Neuroprotective effect of Deliberate Thiopental Coma as

demonstrated by Preservation of the Plasma Antioxidant

Potential during Carotid Endarterectomy

Authors:

HISHAM E.T. SOLIMAN, MD, FRCS *

ASHRAF A. ABDELBASET, MD **

SOMAYA E.T. SOLIMAN, MD ***

*Correspondence and Reprints:

HISHAM E.T. SOLIMAN, MD, FRCS
Saudi Aramco - Dhahran Health Center
Surgical Services Division, Room A-422
POBox 76, Dhahran 31311
Saudi Arabia
abustait@hotmail.com
Phone: 966-3-8778138 Fax: 966-3-8773695

* HISHAM E.T. SOLIMAN

Assistant professor, Surgical Department, Cairo University

** ASHRAF A. ABDELBASET
Assistant professor, Anesthesia Department, Ain Shams University

*** SOMAYA E.T. SOLIMAN

Lecturer, Clinical Pathology department, Nuclear Research Center, Atomic
energy Authority
ABSTRACT

Background:
Barbiturates and several other anesthetics, intravenous and inhalational agents,
have been shown to have neuroprotective effects against focal and global brain
injury. Among most anesthetics, Thiopental was suggested to have peculiar
neuroprotective mechanisms against free radical production during
ischemia/reperfusion. However, this effect has not been investigated yet in
clinical trials. This study was designed to demonstrate the antioxidant effect of
Thiopental induced coma as identified by changes in plasma antioxidant potential
(PAP) levels.

Patients and Methods:

Thirteen patients underwent carotid endarterectomy (CEA) under thiopental
induced coma were compared with 10 control cases underwent the same
procedure without induced coma. Blood was sampled from a venous catheter
inserted in the ipsilateral jugular bulb and from an arterial catheter to monitor
changes in oxidant stress; a colorimetric assay was used to determine PAP. Brain
ischemia was monitored by continuous jugular venous bulb oxygen saturation
(SjO2).

Results:
Jugular venous PAP decreased significantly from 32.8 (5.42) % inhibition to 28
(6.77) % inhibition among controls (P< 0.05), whereas in the thiopental treated
group, it was dropped from 33.9 (5.4) inhibition to 30.8 (4.5) inhibition (P>0.05).
There was no concomitant change in arterial PAP values, indicating a decrease in
antioxidant capacity across the cerebral circulation. There was a significant
increase in the arterio-jugular venous PAP level difference during reperfusion
compared with baseline among the control group compared to the thiopental
treated group, (P<0.05).

Conclusions:
Thiopental induced coma significantly preserve the plasma antioxidant capacity
during carotid endarterectomy, thus providing a protective mechanism against
delayed neuronal injury from ischemia/reperfusion.
Key words: Neuroprotection-Thiopental–carotid endarterectomy –
ischemia/reperfusion – plasma antioxidant potential
Short title: Neuroprotection during carotid endarterectomy
INTRODUCTION
Carotid endarterectomy has been proven to reduce the incidence of ipsilateral
strokes in patients with symptomatic and asymptomatic carotid stenosis ≥70%.
On average, however, 2% to 6% of all patients undergoing carotid
endarterectomy sustain a stroke in the perioperative period . Intraoperative
(1-3)

embolism and hypoperfusion are possible causes of a perioperative neurological

deficit due to clamping of the carotid artery (4).
Two major hypotheses have been developed to account for the phenomenon of
ischemia/reperfusion–induced neuronal injury. The neurotransmitter hypothesis is
related to the role of excitotoxic amino acids and is preferentially aimed at events
during the acute period of ischemia. The free radical hypothesis is directed at
events during reperfusion. During reoxygenation after cerebral ischemia, oxygen
free radicals are produced (5, 6) ,The generation of free radicals initiates a vicious
cascade of tissue injury, in particular, peroxidation of phospholipids with
consecutive alteration of membrane structure (7).

Direct evidence of oxidant-mediated tissue injury would be the ideal method for
demonstration of this phenomenon in clinical trials of focal cerebral ischemia as
carotid endarterectomy. However, in this type of surgery, brain tissue is not
available for analysis. An alternative method is to obtain evidence of free radical
production from indirect markers of oxidative injury such as changes in plasma
antioxidant potential (PAP). The antioxidant levels across the cerebral circulation
were shown to be significantly decreased during ischemia with a further
significant decrease on reperfusion . This is consistent with the hypothesis that
(8)

cellular changes during ischemia produced free radical species on reperfusion

that consumed antioxidants in plasma (9, 10).

Efforts aimed at preventing or limiting neuronal tissue damage are usually to

optimize cerebral perfusion pressure, decrease metabolic requirements (basal
and electrical), and possibly block mediators of cellular injury. Clearly, the most
effective strategy is prevention, because once injury has occurred measures
aimed at cerebral protection become less effective (11).

General anesthetics, intravenous and inhalational agents, have been shown to

have neuroprotective effects against focal and global brain injury with
controversial results between animal and human studies . Among the
(12-15)

anesthetics, barbiturate was found to have a special neuroprotective effects. The

mechanism of neuroprotection provided by barbiturates has not been fully
determined but has mainly been attributed to a reduction in cerebral metabolic
rate through reduction of calcium influx, sodium channel block, inhibition of free
radical formation, potentiation of GABA-nergic activity and inhibition of glucose
transfer across the blood-brain barrier. Other possible protective mechanisms
are reduction of cerebral blood flow with less edema, inhibition of excitotoxicity (16)
,attenuation of hypoxia-induced increase in blood-brain barrier permeability (17, 18)

,and scavenging of free radicals (19) .

In this study, carotid endarterectomy has been used as a clinical model of focal
cerebral ischemia/reperfusion for studying the influence of thiopental induced
coma on free radical production; identified by changes in plasma antioxidant
potential (PAP) levels.
PATIENTS AND METHODS
The study was carried out between June, 2001 and July, 2004 (3 years). Patients
undergoing carotid endarterectomy were enrolled into the study if they fulfill the
inclusion criteria of, being symptomatic with ipsilateral significant focal stenosis at
the bifurcation of the carotid artery as ascertained by conventional digital
subtraction angiography. Patients with chronic respiratory or renal diseases were
excluded from the study. All patients gave written consent prior to start of the
study.
One hour before surgery, patients received 5mg of oral midazolam as
premedication. General anesthesia was induced with Fentanyl 2 ug/kg and
thiopental 3 to 4 mg/kg. Endo-tracheal Intubation was facilitated with
administration of Atracurium 0.5 mg/kg and additional doses were given guided
by peripheral nerve stimulator (Innervator, Fisher& Paykel healthcare model NS
252A). Anesthesia was maintained with 60% nitrous oxide in oxygen
supplemented with Isoflurane and additional doses of Fentanyl as clinically
indicated. Ventilation was controlled to keep a PaCO2 around 30 mmHg. Routine
intraoperative monitoring includes ECG, oxygen saturation, rectal temperature
and end tidal CO2. Blood pressure was monitored invasively through an arterial
catheter inserted in the redial artery.
A full EEG leads were inserted in the scalp by a neurology technician and a base
line EEG wave form was obtained and recorded (CFM, Lectromed Ltd).
After exposure of the carotid artery, a 5F catheter was inserted into the internal
jugular vein under direct vision through the common facial vein and advanced
until its tip lay in the jugular bulb. SjO2 (jugular bulb venous oxygen saturation)
was continuously measured with an Oximetry system (Drägger infinity modular
monitoring system, SC 7000)
Before arterial clamping, patients were randomly assigned to one of two groups.
Group (A) received Thiopental infusion which was given in small increments (50
mg) using syringe pump 50 cc containing thiopental 20mg/ml (Aitecs plus SEP-
125 syringe pump model 12 PL-1300) till iso-electric EEG was obtained. Group
(B) did not receive thiopental, as a control. Dopamine infusion was used to
support arterial blood pressure to acceptable level, 20% from the baseline.
Jugular venous blood samples were collected for PAP (plasma antioxidant
potential) assay at three fixed points (1) before carotid cross-clamping as
baseline, (2) before re-establishment of the shunt flow “reperfusion”, and (3) 15
minutes after reperfusion, respectively. Samples were also taken from the arterial
line to determine any changes in the PAP across the cerebral circulation.
Changes in jugular bulb venous oxygen saturation (SjO2) were correlated with
indices of free radical production during cerebral ischemia/reperfusion.
All cases were shunted and the carotid arteriotomy was closed with patch
angioplasty. Heparin (50-75 mg/Kg) was given intravenously to all patients before
carotid clamping.
Sample Preparation
For analysis of PAP, 2 ml of blood was sampled into a heparinized syringe. All
samples were immediately centrifuged at 8000 rpm for 5 minutes, and the plasma
layer was aspirated and stored in liquid nitrogen until transfer to -70°C for long-
term storage. All samples were analyzed within 1 month of collection at the
Nuclear Research Center, Atomic Energy authority, Egypt.

Materials
Myoglobin type III and Sephadex G-15-120 (40 to 120 µm) were purchased from
Sigma and ABTS, hydrogen peroxide, potassium ferricyanide, Trolox (Hoffmann–
La Roche). All chemicals were the highest purity available.

PAP Assay
This method (8) is based on the interaction of the phenothiazine compound ABTS
(2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) with ferryl myoglobin,
produced through the reaction of metmyoglobin with hydrogen peroxide. This
leads to the production of the ABTS+ free radicals, which can be measured
spectrophotometrically at 650, 734, and 820 nm. In the presence of antioxidants
the absorbance of this radical cation is quenched to an extent directly related to
the concentration of antioxidants present in the added fluid.
Briefly, ABTS (300 µL, 500 µmol/L), metmyoglobin (36 µL, 70 µmol/L), and 497
µL of buffer (of which 8.4 µL was replaced when a sample was being
investigated) were mixed with 167 µL of H2O2 (450 µmol/L) to initiate the reaction.
Six minutes after addition of the hydrogen peroxide, the absorbance of the
solution was read at 734 nm, along with a buffer blank that did not contain the
antioxidant solution or fluid to be tested. The extent to which the antioxidants in
the sample solution inhibited the reaction was calibrated with the use of a
standard antioxidant solution, Trolox (2.5mmol/L). Results were reported as
percentage inhibition of the color intensity for statistical analysis purposes.
Samples were run in triplicate to ensure intra-assay precision.
After completion of surgery, group (A) patients were transferred to ICU intubated
and mechanical ventilation was continued until recovery from thiopental effects.
Group (B) patients were extubated in the theatre and transferred to ICU for 24
hours observation. All patients were thoroughly examined neurologically after
awakening, four hours later and daily until discharge from the hospital. CT scan
for the brain was done if the patient developed any neurological symptoms.

Statistical Analysis
Nonparametric statistics were used to compare changes in PAP during surgery
(Wilcoxon rank sum test) with the use of Stat View 4 (Abacus). Parametric-
numeric data were analyzed by t-student test where as Non-numerical data were
compared with Exact Fischer test.
RESULTS

Between June 2001 and July 2004, 23 patients were enrolled into this
prospective randomized study. Thirteen cases included in group (A) and 10
patients included in group (B). The mean age of the patients was 66.2 (7.5) years
(range, 51- 77) and 64.9 (8.8) years (range, 54 to 76 years), respectively. The
male to female ratio was 2.3:1 in both groups. Table (1) shows insignificant
differences among the two study groups (P> 0.05)

Chart (1) shows the change in SjO2 in the two groups during the surgical
procedure. A significant decrease was seen during arterial clamping and
continued to fall with time compared with the pre-clamping value (P<0.05). The
saturation recovered to almost the pre-clamping levels after reperfusion of the
brain.

Chart (2) shows the changes in jugular venous and arterial mean PAP among the
study groups throughout the procedure. Arterial samples were analyzed to
ensure that the decrease in PAP was due to consumption across the cerebral
circulation by oxidant production rather than a dilution effect. In the control group
(B), baseline jugular bulb venous PAP was 32.8 (5.4); this decreased to 32.0 (6.0)
during ischemia and showed a further decrease to 28.0 (6.8) on reperfusion
(P<0.01). There was no concomitant reduction in arterial PAP during ischemia
30.3 (9.9) and on reperfusion 29.1(10.5) compared with baseline 30.1 (8.4). In
the Thiopental treated group (A), similar drop in the jugular venous PAP inhibition
from the baseline level 33.9 (5.4) to 31.5 (5.0) during ischemia and further to 30.8
(4.5) during reperfusion, however the drop was not statistically significant
indicating less consumption of plasma antioxidants during the
ischemia/reperfusion period of the procedure.
The change in the arterio-jugular venous PAP difference, which reflects the
preserved plasma antioxidant capacity across the cerebral circulation, is shown
in Chart (3). There was a significant reduction in this antioxidant capacity among
the control group compared to the thiopental treated group, during reperfusion
compared with baseline (P<0.05).

TABLES

Table (1): Patients Data and indications for CEA:

Group A Group B P
Criteria
(n=13) (n=10) value
SEX NS*
Males 9 (69.3%) 7 (70%)
Females 4 (30.7%) 3 (30%)
AGE years NS¥
Average 66.2 (7.5) 64.9 (8.8)
Median 68 66
Range (51 -77) (49 -87)
SYMPTOMS NS*
Stroke 11 (84.6%) 6 (60%)
TIAs 2 (15.4%) 4 (40%)
NS,( p> 0.05); S, (p< 0.05); HS, (p<0.01); * chi-square test (exact Fischer), ¥t-
student test
CHARTS

Chart (1): Changes in SjO2 during Carotid endarterectomy *

80

78

76

74

72
Thiopental
Oxygen saturation (%)

70

68

66
Control
64

62

60

58

56

54

52

50
Baseline preclamping predeclamping Reperfusion

Sampling time points

Decrease in SjO2 during the clamping time compared with the baseline both in the thiopental
group and control (P< 0.05).

Chart (2): Changes in the PAP levels during CEA

35
Group (A) Thiopental Venous
34

Group (B) Control Venous

33

32
Group (A) Thiopental Arterial Group (A) Thiopental Venous

31
PAP levels

Group (B) Control Arterial

30 Group (A) Thiopental Arterial

29
Group (B) Control Arterial

28 Group (B) Control Venous

27

26

25
Baseline Ischemia Reperfusion
Sampling Points
Venous PAP levels are significantly reduced during ischemia and reperfusion among controls (P<
0.05), but not in the thiopental group (P>0.05). Wilcoxon rank sum test

Chart (3): Changes in the PAP arteriovenous differences during CEA

3.0

Group B (Control)
2.5

2.0 Group A (Thiopental)

Group B (Control) Group A (Thiopental)
Percentage difference

1.5
Group A (Thiopental)

1.0

0.5

0.0
Baseline Ischemia Reperfusion

-0.5

-1.0

-1.5

Sampling time points

PAP arteriojugular venous difference was significantly reduced among controls during
reperfusion phase (P< 0.05).
DISCUSSION

The brain is at special risk from oxidant-mediated injury because of its large iron
stores , high level of polyunsaturated lipids, and poor antioxidant defenses
(20) (21)

.Consequently, oxidant mechanisms have been implicated in cerebral

ischemia/reperfusion injury, and such injury in animal models has been shown to
be prevented or ameliorated by antioxidant therapy (22-24) .
The neuroprotection mechanisms provided by different anesthetic agents has
been under a lot of experimental investigations. Anesthetics could achieve
antioxidant effects by preventing the initiation of free radical chain reactions, by
terminating the propagation of highly reactive radicals, and by modifying cellular
responses to oxidative stress (25).
Barbiturates neuroprotective mechanisms are multifactorial and yet not
determined. First they may inhibit free radical generation by inducing dose-
dependent decrease in cerebral metabolic rate and cerebral blood flow until the
EEG becomes isoelectric. At that point, maximum reductions of nearly 50% are
observed, additional barbiturate does not reduce metabolic rate further .
(26)

Subsequently reduction of cerebral metabolism result in slowing of cerebral

utilization of oxygen and glucose, inhibit oxidative metabolism in neutrophils (27),
and prevent redox changes in hemoglobin (28) .Unlike isoflurane, however,
barbiturates reduce metabolic rate uniformly throughout the brain (29) .However,
Warner et al. demonstrated that electroencephalographic burst suppression is
not required to elicit maximal neuroprotection from pentobarbital in a rat model of
focal cerebral ischemia (30) .
Second, antioxidant anesthetics directly scavenge reactive oxygen species and
inhibit lipid peroxidation. While Phenobarbital (10-400 μM) and
Pentobarbital (10-50 μM) increase cell death in neuronal cultures
exposed to oxygen and glucose deprivation, Thiopental was found to
prevent lipid peroxidation and membrane disruption(31) .Thiopental scavenges lipid
peroxyl radicals .Moreover, it protects cultured neurons from damage by nitric
(28)

oxide.These effects are caused by scavenging of the propagating radicals (such

as nitric oxide radical) by the sulfhydryl group (-SH) present in dissolved
thiopental because pentobarbital, which does not contain -SH, is not protective in
this respect in vitro system in which temperature and blood flow rate are not
variables .Finally, anesthetics may inhibit the activation of excitatory
(32)

glutamatergic receptors that augment oxidative stress after ischemia. Zhu and
colleagues demonstrated that thiopental, unlike propofol, attenuates glutamate
excitotoxicity (33).

After careful midline search, we could not find any article demonstrating the use
barbiturate as a neuroprotective agent to reduce the associated ischemia-
reperfusion injury during carotid endarterectomy as a model of focal brain
ischemia. Carotid endarterectomy has the disadvantage of the lack of severity of
the insult, since the short period of ischemia, presence of collateral circulation,
and use of shunts during surgery all combine to reduce the extent of injury, and
clinically significant neuronal injury is rare (1) .However, major advantages of this
model include its clinical substrate, controllability, and ability to estimate the
severity of the ischemic insult with multimodality monitoring, based on
transcranial Doppler, jugular venous saturation, and electrophysiological
detection of the consequences of cerebral ischemia (34) .Furthermore, the ability to
sample arterial and jugular bulb venous blood provides us with a unique
opportunity to study changes in levels of biologically significant molecules across
the cerebral circulation. Reactive oxygen species such as superoxide anions,
hydrogen peroxides, and the extremely toxic hydroxyl radical are difficult to detect
in patients because of their short half-life. Therefore, byproducts of lipid
peroxidation (7, 10) .or depletion of endogenous antioxidants have often been used
as indirect markers for free radical generation (8, 9) .Plasma antioxidant potential
(PAP) assay has been successfully used to demonstrate an evidence of oxidant
injury in clinical situations (9, 35, 36) .

In our study the PAP levels across the cerebral circulation decreased during
ischemia with a further significant decrease on reperfusion (P<0.05) in the control
group who were not subjected to Thiopental induced coma. This is consistent
with the hypothesis that cellular changes during ischemia produced free radical
species on reperfusion that consumed antioxidants in plasma. The marked
changes during reperfusion rather than ischemia also suggest that the reduction
in PAP was not merely due to a dilution effect caused by contamination from
extra cranial blood such as facial blood.
In the thiopental group, this drop in PAP levels was not obvious as in the control
group as shown chart (2), which indicates less consumption of plasma
antioxidants during a period of relative rather than complete ischemia.
The role of Thiopental in preservation of the plasma antioxidant capacity is well
demonstrated by chart (3) which shows the significant difference between the
arterio-jugular venous difference in PAP levels of inhibition between the control
group and the thiopental group (P< 0.05).
This pilot study demonstrated that induced thiopental coma during carotid
endarterectomy could be used as a neuroprotection technique if titrated in doses
which can achieve isoelectric EEG. However, the implication of its use on the
clinical outcome of carotid endarterectomy requires more cases to study the
influence of these drugs on the perioperative brain functions and morphology.
We believe that these results have two important implications. First, they provide
supportive evidence for oxidant production during cerebral ischemia/reperfusion
during carotid endarterectomy even in shunted cases. Second, thiopental (high
doses) could be neuroprotective agent against associated ischemia reperfusion
injury as long as cerebral perfusion pressure is maintained in this clinical setting.
CONCLUSIONS
Thiopental induced coma significantly preserve the plasma antioxidant capacity
during carotid endarterectomy, thus providing a protective mechanism against
delayed neuronal death from ischemia-reperfusion.
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