(Stroke. 1999;30:1464-1471.)
© 1999 American Heart Association, Inc.
Original Contributions |
Effects of Citicoline Combined With Thrombolytic Therapy in a Rat Embolic Stroke Model
From the Neurovascular Research Laboratory, Rigshospitalet (M.A.), and Department of Neurology, Bispebjerg Hospital (K.O., P.M., G.B.), Copenhagen University (Denmark).
Correspondence to Karsten Overgaard, MD, Department of Neurology, Copenhagen University Hospital, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. E-mail karsten overgaard{at}dadlnet.dkovergaard@dadlnet.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Background and Purpose—We sought to evaluate the effects of the combination of cytidine-5'-diphosphocholine (citicoline) and thrombolysis on infarct size, clinical outcome, and mortality in a rat embolic stroke model.
Methods—Eighty-three Sprague-Dawley rats were embolized in the carotid territory with a single fibrin embolus and randomly assigned to the following treatment groups: (1) control (saline), (2) citicoline 250 mg/kg, (3) citicoline 500 mg/kg, (4) recombinant tissue plasminogen activator (rtPA) 5 mg/kg, (5) rtPA 5 mg/kg plus citicoline 250 mg/kg, and (6) rtPA 5 mg/kg plus citicoline 500 mg/kg. rtPA was administered as a continuous intravenous infusion over 45 minutes starting 45 minutes after embolization; citicoline was given intraperitoneally 30 minutes and 24, 48, and 72 hours after embolization. At 96 hours, the brains were fixed and stained by hematoxylin-eosin, and infarct volumes were measured. Neurological scores were determined daily.
Results—The median infarct size, measured as percentage of the affected hemisphere, in the control group was 37% (interquartile range, 26% to 69%) compared with 22% (5% to 52%; P=NS) in group 2, 11% (5% to 34%; P=NS) in group 3, 24% (12% to 31%; P=NS) in group 4, 11% (3% to 22%; P=0.02) in the combined group 5, and 19% (9% to 51%; P=NS) in group 6. The infarct size was significantly reduced in the combined citicoline+rtPA–treated groups to a median of 13% (5% to 30%; P<0.01). Citicoline 500 mg/kg and citicoline combined with rtPA also promoted functional recovery.
Conclusions—These results demonstrate that the combination of low-dose citicoline and rtPA significantly reduced infarct size in this focal ischemia model.
Key Words: cerebral ischemia, focal • neuroprotection • thrombolytic therapy • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Cytidine-5'-diphosphocholine (citicoline; CIT) administered exogenously provides both choline and cytidine, which serve as substrates for the synthesis of phosphatidylcholine, a primary neuronal membrane component. It is thought to promote membrane synthesis and repair, which is essential for recovery from stroke.1 2 CIT and its metabolites (choline and cytidine) contribute to the formation of acetylcholine, nucleic acids, and proteins.3 In models of transient global ischemia, CIT reduced production of free fatty acids, a tissue-toxic byproduct of membrane breakdown secondary to an ischemic insult.3 Additionally, CIT improved neurological outcome4 and increased survival time in global chronic hypoxia.5 In models of temporary focal ischemia, CIT reduced the size of cerebral infarcts6 7 and extended the duration of ischemia required to produce a given behavioral deficit or infarct size.8 A neuroprotective effect of CIT was reported in clinical studies of CIT-treated older (aged 50 to 85 years) subjects in which cognitive9 10 11 and behavioral parameters improved.12 In patients with stroke, CIT was associated with neurological improvements.1 13 14 15 16 Administration of CIT to stroke patients within 24 hours of symptom onset was associated with a more rapid recovery of neurological function and a greater proportion of patients with no residual neurological deficit in a recently completed clinical trial.17 In a subset of these patients, a reduction of the volume of the ischemic injury after stroke measured by diffusion-weighted MRI was demonstrated in 7 of 8 patients treated with CIT compared with 1 of 4 placebo-treated patients.18
Thrombolytic therapy has been studied extensively in experimental stroke and clinical trials19 20 21 22 23 and was approved in the United States for stroke treatment within 3 hours of symptom onset in selected patients in 1996.24 The results from the clinical trials were summarized in a meta-analysis; the Cochrane Systematic Reviews suggested a net long-term benefit for death and dependency with thrombolysis.25 However, the short time window and the increased risk of severe intracranial bleeding complications reported in a number of trials26 27 28 29 30 tend to limit the clinical use of thrombolytic therapy.
It therefore seems rational to explore the combination therapy of recombinant tissue plasminogen activator (rtPA), given at doses that are less likely to cause serious adverse events, and a neuroprotective agent that may widen the therapeutic window.31 The combination of thrombolytic agents with neuroprotective agents has been attempted, and some encouraging results have been reported in experimental stroke,32 33 34 although there is no convincing clinical evidence that any neuroprotective drug is effective in reducing size of infarction.35 36 37 To evaluate the effect of the combination of CIT and thrombolysis, CIT was administered in 2 different doses, alone and in combination with rtPA, and the effect on infarct size was compared in a rat embolic stroke model.38 39 The doses and time of administration of CIT and rtPA were chosen at suboptimal levels based on prior studies6 7 8 19 20 21 33 34 38 to evaluate a potential synergy between the drugs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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All experiments were approved and conducted according to the guidelines by the Animal Research Committee of Copenhagen University (No. 1996–101-103). Ninety male Sprague-Dawley rats (Møllegaard Breeding Center, Copenhagen) weighing 340 to 380 g were used. The rats were kept on a 12-hour light/12-hour dark cycle at a constant temperature of 22±1°C and were fasted overnight with free access to water before the surgical procedures.
Anesthesia and Surgical Procedure
Anesthesia was induced with subcutaneous injection
of fluanison (3 mg/kg) and fentanyl (1 mg/kg) (Hypnorm; fluanison 10
mg/mL and fentanyl 0.315 mg/mL; Janssen-Cilag) followed by a
subcutaneous injection of 0.015 mg atropine and an
intraperitoneal injection of 2.5 mg/kg diazepam
(Apozepam 5 mg/mL; Apothekernes Laboratorium). When necessary,
anesthesia was prolonged with one third of the initial dose
of fluanison and fentanyl. The right femoral artery was catheterized
with a polyethylene tube (PP25; ID, 0.4 mm) for monitoring blood
pressure and for sampling blood for analysis of
arterial blood gases and glucose, and the right femoral
vein was catheterized for intravenous drug administration.
During the operation and for the first half hour after reversal of the
anesthesia, body temperature was maintained at
37.0±0.5°C with a heating lamp controlled by a thermostat connected
to a temperature probe in the rectum.
The carotid operation was performed as described previously.38 Briefly, the right common carotid artery and the right external carotid artery (ECA) were exposed, and after ligation of extracranial branches of the internal carotid artery (ICA) and ECA, a polyethylene catheter (PP25) was inserted in the ECA. Clotting of the ECA was avoided by the continuous infusion (0.5 mL/h) of heparinized saline (5 IU/mL). After the second angiography, catheters were removed and vessels ligated. The femoral and neck wounds were closed, and the animals received isotonic saline 3.5 mL IP. Anesthesia was reversed with an injection of naloxone 0.3 mg/kg SC (Narcanti 0.4 mg/mL; Du Pont Pharma).
Embolization
Blood (250 µL) was aspirated from the femoral artery into a
1.0-mL 28-gauge disposable insulin syringe and interconnected by a
15-cm-long polyethylene catheter (PP10; ID, 0.28 mm; Polystan,
Værløse) with another syringe containing 50 µL of human thrombin
(Topostasin 2.5 IU/mL; Roche). A clot suspension was prepared by mixing
the rat arterial blood with thrombin. The suspension was
kept in constant motion by alternate movements from one syringe to the
other for 3 minutes. By this procedure, small fibrin-rich
arterial-like microthrombi were produced under
high-pressure conditions.38 The syringes were left
standing with closed compartments for 20 minutes. Then the suspension
of microclots was injected into a 1.0-m-long air-filled polyethylene
catheter (PP10; ID, 0.28 mm). By this procedure, the microclots in
the suspension aggregated into several visible clots of variable
length.39 Clot material was transferred through a 60-mm
piece of silicone (ID, 0.5 mm) to another 1.0-m-long polyethylene
catheter (PP10; ID, 0.28 mm) filled with saline. A piece of the
catheter (100 mm) containing a single clot with a length of 4
mm (0.25 mm3) was selected for embolization.
The obtained macroclot consisted of a fibrin mesh with a few
corpuscular blood elements as determined by light and electron
microscopy.40 The embolus was then injected through the
ECA catheter, which was flushed gently for 30 seconds with 0.3 mL of
isotonic saline. The common carotid artery was occluded by a temporary
ligature, and the carotid bifurcation was inspected to avoid
excessive dilatation of the ECA during the launching and
flushing procedure.
Angiography
Carotid angiography was performed with the use of a
high-resolution angiographic system (Philips SRO 03/100) with a small
focus spot of 0.15 mm2 and a large focus
spot of 1.5 mm2. Exposure data were 70 kV,
14 mA, and 0.4 seconds. One anteroposterior view with a focus object
distance of 31.5 cm and a focus film distance of 141.5 cm, giving a
linear magnification of 4.5, was done immediately and 2 hours after
embolization. At each time point, a bolus of 0.20 mL heparinized (5
IU/mL) iohexol (Omnipaque, 300 mg/mL; Nycomed) was injected
through the ECA catheter.38 Angiograms were processed on
high-resolution films (Retina XOE; Fotochemische Werke). The occlusions
were classified according to the following categories: 0, patent
arteries; 1, middle cerebral artery (MCA) branch occlusion; 2, MCA stem
occlusion; and 3, ICA occlusion at the entrance of MCA distal to the
posterior communicating artery. Only animals with occlusion types 2 and
3 were included in the study. In addition, a post hoc evaluation of the
angiograms was conducted by raters blind to the treatment regimen, and
animals not fulfilling the angiographic inclusion criteria were
excluded from the final analysis.
Physiological Parameters
Blood samples were drawn from the femoral artery for
analysis of pH, PaO2,
PaCO2, O2
saturation (ABL2; Radiometer), and plasma glucose (B-glucose
photometer; Hemocue AB) before embolization and 2 hours after
embolization. Rectal temperature was continuously monitored, and mean
arterial blood pressure was measured before embolization
and after treatment.
Experimental Protocol
The rats were randomly assigned to 6 groups (n=15 per group).
Group 1 served as a control and was treated with saline as a continuous
intravenous infusion (5 mL/kg) over 45 minutes starting 45
minutes after embolization and with saline administered
intraperitoneally (1 mL/kg) 30 minutes and 24, 48,
and 72 hours after embolization. Group 2 (CIT250) received saline as a
continuous intravenous infusion (5 mL/kg) over 45 minutes
starting 45 minutes after embolization and 250 mg/kg of CIT
intraperitoneally (250 mg/mL) 30 minutes and 24,
48, and 72 hours after embolization. Group 3 (CIT500) received saline
as continuous intravenous infusion (5 mL/kg) over 45
minutes starting 45 minutes after embolization and 500 mg/kg of CIT
intraperitoneally (500 mg/mL) 30 minutes and 24,
48, and 72 hours after embolization. Group 4 (rtPA) received 5 mg/kg of
rtPA (Actilyse, Boehringer Ingelheim) as continuous
intravenous infusion (1 mg/mL) over 45 minutes starting 45
minutes after embolization and saline
intraperitoneally (1 mL/kg) 30 minutes and 24, 48,
and 72 hours after embolization. Group 5 (CIT250+rtPA) received CIT as
in group 2 combined with rtPA as in group 4. Group 6 (CIT500+rtPA)
received CIT as in group 3 combined with rtPA as in group 4.
Clinical Evaluation
Neurological examination and weighing were performed after the
animals recovered from anesthesia and over the next 4 days
once daily at 24-hour intervals. Neurological status was evaluated
according to Bederson et al,41 modified by adding grade 4
for death to a 5-point scale: 0=no neurological motor deficit,
1=flexion of the forelimb contralateral to the injured hemisphere,
2=reduced resistance against push toward the paretic side,
3=spontaneously circling toward the paretic side, and 4=death within 96
hours. Animals dying before 96 hours were autopsied to establish the
cause of death.
Neuropathological Examination
After the neurological examination was completed on day 5, the
animals were anesthetized with halothane and perfused
transcardially with 4% phosphate-buffered formalin (pH 7.2). Brains
were carefully removed, post-fixed in a 4% phosphate-buffered formalin
solution, dehydrated, embedded in paraffin, and cut at a 3-µm section
thickness. From each brain, 
15 coronal sections with a distance of
0.6 mm between slices were obtained and stained with
hematoxylin-eosin. Brains from animals dying spontaneously >8 hours
after embolization were removed and immersion-fixed in 4% buffered
formalin solution. Brains from animals dying before 8 hours after
embolization were considered as having a nonevaluable infarct size and
were only evaluated by a freeze technique to verify pathological
changes. The histology of the affected hemisphere and infarcts was
examined blind to treatment with a Leica M3Z stereo microscope with
x10 to x40 magnification, depending on the need for necessary
details. The infarct was defined as an infarct regardless of whether it
was pannecrotic or consisted of selective neuronal necrosis.
Eosinophilic neurons in the periphery of pannecrotic areas were
considered tissue damaged by ischemia and were included in the
infarcted area. To distinguish the acidophilic neurons from other
neurons and get an impression of the existence of light and dark
neurons, a magnification of x40 was often supplied with a
magnification of x100. Areas with dark neurons without eosinophilic
neurons were not considered infarcted tissue. The borders of the
infarcted areas were determined and delineated with a pen, and the
delineated sections belonging to each brain were then scanned with a
flat-bed scanner (HP ScanJet 4P) linked to a specially designed image
analysis program (Sidney Data) in an IBM Pentium personal
computer. The volumes of the hemisphere and infarcts were determined
semiautomatically by the computer program detecting the circumference
of the brain and the delineated borders in each section. The spatial
resolution on the digitized images of the brain was set to 81 dots per
square millimeter for area calculations. Adjustments and verification
of the different areas were performed manually on enlarged digitized
computer images. The volumes were then calculated as the respective
areas multiplied by the distance between sections in percentage of the
affected right hemisphere. The infarcts were measured without
correcting the infarct volume for edema because the
histological preparation technique dehydrated the
tissue, reducing the influence of edema.34
Statistical Analysis
Nonparametric analyses were used because a
nonnormal distribution of data was observed in subsets of data. The
Kruskal-Wallis test was applied to test for overall significant
differences of ordinal data between groups. If significant differences
were detected by the Kruskal-Wallis test, a Mann-Whitney test was
performed to identify which group was different from controls. For
comparisons of 2 groups, the Mann-Whitney test was performed for
unpaired observations and the Wilcoxon signed rank test for
paired observations. Binomial data (mortality rates, angiographic data)
were compared by Fisher's exact probability test. All probability
values were corrected for multiple comparisons with the Bonferroni
adjustment. The Spearman rank order correlation test was used for
ranked paired observations. Corrected values of P<0.05 were
considered significant. All calculations were performed on an IBM
Aptiva personal computer with the use of a commercially available
statistical software program (Sigma Stat version 2.03 for Windows).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Ninety embolized animals were included in the study and randomly assigned to 1 of 6 treatment groups, as described in Materials and Methods. Of the 90 animals, 83 animals completed the study. Seven animals were excluded after embolization: 2 animals were excluded because of complications during surgery not related to treatment, 3 did not fulfill the angiographic inclusion criteria at a second blind angiographic review of the primary angiography, 1 animal was excluded because of missing second angiographic data, and 1 was excluded because of abnormal physiological variables.
All animals developed right hemisphere infarcts predominantly located in the right MCA territory. Infarction was less frequent in the hippocampus supplied by the posterior cerebral artery. Infarction was not found in the right cingulate cortex supplied by the anterior cerebral artery, except in 2 spontaneously dead animals with edema. In 1 animal, infarction was observed in the basal ganglia contralateral to the embolized hemisphere. No other contralateral infarction was observed. Petechial hemorrhagic transformation of the infarcts was observed in 7 animals, including 4 that died prematurely; 6 of the animals were in the rtPA-treated groups, and 1 was in the control group.
Clinical Outcome
Premature death occurred in 5 animals in the control group (36%),
compared with 3 in the CIT250 group (20%), 2 in the CIT500 group
(15%), 2 in the rtPA group (15%), 1 in the CIT250+rtPA group (7%),
and 3 in the CIT500+rtPA group (21%). Except for 1 animal in the rtPA
group that died within 6 hours after embolization, all animals died
between 12 and 48 hours. There was no significant difference between
the mortality rates in the different groups. The pooled groups treated
with CIT alone (group 2+3) and the CIT+rtPA combined groups showed a
tendency toward a lower mortality.
No significant differences between the groups were observed in
the neurological scores after the animals recovered from
anesthesia (P>0.9, Kruskal-Wallis test), but an
insignificant trend was found among treated animals compared with
controls on day 5 (P<0.08, Kruskal-Wallis test; Figure 1
).
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All animals improved clinically during the study period. Within each
group, including controls, the median clinical score on day 5 was
significantly better than the median clinical score at day 1
(P<0.01, Wilcoxon test). The recovery was faster in
the CIT500 and the CIT250+rtPA groups (Figure 1
).
The body weight of animals in all groups declined from day 1 to day 3,
then remained stable or improved (Figure 2). No significant differences in
preoperative body weight were observed between the groups
(P>0.5, Kruskal-Wallis test). On day 5 the Kruskal-Wallis
test showed no significant differences of body weight between groups
(P>0.3).
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Infarct Volume
To obtain a complete evaluation of the effect of the different
treatments, the results were analyzed both including and
excluding the animals dying prematurely (Figure 3). The median values of the
cortical, subcortical, and total infarct volumes as a percentage of the
affected hemisphere and for animals surviving until euthanasia are
shown in Table 1. There was no
significant group difference of the median total infarct size among
rats surviving 96 hours (P>0.13, Kruskal-Wallis test).
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However, when the data were analyzed including the prematurely
dead animals, significant group differences were found
(P<0.05, Kruskal-Wallis test; Table 1 and Figure 3). Compared with controls (37% [range, 26% to 69%]), a
significant reduction of total infarct size was observed in the
CIT250+rtPA group (11% [range, 3% to 22%]; P=0.02,
Mann-Whitney test corrected by Bonferroni adjustment); no significant
reduction in infarct volume was observed in the other treatment groups.
When we pooled the data of all the CIT alone–treated animals
(CIT250+CIT500), CIT given alone reduced the total infarct size from
37% (range, 26% to 69%) in controls to 19% (range, 5% to 50%;
n=28; P=0.054, Mann-Whitney test adjusted by Bonferroni
method), and CIT combined with rtPA reduced the infarct sizes even
further to a median of 13% (range, 5% to 30%; n=27;
P=0.024, Mann-Whitney test adjusted by Bonferroni method).
One animal in the rtPA group died without having the brain fixed. The
median infarct size in the spontaneously dead control animals was 71%
(range, 55% to 72%; n=5; P<0.01, Mann-Whitney test) and
significantly larger than the median infarct size in surviving controls
(P<0.01, Mann-Whitney test). The median infarct size for
all measured brains from spontaneously dead animals was 69% (range,
54% to 72%; n=15), which was significantly larger than for the
surviving control animals (27% [range, 11% to 36%]; n=9;
P<0.001, Mann-Whitney test).
Evaluation of Angiograms
The distribution of the occlusions immediately after embolization
is illustrated in Table 2.
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Table 2 shows an uneven number of MCA stem occlusions in
the different groups immediately after embolization, but the difference
in distribution was not statistically significant.
Recanalization and redistribution of emboli
distally in the cerebral arteries occurred spontaneously in both
controls and treated animals during the first 2 hours of the
experiment. This was most noticeable in the CIT500, rtPA, and
CIT250+rtPA groups, especially in the CIT250+rtPA group, in which the
frequency of complete recanalization compared with
the control group increased from 0% (0/14) to 57% (8/14)
(P<0.01, Fisher test corrected by Bonferroni
adjustment).
When the 3 groups not receiving rtPA (including controls) were compared with the pooled rtPA-treated groups, the frequency of complete recanalization increased from 12% (5/42) to 37% (15/41) (P<0.01, Fisher test).
Correlations Between Clinical Outcome and Neuropathological
Damage
The total infarct volume was highly correlated to the
Bederson score on day 5 (all 96-hour animals; r=0.686,
P<0.001) and to the net weight reduction on day 5 (all
96-hour animals; r=0.410, P<0.001). In other
words, animals with smaller infarcts fared better functionally and
regained weight more quickly.
Physiological Parameters
There were no systematic intergroup differences in the
physiological parameters (all
P>0.1, Kruskal-Wallis tests) except for the second
measurement of pH (P<0.001, Kruskal-Wallis test). The
CIT250+rtPA group was the only group with a significant
(P<0.01, Mann-Whitney test adjusted by the Bonferroni
method) lower second pH (median of 7.33 compared with a median of 7.40
in the control group). There was no significant difference between the
physiological parameters of the animals
that died prematurely and those that survived 96 hours (Table 3). When we compared the first set of
parameters with the second, a fall in
PaCO2 and an elevation in
PO2 values in the CIT250+rtPA
group was observed (P<0.05, Wilcoxon test). Similar
changes were not observed in the other groups. There were no
correlations between the physiological
parameters and infarct size. No overt side effects of
either rtPA or CIT were observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The present study evaluated the effects of CIT given alone and combined with rtPA in an animal model of focal cerebral ischemia induced by embolization with autologous clots. The embolic model used in this study was created to imitate embolic human stroke, which varies in severity from transient ischemic attack to malignant MCA infarction. Even though the emboli used were of uniform size, the resulting arterial occlusions varied from no apparent vascular occlusion in the MCA branch to MCA stem or ICA occlusion. The ensuing infarcts varied from small lesions to infarctions of the whole MCA territory with fatal edema formation. As in humans, occlusions of ICA could cause a range of infarcts from minimal to very large. This similarity to human stroke has the disadvantage of requiring large groups to obtain statistically significant effects.
The initial angiograms allowed selection of animals with occlusion of the MCA stem and the ICA, but the distribution of these 2 types of occlusion was unevenly balanced, with more initial MCA occlusion in groups 2 to 5. One could speculate that this may have influenced the finding of significant treatment effect. However, if the data analysis was limited to the animals with ICA occlusions on the primary angiogram, the median total infarct volume was 41% in controls (n=11) and 22% in the combination group with CIT250 and tPA (n=6; P=0.08, Mann-Whitney test). A similar trend was seen if the data analysis was restricted to the animals with MCA occlusion on the primary angiogram, where the median infarct volume was 27% in controls (n=3) and 5% in the CIT250+rtPA group (n=8; P=0.10, Mann-Whitney test). Thus, the significant reduction of median infarct volume in the combination therapy group 5 was not due to a favorable outcome in a subset of animals but was present regardless of the type of occlusion on the first angiogram.
The animals that died prematurely were evaluated to ensure that no treatment would cause an excess fatality rate and result in false-positive treatment effect by allowing only subjects with small infarcts to survive. Characteristically, the brains from the animals that died prematurely had edema with compression of the ventricles, and premature death was probably caused by large infarcts with brain swelling. Small hemorrhagic transformations, observed primarily in the rtPA-treated animals, did not seem to contribute to fatal outcomes.
To more easily evaluate potential synergy between the 2 drugs, the doses for each drug were selected to have a suboptimal effect on infarct volume given alone. At the same time, a parallel series of experiments was conducted to evaluate the effect of a fully effective dose of CIT on infarct size, given alone and in combination with rtPA. In previous studies using a temporary focal occlusion model in rats, CIT significantly reduced infarct size at a dose of 500 mg/kg IP6 but not at a dose of 250 mg/kg IP.7 The findings with these doses of CIT in the present study are consistent with those in the temporary focal occlusion model. In the rat, rtPA needs to be given at doses 10 times higher than in humans to have equal effect.42 43 In our previous studies with rtPA, doses of 10 to 20 mg/kg were used.19 20 21 33 34 38 The 5-mg/kg dose used in this study was considered suboptimal in terms of recanalization capability, but with reduced risk of bleeding.
When a suboptimal treatment regimen of CIT (250 mg/kg) and rtPA were combined, the result was a significant reduction in infarct volume and a tendency for improvement in clinical outcome. This interaction between the 2 agents appears to be additive. It is unclear why the combination of a more effective dose of CIT (500 mg/kg) with the suboptimal rtPA treatment regimen did not produce a similar or greater infarct size reduction than the lower dose combination. One explanation may be the large variation in infarct size within this group (9% to 51%) compared with other treatment groups (eg, 3% to 22% for the CIT250+rtPA group). Acknowledging the wide range of infarct sizes with this model and the need for large groups of animals, we also compared the effects of the combined CIT-alone groups with the combined CIT+rtPA groups. The combined groups treated with CIT alone showed a tendency to reduce infarct size, but infarct size was significantly reduced in the combined CIT+rtPA-treated groups when the dead animals were included in the analysis.
In conclusion, this study provides a basis for further investigation of the effects of CIT combined with rtPA for the treatment of ischemic stroke. Although histological assessment of neuropathological changes is ultimately the most predictive measure of potential therapeutic value of a drug,31 the demonstration that CIT+rtPA also promotes functional recovery supports the assumption of a potential benefit of this combined therapy.
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Acknowledgments |
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This study was supported by Interneuron Pharmaceuticals, the Søren and Helene Hempel Foundation, the E. Danielsen and Wife Foundation, and the Danish Research Council (120-1645-1 and 95-0293-4). Kristine Dahlin and Annika Rasmussen are thanked for excellent technical assistance. The authors would like to express their gratitude to Kenneth W Locke, PhD, for assistance in the preparation of the manuscript.
Received October 12, 1998; revision received March 16, 1999; accepted April 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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1. D'Orlando KJ, Sandage BW Jr. Citicoline (CDP-choline): mechanisms of action and effects in ischemic brain injury. Neurol Res. 1995;17:281–284.[Medline] [Order article via Infotrieve]
2. López-Coviella I, Agut J, Savci V, Ortiz JA, Wurtman RJ. Evidence that 5'-cytidinediphosphocholine can affect brain phospholipid composition by increasing choline and cytidine plasma levels. J Neurochem. 1995;65:889–894.[Medline] [Order article via Infotrieve]
3. Weiss GB. Metabolism and actions of CDP-choline as an endogenous compound and administered exogenously as citicoline. Life Sci. 1995;56:637–660.[Medline] [Order article via Infotrieve]
4.
Kakihana M, Fukuda N, Suno M, Nagaoka A. Effects of
CDP-choline on neurologic deficits and cerebral glucose
metabolism in a rat model of cerebral ischemia.
Stroke. 1988;19:217–222.
5. Hamdorf G, Cervos-Navarro J, Müller R. Increase of survival time in experimental hypoxia by cytidine diphosphate choline. Arzneimittelforschung. 1992;42:421–424.[Medline] [Order article via Infotrieve]
6. Schäbitz WR, Weber J, Takano K, Sandage BW Jr, Locke KW, Fisher M. The effects of prolonged treatment with citicoline in temporary experimental focal ischemia. J Neurol Sci. 1996;138:21–25.[Medline] [Order article via Infotrieve]
7.
Önal MZ, Li F, Tatlisumak T, Locke KW, Sandage
BW Jr, Fisher M. Synergistic effects of citicoline and MK-801 in
temporary experimental focal ischemia in rats.
Stroke. 1997;28:1060–1065.
8. Aronowski J, Strong R, Grotta JC. Citicoline for treatment of experimental focal ischemia: histologic and behavioral outcome. Neurol Res. 1996;18:570–574.[Medline] [Order article via Infotrieve]
9.
Spiers PA, Myers D, Hochanadel GS, Lieberman HR,
Wurtman RJ. Citicoline improves verbal memory in aging. Arch
Neurol. 1996;53:441–448.
10. Di TG, Fioravanti M. Citicoline in the treatment of cognitive and behavioral disorders in pathologic senile decline [in Italian with English abstract]. Clin Ter. 1991;137:403–413.[Medline] [Order article via Infotrieve]
11. Alvarez XA, Laredo M, Corzo D, Fernández-Novoa L, Mouzo R, Perea JE, Daniele D, Cacabelos R. Citicoline improves memory performance in elderly subjects. Methods Find Exp Clin Pharmacol. 1997;19:201–210.[Medline] [Order article via Infotrieve]
12. Serra F, Diaspri GP, Gasbarrini A, Giancane S, Rimondi A, Tame MR, Sakellaridis E, Bernardi M, Gasbarrini G. Effect of CDP-choline on senile mental deterioration: multicenter experience on 237 cases [in Italian with English abstract]. Minerva Med. 1990;81:465–470.[Medline] [Order article via Infotrieve]
13.
Tazaki Y, Sakai F, Otomo E, Kutsuzawa T, Kameyama M,
Omae T, Fujishima M, Sakuma A. Treatment of acute cerebral infarction
with a choline precursor in a multicenter double-blind
placebo-controlled study. Stroke. 1988;19:211–216.
14. Bruhwyler J, Vandorpe J, Géczy J. Multicentric open label study of the efficacy and tolerability of citicoline in the treatment of acute cerebral infarction. Curr Ther Res Clin Exp. 1997;58:309–316.
15. Hazama T, Hasegawa T, Ueda S, Sakuma A. Evaluation of the effect of CDP-choline on poststroke hemiplegia employing a double-blind controlled trial: assessed by a new rating scale for recovery in hemiplegia. Int J Neurosci. 1980;11:211–225.[Medline] [Order article via Infotrieve]
16. Pettigrew LC, Clark WM, Warach S, Sabounjian LA. Effect of citicoline on cognitive function in acute stroke. Stroke. 1997;28:233. Abstract.
17.
Clark WM, Warach SJ, Pettigrew LC, Gammans RE,
Sabounjian LA. A randomized dose-response trial of citicoline in acute
ischemic stroke patients. Neurology. 1997;49:671–678.
18. Warach S, Sabounjian LA. Effects of citicoline on the evolution of lesion volume as measured by diffusion-weighted imaging. Stroke. 1997;28:271. Abstract.
19.
Overgaard K, Sereghy T, Boysen G, Pedersen H, Diemer
NH. Reduction of infarct volume and mortality by
thrombolysis in a rat embolic stroke model.
Stroke. 1992;23:1167–1173.
20. Overgaard K, Sereghy T, Pedersen H, Boysen G. Effect of delayed thrombolysis with rt-PA in a rat embolic stroke model. J Cereb Blood Flow Metab. 1994;14:472–477.[Medline] [Order article via Infotrieve]
21. Overgaard K. Thrombolytic therapy in experimental embolic stroke. Cerebrovasc Brain Metab Rev. 1994;6:257–286.[Medline] [Order article via Infotrieve]
22.
The National Institute of Neurological Disorders and
Stroke rt-PA Study Group. Tissue plasminogen
activator for acute ischemic stroke. N
Engl J Med. 1995;333:1581–1587.
23.
The NINDS t-PA Stroke Study Group.
Intracerebral hemorrhage after
intravenous t-PA therapy for ischemic stroke.
Stroke. 1997;28:2109–2118.
24. Viste KMJ. rt-PA approved for stroke. World Neurol. 1996;11:10. Abstract.
25. Wardlaw JM, Warlow CP. Systematic review of evidence on thrombolytic therapy for acute ischaemic stroke. Lancet. 1997;350:607–614.[Medline] [Order article via Infotrieve]
26.
Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von
Kummer R, Boysen G, Bluhmki E, Höxter G, Mahagne MH, Hennerici M.
Intravenous thrombolysis with recombinant
tissue plasminogen activator for acute
hemispheric stroke: the European Cooperative Acute Stroke Study
(ECASS). JAMA.. 1995;274:1017–1025.
27.
Donnan GA, Davis SM, Chambers BR, Gates PC, Hankey GJ,
McNeil JJ, Rosen D, Stewart WE, Tuck RR. Streptokinase for acute
ischemic stroke with relationship to time of administration:
Australian Streptokinase (ASK) Trial Study Group. JAMA. 1996;276:961–966.
28.
The Multicenter Acute Stroke Trial–Europe Study Group.
Thrombolytic therapy with streptokinase in acute
ischemic stroke. N Engl J Med. 1996;335:145–150.
29. Multicentre Acute Stroke Trial–Italy (MAST-I) Group. Randomised controlled trial of streptokinase, aspirin and combination of both in treatment of acute ischemic stroke. Lancet. 1995;346:1509–1514.[Medline] [Order article via Infotrieve]
30. Hacke W, Kaste M, Fieschi C, Von Kummer R, Davalos A, Meier D, Larrue V, Bluhmki E, Davis S, Donnan G, Schneider D, Diez-Tejedor E, Trouillas P. Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischemic stroke (ECASS II). Lancet. 1998;352:1245–1251.[Medline] [Order article via Infotrieve]
31.
Fisher M, Bogousslavsky J. Evolving toward effective
therapy for acute ischemic stroke. JAMA. 1993;270:360–364.
32.
Zivin JA, Mazzarella V. Tissue plasminogen
activator plus glutamate antagonist improves
outcome after embolic stroke. Arch Neurol. 1991;48:1235–1238.
33. Meden P, Overgaard K, Sereghy T, Boysen G. Enhancing the efficacy of thrombolysis by AMPA receptor blockade with NBQX in a rat embolic stroke model. J Neurol Sci. 1993;119:209–216.[Medline] [Order article via Infotrieve]
34. Meden P, Overgaard K, Pedersen H, Boysen G. Effect of early treatment with tirilazad (U7400F) combined with delayed thrombolytic therapy in rat embolic stroke. Cerebrovasc Dis. 1996;6:141–148.
35.
Pessin MS, Adams HP, Adams RJ, Fisher M, Furlan AJ,
Hacke W, Haley C, Hazinski MF, Helgason CM, Higashida RT, Koroshetz W,
Marler JR, Ornato JP. Acute interventions. Stroke. 1997;28:1518–1521.
36. Lees KR. Clinical trials for acute cerebral ischaemia. In: Krieglstein J, ed. Pharmacology of Cerebral Ischemia 1996. Stuttgart, Germany: Medpharm Scientific Publisher; 1996:691–699.
37. Grotta J, Chiu D. Current clinical status of cytoprotection. In: Welch KMA, ed. Primer on Cerebrovascular Diseases. New York, NY: Academic Press; 1997:731–737.
38. Overgaard K, Sereghy T, Boysen G, Pedersen H, Høyer S, Diemer NH. A rat model of reproducible cerebral infarction using thrombotic blood clot emboli. J Cereb Blood Flow Metab. 1992;12:484–490.[Medline] [Order article via Infotrieve]
39. Meden P, Overgaard K, Pedersen H, Boysen G. Increased mean arterial blood pressure during the first 21/2 hours of infarct development significantly decreased infarct volume in rat embolic stroke. J Cereb Blood Flow Metab. 1995;15(suppl 1):S394. Abstract.
40. Hoffmann P, Sainte Marie M, Bernat A, Roque C, Fedeli O, Volk A, Meric P, Herbert JM. Stroke outcome determination with a prefabricated fibrin-rich thrombus in a thromboembolic rat MCA occlusion model. Cerebrovasc Dis. 1997;7(suppl 4):79. Abstract.
41.
Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL,
Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the
model and development of a neurologic examination. Stroke. 1986;17:472–476.
42. Korninger C, Collen D. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thromb Haemost. 1981;46:561–565.[Medline] [Order article via Infotrieve]
43. Niewenhuizen W, Keyser J. Species specificity in the acceleration of tissue-type plasminogen activator-mediated activation of plasminogens, by fibrinogen cyanogen bromide fragments. Thromb Res. 1985;38:663–670.[Medline] [Order article via Infotrieve]
Editorial Comment
Department of Biostatistics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The accompanying article reports experimental results of combined neuroprotective agent and thrombolytic therapy in the embolic stroke model. The study design seems to be carefully considered, so that one can examine the effect of various combination doses of rtPA and citicoline (CIT). In addition, appropriate statistical analyses were performed, including adjustments for multiple tests followed by an overall test for each variable.
The median infarct volumes reported by the authors can be conveniently
displayed in the following 2x3 table. The Table allows
the readers to see an apparent effect of CIT dose alone, rtPA dose
alone, and combination rtPA and CIT dose on median infarct
volumes.
Although the sample size in each group is not large, the results indicate linear effects of rtPA alone, CIT alone, and CIT and rtPA combination, except for the CIT 500 mg/kg and rtPA combination. First, although only the rtPA+CIT250 group is significantly different from the control, it is certain that the difference between the CIT500 group and the rtPA+CIT250 group is not significant. Second, the result seems to suggest an interaction effect of CIT and rtPA. Similar results are observed for body weight change and Bederson scores. We hope that authors will perform further studies to confirm the superiority of the combination of the 2 agents and the possible interaction effect. Incidentally, it is interesting to note that spontaneously dead animals had large infarct volumes in each group. Consequently, the nonparametric analysis used by the authors would not have changed the results even if dead animals survived 4 days.
A concern the readers might have is the large variance in infarct volumes. Sometimes, part of a large variance may be due to the variances of uncontrolled factors in the model, such as age and weight of animals. Another reason is the large positive skew in the data. Note that the shape of distribution of the infarct volume for the 6 groups is about the same with a large positive skew. A positively skewed distribution is to be expected in infarct volumes.
Received October 12, 1998; revision received March 16, 1999; accepted April 12, 1999.
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