(Stroke. 1997;28:1060-1065.)
© 1997 American Heart Association, Inc.
Articles |
Synergistic Effects of Citicoline and MK-801 in Temporary Experimental Focal Ischemia in Rats
From the Department of Neurology, Medical Center of Central Massachusetts, Worcester (M.Z.Ö., F.L., T.T., M.F.); Departments of Neurology and Radiology, University of Massachusetts Medical School, Worcester (M.F.); and Interneuron Pharmaceuticals Inc, Lexington, Mass (K.W.L., B.W.S.).
Correspondence to Marc Fisher, MD, Department of Neurology, Medical Center of Central Massachusetts, 119 Belmont St, Worcester, MA 01605-2982.
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Abstract |
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Background and Purpose Citicoline, a naturally occurring precursor of phosphatidylcholine, is neuroprotective and is currently being assessed in clinical trials. To evaluate potential synergistic neuroprotective effects of prolonged citicoline treatment and early N-methyl-D-aspartate (NMDA) antagonist therapy, suboptimal treatment regimens of citicoline and MK-801 were tested alone and in combination in a rat model of temporary focal ischemia.
Methods Four groups of Sprague-Dawley rats (n=12 per group) underwent 90 minutes of temporary middle cerebral artery occlusion (MCAO) with the suture model. Animals were randomly and blindly assigned to one of four treatment groups: (1) saline, vehicle; (2) MK-801, 0.5 mg/kg IV bolus at 60 minutes after MCAO followed by saline 1 mL/kg IP daily for 7 days; (3) saline IV at 60 minutes after MCAO followed by citicoline 250 mg/kg IP daily for 7 days; or (4) both MK-801 and citicoline (daily for 7 days) active treatment. Triphenyltetrazolium chloride staining was used to assess postmortem infarct volume. Neurological scores were determined daily.
Results Premature mortality between days 2 and 4 was 33.3% in group 1, 41.7% in groups 2 and 3, and 25.0% in group 4. Mean corrected infarct volume was significantly reduced in group 4 compared with the others (175.2±89.3 mm3 in group 1, 179.1±78.5 mm3 in group 2, 163.9±73.7 mm3 in group 3, and 84.7±56.8 mm3 in group 4 [P<.02, ANOVA and P<.05, Scheffé's test for group 1 versus group 4]). Mean infarct volume in animals dying prematurely was significantly (P<.05, Student's t test) larger in group 1 than those surviving for 7 days (247.2±89.5 versus 139.2±68.2 mm3), but there was no significant difference in infarct volume in groups 2, 3, and 4 between animals dying prematurely and those surviving for 7 days.
Conclusions These results demonstrate synergistic neuroprotective effects of citicoline and an NMDA antagonist in temporary experimental focal ischemia.
Key Words: citicoline • MK-801 • cerebral ischemia, focal • neuroprotection • rats
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Introduction |
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Therapeutic agents for AIS typically affect only one of several mechanisms in the ischemic cascade, and multiple therapies are likely to be more effective than monotherapy.1 2 Neuroprotective and thrombolytic therapies are recent advances in the management of AIS that are likely to be effective.2 Citicoline (CDP choline, cytidine 5'-diphosphocholine) and MK-801 (dizocilpine maleate, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) are two neuroprotective agents with different mechanisms of action.3 4
Citicoline is a naturally occurring, endogenous compound that is an essential intermediate in the synthesis of phosphatidylcholine, a major neuronal membrane lipid.5 Citicoline and its hydrolysis products (cytidine and choline) play important roles in the generation of phospholipids (through the Kennedy cycle), which are involved in membrane formation and repair.5 Cytidine and choline also contribute to critical metabolic functions such as the formation of nucleic acids, proteins, and acetylcholine.3 Experimental studies of focal and global ischemia in citicoline-treated animals showed improvement of neurological outcome and reduced infarct size.6 7 8 9 In clinical studies of citicoline in Europe and Japan, improvement of neurological outcome was observed with mild adverse effects.10 11 12 Recently, a double-blind, placebo-controlled, randomized, dose-ranging study with citicoline was completed with favorable outcomes.13
Through the ischemic injury cascade, excitotoxic glutamate receptor activation causes intracellular entry of cations and water.14 15 MK-801 is a potent, noncompetitive NMDA receptor antagonist that blocks the excitotoxic sequelae of ischemia in tissue culture and animal models of cerebral ischemia, reduces infarct size, and improves neurological outcome.16 17 18 19 20 21 MK-801 was developed for neuroprotection in AIS, but side effects such as cataplexy, hallucinations, locomotor disturbance, reduced spatial learning, hypotension, and dose-dependent depression of consciousness limited its clinical utility.22 23 24 25 MK-801 remains helpful in AIS research because it serves as a reference compound of neuroprotective efficacy.
Citicoline and MK-801 presumably have different mechanisms for preventing ischemic neuronal injury. We investigated the therapeutic effect on infarct size and mortality of a combination of a single low dose of MK-801 (0.5 mg/kg) delivered intravenously 1 hour after arterial occlusion and low-dose citicoline (250 mg/kg) delivered intraperitoneally beginning 75 minutes after ischemia induction and then daily for an additional 6 days in a model of temporary focal cerebral ischemia. The dose and time of administration of MK-801 and citicoline were chosen because they were unlikely to have significant neuroprotective effects based on prior studies.9 19 25
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Materials and Methods |
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Normotensive Sprague-Dawley rats (n=48; weight range, 290 to 350 g) were assigned to four groups (n=12 per group): vehicle (saline), MK-801 alone, citicoline alone, and a combination of MK-801 and citicoline. Animals were maintained on a 12-hour light/12-hour dark cycle and had free access to food and water before and after the surgical procedures. All experimental procedures, including surgery, daily drug administration, daily monitoring, digitizing, and evaluation of postmortem brain slices, were conducted in a blinded, randomized manner. Our procedures were approved by the Animal Research Committee of the University of Massachusetts Medical School (ARC Protocol No. 821).
Rats were anesthetized with chloral hydrate (400 mg/kg body wt IP). The femoral vein was cannulated with polyethylene tubing (PE 50) (Intramedic, Clay Adams) for intravenous drug administration. The left femoral artery was also cannulated with PE 50 polyethylene tubing for continuous monitoring of MABP (78905A, Hewlett-Packard) and blood sampling for analysis of pH, PaCO2 (millimeters of mercury), and PaO2 (millimeters of mercury) (Corning 178 pH/Blood Gas Analyzer, Corning). Body temperature was maintained at 37°C by a rectal probe (series 400, Yellow Springs Instruments) connected to a feedback telethermometer (model 73ATD, Yellow Springs Instruments) during the surgery until recovery from the anesthesia. A commercially available heating lamp was used as the heat source. Physiological measurements were obtained before induction of ischemia, after 60 minutes of vessel occlusion, and 5 minutes after reperfusion.
Focal brain ischemia was induced by the intraluminal suture MCAO model, which has been described previously in detail.26 27 28 Briefly, the right CCA and the right ECA were exposed by a ventral midline neck incision. The proximal portion of the CCA and the origin of the ECA just after the bifurcation were ligated with a 3-0 silk suture. The CCA was loosely ligated with a 6-0 silk suture 3 mm proximal to the bifurcation to mark the arteriotomy site. To prevent bleeding, the CCA bifurcation was also loosely ligated with a 3-0 silk suture before arteriotomy. The tip of a 40-mm 4-0 monofilament nylon suture (Ethilon, Ethicon) was rounded with a soldering pencil (model TC 201 S/WTL 24, Weller) under a microscope for use as an intraluminal occluder of the MCA and then introduced through a small incision in the right CCA just beneath the 6-0 suture. The suture was carefully advanced intracranially, approximately 17 mm from the carotid bifurcation. Mild resistance to this advancement indicated that the intraluminal occluder had entered the anterior cerebral artery and occluded the origins of the anterior cerebral artery, the MCA, and posterior communicating artery.26 27 28 After the advancement of the nylon suture, loosely ligated 3-0 and 6-0 silk sutures were firmly ligated.
The treatment protocol was as follows. Sixty minutes after MCAO, the first treatment (either saline or MK-801 [0.5 mg/kg]) was given intravenously. Seventy-five minutes after MCAO, the second treatment (either saline or citicoline [250 mg/kg]) was given intraperitoneally. Citicoline or saline was given intraperitoneally daily for 6 additional days or for as long as the animal survived. Both of the agents, MK-801 and citicoline, were dissolved in 1 mL of saline and administered in a volume of 1 mL/kg. The same volume of the saline was administered as the placebo.
The MCA occluder was removed after 90 minutes of ischemia. Five minutes after reperfusion, the femoral vein and arterial catheters were also removed. All the incisions were closed by 1-0 silk sutures. The animals were allowed to recover from the anesthesia and to eat and drink freely. Over the next 6 days, neurological examinations and intraperitoneal treatments were performed once daily at 24-hour intervals. Neurological evaluations were performed according to a six-point scale: 0=no neurological deficit, 1=failure to extend left forepaw fully, 2=circling to the left, 3=falling to left, 4=no spontaneous walking with a depressed level of consciousness, and 5=death.28 29
After completing the final treatment and neurological examination on day 7, the animals were reanesthetized with 400 mg/kg IP of chloral hydrate and decapitated. The brains were then removed quickly, inspected to confirm that no subarachnoid hemorrhage had occurred, and coronally sectioned into six 2-mm-thick slices (from rostral to caudal, first to sixth). The brain slices were incubated for 30 minutes in a 2% solution of 2,3,5-TTC (Sigma) at 37°C and fixed by immersion in a 10% buffered formalin solution (Sigma). Six TTC-stained brain sections per animal were photographed by a charge-coupled device camera (EDC-1000HR Computer Camera, Electrim Corp), and the images were stored on a microcomputer. The unstained area of the fixed brain sections was defined as infarcted. Cortical and subcortical uncorrected infarcted areas and total hemispheric areas were calculated separately for each coronal slice by an image processing software package (Bioscan OPTIMAS).30 31 A corrected infarct volume was calculated to compensate for the effect of brain edema. The corrected infarct area for each slice was calculated according to the following equation: Corrected Infarct Area=Left Hemisphere Area-(Right Hemisphere Area-Infarct Area). Total cortical and subcortical uncorrected and corrected infarct volumes were calculated by multiplying the infarcted area by the slice thickness and summing the volumes of the six slices. Brain edema was calculated by subtracting the corrected from uncorrected infarct volume. Animals that survived beyond the second intraperitoneal injection of citicoline were included in the study. The brains of animals that died prematurely were harvested within 60 minutes and evaluated in a manner similar to those who were electively killed.
The randomization code was broken after all data were acquired. An ANOVA and subsequent post hoc Scheffé's test were used to determine the statistical significance of parametric variables, and a Kruskal-Wallis test was performed for nonparametric variables. Student's t test was used to compare the corrected infarct volumes from animals dying prematurely and 7-day survivors in each group. Values are presented as mean±SD. For all tests, values of P<.05 were considered significant.
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Results |
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Body weight and physiological parameters such as temperature, MABP, arterial pH, PCO2, PO2 at baseline, 60 minutes after MCAO, and 5 minutes after reperfusion are shown in the Table
and revealed no significant
differences among the four groups at any time point (P>.05,
ANOVA). The body weight declined from day 1 to day 3 and then remained
stable after day 4. The percent decline in body weight from baseline to
day 7 in those surviving for 7 days was similar (12.8% to 15.5%) in
all four treatment groups.
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Four animals in the placebo group (33.3%), 5 animals in the MK-801
group (41.7%), 5 animals in the citicoline group (41.7%), and 3
animals in the combination group (25.0%) died prematurely (between 24
and 96 hours after MCAO). There were no observable behavioral side
effects in any of the groups. The mean neurological scores from all
daily examinations were not significantly different among the four
groups, but a trend toward a lower mean neurological score was seen in
the combination treatment group (Fig 1).
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The mean corrected infarct volumes were 175.2±89.3
mm3 in the placebo group, 179.1±78.5 mm3
in the MK-801 group, 163.9±73.7 mm3 in the citicoline
group, and 84.7±56.8 mm3 in the combination treatment
group (Fig 2). Mean corrected infarct volumes of the
groups were significantly different (F=4.16, P<.02, ANOVA).
The difference of the mean values of corrected infarct volume was
significant for the combination treatment group (P<.05,
Scheffé's test), but not significant for the MK-801 and
citicoline groups (P>.05, Scheffé's test), compared
with the placebo group.
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The mean infarct volume in the animals who died prematurely tended to be larger than the 7-day survivors in the placebo, MK-801, and citicoline groups (247.2±89.5 mm3 [n=4], 229.9±82.4 mm3 [n=5], and 171.7±43.3 mm3 [n=5], respectively). In contrast, the mean infarct volume of the three animals dying prematurely in the combination treatment group was smaller than the overall group mean (40.2±29.5 mm3). Mean infarct volume in animals dying prematurely was significantly (P<.05, Student's t test) larger in group 1 than those surviving for 7 days (247.2±89.5 versus 139.2±68.2 mm3), but there was no significant difference in the infarct volume in group 2 (229.9±82.4 versus 142.7±55.4 mm3), group 3 (171.7±43.3 versus 157.4±92.7 mm3), and group 4 (40.2±29.5 versus 99.5±56.9 mm3) between animals dying prematurely and those surviving for 7 days.
The mean uncorrected infarct volume of the cortex and the subcortex
showed the same results as total corrected infarct volume in favor of
the combination group (Fig 3). The mean uncorrected
cortical and subcortical infarct volumes were 147.2±103.5 and 72.6±
23.3 mm3 in the placebo group, 166.1±79.1 and
78.8±43.3 mm3 in the MK-801 group, 161.7±84.5 and
52.8±28.5 mm3 in the citicoline group, and 78.1±65.7
and 36.0±18.7 mm3 in the combination group. Cortical
(F=2.82, P<.05, ANOVA) and subcortical (F=5.08,
P<.005, ANOVA) mean uncorrected infarct volumes were
significantly different among the groups. Post hoc analysis of
cortical infarct volume revealed no significant difference among groups
but a trend toward a smaller infarct volume in the combination group.
Post hoc analysis of subcortical infarct volume was
significantly different for the combination group versus placebo group
(P<.05, Scheffé's test) and the combination group
versus the MK-801 group (P<.02, Scheffé's test).
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P<.05, Scheffé's test).
Abbreviations are as in Fig 1The mean brain edema volume was 40.7±36.5 mm3 in the placebo group, 65.5±50.7 mm3 in the MK-801 group, 57.4±41.8 mm3 in the citicoline group, and 33.3±37.1 mm3 in the combination group. No significant difference of the mean brain edema volumes among the four groups was seen, although there was a trend toward less brain edema volume in the combination group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrates that the combination of citicoline and MK-801 reduces infarct volume in an animal model of temporary focal cerebral ischemia. The two drugs were ineffective at the doses used when given alone. Nevertheless, the combination of ineffective doses of the two drugs produced a significant (52%) reduction in infarct volume. The neuroprotective effect observed in the current and prior experiment with citicoline occurred in temporary focal ischemia, and the effect of citicoline in permanent focal ischemia is unknown.9
In a previous study in this laboratory, citicoline effectively reduced infarct volume at a dose of 500 mg/kg IP daily administered for 7 days in an experimental paradigm similar to the one used currently.9 Similarly, MK-801 alone reduced ischemic infarct volume by approximately 50% when given 5 minutes after the onset of permanent focal ischemia, with death at 24 hours.19 The present experiment used a dosing paradigm for each drug alone that was likely to have little or no effect on infarct volume to more easily evaluate potential synergy between these classes of drugs. The effects of a combination of fully effective doses of these drugs on infarct volume remain uncertain but may demonstrate even greater reductions of infarct volume than either drug by itself.
The present results demonstrate that a synergistic reduction in ischemic infarct volume can be achieved by combination therapy with neuroprotective agents that act through different mechanisms. NMDA receptor antagonists, such as MK-801, are believed to produce their neuroprotective effects by preventing the intracellular entry of cations and water caused by excitotoxic glutamate release.14 15 Although the exact mechanisms by which citicoline produces its neuroprotective effects are unknown, it has been shown to reduce the accumulation of free fatty acids, arachidonic acid, and diglycerides at the site of ischemic injury.32 33 In addition, citicoline may prevent membrane breakdown and promote membrane repair as an essential intermediate in the synthesis of phosphatidylcholine.12
Mortality rates were similar in the four treatment groups, although there was a trend toward reduced mortality in the combination treatment group. The daily neurological score also tended to show a benefit in the combination treatment group. All animals survived for at least 24 hours after the onset of focal ischemia, and the brains were harvested within 60 minutes of death in animals dying prematurely. This relatively short time interval to brain harvesting in animals who died prematurely is unlikely to produce enlarged TTC-detected lesions because of mitochondrial enzyme degradation, but this possibility could have been associated with larger lesions in some animals. Since TTC staining maximizes detection of infarction between 12 and 24 hours after onset,1 34 35 it is possible that TTC staining at 7 days might underestimate the infarct volume because of active gliosis, macrophage infiltration, and other proliferative responses in the infarct periphery.31 However, Lin et al31 did not observe a significant reduction of corrected infarct volume with TTC staining at 7 days compared with TTC staining at 24 or 72 hours in rats undergoing 90 minutes of temporary ischemia. They did observe a significant reduction of hemispheric volume and brain edema at 7 days. Additional evidence that delayed TTC staining at day 7 does not underestimate infarct volume can be obtained from the observation that brain TTC-derived infarct volume at 24 hours after 90 minutes of temporary MCAO was 179.0±43.0 mm3 (mean±SD) in the control group (n=10) of another recent treatment study in our laboratory.36 This value was very similar to that seen at 7 days in the current placebo group. In the placebo, MK-801, and citicoline groups, the infarct volumes in the animals dying prematurely tended to be greater than those in animals who survived for 7 days, suggesting that larger infarcts led to premature mortality. In the citicoline group, MK-801 group, and combined treatment group, infarct size was not significantly different between the 7-day survivors and those animals dying prematurely. In the combined treatment group there were only three premature deaths, and in these animals there was a tendency for somewhat smaller lesions than in the group overall. The cause of death in these animals is uncertain, since autopsies were not performed. However, death was apparently not caused by large infarctions. Despite these observations, the trend toward a reduction in mortality in the combination therapy group may be related to a reduction in infarct volume.
In summary, the results of this study provide a rational basis for the clinical investigation of the effects of combination therapy with neuroprotective agents that act by different mechanisms for the treatment of ischemic stroke. Synergistic effects on infarct volume can be achieved with suboptimal doses of one or both agents; whether synergism can be achieved with fully effective treatment regimens of both agents remains to be determined. The results of this study also suggest that citicoline may allow a reduction in the dose of an NMDA antagonist and hence reduce unacceptable side effects (eg, hallucinations, locomotor and learning disturbances, hypotension, altered consciousness) while maintaining or augmenting neuroprotective activity.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by the Harrington Neurological Research Fund and Interneuron Pharmaceuticals Incorporated, Lexington, Mass. Dr Li was supported by an international fellowship (F05 TW/NS05272-01) from the Fogarty International Center of the National Institutes of Health. Citicoline was provided by Interneuron Pharmaceuticals Incorporated, Lexington, Mass. We would like to thank Kentaro Takano, MD (Kyushu, Japan).
Received October 2, 1996; revision received February 10, 1997; accepted February 11, 1997.
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References |
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