Background: of 10 rats. Group II: ? (Positive control

Background: Tramadol is available worldwide as a synthetic centrally acting
analgesic for treatment of moderate to severe acute or chronic pain. In
tramadol overdose, the most common findings which have been reported in many
cases are seizures. The aim of this work was to evaluate the role
of ILE in acute tramadol toxicity as regarding seizures in adult male albino rats.   Materials and
methods: The study was
carried out on eighty male albino rats weighted 180 – 220 g for 2 days. Rats
were divided into five groups as follows: Group I: ? (Negative control
group) consisted of 10 rats. Group II: ? (Positive control group)
contained 30 rats; normal saline group received 1.5 mL/min for 30 minutes,
ILE10 group received 10 ml/kg of Intravenous lipid emulsion, ILE19 group
received 19 ml/kg of Intravenous lipid emulsion. Group III: ? (tramadol
treated group) Rats treated with tramadol (150mg/kg) once (1/2 of LD50) orally
by gavage. Group IV: ? (tramadol and normal saline treated group)
contained 10 rats received tramadol & normal saline in the previous doses. Group
V: ? contained 20 rats; tramadol+ILE10 group and tramadol+ILE19 group.Throughout
the experiment, rats from each group were observed for occurrence of seizures. Also
Creatinine phosphokinase was estimated at end of the experiment. Results: The present study revealed No seizures in control groups (Negative
control, Normal saline, ILE10 & ILE19). While, seizures occurrence was
observed in 50% of the group treated with tramadol alone, while there was a
considerable decrease in seizures occurrence in groups that were treated with
ILE. However, this effect of ILE was best revealed in the dose of 10 mL/kg.  Also, serum CPK levels were dramatically raised after tramadol
toxicity, whereas ILE could powerfully reduce it to normal ranges when
administered in both doses (10 and 19 mL/kg), but the decrease was more
significant with dose of 10 mL/kg. Conclusion: There was a considerable decrease in seizures occurrence and
Creatinine phosphokinase level in tramadol-intoxicated groups that were treated
with Intravenous lipid emulsion.

Key words:
Tramadol, Intravenous lipid emulsion, seizure, Creatinine Phospho Kinase.

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1.     
INTRODUCTION

     Tramadol is a commonly
prescribed as analgesic that is structurally similar to morphine and codeine.
Its analgesic effects are recognized as a weak ?-opioid receptor agonist with
norepinephrine and serotonin reuptake inhibitor. Due to its opioid-like effect,
tramadol misuse is a public health concern (Vahabzadeh et al., 2013 &
Nazarzadeh et al., 2014).

   Shipton (2000)
mentioned that tramadol efficacy in a wide range of acute and chronic pain
states, its multi-formulation availability and its low serious side effect
except at high doses and in prolonged use, increased its short and long-term
use in hospitals and communities.

   The most serious and
significant adverse effects of tramadol are those of the central nervous system
including dizziness, headache, agitation, movement disorders and seizures (Kathy
et al., 2005(.

   In tramadol overdose, the
most common findings that have been reported in many cases are seizures and
hypotension. Less common findings are central nervous system depression,
tachycardia, nausea and vomiting (Shadnia et al., 2008(.

   Intravenous lipid emulsion
(ILE) is composed of triglycerides and a phospholipid emulsifier. It provides
calories and essential fatty acids within total parenteral nutrition (TPN) (Bania
et al., 2007).

   As local anesthetics are
commonly used in the emergency department, overdoses can lead to disastrous
complications including cardiac toxicity and arrest (Tierney et al., 2016).

   So that, ILE has been
reported to reverse cardiovascular collapse in overdoses of local anaesthetic
agents in addition to other medications (Jamaty et al., 2010).

   The aim of this study was
to study the effect of ILE in amelioration of tramadol-induced seizures in
adult male albino rats through observations of its occurrence and estimation of
Creatine phosphokinase.

                                          

2.     
Materials
and Methods:

 

2.1Chemicals:

1-Tramadol tablets: each one contains 225 mg tramadol hydrochloride
obtained from October Pharma Company.

2- Intravenous lipid emulsion (ILE): SMOFlipid™ 20%, 500 mL of
injectable emulsion that is a white homogeneous emulsion, obtained from
Fresenius Kabi Company, Austria.It is composed of (6% soybean oil, 6% medium
chain triglycerides, 5% olive oil and 3% fish oil).

2.2 Animals:

    The 
rat  has  become 
a species  of  choice 
in  toxicology  research because  of 
metabolic  similarities,  as 
well  as  their 
small  size, relatively quiet
nature, short life span and short gestation period (Johnson, 2007).

   Its numerical availability
provides grounds for its use in order to obtain relevant statistical evaluation
(Ghosh, 1984). 

   All  animals 
received  human  care 
in  compliance  with 
the Animal  Care  Guidelines 
and  Ethical  Regulations 
in  accordance  with ”The 
Guide  for  the 
Care  and  Use 
of  Laboratory  Animals” (Institute of  Laboratory 
Animal  Resources, 1996). 

2.3Experimental design:

    The 
study  was  carried 
out  on  80 
adult  male  albino 
rats,  their weights  ranging 
from 180- 220 gm for each, they 
were  obtained  from 
the  Animal  House 
of  the Faculty  of 
Veterinary  Medicine , Zagazig
University. The  study  had been conducted  in 
Animal  House  of 
the  Faculty  of 
Medicine, Zagazig University. 
Before  commencing  the 
experiment, all  animals  were 
subjected to 14  days  period 
of  passive  preliminaries 
in  order  to 
adapt themselves  to  their 
new  environment,  to 
ascertain  their  physical 
well-being  and to exclude any
diseased animals. The animals were housed 
in  plastic cages free  from 
any  source of chemical
contamination  under  controlled 
conditions  with  an 
ambient temperature  range  of  22
± 2 oC,  relative  humidity 
of  50 ±5%  and  a
12 hours  light–cycle.  Soft 
wood  shavings  were 
used for  bedding  and changed 
during  the  cleaning 
of  cages  on 
alternate  days. The  rats 
received  balanced  food 
rich  in  all stuffs 
necessary  to maintain  their 
health  before  and 
during  drug  administration.  It consisted of bread, barley and milk. Water
was offered in separate clean containers.

 The rats were divided into
five groups as the following: Group I (Negative control group): It
consisted of 10 adult male albino rats. Each rat of this group received only
regular diet and tap water for 48 hours. Group II (Positive control group):
It consisted of 30 adult male albino rats. Rats in this group were subdivided
into 2 subgroups:  Subgroup IIA
(Normal saline group): It consisted of 10 adult male albino rats. Each
rat of this group received normal saline 0.9% NaCl I.V. infusion with infusion
rate of 1.5 mL/min for 30 minutes (Jamaty et al., 2010) once. Subgroup
IIB (Intravenous lipid emulsion group/ILE): It consisted of 20 adult male
albino rats. Rats in this group further equally subdivided into 2 subgroups: Subgroup
IIB1: It consisted of 10 adult male albino rats. Each rat of this group
received 10 mL/kg of ILE (Carreiro et al., 2013) by I.V. infusion once. Subgroup
IIB 2: It consisted of 10 adult male albino rats. Each rat of this group
received 19 mL/kg of ILE (Carreiro et al., 2013) by I.V. infusion once. Group III (Tramadol group): It consisted of 10
adult male albino rats. Each rat of this group received a toxic dose of 150
mg/kg of tramadol (1/2 of LD50) by oral gavage once (Matthiessen et al.,
1998). Group IV (Tramadol + Saline group): It consisted of 10 adult
male albino rats. Each rat of this group received 150 mg/kg of tramadol   (1/2 of LD50) by oral gavage once (Matthiessen
et al., 1998). Thirty minutes later (Vahabzadeh et al., 2013), each
rat received normal saline 0.9% NaCL I.V. infusion with infusion rate of 1.5
mL/min for 30 minutes (Jamaty et al., 2010). Group V (Tramadol + ILE
group): It consisted of 20 adult male albino rats. Rats in this group
subdivided equally into 2 subgroups: 
Subgroup VA: It consisted of 10 adult male albino rats. Each rat of
this group received 150 mg/kg of tramadol (1/2 of LD50) by oral gavage once.
Thirty minutes later, each rat received 10 mL/kg of ILE (Carreiro et al.,
2013) by I.V. infusion once. Subgroup VB: It consisted of 10 adult
male albino rats. Each rat of this group received 150 mg/kg of tramadol (1/2 of
LD50) by oral gavage once. Then 30 minutes later, each rat received 19 mL/kg of
ILE (Carreiro et al., 2013) by I.V. infusion once.

   All
of the control and treated rats were observed for 48 hours for occurrence of
seizures using camera (Van Vliet et al., 2006) and digital video
recorder.

     Racine’s
scale, originally developed for the amygdala-kindling model, is also frequently
used as an intensity measurement in other experimental seizure or epilepsy
models (Lüttjohann et al., 2009).

 According to Racine
(1972), Classical stages of seizures are:

– Mouth
and facial movement.

– Head
nodding.

– Forelimb
clonus.

– Rearing
with forelimb clonus.

– Rearing and falling with forelimb clonus.   

   After
that rats were anesthetized by ether anesthesia and blood samples were
collected from the retro-orbital plexuses for estimation of creatinine
phosphokinase by spectrophotometry according to Graeber et al. (1981).

2.4 Statistical analysis

  
Data were analyzed by Statistical Package of Social Science (SPSS),
software version 22.0 (SPSS Inc., 2013).1.     
DISCUSSION

   Tramadol is an available
worldwide synthetic centrally acting analgesic for treatment of moderate to
severe acute or chronic pain (Nossaman et al., 2010), with effects
similar to those of codeine and 10 times less than  morphine (Marquardt et al., 2005).

   The mechanism of action of
tramadol has not yet to be fully clarified (Mehrpour, 2005).

  Most  reports 
suggested  that  the 
analgesic  activity and other
clinical effects of  tramadol  are  a
result  of  opioid 
and  non-opioid  mechanisms. Tramadol binds to the ? -opioid
receptors in the brain, though much more weakly than morphine. It is only
1/6000 of that of morphine although its selectivity for this sub-type of
receptor is greater than that of morphine (Volpe et al., 2011). 

   Tramadol, and its
metabolite, known as M1, have been found to bind to ?-opioid receptors thus
exerting their effect on GABAergic transmission, and to inhibit reuptake of
5-hydroxytryptamine (5-HT) and noradrenaline. The second mechanism is believed
to be important since the analgesic effects of tramadol are not fully
antagonized by the ? opioid receptor antagonist naloxone (Gasse et al.,
2000).

   Intravenous lipid emulsion
is used as a source of calories and essential fatty acids in patients who
require parenteral nutrition (Arslan et al., 2013).

  Intravenous lipid emulsion
has been promoted in the resuscitation of local anesthetic toxicity refractory
to conventional modes of resuscitation (Picard et al., 2009). 

   Also, its successful use
in case reports and animal studies of drug toxicity other than local
anesthetics has been reported (Arslan et al., 2013). 

   Kosh et al. (2010) recommended that ILE 20% should be available whenever patients
receive large doses of local anesthetics in operating rooms and emergency
departments.

   The results of present
study showed that seizures’ occurrence was observed in 50% of the group treated
with tramadol alone, while there were a considerable decrease in seizures
occurrence in groups that were treated with ILE. However, this effect of ILE
was best revealed in the dose of 10 mL/kg.

   The observations of the
present study were agreed with the results of Vahabzadeh et al. (2013),
who showed that ILE prevented tramadolrelated seizures in doses of 6 and 12
mL/kg.

   It has been shown that the
appearance of seizures with tramadol is not dose dependent (Talaie et al.,
2009). Seizures due to tramadol use may occur in both overdose and within
therapeutic dose range (Jick et al., 1998).

   Fewer  than 1% 
of  consumers  have  a
presumed  incident  seizure 
right  after their  first 
tramadol   prescription.  Risk of  
seizure claim increases two- to six-fold between users adjusted for
selected comorbidities and concomitant drugs. Risk  of 
seizure is highest among those aged 25-54 years, those with more than
four tramadol  prescriptions, and  those 
with  a history  of 
alcohol  abuse, stroke, or
head  injury (Gardner, 2000).

   Seizures caused by
tramadol are most often tonic-clonic seizures, more commonly known as grand-mal
seizures. Also when taken with Selective serotonine reuptake inhibitors, there
is an increased risk of serotonin toxicity, which can be fatal (Gillman,
2005). 

   The GABA receptor
inhibition induced by tramadol can be secondary to its opioid receptor agonist
activity (Rehni et al., 2008), and continuing this agonist activity on
opioid receptor has been proven to precipitate seizure due to inhibition of
GABA pathways (Miura et al., 2007(.

   The most severe
complications of tramadol overdose include refractory seizures, rhabdomyolysis
and renal failure (Taghaddosinejad et al., 2011).

   Tramadol may increase the
seizure risk in patients receiving other medications such as tricyclic
antidepressants, phenothiazines, and selective serotonin reuptake inhibitors (Kroenke
et al., 2009).

   While tramadol-related
seizures can be controlled by diazepam, they are not responsive to naloxone,
and tramadol-induced seizures can be precipitated by administration of
naloxone, at high tramadol doses (Raffa and Stone, 2008).

   Corman and Skledar (2007) reported that no further convulsions were observed after ILE
infusion in Local Anesthetic-induced CNS toxicity. 

   Levine et al. (2012) stated that ILE infusion seizures and dysrhythmias experienced in
patient with TCA toxicity were terminated within 2 minutes of the second ILE
bolus; however, they weren’t rapidly to traditional treatment represented by
benzodiazepines and sodium bicarbonate.

   The clinical effect of ILE
infusion on reversal of CNS and cardiac toxicity with serial Local anesthetics
plasma concentrations or other lipophilic drugs is mainly attributed to its
mechanism of action by reestablishing equilibrium within an expanded plasma
lipid phase (sink) with subsequent reduction in free drug levels (Cave and
Harvey, 2009), also by extracting lipophilic drugs from tissues or by
counteracting local anesthetic inhibition of myocardial fatty acid oxygenation (Corman
and Skledar, 2007).

   Regarding effect of ILE on tramadol-induced
seizures can be also attributed to the lipid sink theory as ILE removes
tramadol from the tissues preventing it from coupling to its receptors (Vahabzadeh
et al., 2013).

   In this study, Serum CPK
levels were dramatically raised after tramadol toxicity, whereas ILE could
powerfully reduce it to normal ranges when administered in both doses (10 and
19 mL/kg), but the decrease was more significant with dose of 10 mL/kg.

   The previous result agreed
with Afshari and Ghooshkhanehee (2009) & Tashakori and Afshari (2010),
who reported dramatic rise of CPK in cases with tramadol overdose because of
frequent seizures.

   While Vahabzadeh et al.
(2013) stated that ILE could powerfully
reduce it to normal ranges when administered in high dose.

   The serum level of
skeletal muscle enzymes is a marker of the functional status of muscle tissue
and varies broadly in both pathological and physiological conditions. Upsurge
in these enzymes may represent an index of cellular necrosis and tissue damage following
acute and chronic muscle injuries (Brancaccio et al., 2007(.

    In tissues and cells that consume ATP rapidly,
especially skeletal muscle, brain, photoreceptor cells of the retina, hair
cells of the inner ear, spermatozoa and smooth muscle, CPK serves as an energy
reservoir for the rapid buffering and regeneration of ATP in situ, in addition
to intracellular energy transport by the CPK circuit or shuttle. Therefore, CPK
is an important enzyme in such tissues (Wallimann and Hemmer 1994).

   Total CPK levels depend on
age, gender, race, physical activity, muscle mass, and climatic condition (Stomme
et al., 2004).

   Young adult males have
high serum levels of CPK, which decline to some extent with age during the
geriatric period (Borges and EssenGustavsson, 1989 & Tietz et al., 1992).

   The serum CPK level can be
elevated from the damage of the muscle tissue because of intense prolonged
training. This may be a consequence of both metabolic and mechanical causes.
Certainly, metabolically exhausted muscle fibers exhibit a decrease in the
membrane resistance following an increase in the internal free calcium ions,
which promotes the activation of the potassium channel (Fink et al., 1983).

    Another mechanism could be the local tissue
damage with sarcomeric degeneration from Z-disk fragmentation. CPK is an
indicator of muscle necrosis, increasing with its extent (Nakada et al.,
1984 & Martinez-Amat et al., 2008).

   Creatine phosphokinase
acts as an enzyme in the conversion of creatine to phosphocreatine, producing
adenosine diphosphate and consuming ATP in between.           Such enzyme reaction is reversible;
therefore, ATP can be produced from phosphocreatine and adenosine diphosphate (Goldblatt
et al., 1969 & Vahabzadeh et al., 2013).

   In normal cellular
conditions, fatty acids are the preferred substrate for oxidative
phosphorylation, providing up to 90% of ATP particularly for myocytes. One
alternative mechanism of action suggested for ILE is that it can provide cells
with ATP (Rothschild et al., 2010).

    Use of ILE as antidote
remains a promising field demanding further preclinical study and systematic
reporting of human cases of use. Also, Further
studies are needed to establish the most effective dose of ILE in order to
minimize the risk of using higher doses.