Dynamic of Cytokine Gene Transcription (TNF-α, IL-1β, IL-6, IL-8) in Surgically Treated Colic Horses by Use of Real-Time PCR

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Dynamic of Cytokine Gene Transcription (TNF-α, IL-1β, IL-6, IL-8) in
Surgically Treated Colic Horses by Use of Real-Time PCR (RT-PCR)
Epstein, A.,1* Nir, E.,1 Eyngor, M.,2 Eldar, A.,2 Bdolah-Abram, T.,2 Kelmer, E.,1 Steinman, A.1 and
Bruchim, Y.1
The Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew
University of Jerusalem, Israel.
Department of Poultry and Fish Diseases, The Kimron Veterinary Institute, Israel.
* Corresponding author: Dr. Ana Epstein, DVM, The Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100,
Israel, Tel: +972-3-9688588 Fax: +972-3-9604079. Email address :ana6@hotmail.com
Horses are highly sensitive to endotoxemia and its complications. During systemic inflammatory response
syndromes such as seen in colic, acute phase proteins and cytokines enter into the circulation. In this study,
gene expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6)
and interleukin 8 (IL-8) were quantified using real-time PCR (RT-PCR) in 15 adult horses undergoing
colic surgery at different points of time (0, 6, 12, 24, 48 hours), before and after surgical intervention.
Gene expression of TNF-α was down-regulated while gene expression of IL-1β, IL-6 and IL-8 were
up-regulated in the first 12 hours. This study shows that RT-PCR is reliable and sensitive method for
detection of the changes in cytokines serum levels in horses with colic. This study demonstrated the presence
of inflammatory processes mediated by cytokines upon admission of horses for medical treatment before
surgical intervention.
Keywords: Horse; Colic Endotoxemia; Cytokines; RT-PCR; TNF-α.
Colic is one of the most common pathologies in equine
medicine requiring immediate medical and in some cases
surgical intervention (1, 2). During a colic event the horse
maybe suffering from a combination of intestinal hypoperfusion, stasis, ischemia and increased permeability, resulting in
transmural and transvascualar bacterial and their associated
endotoxin migration (4). Colic and its associated endotoxemia has been found to induce release of acute phase proteins
and cytokines resulting in cardiovascular and gastrointestinal
dysfunction complement activation, organ failure and death
(1, 4, 5, 6).
Real-time PCR (RT-PCR) methodology is increasingly used for determining cytokine production in humans
and animals (7, 8) due to the accuracy and reliability of
Epstein, A.
March 2016.indb 24
the method (7, 9). Results from human and laboratory
rodent studies suggest that cytokine expression levels along
with clinical signs can improve diagnosis, treatment and
prognosis of patients with endotoxemia and sepsis (10,
11, 12). This method has been described in septic foals
and in horses with infiltrative inflammatory bowel disease
and horses with herpes virus infection (13, 14, 15, 16, 17).
Expression of cytokine mRNA in neonatal foals has been
used as a surrogate for sepsis and bacterial virulence (13).
To the best of knowledge of the authors, measurement
of cytokine gene transcription has not been reported in
adult horses undergoing colic surgery. We hypothesized
that gene expression of selected blood biological markers
(i.e. TNF-α, IL-1β, IL-6, IL-8) can be detected by using
RT-PCR technique.
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Fifteen adult horses between the ages of 2-23 years of age
were included in this study. All the horses were presented
to the Koret School of Veterinary Medicine – Veterinary
Teaching Hospital (KSVM-VTH) between the years
2009-2011 with abdominal colic and were treated surgically. The study was approved by the Ethics Committee for
Animal Experimentation, Faculty of Agriculture, Food and
Environment, Hebrew University of Jerusalem.
Study design
Data retrieved from files which included physical examination parameters, routine blood analysis [complete blood
count (CBC), packed cell volume (PCV), total solids (TS),
urea and lactate] upon admission and during the following
48 hours of the hospitalization period. Five blood samples
were collected from the jugular vein in Tempus TM Spin
RNA isolation blood tubes (Applied Biosystems, Foster City,
California) during the hospitalization period; at induction
of general anesthesia before surgery (time 0) and after 6, 12,
24 and 48 hours. Blood samples were kept at -20°C until
The decision to perform surgical intervention was carried
out by an expert board certified equine surgeon based on
clinical signs and clinicopathological findings. Severity of
the disease was graded as mild, moderate or severe based on
intraoperative findings (perfusion state of intestine, motility,
and invasiveness of surgery procedure)
Anesthetic protocol
The anesthesia protocol was similar in all horses and included
premedication with xylazine (AnaSed, Lloyd, Shenandoah,
Iowa, USA) 0.8 mg/kg intravenously, induction with ketamine (Clorketam, Vetoquinol, Paris, France) 2.2mg/kg and
diazepam (Assival, Teva pharmaceuticals, Petach-Tikva,
Israel) 0.05 mg/kg intravenously, and maintenance with
isoflurane inhalant anesthesia (Nicholas Piramal, Andhra
Pradesh, India) delivered in 100% oxygen. All horses were
mechanically ventilated during the anesthetic period.
Monitoring during anesthesia included; respiratory rate,
pulse rate and quality, direct blood pressure, ECG and end
tidal CO2 (ETCO2). Dobutamine (Taro Pharmaceutical
Industries Ltd., Haifa, Israel) was administered in constant
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rate infusion in order to maintain mean arterial blood pressure above 80 mmHg. Lidocaine (Esracaine, Rafa, Jerusalem,
Israel) was administrated to 8/15 (53.3%) of the horses as
bolus of 1.8 mg/kg immediately after induction of anesthesia
followed by constant rate infusion of 50 µg/kg/min for the
next 24 hours based on the preference of the anesthesiologist.
PCR (RT-PCR) for relative measurement of
cytokine-specific mRNAs
Since a detection system that quantifies horse cytokines is
not available, and as most cytokines are transcriptionally
regulated, cytokine induction and quantification was assessed
through cytokine messenger RNA (mRNA) transcripts levels. Unrelated studies, comparing between the rise of mRNA
transcripts and the (ELISA) quantification of cytokines have
shown that these correlate, and that the assay is reproducible
(18, 19, 20).
cDNA extraction
Extraction of RNA was performed according to the protocol
of Applied Biosystems Tempus TM Spin RNA Isolation
by Nanodrop (ND 1000), and storage at -80°C up to use.
Dilution of samples was performed in order to obtain similar
concentrations of RNA in all samples (400ηg/µl). Ten µl was
taken from each sample and diluted with 10 µl of standard
reagents (buffer 4 µl, H2O 1 µl, DNTP 2 µl, Enhancer1 µl,
enzyms1 µl and primer 1 µl) (verso cDNA kit, Thermo Fisher
Scientific). RT-PCR reaction to product cDNA was carried
out with PTC-200 DNA engine according to protocol of
P7S1; first cycle for 30 minutes in 42°C and second cycle for
2 minutes at 95°C. Final volume of 20 µl of cDNA was kept
at -20°C. To normalize differences in quantity, stability and
expression between the samples, the reference, housekeeping gene GAPHD used, which is in wide use for RT-PCR
in horses. Primers for RT-PCR readings were stabilized to
between 100-200 base ranges. Each gene primer was read in
both directions in order to maximize sensitivity of the test.
RT-PCR reaction
The amount of expression of cytokines was determined by
the RT-PCR method used by Absolute Blue SYBR Green
Rox Mixes (Thermo scientific) according to the manufactor
instructions. Mixture for PCR included SYBR Green Mix
5 µl 2.5 µl cDNA (constant concentration of 1.4ηg/µl), 0.15
µl forward primer, 0.15 µl reverse primer and 2.2 µl of water
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(final concentration 300nM). Each well contained 10 µl.
Each plate had two rows for the same sample in order to
double-check results.
The reaction was carried out using StepOne Real Time
PCR (Applied Biosystems, USA), one cycle 95°C for 3
minutes, 35 cycles 95°C 30 seconds, 55°C for 30 seconds
and 72°C for 50 seconds. Finally, 45 cycles at 72°C for 5
seconds with increase of temperature every two cycles by
half a degree centigrade for determination of temperature
for increasing given gene.
Analysis of results
The amplification efficiency of each cytokine was reported
compared to GADPH mRNA expression (internal control),
through evaluating and analyzing comparative threshold
method (∆Ct) variation (final amount of cDNA template
= 25ηg/well). Relative quantification (RQ) was obtained
using the 2-∆∆Ct method (21) by adjusting the mRNA
cytokine expression to the expression of β-actin mRNA and
considering the adjusted expression in the control group as
reference (relative quantification RQ = 1). Data were analyzed by the Applied Biosystems StepOneTM software v2.0
and expressed as RQ. Descriptive statistics (mean ± standard
deviation of mean) was carried out to describe RQ in both
in vivo and in vitro experiments.
All parameters were normally distributed and reported as
mean ± standard deviation (SD), were compared between
the expressions of different cytokines by Student’s t-test.
Repeated measures ANOVA were used to assess changes in
continuous variables over time. For determination of influence of onset of clinical signs to surgery, Fisher’s Exact Test
was used. For determination if the severity of disease has
influence of cytokine expression, Kruskal-Wallis Test was
used. Spearman’s rank correlations were used to assess the
correlation between continuous variables of the cytokine
level in the different sampling times. For all test applied
P<0.05 was considered statistically significant. All calculation was performed using statistical software. (SPSS 17.0 for
Microsoft Windows, SPSS Inc., Chicago, IL, USA.)
Fifteen horses, 2-23 years of age participated in the study,
with mean age of 11 years (SD ± 6.81 years). Mean lag time
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from the onset of clinical signs to the surgery was 13.4 hours
(SD ± 16.99 hours). Data on admission for all the horses are
presented in Table 1. There was no correlation between all
the measured cytokine expression (TNF-α p=0.949, IL-1β
p =0.820, IL-6 p=0.102, IL-8 p=0.151) and lag time from
the onset of clinical signs to surgery. There was no correlation
between the severity of disease and cytokine expression at the
first sampling time during induction before surgery (TNF-α
p=0.322, IL-1 β p=0.747, IL-6 p=0.396, IL-8 p=0.946).
Lactate levels upon admission was not statistically significant
(p= 0.903) different between mild to moderate and severe
classified severity groups (26.80±13.72, 30.45±17.00 and
39.25±25.85, respectively).
Mean heart rate upon admission was 54.1 beats per
minute (SD ± 11.5 BPM) and mean respiratory rate of 22.7
breaths per minute, (SD ± 10.7 breaths per minute). There
was no correlation between clinical signs (heart and respiratory rate, temperature, and mucosal membrane color), and
cytokine expression at admission and during hospitalization
In 8 of 15 (53%) horses, increased expression of IL-1β
was seen at induction before surgery (time 0) comparing
with reference gene GAPDH (RQ>1). In 11 of 15 (73%)
and in 13 of 15 (87%) horses IL-6 and IL-8 were increased
at the first sampling time at induction before surgery All
three interleukins reached peak levels 6 hours post induction
and then gradually decreased over time (Figure 1). In contrast, TNF-α levels in 13/15 (87%) horses, were lower than
GAPDH (RQ>1) at anesthetic induction, reaching minimal
level after 12 hours and then gradually increasing over time
but staying below the reference range for all measurements
(Figure 1). There was no difference in cytokine expression
profile between horses treated with lidocaine and untreated
horses for all measured points during 48 hours of hospitalization period.
The pathophysiology of colic is characterized by disturbances in gastrointestinal blood flow, leading to an increase
in intestinal permeability (22). This may result in leakage
of endotoxins into the blood stream and consequently
may lead to systemic endotoxemia, systemic inflammatory
response syndrome (SIRS), multiple organ dysfunction syndrome (MODS) and death (23). On the other hand, antiinflammatory cytokines that are released later in the course
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Table 1: Age, basic physical parameters, lactate level, diagnosis, severity of the colic and cytokine
level in 15 horses surgically treated for colic upon admission.
hyperemic 12 hrs
Operative diagnosis
Large intestine impaction
Pale dry
24 hrs
Cecum torsion
6 hrs
Large colon torsion
48 hrs
Large colon impaction
6 hrs
Pelvic flexture impaction
Pale dry
4 hrs
Large colon volvulus
6 hrs
Pelvic flexture sand
6 hrs
Epiploic entrapment
8 hrs
Large intestine torsion
8 hrs
Small colon impaction
12 hrs
Nephrospl. entrapment
20 Yellow pale 12 hrs
hyperemic 12 hrs
Diaphragm. hernia
Nephrospl. entrapment
Pale dry
6 hrs
Nephrospl. entrapment
Pale dry
12 hrs
Lactate Severity** TNFα4 IL-1β5 IL-66 IL-87 Lidocaine***
0.236 0.48 0.461
0.752 2.63
0.405 42.48 6.33
3.966 0.33
0.664 0.829 1.575
0.852 1.509 2.96
1.616 2.083
1. heart rate per minute; 2. respiratory rate per minute; 3. mucous membrane color; * lag time from the onset of clinical signs to admission to the
hospital; ** based on intraoperative findings; 4. tumor necrosis factor α; 5. interleukin1β; 6. inteleukin 6; 7.interlukin 8; *** treatment with IV. lidocaine
in constant rate infusion.
Figure 1: Relative gene expresion of cytokines (TNF-α, IL-1β, IL-6,
IL-8) over the time in horses undergoing colic surgery
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of the disease confront pro-inflammatory effects and in so
doing limit the inflammatory response (6). In case of failure,
pro-inflammatory states will predominate and may lead to
severe complications (6). In this study, we have followed the
dynamics of expression of TNF-α, IL-1β, IL-6 and IL-8
which have been previously shown to have a role during
sepsis and surgical interventions (23, 24, 25).
In the present study, levels of TNF-α were decreased
from the first sampling before surgery at the induction of
anesthesia continuing to decrease over the next 12 hours
and then gradually increasing over time and staying below
reference range even after 48 hours in all horses. TNF-α
was the first cytokine to be detected after exposure to LPS
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(26). In experimental studies, TNF-α reach peak expression
60 minutes after exposure to LPS, returning to basal levels
at 180 minutes and kept decreasing due to initiation of the
production of anti-inflammatory cytokines in the latter stage
of disease (1, 14, 26, 28, 29). It acts as a potent activator
of neutrophils by mediating their adherence, chemotaxis
and degranulation, by vasodilatation and increased vascular
permeability and negative inotropic effects during toxic
and septic sepsis (27). Expression of TNF-α can peak early
during an inflammatory response and can decrease rapidly
after 1 to 2 hours, even during continuous infusion of LPS
and persistence of fever (14, 28, 29). Decreased expression of
TNF-α seen in this study indicates the relatively early stage
in the activation of inflammation and correlates with the
decreasing phase of TNF-α.
In contrast to TNF-α, the IL-1β levels were elevated from
the first sampling before surgery and 6 hour later, decreasing over time and reaching reference values after 48 hours.
Interleukin-1 beta is a key mediator of acute inflammatory
response to microbial invasion, inflammation, immunological
reactions, fever and tissue injury (27). IL-1β production may
be stimulated by a variety of agents, including endotoxin,
other cytokines, primarily TNF-α, microorganisms, and antigens (27). One of the most important biological activities
of IL-1β is its ability to activate T lymphocytes by enhancing
the production of IL-2 and expression of IL-2 receptors (27).
In addition, IL-1β elicits the release of histamine from mast
cells at the site of inflammation (30). Moreover, TNF-α and
IL-1β act in synergism in the initiation phase of the inflammatory reaction (31) sharing numerous biological activities.
Again, results of this study points on activation of IL-1β by
TNF-α in the early proinflamatory stage and endotoxemia.
Similarly, levels of the IL-6 were increased on the first
sampling before surgery and 6 hour later. It then started to
decrease being slightly below reference interval after 48 hours
from the time of the first sampling. IL-6 expression is in
part under the control of several endogenous pyrogens well
as by IL-1β and TNF-α (32). The members of this cytokine
family have pro- as well as anti-inflammatory properties and
are major players in acute-phase and immune responses of
the organism. Under the influence of IL-6, B lymphocytes
differentiate into mature plasma cells and secrete immunoglobulins with T-cell activation, growth, and differentiation
(23). In contrast to these pro-inflammatory effects, it shares
anti-inflammatory effects such as down-regulation of the
Epstein, A.
March 2016.indb 28
inflammatory cascade by inhibition of TNF- α synthesis (27,
32). Well established production of IL-6 in the first measurement in this study can explain the low levels of TNF-α
production and its decreased expression.
Levels of IL-8 were increased on first sampling before
surgery, peaking after 6 hours and then decreasing over time
and staying elevated compared to reference value after 48
hours. Interleukin-8 stimulates polymorphonuclear leukocyte
function by attracting these cells to sites of inflammation and
inducing expression of cell surface markers (30). It belongs
to a chemotactic cytokine family and it is responsible for
the chemotactic migration and activation of neutrophils and
other cell types (such as monocytes, lymphocytes, basophils,
and eosinophils) at sites of inflammation (30). Synthesis of
IL-8 is stimulated by TNF-α and IL-1β (32). This cytokine
is been used in the prognosis of critically ill patients (34, 35)
by itself or in combination with other inflammatory cytokines
such TNF-α, IL-1β an IL-6 (36). However in this study no
correlation was found between IL-8 and clinical presentation
or postoperative recovery from colic surgery.
Lidocaine has been shown to have anti-inflammatory
properties in the course of endotoxemia, due to a decrease
in production of pro-inflammatory cytokines (37, 38, 39,
40, 41). In order to achieve this effect, lidocaine was given
in experimental and clinical studies before or immediately
after induction of endotoxemia (37, 38, 39, 40, 41). Recently
experimental LPS injection to horses followed by a bolus
of lidocaine and constant rate infusion has shown that
horses treated with lidocaine had significantly lower clinical
scores and serum and peritoneal fluid TNF-α activity (39).
In the present study there was no significant difference
in the measured cytokines between lidocaine treated and
non- lidocaine treated horses. We assume that the timing
of lidocaine administration is crucial for its efficacy. In the
present study horses were already in advanced stages of the
clinical and inflammatory process in contrast to the above
mentioned experimental study (39) in which lidocaine was
administrated 20 minutes after the induction of the endotoxemia. Another two clinical studies evaluating the efficacy
of the treatment of lidocaine in canine gastric dilatation and
volvulus have shown results that support this theory (42, 43).
In the first retrospective study there was no difference in any
clinical, complication and outcome between treatment and
non-treatment groups (42), while in the second study there
was significant reduction in the complication occurrence (e.g
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arrhythmias and acute kidney injury) (43). It was concluded
by the authors of these two studies that the rate and timing
of the lidocaine administration was probably the reason for
the lack of influence in the first study (42).
This study has a few limitations. First, the number of
the horses was not large enough to reach any statistical
significance between treatment groups. Secondly, the retrospective nature of the study and lack of data made the
analysis weaker and less significant. In the present study
the presence of endotoxemia was not evaluated by specific
biomarkers (e.g LPS). However, in all the horses in the present study changes in the cytokine were noted in most of
the cases, emphasizing the inflammatory reaction in equine
colic with or without endotoxemia. Finally, screening was
done mainly for pro-inflammatory cytokines and inclusion
of anti-inflammatory cytokines such as IL-4, IL-5, IL-10
and IL-13 may contribute to better understanding of the
dynamic of the inflammatory process.
In conclusion, in this study we present the dynamics
of the expression of inflammatory cytokines over time in a
clinical setting of horses with colic. Cytokine production was
uniform and confirms that all acute abdominal diseases in
horses have similar pathophysiology, regardless of its primary
etiology. Decreased levels of TNF-α together with elevated
levels of Il-1, IL-6 and IL- 8 upon admission suggest a late
intervention in the course of inflammation. Further, larger
scale studies are needed to gain a better understanding of
the dynamics of cytokine in horses with colic production in
order to established prognostic and treatment guidelines.
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Israel Journal of Veterinary Medicine  Vol. 71 (1)  March 2016
17/03/2016 11:04:16

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