, p > 0.05).
In the short-term (1 week post-injury) MWM of the single hit male mice (see figure 4), there were no significant differences in hidden trials or probe trial (sham 18 ± 6.8 seconds vs CHI 14.2 ± 4.8 seconds, p = 0.31), visible 1 (sham 9.7 ± 2.2 seconds vs CHI 9.5 ± 1.9 seconds, p = 0.9), or visible 2 (sham 6.3 ± 0.7 seconds vs CHI 6.8 ± 0.9 seconds, p = 0.34).
Figure 4. Short-term Morris water maze performance for CHI male mice that received one hit and sham mice. There were no significant differences in all hidden trials (H1-5, p > 0.05) and visible trials (V1-2, p > 0.05).
In the short-term (1 week post-injury) MWM of the 3HD female mice (see figure 5), there were no significant differences in hidden trials. There were no significant differences in the probe trial (sham 16.5 ± 8.8 seconds vs CHI 14.3 ± 2.8 seconds, p = 0.61) or visible 1 (sham 10.5 ± 2.7 seconds vs CHI 11.7 ± 6.7 seconds, p = 0.71). There was a significant difference between groups for visible 2 (sham 9 ± 2 seconds vs CHI 6.8 ± 0.6 seconds, p = 0.046).
Figure 5. Short-term Morris water maze performance for CHI female mice that received three hits and sham mice. There were no significant differences in all hidden trials (H1-5, p > 0.05) and the first visible trial (V1-2, p > 0.05). There was a statistically significant difference between groups for V2 (p = 0.046).
In the short-term (1 week post-injury) MWM of the single hit female mice (see figure 6), there were no significant differences in hidden trials. There were no significant differences in the probe trial (sham 16.5 ± 8.8 seconds vs CHI 11.9 ± 4.3 seconds, p = 0.33) or visible 1 (sham 10.5 ± 2.7 seconds vs CHI 9.2 ± 4.1 seconds, p = 0.58). There was a significant difference between groups for visible 2 (sham 9 ± 2 seconds vs CHI 6.5 ± 1 second, p = 0.039). 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 H1 H2 H3 H4 H5 V1 V2 La te n cy ( s) Trial Sham 3HD Females
Figure 6. Short-term Morris water maze performance for CHI female mice that received one hit and sham mice. There were no significant differences in all hidden trials (H1-5, p > 0.05) and the first visible trial (V1-2, p > 0.05). There was a statistically significant difference between groups for V2 (p = 0.039).
Motor Testing
In the motor testing, there were no significant differences between groups for the average score (sham 3.8 ± 1.2 vs CHI 3.4 ± 1.4, p=0.533; see figure 7) and average hold time on the apparatus (sham 56.2 ± 7.7 seconds vs CHI 52.3 ± 9 seconds, p=0.3; see figure 8).
Figures 7 (left) and 8 (right). Motor testing performance for CHI mice that received one hit daily for 3 consecutive days and sham mice. As shown in figure 7, there is no significant difference in motor testing score between the sham and CHI mice (p=0.533). As shown in figure 8, there is no significant difference in maximum hold time on the trapeze between sham and CHI mice (p=0.3).
Elevated Plus Maze
In the elevated plus maze testing, there were no significant differences between groups for mean speed (sham 0.026 ± 0.006 m/s vs CHI 0.028 ± 0.002 m/s, p = 0.465; see figure 9) and the percentage of time in the open arms (sham 14.6 ± 7.1% vs CHI 14.1 ± 6.9%, p = 0.884; see figure 10).
Figures 9 (left) and 10 (right). Elevated plus maze performance for CHI mice that received one hit daily for 3 consecutive days and sham mice. As shown in figure 9, there is no significant difference in mean speed between sham and CHI mice (p=0.465). As shown in figure 10, there is no significant difference in percentage of time in open arms between sham and CHI mice (p=0.884).
Overnight Open Field
In the overnight open field testing, there were no significant differences between groups for distance traveled (sham 359.8 ± 332.4 m vs CHI 365 ± 298 m, p=0.971; see figure 11) and mean speed (sham 0.013 ± 0.01 m/s vs CHI 0.013 ± 0.01 m/s, p=0.975; see figure 12).
Figures 11 (left) and 12 (right). Overnight open field performance for CHI mice that received one hit daily for 3 consecutive days and sham mice. As shown in figure 11, there is no significant difference in distance traveled between sham and CHI mice (p=0.971). As shown in figure 12, there is no significant difference in mean speed between sham and CHI mice (p=0.975).
Y-Maze
In the Y-maze testing, there was no significant difference between groups for the overall score (sham 55.5 ± 10.2 vs 57.2 ± 11.7, p=0.732; see figure 13).
0 0.005 0.01 0.015 0.02 0.025 0.03 M ea n S p ee d ( m /s) Sham CHI
Figure 13. Y-maze performance for CHI mice that received one hit daily for 3 consecutive days and sham mice. There is no significant difference in overall score between sham and CHI mice (p=0.732).
Porsolt Forced Swim
In the Porsolt forced swim test, there were no significant differences between groups for the first freeze time (sham 73.9 ± 22.9 seconds vs CHI 61.3 ± 33.1 seconds, p=0.319; see figure 14) and the total freezing latency (sham 145.9 ± 25.5 seconds vs 132.4 ± 39.5 seconds, p=0.359; see figure 15).
Figures 14 (left) and 15 (right). Porsolt forced swim performance for CHI mice that received one hit daily for 3 consecutive days and sham mice. As shown in figure 14, there is no significant difference latency to first freeze between sham and CHI mice (p=0.319). As shown in figure 15, there is no significant difference in total freezing latency between sham and CHI mice (p=0.359).
Fear Conditioning and Extinction
There were no statistically significant differences between groups for freezing latency at any stage of fear conditioning and extinction (see figure 16, p = 0.987).
Figure 16. The freezing latencies of the fear conditioning and extinction tests for CHI mice that received one hit daily for 3 consecutive days and sham mice. There were no significant differences between groups for any of the cue tones (p > 0.987).
Immunohistochemistry PI-Positive Cells
There was a positive signal as indicated by the red staining in the choroid plexus of the sham and CHI mice (see image 4). There was a negative signal for PI cells in the cortex, hippocampus, sub-hippocampus, and thalamus for both the sham and CHI mice (see image 5).
Image 4. The choroid plexus of a CHI mouse. The red staining indicates PI permeated the choroid epithelium and is present in the brain. This picture serves as a positive control.
Fluoro Jade-B
The cortex, denate gyrus, and three regions of the hippocampus of a CCI brain showed fluorescent green cells indicating cell death as the positive control (see images 6 and 7). There was a negative signal for cell death in both sham and CHI mice in the same regions (see images 8 and 9).
Image 6. A Fluoro Jade-B stained denate gyrus section from a CCI mouse brain. The fluorescent green cells indicate degenerating neurons and serves as a positive control.
Image 7. A Fluoro Jade-B stained denate gyrus section from a CCI mouse brain at a higher magnification. The fluorescent green cells indicate degenerating neurons and serves as a positive control.
Image 8. A Fluoro Jade-B stained denate gyrus section from a sham mouse brain. The lack of fluorescent green cells indicates no degenerating cells.
Image 9. A Fluoro Jade-B stained denate gyrus section from a CHI 3-hit mouse brain. The lack of fluorescent green cells indicates no degenerating cells.
Silver Staining
There was a negative signal of neuronal degeneration in the cortex, hippocampus, and thalamus of both sham and CHI brains as indicated by the lack of silver precipitates (see images 10 and 11).
Image 10. A silver stained denate gyrus section of a sham brain. The lack of silver precipitates indicates no neuronal degeneration.
Image 11. A silver stained denate gyrus section of a CHI 3-hit brain. The lack of silver precipitates indicates no neuronal degeneration.
Hematoxylin and Eosin
No signs of intracranial hemorrhages or necrotic cells were found in either sham or CHI brain. The CHI brain (see image 13) had the same gross appearance as the sham brain (see image 12).
Image 12. An H&E stained denate gyrus section of a sham brain. There were no signs of hemorrhagic lesion or necrotic cells in this region.
Image 13. An H&E stained denate gyrus section from a CHI 3-hit mouse brain. There were no signs of hemorrhagic lesion or necrotic cells in this region.
DISCUSSION Interpretation of Results
We found that 3 hits on postnatal day 38 male mice produced cognitive deficits in both the short- and long-term MWM. There was a statistically significant difference of sham and CHI mice over time in both the short- and long-term MWM, indicating the mice learned and recalled where the platform was located. However, the CHI mice performed worse as a group, indicating a deficit in their learning and memory function. These behavioral deficits are two of the same cognitive symptoms that concussed athletes commonly suffer from (Simma, Lütschg, and Callahan, 2013). The lack of difference in visible trials between groups indicates the hidden trial performance was due to learning and memory deficits, not from visual impairments (Creed et al., 2011). The Mannix et al. (2013) study also demonstrated an acute cognitive deficit in adult mice that received 5 hits daily for 5 days when tested in the MWM 3 days post-injury. While these mice had 2 more hits with an impact of the same weight as our study, the drop height was shorter at 28 inches thus making the impact less severe compared to our hits.
The long-term MWM had an even larger statistical difference between groups, demonstrating a lasting cognitive deficit that worsens over time. Since the platform was moved to the opposite side of the tank, the mice’s working memory was tested since they had to learn and remember the new location. Procedural learning was not tested in this case since the mice already learned how the task worked in the first MWM. In the Hamm et al. (1996) study using a fluid-percussion model to induce a moderate TBI, they demonstrated the same spatial working memory deficit for the injured mice when they
moved the location of the platform of the second trial. The Meehan et al. (2012) study with repetitive hits on adult mice demonstrated the same long-term cognitive deficit tested both a month and year post-injury when the platform was moved to a new location. Despite the spatial learning and memory deficits between groups in the MWM, there were no statistically different results in the Y-maze, elevated plus maze, forced swim, or fear conditioning and extinction tests. No difference between groups suggests that the CHI mice do not express long-term depression and anxiety side effects from injury. In contrast, the Heldt et al. (2014) study used an air pressure wave to induce a single TBI on adult mice and found the injured mice expressed anxiety, depression, and the perseverance of learned fear, but only at the highest level of pressure blast. Despite our model lacking these behavioral deficits, it can now be used to study how pre-existing psychiatric problems affect behavioral outcome post-injury. How depression and anxiety affect post-concussed individuals has been a topic of interest due to its high prevalence in the population (Lange, Iverson, and Rose, 2011). In our current study, the behavioral outcome is a function of injury rather than co-morbid psychiatric/neurological problems. These pre-existing issues make human studies controversial and thus difficult to interpret for the advancement of treatment and knowledge of future generations. In the Hutchinson et al. (2014) study, they found a longer recovery time for athletes who had pre-existing psychosocial (i.e. attention deficit hyperactivity disorder, anxiety, depression) and/or learning disabilities. However, no human study can infer a cause and effect relationship between psychiatric problems and TBIs due to confounding variables in diagnosis. A future animal study can study this by stressing the mice prior to the injury, then
conducting the same behavioral tests as our current study. It is hypothesized that the stressors prior to injury will have more robust cognitive and emotional deficits during these behavioral tasks than the solely injured mice.
The pathophysiology of the 3-hit CHI male mice helps support an animal model for repetitive mild TBIs. The PI, Fluoro Jade-B, and silver stain all have negative signals in the cortex, three regions of the hippocampus, and thalamus, indicating a non-cell death model. Thus, we can conclude that there are no confounding effects of cell death on the CHI cognitive deficits. Based on post-mortem examination of human brains that have received repetitive mild TBIs, brains appear grossly normal with mild neuronal loss (Khurana and Kaye, 2012). The Prins et al. (2010) study also had a negative Fluoro-Jade staining in the cortex and hippocampus in their 2-hit adolescent rat CHI model. In the Creed et al. (2011) study, there was a positive Fluoro-Jade signal in the cortex and dentate gyrus of single-hit mice that were perfused one day later. Despite being a closed head injury model, the injury induced a skull fracture in the mice, which is not commonly found in concussed athletes and may account for this cell death (Khurana and Kaye, 2012). In addition, our study perfused the mice 4 hours post-injury of the third hit, showing only short-term effects. Future studies can postpone the perfusion to study chronic effects of injury on cell death.
Interpretation of the Model
The parameters of our model mimic key aspects of a repetitive concussion injury. Unlike a CCI-induced or fluid percussion-induced TBI, our weight drop model delivers
the traumatic blow on a closed head, similar to athletes who do not wear a helmet such as soccer players. Every concussion occurs in a different circumstance, whether it happens accidentally in practice by a teammate or intentionally during a game by an opponent. In the Lighthall (1988) CCI model, the mechanized piston delivered the traumatic blows in a precise location. While this model demonstrated cognitive deficits, the CCI model creates a cerebral contusion that is not commonly found in concussions. Concussions rarely occur in the same manner, and to have two concussions occur in the same location with the same biomechanics is highly unlikely. The manual placement of the mouse’s head under the pipe with alternating side of hits in our model includes for this variability of location in concussions. Lastly, the anesthesia prevented the mouse to prepare for the hit, similar to an athlete who received an abrupt blow. Despite the emphasis on keeping athletes’ heads looking up to be aware of their surroundings, hits do occur blind-sided. The anesthetized mouse lacks the same preparation as an athlete might.
One question that arises from our 3-hit model is whether the effect of injury is due to time elapsed after the hits or the number of hits. The single-hit male cohort was tested in a short-term MWM under the same circumstances as the 3-hit male mice in order to address this concern. Based on the results, there is no significant difference between the sham and the single-hit mice, suggesting it is the effect of injury due to the number of hits that gives the 3-hit mice the cognitive deficits. One hit at 48 inches is not enough to produce the same cognitive deficits as three hits over the course of 3 days when measured a week after injury, suggesting a higher force of an single impact is necessary to induce cognitive deficits.
The cohorts of single and 3-hit female mice were to assess possible gender differences after injury. There were no significant differences in either group in all trials except for the last visible trial for each group. This suggests that adolescent female mice are protected against this level of injury and several factors may account for this. Female mice at postnatal day 38 on average weigh less compared to male mice, potentially affecting the biomechanics of the impact. Female hormones or differing cerebral blood flow may also play a role in this apparent protection, so further studies are necessary to understand this gender difference (Harmon et al., 2013).
The idea that there is a higher risk of a second concussion after the initial one brings up the question of the cause for this increased vulnerability. There have been studies focusing on polymorphisms and their associated risks with a history of concussions (Tierney et al., 2010; Terrell et al., 2008). In the Tierney et al. (2010) study on college athletes, they found an association between athletes with a history of one concussion and all of the rare apolipoprotein E (APOE) alleles, and an association between two or more concussions with the APOE promoter minor allele. Terrell et al. (2008) found a similar finding where college athletes who have the APOE promoter G-219T TT genotype have a higher probability with a history of concussions, especially severe ones. Having a past with concussions increases the risk of the next by up to 5.8 fold, thus suggesting a biochemical response to the initial concussion (Harmon et al., 2013).
Limitations of the model
A key difference between concussions in sports and in our model is the state of the subject. Athletes are fully conscious when the traumatic impact occurs, whereas the mice are lightly anesthetized. Anesthesia puts the mouse in a state of unconsciousness and the mouse would not have the same reaction to a blow like an athlete who is prepared for the hit such as a tackle or guard in football. This could include tensing of the neck muscles that could affect the biomechanics of the concussion. In addition, past literature has shown the effect of anesthesia on long-term spatial memory performance (Lin and Zuo, 2011). While there is still a significant difference between groups in our model, it would be more ideal to have anesthesia with no potentially confounding effects.
Our CHI model represents athletes who play sports such as soccer that do not use equipment. However, the sports that involve more contact, such as ice hockey and football, take precautions by requiring the use of helmets and mouth guards. While this does offer protection, there still a high prevalence of repetitive TBIs in these athletes (Khurana and Kaye, 2012). A modified model incorporating these aspects would be necessary in order to apply to these athletes.
Conclusion
The development of a reliable adolescent animal model is a required and important step towards understanding the association between repetitive injuries in adolescence and behavioral outcome. Despite being considered “mild” in respect to injury level and the apparent brief recovery period from blatant symptoms (Prins et al.,
2010), these traumatic injuries create detrimental short- and long-term cognitive deficits. Based on our results, we have demonstrated a translational model that gives consistent spatial memory and learning deficits in repetitively injured male mice. With the injured mice failing to show behavioral deficits in the depression and anxiety tests, this study sets up a solid foundation for studying how pre-existing psychiatric conditions can affect behavioral outcomes from repetitive mild TBIs.
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CURRICULUM VITAE
Shivani Marguerite Saith
15 N Beacon St, Apt 615 Allston, MA 02118 269.830.3465 [email protected] Born: 1990
Education Boston University, Boston, MA Sept 2012 - Present Masters of Arts in Medical Sciences
Cumulative GPA: 3.51/4.00
University of Michigan, Ann Arbor, MI Sept 2008 - Aug 2012 Bachelor of Science in Neuroscience
Cumulative GPA: 3.27/4.00
Research Neurosport, University of Michigan-Ann Arbor
Research Assistant Volunteer June 2011 – Aug 2012 o Performed various baseline concussion tests on high school and college
athletes
Sarter Laboratory, Department of Psychology, University of Michigan-Ann Arbor
Employment June 2010 – June 2011
Research Scholars Program June 2009 – May 2010 o Selected to participate in the second-year accelerated research program Undergraduate Research Opportunity Program (UROP) Sep 2008 – May 2009
o Investigated the influence of NMDA in the shell of the nucleus
accumbens on attentional performance the sustained attention task (SAT) o Awarded the UROP Spring Symposium Poster Award for overall
presentation
Athletics Women’s Club Ice Hockey Team, University of Michigan-Ann Arbor
Vice President Mar 2011 – Mar 2012
o Member of the executive board that organized travel, accommodations, and referee payments
Member Sept 2009 – Mar 2012
Club Sports Council, University of Michigan-Ann Arbor
Executive Board Member- Secretary, Women’s Ice Hockey Representative April 2011 – April 2012
o Provided feedback on new policies and devised methods to improve the club sports program as an officer of the executive board
Student-Athlete Advisory Committee, University of Michigan-Ann Arbor
Women’s Ice Hockey Representative Sept 2010 – Sept 2011 o A member of the NCAA-recognized committee that offers review of
