Brain, Behavior, and Immunity 60 (2017) 304–311

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Full-length Article

Binge ethanol in adulthood exacerbates negative outcomes following

juvenile traumatic brain injury
Kate Karelina ⇑, Kristopher R. Gaier, Maya Prabhu, Vanessa Wenger, Timothy E.D. Corrigan, Zachary M. Weil
Department of Neuroscience, Group in Behavioral Neuroendocrinology and Center for Brain and Spinal Cord Repair, Ohio State University Wexner Medical Center, Columbus,
OH 43210, USA

a r t i c l e i n f o a b s t r a c t

Article history: Traumatic brain injuries (TBI) are a major public health problem with enormous costs in terms of health
Received 5 August 2016 care dollars, lost productivity, and reduced quality of life. Alcohol is bidirectionally linked to TBI as many
Received in revised form 4 October 2016 TBI patients are intoxicated at the time of their injury and we recently reported that, in accordance with
Accepted 8 November 2016
human epidemiological data, animals injured during juvenile development self-administered signifi-
Available online 12 November 2016
cantly more alcohol as adults than did sham injured mice. There are also clinical data that drinking after
TBI significantly reduces the efficacy of rehabilitation and leads to poorer long-term outcomes. In order to
Keywords:
determine whether juvenile traumatic brain injury also increased the vulnerability of the brain to the
Traumatic brain injury
Alcohol
toxic effects of high dose alcohol, mice were injured at 21 days of age and then seven weeks later treated
Axon degeneration daily with binge-like levels of alcohol 5 g/kg (by oral gavage) for ten days. Binge-like alcohol produced a
Microglia greater degree of neuronal damage and neuroinflammation in mice that sustained a TBI. Further, mice
Neuroinflammation that sustained a juvenile TBI exhibited mild learning and memory impairments in adulthood following
Learning and memory binge alcohol and express a significant increase in hippocampal ectopic localization of newborn neurons.
Neurogenesis Taken together, these data provide strong evidence that a mild brain injury occurring early in life renders
the brain highly vulnerable to the consequences of binge-like alcohol consumption.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction
Traumatic brain injuries (TBI) are a major public health problem
with millions of injuries and tens of thousands of deaths occurring
annually in the US alone (Langlois et al., 2006). Further, the long-
term health consequences and economic costs in terms of health
spending and loss of productivity are staggering (Coronado et al.,
2012).
Alcohol use is tightly linked to traumatic brain injuries as alco-
hol intoxication is linked to, by some estimates, more than half of
all traumatic brain injuries (Tagliaferri et al., 2006). Much of the
research into this relationship therefore has focused on the role
of alcohol as a risk factor for TBI, and intoxication at injury as a
variable in functional outcomes and recovery (Berry et al., 2011;
Chen et al., 2012; Opreanu et al., 2010; Pandit et al., 2014). How-
ever, there is also some experimental and clinical evidence that
Abbreviations: TBI, traumatic brain injury; DCX, doublecortin; Iba-1, ionized
calcium binding adaptor molecule 1; TLR4, toll like receptor 4; IL-1b, interleukin 1
beta.
⇑ Corresponding author at: Biomedical Research Tower #618, 460 West 12th
Ave., Columbus, OH 43210, USA.
E-mail address: Ekaterina.karelina@osumc.edu (K. Karelina).
TBI itself could serve as an independent risk factor for substance
abuse issues, particularly when injuries occur early in development
(Bjork and Grant, 2009; Corrigan et al., 2013; Weil et al., 2016a).
Most epidemiological studies have reported that although there
is a population of patients that abstain from alcohol after TBI there
is also a substantial number that resume drinking in the months
after injury (Bombardier et al., 2003; Kreutzer and Harris, 1990;
Ponsford et al., 2007). There is some limited, though controversial,
evidence that intoxication at the time of TBI can be neuroprotec-
tive (Berry et al., 2011; Chen et al., 2012; Opreanu et al., 2010;
Pandit et al., 2014), but alcohol abuse following TBI is clearly prob-
lematic as it produces significantly poorer outcomes including:
impairing the efficacy of rehabilitation programs; reducing voca-
tional opportunities; and substantially increasing the risk for psy-
chiatric disorders, post-traumatic seizures, and additional injuries
(Corrigan, 1995; Corrigan et al., 2014; Jorge et al., 2005; Vaaramo
et al., 2014a,b).
Recently, we reported that female, but not male, mice that
received a single mild traumatic brain injury at 21 days of age
self-administered significantly more alcohol in adulthood than
did sham-injured mice or those injured in adulthood (Weil et al.,
2016b). These data revealed that injuries occurring at a critical

http://dx.doi.org/10.1016/j.bbi.2016.11.009
0889-1591/Ó 2016 Elsevier Inc. All rights reserved.
 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311
developmental time point in pre-adolescent mice resulted in long-
lasting consequences that contributed to voluntary alcohol abuse
in adulthood.
Although it is clear that drinking after traumatic brain injury
reduces the effectiveness of rehabilitation therapy and produces
poorer outcomes, the precise mechanisms that link drinking to
impaired neuronal recovery and function in this clinical population
are not fully understood. However, there is mounting evidence
from both the experimental and clinical literature that heavy or
binge-like drinking, even in the absence of other insults, induces
neuroinflammation and degeneration (Alfonso-Loeches et al.,
2010; Crews et al., 2011; Hayes et al., 2013; Qin and Crews,
2012; Vetreno and Crews, 2012). Further, TBI itself induces long
lasting impairments in neuronal structure and function and also
induces neuroinflammatory responses. Additionally, inflammatory
events early in life appear to prime the neuroimmune system to
exhibit greater inflammatory responses to other stimuli, such as
high-dose alcohol, later in life (Bilbo et al., 2010). Thus, heavy
drinking by TBI survivors may impair functional outcomes, at least
in part, by inducing inflammatory responses and further impairing
already dysfunctional neuronal circuits.
Our previous study using the self-administration paradigm
allowed us to investigate injury-induced alterations in alcohol
reward processing, but it did not allow us to investigate the func-
tional consequences of alcohol consumption after injury because
sham-injured mice did not voluntarily self-administer high doses
of ethanol (Weil et al., 2016a). Thus, in order to determine whether
heavy drinking after TBI exacerbates the behavioral, neuropatho-
logical, and inflammatory consequences of juvenile injuries in
female mice, we administered ten daily binge-like doses of ethanol
via oral gavage to adult females that had sustained a TBI or sham
injury in early life. The goals of this study were to 1) determine
whether juvenile TBI increased the functional deficits and tissue
damage induced by binge-like levels of alcohol and 2) to begin to
uncover the mechanisms that link TBI, binge-like alcohol, and tis-
sue damage. We hypothesized that binge-like exposure to alcohol
would exacerbate tissue damage and functional deficits in previ-
ously injured female mice in part by inducing neuroinflammation
and impairing neuroplasticity.
2. Methods
2.1. Animals
All procedures were conducted on female Swiss-Webster mice
derived from breeders purchased from Charles River (Wilmington,
MA) and bred at the Ohio State University. Pups were weaned at
21 days of age into a standard mouse cage (32  16  12 cm) with
ad libitum access to food and filtered tap water. All animals were
housed in a 14:10 light-dark cycle. All procedures were approved
by the OSU Institutional Animal Care and Use Committee, and were
conducted in accordance with the National Institute of Health
guidelines.
2.2. Traumatic brain injury
On the day of weaning, 21 day-old female mice sustained a mild
closed-head injury or a sham injury as described previously (Weil
et al., 2016b). All mice were anesthetized with inhaled isoflurane,
secured in a stereotaxic frame, and a round 2 mm impactor (Impact
One device, Leica Biosystems, Richmond IL) was placed on the sur-
face of the exposed skull ( 1 mm AP, 1 mm ML). TBI mice under-
went an impact at 3 mm/s (dwell time 30 ms) to a depth of 1 mm,
while sham mice were exposed to an equivalent amount of anes-
thesia in the absence of impact. The skin was closed with nylon
suture and mice were returned to their home cages.
305
2.3. Alcohol administration
Beginning seven weeks following TBI or sham injury, all mice
were administered 5 g/kg of a 31.5% solution of ethanol or water
control (dosing based on (Bertola et al., 2013)). Ethanol or water
was administered via daily oral gavage for 10 consecutive days.
Mice were weighed daily to account for body mass fluctuations
in the dosing. This paradigm resulted in 4 groups (sham/water,
TBI/water, sham/ethanol, TBI/ethanol), which were divided into 3
separate cohorts. One cohort was used for assessment of patho-
physiology only (n = 8–10 per group), one for microglial extraction
(n = 6–8 per group), and another cohort underwent behavioral
testing followed by histological assessment (n = 9–12 per group).
See Fig. 1 for experimental timeline.
2.4. Blood ethanol concentration
Blood ethanol concentration (BEC) was assessed via a colori-
metric 96-well plate assay (modified from Prencipe et al., 1987).
Blood was drawn from the retro-orbital sinus 90 min after the final
oral gavage. Blood samples were centrifuged and serum stored at
80 °C until use. A BEC assay buffer (100 mM KH2PO4; 100 mM
K2HPO4; 0.7 mM 4-aminoantopyrine; 1.7 mM chromotropic acid
disodium salt; 50 mg/L EDTA; 50 mL/L triton X-100) was used to
dilute ethanol standards and serum and all wells were assessed
in duplicate. A reaction mixture of alcohol oxidase (Sigma A2404)
and peroxidase from horseradish (Sigma P8375) diluted in BEC
assay buffer was added to all wells and the plate was read at
600 nm.
2.5. Histology
Axon damage was assessed via silver staining using the
NeuroSilver kit (FD Neurotechnologies, PK301) per manufacturer’s
instructions. Mice were overdosed with sodium pentobarbital
(200 mg/kg) and transcardially perfused with 4% paraformalde-
hyde. Following an overnight postfix and cryopreservation, brain
sections throughout the forebrain were sliced on a cryostat,
(40 lm) and collected into a 24-well culture plate for free-
floating immunohistochemistry and silver staining. For immuno-
histochemistry, tissue was washed with 0.1 M phosphate-
buffered saline, quenched in hydrogen peroxide, and incubated in
primary antibody overnight (rabbit anti-Iba1, Wako 019-19741;
goat anti-doublecortin, Santa Cruz sc-8006). Biotinylated sec-
ondary antibodies (goat anti-rabbit and donkey anti-goat, Vector
Laboratories BA-1000 and BA-5000) were applied the following
day, and staining was visualized using the avidin biotin-
diaminobenzidene staining method (Vector Laboratories PK-6100
and SK-4100).
2.6. Histological analysis
Axonal degeneration (silver staining) was assessed qualitatively
as previously reported (Weil et al., 2014). Briefly, representative
tissue throughout the entire forebrain were assessed and scored
on a 4-point scale (0 = few axonal profiles, 3 = dense axonal degen-
eration throughout white matter tracts bilaterally).
Microglial cells (Iba1-positive staining) were assessed in the
prefrontal cortex. Morphological analysis was conducted using
Neurolucida software (MicroBrightfield). Briefly, 2–3 cells per
hemisphere from each brain were selected using strict criteria as
previously reported (Karelina et al., 2016) and traced for subse-
quent sholl analysis. Using NeuroExplorer (MicroBrightfield) con-
centric sholl rings spaced 5 lm apart were centered on the cell
body, and the number of intersections, total process length, and
cell body surface area were obtained for each cell. Total microglial
306 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311

Fig. 1. Experimental timeline. All animals underwent TBI or sham injury at 21 days of age and were returned to their home cages for seven weeks. In adulthood, all mice were
administered ethanol or water via oral gavage daily for ten consecutive days. These studies were conducted on three cohorts, cohort 1 was perfused for tissue collection
immediately after the final gavage, cohort 2 was used for microglia isolation, and cohort 3 underwent behavioral testing before tissue collection. Passive avoidance testing
was begun twenty-four hours following the final gavage and brain tissue collection was conducted the day after testing was completed.

numbers were counted in a 0.26 mm2 region of interest using Ima-
geJ software (Abràmoff et al., 2004) and converted to cell number/
mm2.
Doublecortin-positive neurons were counted in the hippocam-
pus dentate gyrus, sections spanning approximately 1.94 mm
through 2.18 mm from bregma. Total cell counts were collected
in the granule cell layer in both the top and bottom blades of the
dentate, as well as the molecular layer and dentate hilus.
stepped into the dark chamber. Once the mouse entered the dark
chamber, the door closed, a brief shock (1 mA, 2 s) was delivered
to the grid floor, and the mouse was immediately replaced into
its home cage. Twenty-four hours following the training, each
mouse was again individually placed into the illuminated chamber
for 1 min. Once the guillotine door was open, latency to enter the
dark chamber was recorded (with an upper limit of 300 s). No
shock was administered on testing day.

2.7. Microglial isolation 2.10. Statistical analysis

Microglia were extracted from bilateral frontal lobes of sham
and injured mice 24 h following their final oral gavage. Fresh
brains were collected, olfactory bulbs were removed and tissue
was dissected (approximately bregma 0.56 mm through
3.56 mm) and homogenized in Dulbecco’s phosphate buffer solu-
tion (DPBS). The homogenate was centrifuged and the pellet resus-
pended in 70% isotonic Percoll (GE Healthcare Life Sciences #17-
0891-01). A discontinuous Percoll gradient was created by layering
50% and 35% Percoll and topping off with (DPBS). The gradient was
then centrifuged and the microglia-containing layer in the 50%/70%
interface was collected and washed.
Qualitative analysis of silver staining was assessed via the non-
parametric Mann-Whitney U and Kruskall-Wallis tests. Significant
overall Kruskall-Wallis results were followed up by pairwise com-
parisons. Microglial sholl analysis was conducted using a repeated
measures ANOVA. All histology analyses (cell counts, process
length, cell body area), behavioral data, and gene expression data
were assessed via a 2-way ANOVA (injury  ethanol administra-
tion). All statistics were analyzed using the IBM SPSS V.23 soft-
ware. All significant overall ANOVA results were followed by the
Least Significant Differences posthoc test. Data were considered
significant for p-values <0.05.

2.8. qPCR 3. Results

Extracted microglia were lysed and cDNA synthesized using the
SuperScript III CellsDirect cDNA synthesis kit (Invitrogen
#11739010 and #18080200) according to the manufacturer’s pro-
tocol. A TaqMan 18S ribosomal RNA primer/probe set (Applied
Biosystems #4319413E) was used as an internal control. Primer/
probe sets for the target genes were also purchased at Applied
Biosystems (TLR4: Mm00445273_m1; IL1b: Mm00434228_m1)
and amplification was performed on an ABI 7500 Sequencing Fast
System using the TaqMan Fast Advanced Master Mix (#4444963).
The universal two-step RT-PCR cycling conditions used were: 50 °C
for 2 min, 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and
60 °C for 30 s. Gene expression was analyzed relative to a standard
curve and normalized to the sham/water group.
2.9. Passive avoidance
Learning and memory was evaluated via the passive avoidance
task. The task consisted of a 2-chambered testing environment
(one chamber was illuminated and the other was dark) with a grid
floor and guillotine door separating the chambers. On training day,
mice were placed individually into the illuminated chamber for
1 min; the door was then lifted and remained open until the mouse
3.1. Binge-like alcohol administration exacerbates TBI-associated
pathology
Blood ethanol concentrations were assessed on the final day of
oral gavage (90 min following the gavage) and indicate a high level
of ethanol in the blood (0.4 g/dL) which did not differ between
sham and TBI mice (Fig. 2A). In order to determine the pathological
impact of high-dose alcohol exposure, we examined whether
binge-like alcohol levels further exacerbate axonal degeneration
in previously injured mice (Fig. 2B–C). As expected, TBI sustained
in pre-adolescence led to a significant degree of axonal degenera-
tion (U = 14.0, p < 0.05). However, a 10-day period of binge-like
alcohol administration in adulthood significantly exacerbated axon
damage in mice that had sustained a TBI as juveniles (H3 = 20.009,
p < 0.05). The overall significant Kruskall-Wallis test was followed
up by pairwise comparisons which indicated significant differences
between the sham/water and TBI/water groups, as well a signifi-
cant increase in degeneration in the TBI/ethanol mice compared
to TBI/water (all p < 0.05).
We further examined microglial morphology as a measure of
neuroinflammation. Brain tissue from all four groups was
processed for Iba1 immunohistochemistry. Sholl analysis of
 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311
Fig. 2. Binge alcohol administration exacerbates TBI-associated pathology. A) Blood
ethanol concentrations in sham and TBI mice 90 min after a gavage. B) Analysis of
silver staining in the forebrain white matter reveals that juvenile TBI-induced axon
degeneration is further exacerbated by repeated high dose administration of
alcohol in adulthood. C) Representative staining in the genu of the corpus callosum
(bregma 0.50 mm). Scale bar = 50 lm, an asterisk (⁄) indicates statistically signif-
icant differences between indicated groups (p < 0.05).
representative microglia in the prefrontal cortex revealed a
significant effect of injury history on microglial morphology
(F1,141 = 194.942, p < 0.01) such that mice that had sustained a
TBI exhibited microglia with fewer sholl ring intersections
(reduced process complexity), indicative of an activated state
(Fig. 3A). TBI also significantly reduced overall microglial process
length compared to sham injury (F3,145 = 3.346, p < 0.05; Fig. 3C).
An analysis of the total number of microglia in the prefrontal cor-
tex revealed main effects of both injury (F1,29 = 6.294, p < 0.05) and
ethanol treatment (F1,29 = 80,959, p < 0.01), as well as an interac-
tion (F1,29 = 8.382, p < 0.01) such that the ethanol-administered
TBI mice exhibited the greatest number of cortical microglia
(Fig. 3D). Finally, a measure of microglial cell body area revealed
a main effect of injury (F1,27 = 8.879, p < 0.001), and an interaction
(F1,27 = 5.427, p < 0.05) such that ethanol significantly increased
cell body area in TBI mice compared to all other groups (Fig. 3E).
3.2. Microglia from injured mice treated with alcohol show increased
pro-inflammatory gene expression
Given that both TBI and alcohol resulted in activated microglia,
and the increased total number of microglia in the TBI/ethanol
group, we examined the microglia directly in order to determine
whether the activational state corresponds to increased neuroin-
flammation. Both Toll-like receptor 4 (F3,16 = 3.516, p < 0.05; Fig. 4A)
and interleukin-1 beta (F3,19 = 3.311, p < 0.05) gene expression were
significantly increased in the TBI/ethanol group relative to all other
groups assessed.
3.3. High doses of alcohol impair learning and memory in previously
injured mice
Learning and memory was assessed via the passive avoidance
test in all four groups beginning 24 h after the final ethanol (or
water) administration day. This test takes advantage of the fear-
motivated tendency of mice to avoid brightly illuminated areas.
307
Latency to enter the dark chamber on the training day was
recorded and indicates no significant difference between groups
(Fig. 5A). However, analysis of the latency to enter the dark cham-
ber (in which each mouse had previously endured a painful shock)
revealed an interaction of injury history and ethanol treatment
(F1,34 = 5.247, p < 0.05). These data indicate that the TBI/ethanol
group exhibited a significantly shorter latency to enter the
shock-associated chamber compared to the TBI/water group.
Importantly, this difference is not likely to be a result of increased
anxiety in the TBI/ethanol group as all mice had similar latencies to
escape the light chamber on the training day.
3.4. Binge alcohol and TBI interact to induce ectopic expression of
doublecortin-positive cells in the hippocampus
Given the learning/memory impairment observed in the TBI/
ethanol mice, we conducted an immunohistochemical analysis of
doublecortin in the hippocampus. Doublecortin (DCX) is tran-
siently expressed in progenitor cells committed to the neuronal
lineage and serves as an indirect marker of hippocampal neuroge-
nesis. DCX is expressed for approximately 2–3 weeks in newborn
neurons before it is replaced by mature neuronal markers
(Brown et al., 2003), thus brain tissue fixed 13 days after the start
of the ethanol administration paradigm is likely to contain a sub-
stantial subset of neurons developing during our experimental
manipulation. Here, we assessed DCX-positive cells in the dentate
gyrus (Fig. 6). The total number of DCX-positive cells in the dentate
gyrus granule cell layer did not differ significantly by injury or
ethanol manipulations. However, we identified a significant
increase in the ectopic expression of DCX-positive neurons in
ethanol-administered TBI mice. Specifically, the TBI/ethanol group
had significantly more DCX-positive neurons in the dentate hilus
(F1,33 = 7.129, p < 0.05) than any other group. Moreover, among
mice that sustained a TBI, binge ethanol significantly increased
DCX expression in the molecular layer (T14 = 2.420, p < 0.03).
4. Discussion
Binge-like levels of alcohol produce significant neuropathology,
neuroinflammation, and functional deficits in mice that had expe-
rienced a traumatic brain injury during juvenile development.
Specifically, alcohol administration led to greater axonal degener-
ation and microglial reactivity in mice that had been previously
injured. Further, microglia isolated from previously injured ani-
mals exhibited significantly increased gene expression for both
TLR4 and IL-1b following alcohol administration. The combination
of binge-like levels of alcohol and TBI also impaired normal neuro-
genesis, resulting in increased ectopic expression of DCX-positive
cells in the hippocampus. Finally, the combination of high-dose
alcohol and juvenile TBI produced impairments in learning and
memory as assessed in the passive avoidance paradigm. Taken
together, these data indicate that a history of traumatic brain
injury significantly alters the neural and functional responses to
binge-like levels of alcohol and that the deleterious consequences
of high levels of alcohol in human populations may be related to
inflammatory responses and impairments in neural plasticity.
Imaging and post-mortem studies have revealed pronounced
neuroinflammation and degeneration, particularly of white matter,
in the brains of alcoholics. Specifically, there is evidence of ongoing
inflammation, microglial activation, and cortical and subcortical
atrophy (Harper et al., 2003; He and Crews, 2008; Ikegami et al.,
2003). The neurotoxicity of alcohol varies across the lifespan, but
is most prominent in cases of long term heavy drinking. Similarly,
axonal degeneration is a key pathological marker of TBI, however
this pathology resolves over time following a mild injury. Here
308 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311

Fig. 3. Binge alcohol and TBI alter microglial morphology. A) Sholl analysis in the prefrontal cortex reveals that both alcohol and TBI reduce microglial process complexity,
indicative of an activated state, as seen in B) representative images, scale bar = 50 lm. C) TBI also significantly reduced process length. D) The total number of microglia were
significantly increased in ethanol-treated mice that sustained a juvenile TBI. E) The cell body area of cortical microglia were also significantly increased in ethanol-treated TBI
mice. An asterisk (⁄) indicates statistically significant differences between the indicated groups (p < 0.05).

Fig. 4. TBI and alcohol administration increase proinflammatory gene expression. Relative mRNA gene expression is shown relative to the sham/water group and indicates
that A) TLR4 and B) IL-1b is exacerbated by alcohol in mice that underwent juvenile TBI. An asterisk (⁄) indicates statistically significant differences between the indicated
groups (p < 0.05).

we report that chronic exposure to binge-like levels of alcohol con-
tribute to sustained axonal damage even when the drinking occurs
many weeks after the actual injury. Importantly, the binge-like
alcohol administration paradigm used in this study was not suffi-
cient to induce axonal degeneration in mice that had not experi-
enced a prior TBI; rather TBI renders axons vulnerable to the
toxic effects of high dose alcohol.
Mild traumatic brain injury, such as that used in the current
study, is characterized by a significant but transient inflammatory
response and a number of cognitive and emotional disruptions that
occur in the acute phase following a TBI typically improve as the
pathophysiology resolves. However, even mild neuroinflammation,
particularly when it occurs during development, has been shown
to prime or sensitize the neuroimmune system to later inflamma-
tory events. For instance, neonatal E. coli infection enhances the
cognitive deficits and microglial responses to lipopolysaccharide-
induced inflammation later in life (Bilbo et al., 2008, 2010). Fur-
ther, TBI itself can act as a priming stimulus as microglial reactivity
and behavioral depression induced by experimental inflammation
is enhanced in mice with a history of mild traumatic brain injury
(Fenn et al., 2014). A primary goal of the work presented here
was to determine whether an early life brain injury could similarly
potentiate the consequences of repeated high dose alcohol expo-
sure in adulthood.
 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311 309

Fig. 5. Learning and memory are mildly impaired in alcohol-treated mice that sustained a juvenile TBI. A) All mice exhibit a similar latency to enter the dark side of the
passive avoidance chamber during training. B) However, the latency to re-enter the dark, shock-associated, side 24 h later was considerably longer, but was significantly
reduced in alcohol-treated mice that sustained a juvenile TBI. An asterisk (⁄) indicates statistically significant differences between the indicated groups (p < 0.05).

Fig. 6. Binge alcohol increases the number of ectopic doublecortin-positive cells in the hippocampus of mice that sustained a juvenile TBI. A) While the number of total DCX-
positive cells did not differ in the granule cell layer, high doses of alcohol significantly increased the number of ectopic newborn neurons in the B) hilus and C) molecular cell
layers of the hippocampus (white arrows). D) Representative images of the dentate gyrus, scale bar = 100 lm. An asterisk (⁄) indicates statistically significant differences
between the indicated groups (p < 0.05). ML = molecular layer, GCL = granule cell layer.
310 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311
Neuroinflammatory responses are bidirectionally linked to alco-
hol abuse as neuroinflammation can increase drinking behavior
(Crews et al., 2011). Conversely, chronic exposure to high levels
of alcohol is neuroinflammatory as alcohol can activate glial cells
and promote the expression of cytokine and chemokine molecules
(He and Crews, 2008). Although the mechanisms of alcohol-
induced neuroinflammation are incompletely understood, alcohol
does appear to signal in part through toll-like receptors (Alfonso-
Loeches et al., 2010; Fernandez-Lizarbe et al., 2009). For instance,
the long term behavioral and neurochemical effects of adolescent
alcohol exposure require TLR4 (Montesinos et al., 2016).
Microglia isolated from the injured frontal lobes following the
binge-like administration of alcohol exhibited marked upregula-
tion of mRNAs encoding the genes toll-like receptor 4 and IL-1b.
Critically, this increase in expression of TLR4 and IL-1b only
occurred in mice that had been previously injured and treated with
binge-like levels of ethanol in adulthood. We have hypothesized
that TBI sets up a vicious cycle wherein TBI during development
primes inflammatory responses to exhibit exaggerated inflamma-
tory responses to alcohol, which in turn promotes increased drink-
ing (Weil et al., 2016a). As an extension of this theory, the
neurotoxic effects of juvenile TBI could also be exacerbated by high
doses of alcohol. The up-regulation of both TLR4, a potential medi-
ator of alcohol-induced inflammation, and IL-1b, a key proinflam-
matory cytokine, only in mice that had been injured and exposed
to high levels of alcohol would tend to support this hypothesis. It
remains unspecified why we did not detect changes in gene
expression in microglia from sham-injured mice although this lar-
gely agrees with our histological evidence including reduced
microglial numbers, activated morphology and axonal
degeneration.
In the current study, we report exacerbation of the TBI-induced
damage to white matter tracts by binge-like levels of alcohol in
accordance with increased numbers and activational state of
microglia as well as exaggerated inflammatory gene expression
profiles. Given that much of the tissue damage induced by chronic
exposure to high levels of alcohol appears to require microglial-
mediated inflammatory responses (Boyadjieva and Sarkar, 2010;
Chastain and Sarkar, 2014; Yang et al., 2014), we conclude that a
transient period of TBI-induced inflammation in juvenile develop-
ment is sufficient to significantly exacerbate the neurotoxic effects
of binge alcohol in adulthood.
Interestingly we report a differential effect of TBI and alcohol
exposure on microglial morphology, counts and gene expression
profiles. Specifically, both TBI and alcohol exposure reduced the
length of microglial processes as assessed by Sholl analysis but
only microglia from mice that were exposed to alcohol and were
previously injured were more numerous, had larger cell bodies
and upregulated TLR4 and IL1b. That microglial morphology does
not fully track gene expression profiles is consistent with previous
reports (McClain et al., 2011) and suggests that both alcohol expo-
sure and TBI are independently associated with changes in micro-
glial physiology and morphology but that can induce a fuller
activation when combined.
Moreover, exposure to binge-like levels of alcohol greatly
increased the presence of immature doublecortin-positive neurons
in the non-neurogenic molecular layer and hilus of the dentate
gyrus but did so only in mice that had experienced TBI. Impor-
tantly, the exposure to binge-like alcohol in the current study
occurred more than six weeks after the TBI, thus the DCX-
positive cells assessed at this time point were those cells that were
born around the time of alcohol exposure rather than at the time of
injury. Although the combination of injury and alcohol-exposure
increased the number of hilar DCX-positive cells, the total number
was relatively small. Further there were no differences in the num-
ber or localization of DCX-positive cells induced by injury alone
indicating that any acute effects of TBI on this cell population
had resolved by adulthood.
CNS trauma often leads to an increase in the proliferation rate of
neuronal precursors, and this phenomenon has been explored as a
potential replacement source for cells lost to trauma (Dash et al.,
2001; Kernie and Parent, 2010). However, this strategy is compli-
cated by reactive and aberrant neurogenesis that can occur after
injury, seizure, or following withdrawal from heavy drinking which
can render the brain more vulnerable to cognitive dysfunction,
excitotoxicity, and additional seizures (Parent et al., 2006). Ectopic
granule cells that have been identified in the brains of epileptic
animals and humans exhibit abnormal morphology, connectivity
and electrophysiological properties that contribute to hyperex-
citability (Parent et al., 2006; Scharfman et al., 2000). Additionally,
alcohol (and alcohol withdrawal) even in the absence of trauma
modulates neurotransmission and generally biases towards excita-
tory neurotransmission via changes in neuronal and glial neuro-
transmitter metabolism, transport, and receptor expression
(Adermark and Bowers, In Press; Dodd et al., 2000). Clinically,
TBI patients at high risk for post-traumatic epilepsy are advised
to abstain from alcohol as drinking is positively correlated with
the occurrence of seizures (Centers for Disease Control and
Prevention; Corrigan, 1995). The data collected here suggest that
an increased propensity for alcohol-induced ectopic neurogenesis
in the previously injured brain may at least in part, underlie this
relationship. Further, we cannot rule out the possibility that alco-
hol or alcohol-withdrawal induced seizures may also have
occurred and underlies the ectopic neurogenesis rather than the
other way around.
The behavioral results reported here, namely relatively subtle
deficits in simple associative learning and memory in the passive
avoidance paradigm, were conducted on animals experiencing
alcohol withdrawal (retention trials occurred approximately 48 h
after their final dose of alcohol). Importantly, although there were
statistical differences between alcohol-and water-treated injured
mice there were not statistical differences between the sham
injured and TBI groups. In any case, we elected to test animals dur-
ing withdrawal because we wanted to avoid any direct effects of
intoxication on behavioral outcomes. Additionally, it will be
important in future investigations to determine whether the mild
impairments in learning and memory persist or improve beyond
the acute phase of alcohol withdrawal. Given the enhanced axonal
degeneration some degree of long-term functional deficit is likely
and may require more extensive behavioral characterization. In
any case, these data provide further compelling evidence that
binge-like alcohol consumption negatively impacts functional out-
comes in animals that were previously injured. Importantly, over
the 10-day treatment paradigm, mice were administered a single
daily high dose of alcohol every 24 h, thus in effect these animals
experienced repeated cycles of intoxication and withdrawal over
a short period of time. In addition, although we did not assess
the estrous cycle, the prolonged alcohol exposure paradigm did
presumably span at least two cycles. Finally, the paradigm we uti-
lized produced high (but similar between sham-injured and
injured mice) blood alcohol concentrations. Such a high dose of
alcohol was utilized in order to evaluate whether neurotoxic doses
would produce worse outcomes in the previously injured brain,
however, it remains unclear whether a longer exposure to more
moderate levels of alcohol would also be deleterious.
5. Conclusions
The human epidemiological literature has clearly demonstrated
that 1) the TBI patient population consists of a high proportion of
heavy and binge drinkers, 2) some percentage of TBI patients con-
tinue to drink at high levels after TBI, and critically 3) that drinking
 K. Karelina et al. / Brain, Behavior, and Immunity 60 (2017) 304–311
after TBI impairs functional and occupational recovery and puts the
patient at risk for serious complications including additional brain
injuries and seizures. Here we begin to demonstrate the underlying
neurobiological mechanisms that underlie this phenomenon
including alcohol-induced axonal degeneration, ectopic neurogen-
esis, and enhanced neuroinflammation induced by alcohol in pre-
viously injured mice. This vulnerability to alcohol-induced
histopathology and functional impairment occurred despite a long
delay (more than 6 weeks) between the injury and the beginning of
binge-alcohol administration. These data reinforce the importance
of targeting substance abuse in this population and are of even
more importance given our previous observation that TBI can also
increase alcohol self-administration and reward.
Acknowledgments
This work was supported by an OSU Neurological Institute Pilot
Award and the National Institutes of Health (NS045758). The
authors gratefully acknowledge Joseph Abraham, Katie Centner,
Samuel Nicholson, Benjamin Sarac, and Lauren Martin for their
assistance and support on these experiments. We also thank Dr.
Kristina Kigerl for guidance through the microglial extraction pro-
tocol and Dr. Jacqueline Barker for assistance with the BEC assay.
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