J. Comp. Path. 2020, Vol. 179, 45e51 Available online at www.sciencedirect.

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SPONTANEOUSLY ARISING DISEASE

Serum Concentrations of Neuron-Specific Enolase

in Dogs Following Traumatic Brain Injury
O. Chai, M. Mazaki-Tovi, S. Klainbart, E. Kelmer, A. Shipov
and M. H. Shamir
Koret School of Veterinary Medicine, Veterinary Teaching Hospital, Hebrew University of Jerusalem, Rehovot, Israel

Summary
The ability to make an accurate prognosis, which is a prerequisite for treatment decisions, is very limited in
dogs with traumatic brain injury (TBI). To determine whether serum concentrations of neuron-specific
enolase (NSE) have prognostic value in dogs following TBI, we conducted a prospective, observational,
controlled clinical study in an intensive care unit of a university teaching hospital. The study population
comprised 24 dogs admitted to the hospital within 72 h of a known event of TBI between January 2010
and January 2015, as well as 25 control healthy shelter dogs admitted for elective neutering. Seventeen
injured dogs (70%) survived to discharge, four were euthanized and three died within 48 h. Serum samples
were obtained from all dogs (in injured dogs, within 72 h of TBI) and NSE concentrations were measured
using enzyme-linked immonosorbent assay. Associations between NSE levels and outcome, Modified Glas-
gow Coma Scale, time to sampling, age or haemolysis scale were determined. Mean serum NSE concentra-
tions were decreased in dogs with TBI compared with healthy controls (19.4
4.14 ng/ml vs. 24.9
4.6 ng/
ml, P <0.001). No association was found between serum NSE concentrations and either survival or severity
of neurological impairment. A negative correlation was found between serum NSE concentrations and time
from trauma to blood collection (r ¼ 0.50, P ¼ 0.022). These results indicate that serum NSE concentration
in dogs following TBI is not an effective marker for severity or outcome. Further studies are warranted to
standardize serum NSE measurements in dogs and to determine the peak and half-life levels of this potential
biomarker.

Ó 2020 Elsevier Ltd. All rights reserved.

Keywords: dog; neuron-specific enolase; prognosis; traumatic brain injury

Introduction matic injuries in dogs, as indicated by reported mor-

Traumatic brain injury (TBI) can result from
external mechanical forces, such as road traffic acci-
dents, falls from height, penetrating injuries, physical
assaults and crush or missile injuries (Platt et al., 2001;
Simpson et al., 2009; Sande and West, 2010; Lorenz
et al., 2011; Friedenberg et al., 2012; DiFazio and
Fletcher, 2013; Steinmetz et al., 2013; Beltran et al.,
2014; Hall et al., 2014; Sharma and Holowaychuk,
2015; Yanai et al., 2015; Dewey and Fletcher, 2016).
The prognosis for TBI is poor relative to other trau-
Correspondence to: O. Chai (e-mail: orit18m@gmail.com).
tality rates of 15e24% (Platt et al., 2001; Simpson
et al., 2009; Hall et al., 2014; Sharma and
Holowaychuk, 2015). The intensity and type of the
traumatic insult differ greatly, resulting in a variety
of clinical presentations and outcomes (Sande and
West, 2010; Friedenberg et al., 2012; DiFazio and
Fletcher, 2013; Steinmetz et al., 2013; Dewey and
Fletcher, 2016). Consequently, providing an accurate
prognosis is challenging and often impossible. As the
prognosis strongly influences treatment decisions
and course, providing an accurate prognosis is essen-
tial.

0021-9975/$ - see front matter Ó 2020 Elsevier Ltd. All rights reserved.
https://doi.org/10.1016/j.jcpa.2020.06.011
46 O. Chai et al.
Unlike human medicine, in veterinary practice
only a few prognostic indicators of survival following
TBI have been reported. These include the clinically
based Modified Glasgow Coma Scale (MGCS) (Platt
et al., 2001; Sharma and Holowaychuk, 2015)) and
the Animal Trauma Triage (ATT) (Sharma and
Holowaychuk, 2015; Ash et al., 2018), the scores of
which correlate linearly with the probability of sur-
vival or death, respectively (Platt et al., 2001;
Sharma and Holowaychuk, 2015; Ash et al., 2018).
Other predictors of death are laboratory findings sug-
gestive of decreased perfusion and oxygenation,
including base excess, bicarbonate, potassium, lactate
and blood urea nitrogen (BUN) (Sharma and
Holowaychuk, 2015). Recently, several groups pre-
sented magnetic resonance imaging (MRI) and
computed tomography (CT) neuroimaging findings
of potential prognostic value in dogs with TBI
(Beltran et al., 2014; Yanai et al., 2015; Chai et al.,
2017). However, those indicators have relatively low
specificity, are relatively expensive, require anaes-
thesia or sedation and have not been validated pro-
spectively. Therefore, their predictive value is
limited and there is a need for additional prognostic
tests that can be applied in the clinical setting as
sole indicators or in combination with other prog-
nostic indicators.
Several biomarkers for TBI have been examined in
human patients. These include neuron-specific
enolase (NSE), neurofilaments, cleaved-tau, microtu-
bule-associated protein 2, myelin basic protein, S100b,
glial fibrillary acidic protein, ubiquitin carboxyl ter-
minal hydrolase L1 and alpha-II spectrin breakdown
products (Yokobori et al., 2013; Kawata et al., 2016).
NSE, also known as gamma-enolase or enolase 2, is
a 78-kD dimeric isoenzyme of the glycolytic enzyme
enolase that is highly specific to brain tissue. It is local-
ized predominantly to the cytoplasm of neurons and
participates in slow axoplasmic transport (de Kruijk
et al., 2001; Li and Feng, 2009). In healthy people,
NSE is largely confined to neurons; however, baseline
serum concentrations of approximately 10 ng/ml orig-
inate from erythrocytes (Nygaard et al., 1998; Casmiro
et al., 2005). Sudden increases in serum NSE have been
reported after various types of neurological insults,
including TBI (Anand and Stead, 2005; Cheng
et al., 2014). Structural damage to neurons causes
leakage of NSE into the extracellular compartment,
from which it is drained to the bloodstream. Following
neuronal death secondary to TBI, NSE can be de-
tected in the serum (Bandyopadhyay et al., 2005),
where its half-life in humans is 24e72 h (Thelin
et al., 2017). These characteristics make NSE a prom-
ising prognostic biomarker for patients with TBI,
which can also be used as a therapeutic indicator for
neurological monitoring in intensive care settings
(Thelin et al., 2017). Indeed, multiple studies in hu-
mans have shown that serum NSE levels spike after
moderate to severe head injury (Dauberschmidt
et al., 1983; Skogseid et al., 1992; Kuroiwa et al.,
1993; Ingebrigtsen et al., 1995; Rothoerl et al., 1998;
Raabe et al., 1999; Woertgen et al., 1999; Vos et al.,
2004; Guzel et al., 2008; Meric et al., 2010). Moreover,
NSE levels were strongly predictive of either death or
unfavourable outcome post injury (Vos et al., 2004;
Guzel et al., 2008; Meric et al., 2010; Cheng et al.,
2014) and a cutoff value has been proposed for pre-
dicting poor neurological outcome (Vos et al., 2004;
Meric et al., 2010).
Information on serum NSE concentrations in dogs
with TBI or other primary brain lesions is lacking.
The aim of this study was to determine serum NSE
concentrations in dogs with TBI during the first
72 h after trauma and to test the hypothesis that these
levels are predictive of outcome.
Materials and Methods
Dogs admitted to a veterinary teaching hospital within
72 h of a known TBI event between January 2010 and
January 2015 were included. The diagnosis of TBI was
based on a history of recent (up to 72 h) head trauma
and clinical findings of neurological deficits that were
attributed to the head trauma. All dogs were hospital-
ized in the intensive care unit (ICU) and received
treatment according to clinical indications. All pro-
cedures were part of regular treatment and all owners
agreed that a surplus of blood, taken for clinical pur-
poses, could be used for the purposes of the study.
The collection of surplus blood was approved by the
institutional Ethics Review Committee.
Dogs that died were excluded from the study if the
cause of death was considered to be other than TBI.
Data retrieved from the medical records included
age, sex, weight, medical history, cause of trauma,
complete physical and neurological examination,
MGCS on admission (Supplementary Table 1),
time from trauma to admission (TTA), time from
trauma to blood collection and outcome (dead or
alive). The occurrence and frequency of seizures
were recorded and divided into three categories ac-
cording to the time of first occurrence: up to 24 h,
within 1e7 days, and more than 7 days following
the initial trauma.
Survival to discharge was considered as positive
outcome. Death of fatal cases was documented as
either spontaneous or as a result of humane eutha-
nasia.
Serum samples were obtained from all dogs within
72 h of TBI and from 25 healthy shelter dogs admitted
 Serum Concentrations of Neuron-Specific Enolase
to the Koret School of Veterinary Medicine Teaching
Hospital for elective neutering. Samples were sepa-
rated within 30 min and stored at 80 C. Because hae-
molysis can artefactually increase NSE concentrations,
the degree of haemolysis in each serum sample was
determined subjectively on a scale of 0e3 (0 ¼ no hae-
molysis; 1 ¼ mild haemolysis; 2 ¼ moderate haemoly-
sis; and 3 ¼ severe haemolysis). Serum NSE
concentrations were measured using the commercially
available Canine Neuron Specific Enolase Elisa
(enzyme-linked immunosorbent assay) Kit
(MBS736549, MyBioSource, San Diego, California,
USA) according to the manufacturer’s instructions.
This assay utilizes standards of canine origin and the
dynamic range of the assays was 1e5,000 pg/ml.
Serum samples were taken before fluid administration
and diluted 1:50 with 0.9% saline in preparation for
assay. All standards and samples were run in dupli-
cates. Intra- and inter-assay coefficients of variation
were 4e6% and 7e8%, respectively.
Statistical Analysis
Serum NSE concentrations were distributed normally,
as determined by the ShapiroeWilk test and evalua-
tion of QeQ plot. Mean NSE concentrations were
compared using an independent t-test or analysis of
variance with Bonferroni correction for multiple pair-
wise comparisons for two or more than two groups,
respectively. Associations between NSE concentra-
tions and MGCS, TTA, age or haemolysis scale were
determined using the Spearman correlation test, due
to non-normal distribution of the additional variables.
Data were analysed using the commercially available
statistics program SPSS 25.0 for Windows (SPSS
Inc, Chicago, Illinois, USA). Results are reported as
mean
SD or median and range for normally and
non-normally-distributed variables, respectively.
P <0.05 was considered statistically significant.
Results
Population Characterization, Clinical Findings, Modified
Glasgow Coma Scale Scores and Outcome
Twenty-four dogs fulfilled the inclusion criteria, of
which 12 (50%) were males (two castrated, 10 intact)
and 12 (50%) females (four spayed and eight intact).
Twelve dogs (50%) were of mixed breed, three were
Shih Tzu, two Yorkshire terriers, two Maltese
dogs and one of each breed: toy poodle, pug, border
collie, American Staffordshire terrier and Canaan
dog. Median age (n ¼ 22) was 2.75 years (range
0.3e12 years) and median weight was 6 kg (range
1.3e35 kg).
47
The most common cause of head trauma was road
traffic accident (15/24 dogs, 63%) followed by fall
from height (6/24 dogs, 25%), struck by an object
(2/24, 8%) and dog fight (1/24, 4%). Twelve dogs
presented within the first 8 h of the trauma, seven pre-
sented within 8e24 h and two within 24e72 h. The
other three dogs were admitted within 72 h of the
traumatic event, but the exact time interval could
not be determined. Concurrent injuries were docu-
mented in 8/24 (33%) dogs and included vertebral
fractures (2/8), rib fracture (2/8), pelvic fracture (1/
8), humeral fracture (1/8), brachial plexus avulsion
(1/8), haemoabdomen (1/8) and bile peritonitis (1/8).
Mental status and MGCS were documented on
admission in 22/24 dogs and were categorized as
depressed or obtunded in 9/22 dogs (38%), delirium
in 2/22 (8%), stupor or semi-coma in 6/22 (25%)
and coma in 5/22 (20%). Median MGCS score was
15 points (range, 4e17). Seizures were documented
during hospitalization in 3/24 dogs (13%). Two had
seizures in the first 24 h only and one had seizures 3
days after the trauma.
Seventeen dogs (70%) survived to discharge. Me-
dian hospitalization time was 3 days (range, 1e19
days). Of the seven fatal cases, four were euthanized
and three died during the first 48 h of hospitalization.
Serum Neuron-Specific Enolase Concentrations
The mean serum NSE concentration in dogs with TBI
was significantly lower than in healthy controls
Fig. 1. Serum NSE concentrations in healthy (n ¼ 25) and TBI
(n ¼ 24) dogs. Box plots of serum NSE concentrations as
measured by canine neuron-specific ELISA. Middle hori-
zontal bar indicates the median, box ends indicate the
25th and 75th percentiles, whiskers indicate the range and
circle indicates an outlying data point.
48 O. Chai et al.
(19.4
4.14 ng/ml vs. 24.9
4.6 ng/ml, P <0.001)
(Fig. 1). These results remained unchanged following
removal of one outlier in the healthy control group.
No significant correlation was found between serum
NSE concentrations and the haemolysis score.
Following exclusion of nine samples with evident hae-
molysis (haemolysis score >1), the mean serum NSE
concentration remained significantly lower in dogs
with TBI compared with control dogs
(18.7
3.9 ng/ml vs. 25.2
5.0 ng/ml, P <0.001).
There was no significant difference in the mean
serum NSE concentration between survivors and
non-survivors (19.8
3.61 ng/ml vs. 18.4
5.42 ng/
ml). NSE concentrations were not associated with
sex, age, cause of TBI, mental state, presence of con-
current injuries or MGCS score. Similarly, no signifi-
cant difference in the mean serum NSE concentration
was found between dogs with MGCS scores of <15
(n ¼ 11) or $15 (n ¼ 12) (18.7
4.6 ng/ml vs.
20.4
4.7 ng/ml). A moderate significant negative
correlation was found between serum NSE concentra-
tions and time interval between the traumatic event
and blood collection (r ¼ 0.50, P ¼ 0.022).
Discussion
Serum NSE has several potential advantages as a
biomarker for severity of nervous tissue injury. This
enzyme is highly specific to the brain in humans
and elevations were found to correlate with the
severity of neuronal damage, but not with glial cell
pathology (Cicero et al., 1970). Numerous studies in
humans with TBI demonstrated an increased serum
NSE concentration that correlates with poor outcome
(Dauberschmidt et al., 1983; Skogseid et al., 1992;
Kuroiwa et al., 1993; Ingebrigtsen et al., 1995;
Rothoerl et al., 1998; Raabe et al., 1999; Woertgen
et al., 1999; Vos et al., 2004; Guzel et al., 2008; Meric
et al., 2010).
Interestingly, in this study, serum NSE concentra-
tions were decreased in canine patients suffering from
TBI compared with healthy controls. In addition, no
association was found between serum NSE concentra-
tions and either survival or severity of neurological
impairment. We suggest that in contrast to humans,
serum NSE concentration in dogs with TBI is not a
good marker for severity or outcome. The fact that
the difference in the mean NSE concentration be-
tween the groups was statistically significant may
not necessarily represent clinical significance, as
the difference between the groups was small
(19.4
4.14 ng/ml vs. 24.9
4.6 ng/ml).
The reference range of serum NSE concentrations
in healthy humans is 5e12 ng/ml and an increase to
81.3 and 54.52 ng/ml has been reported in severe
and moderate TBI patients, respectively (Guzel
et al., 2008; Meric et al., 2010). A strong correlation be-
tween serum NSE levels and the degree of brain
injury, as assessed by clinical neurological examina-
tions, was also shown (Guzel et al., 2008; Meric et al.,
2010). In our study, the mean serum NSE concentra-
tion in healthy dogs (n ¼ 25) was 24.9
4.56 ng/ml,
which is higher than the mean pretreatment concen-
tration of 8.1
3.3 ng/ml that was previously reported
in 10 healthy dogs (Usui et al., 1994). To our knowl-
edge, this is the only other study to have tested serum
NSE concentrations in dogs. The difference may be
due to the use of different analytical methods in the
two studies. The canine-specific assay used in our
study was not available at the time of the other earlier
study, which used a rat-specific assay. Thus, the higher
reference range could potentially be attributed to a
higher sensitivity of our assay.
Dissimilar serum NSE concentrations, as well as
different release patterns after trauma, were also
noticed when human studies were compared (Thelin
et al., 2017). This finding may imply that the assays
and hence the results are not sufficiently comparable,
stressing the need for standardized testing.
It is assumed that following brain damage, NSE
leaks from injured neurons to the extracellular space,
is drained to the cerebrospinal fluid (CSF) and then
reaches the bloodstream. Although the central nervous
system lacks lymphatic vessels to assist in the removal of
interstitial metabolic waste products, recent work has
led to the discovery of the glymphatic system, a glial-
dependent perivascular network that functions as a
lymphatic-like system in the brain (Plog et al., 2015).
It was shown that CSF and interstitial fluid continu-
ously interchange by convective influx of CSF along
the periarterial space, which is mediated by glial aqua-
porin 4 water channels (Plog et al., 2015). The elevation
of biomarkers such as NSE in the serum necessitates an
intact glymphatic system. However, it has been shown
that in humans, glymphatic function can be suppressed
following TBI, causing reduction in serum NSE levels.
Moreover, the degree of glymphatic function suppres-
sion can cause variations in serum NSE concentrations
among individuals (Jessen et al., 2015; Plog et al., 2015).
One can assume that a similar physiological mecha-
nism exists in dogs; however, the implications of TBI
in dogs remain to be investigated.
The half-life of NSE in dog blood has yet to be
determined. Usui et al. (1994) examined serum NSE
in dogs before and after cardiac arrest to evaluate
brain damage secondary to hypoxia. They showed
that the serum NSE concentration increased slightly,
reaching a plateau after 2 h of reperfusion with a 4-
fold increase at 18 h after reperfusion (8.1
3.3
ng/ml vs. 23.5
7.0 ng/ml). In our study, serum
 Serum Concentrations of Neuron-Specific Enolase
NSE was measured up to 72 h from the initial trauma.
Based on studies in humans, NSE half-life is 24e72 h
(Thelin et al., 2017). NSE levels increase over the first
few hours post injury and are then maintained for
24e72 h, after which they slowly decline. In some
TBI patients, secondary peaks occurred due to sec-
ondary brain injury (Herrmann et al., 2000; Cheng
et al., 2014; Thelin et al., 2017). In our study, serum
NSE concentrations decreased as the time from injury
to blood sampling (up to 72 h) increased.
NSE concentrations measured in human patients
with mild TBI did not differ significantly from the
control group (de Kruijk et al., 2001; Geyer et al.,
2009; Shahim et al., 2014), suggesting a low sensitivity
of this biomarker in mild cases. In our study, 12 out of
the 23 dogs examined (52%) had a MGCS score >15
and were thus considered mild. This finding, together
with the relatively small number of severe TBI cases
in our cohort, could account for the lack of correlation
between NSE concentration and outcome.
This study has several limitations, including a small
sample size, heterogeneity in signalment and causes of
injury, relatively few severe TBI cases and the un-
known effects of the various treatments administered
to the dogs post injury. In addition, because erythro-
cytes contain NSE, haemolysis could have increased
serum NSE concentrations artefactually. Notably,
our results did not change when we excluded haemo-
lyzed samples from the statistical analysis. Neverthe-
less, evaluation of haemolysis was subjective and
therefore could have been inaccurate. Another limita-
tion is the lack of data on sex and age in the control
group. However, mean serum NSE levels in neurolog-
ically healthy human subjects have been reported to
be unrelated to age or sex (Nygaard et al., 1998;
Casmiro et al., 2005). Lastly, we only investigated a
single potential biomarker for neural damage. It is
plausible that measuring the concentrations of several
proteins is required for a meaningful prognosis in dogs
with TBI.
In conclusion, this study demonstrates that a single
measurement of serum NSE taken up to 72 h post
injury may not be a useful prognostic indicator for
dogs following TBI. Future studies are warranted to
standardize serum NSE measurements in dogs and
to investigate temporal changes in NSE concentra-
tions after TBI events, including over a shorter time
period, and thereby, determine the peak and half-
life of NSE in dogs.
Funding
This research did not receive any specific grant from
funding agencies in the public, commercial or not-
for-profit sectors.
49
Conflict of Interest Statement
The authors declare that there were no conflicts of in-
terests relating to this study.
Supplementary data
Supplementary data to this article can be found on-
line at https://doi.org/10.1016/j.jcpa.2020.06.011.
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