Protection With Lubeluzole Against Delayed Ischemic Brain Damage in Rats
A Quantitative Histopathologic Study
Background and Purpose Cerebral ischemia may lead to glutamate-induced excitotoxic damage in vulnerable brain areas. Lubeluzole is not an N-methyl-d-aspartate antagonist but prevents postischemic increase in extracellular glutamate concentrations. The present study examined whether lubeluzole, administered after global incomplete ischemia in rats, is capable of preserving the structural integrity of CA1 hippocampus.
Methods Ischemia was induced by bilateral carotid artery occlusion and severe hypotension for a duration of 9 minutes. Delayed neuronal cell death was histologically evaluated 7 days later. This was done by scoring acidophilic cell change and coagulative necrosis and by counting the number of surviving neurons in the CA1 subfield. Experiments were performed according to a paired design (13 animals per treatment group).
Results Posttreatment with lubeluzole (0.31 mg/kg IV bolus at 5 minutes and 0.31 mg/kg IV infusion during 1 hour) resulted in significant neuroprotection. Whereas in the untreated rats there were 42 (median) viable neurons per millimeter CA1 layer in the left and 69 in the right hemisphere, in the drug-treated rats 99 viable neurons per millimeter were found in the left (P=.002) and 113 in the right hemisphere (P=.013). Histological scores, reflecting altered staining properties of the hippocampal cells, correlated strongly with the quantitative data, reflecting the structural integrity of CA1 pyramidal neurons.
Conclusions Lubeluzole, when administered after an ischemic insult in rats, protects vulnerable brain regions against delayed structural injury. The results support the potential clinical use of this new drug in stroke treatment.
Global forebrain ischemia leads to delayed neuronal necrosis in vulnerable brain areas in experimental animals and humans.1 2 3 4 It is generally accepted that calcium is a key mediator in processes leading to neuronal cell death and that an important link exists between cellular calcium metabolism and excitatory amino acids, particularly glutamate.5 Glutamate is massively released during ischemia.6 7 Excitotoxic mechanisms, triggered by glutamate, are believed to mediate ischemic neuronal damage in vulnerable areas.8 9
Lubeluzole is a new cerebroprotective compound in clinical development for the treatment of acute ischemic stroke.10 The drug improves neurological outcome after photothrombotic infarction of rat sensorimotor cortex.11 It prevents the increase of extracellular glutamate concentrations in the peri-infarct region12 and normalizes neuronal excitability.13 In vitro experiments indicate that the drug also inhibits glutamate-induced nitric oxide–related neurotoxicity in hippocampal neurons14 and that it is able to prevent the pathological consequences of sodium-dependent calcium overload in hippocampal slices.15
The aim of the present study was to determine whether lubeluzole, administered at its optimal dose regimen for protection against photochemical stroke,11 is capable of preserving the structural integrity of vulnerable neurons after a severe ischemic challenge in rats.
Materials and Methods
Induction of Incomplete Ischemia
Animal housing and treatment conditions complied with European Union directive for Animal Welfare (No. 86/609). Incomplete ischemia was induced in fasted male Wistar rats (n=36) weighing approximately 300 g. The method of Smith et al16 was used. Induction of anesthesia and tracheal intubation were performed under 4% isoflurane. During subsequent operative procedures, the animals were kept under 1% isoflurane in a 30% O2 and 70% N2O mixture. Intra-arterial and intravenous catheters were placed for monitoring of mean arterial blood pressure and for drug delivery or blood sampling. The common carotid arteries were isolated by a neck incision. Blood gases were routinely checked (ABL 300, Radiometer). All rats were thermally supported up to 1 hour after recirculation (ie, until the end of drug treatment). Body temperature was maintained by a rectally controlled heating pad. Brain temperature was maintained by a heating lamp controlled by the temporal muscle. Temperature settings for both were 36.7°C. After a steady state of 10 minutes, cerebral ischemia was induced by bilateral clamping of both carotid arteries and simultaneous induction of hypovolemia by rapid bleeding through the jugular vein until mean arterial blood pressure dropped to 35 mm Hg. During ischemia, isoflurane was omitted from the O2/N2O mixture. After an ischemic period of 9 minutes, carotid clamps were released, and the shed blood was reinfused. Sodium bicarbonate (0.5 mL; 0.6 mol/L) was injected intravenously, and the animals were allowed to recover in an acclimatized room. They were killed 7 days after the insult.
Five minutes after termination of ischemia, treatment with lubeluzole (n=18) or its solvent (n=18) was started. Treatment was blinded. Coded solutions of drug or solvent were given alternately. The drug was administered as an intravenous bolus (1 mL/kg) at a dose of 0.31 mg/kg followed by slow intravenous infusion over 1 hour (1 mL/kg) of a dose of 0.31 mg/kg. The solvent was an aqueous solution of 50 mg/mL glucose and 0.392 μg/mL HCl (pH 3.8 to 4.2).
To evaluate possible drug-induced delayed postischemic hypothermia, a limited number of paired animals (n=12) were subjected to similar ischemic conditions as described above. Five minutes after recirculation, they were treated with coded solutions of either lubeluzole (0.31 mg/kg IV bolus followed by 0.31 mg/kg IV infusion; n=6) or vehicle (n=6). Body temperature was rectally controlled in awake animals every hour up to 5 hours after ischemia and at 24 and at 48 hours after ischemia.
Histopathology and Quantitative Evaluation
All brains were fixed by intracardiac perfusion with diluted Karnofsky's fixative (2% formaldehyde/2.5% glutaraldehyde in Sörensen's phosphate buffer; pH 7.4) followed by overnight immersion in the same fixative. Coronal sections of the dorsal hippocampus were prepared stereotaxically 3.6 mm caudally to the bregma (Vibratome 1000, TPI). Sections (100 μm) were mounted in toto and stained with azure-eosin. Alternate 200-μm slices were postfixed with 2% osmium tetroxide, dehydrated in a graded ethanol series, and routinely embedded in epoxy resin. Epoxy resin sections were cut at 2 μm and stained with toluidine blue. An estimate of acidophilic cell change and coagulative necrosis was obtained by scoring ischemic damage in the entire CA1 subfield of left and right hippocampi on 100-μm sections. Damage was graded according to a 5-point scale (0=no damage, 4=maximal damage). In addition, the severity of damage was quantitatively assessed by counting the number of viable pyramidal cells in the middle subsector of the CA1 subfield of the hippocampus. A total of three 2-μm sections randomly selected from each animal were evaluated on a Polyvar microscope (Reichert-Jung) at a final magnification of ×400. The results were expressed as the number of viable neurons per millimeter CA1 layer for the left and right hippocampi. To ensure unbiased evaluation, codes were broken only after termination of all examinations.
Experiments were performed according to a paired design on animals surviving for 7 days. Results were analyzed with the two-sided Wilcoxon matched-pair signed rank test on the difference between the treated and the control animal of each pair. Two-tailed values of P≤.05 were considered to indicate statistical significance. Treatment effects are reported as median values of difference between treated and control animals. Confidence intervals (CIs) for this median difference were constructed by means of the Wilcoxon statistic.17 Relationships between the different variables were evaluated with the Spearman rank correlation and LOESS, a nonparametric curve-fitting technique.18 CIs for the rank correlation coefficient and variability of the curve fitting were assessed by means of bootstrapping.19 Individual data are represented as scatterplots.
Of the 18 lubeluzole-treated rats, 1 died in the early postischemic period as a result of a tracheal tube obstruction. In the solvent group, 4 of 18 rats died at various times after ischemic challenge of an unknown cause. Finally, 13 paired rats were retained from the two groups. The baseline physiological variables before ischemia did not differ between the two groups (Table 1⇓). At 1 hour after recirculation (ie, at the end of drug treatment), significantly higher Paco2 (P=.037) and actual base excess (P=.049) values were found for the lubeluzole-treated group. At 1 hour after treatment, median brain temperatures were 36.6°C in the control group and 36.0°C in the lubeluzole group (n=12 because of failure of registration in 1 rat). The median difference was −0.45°C (95% CI, −1.4°C to 0.1°C; P>.05).
Ischemic injury in 100-μm sections was characterized by coagulative and acidophilic cell change, restricted to the CA1 subfield of the two hippocampi (Fig 1⇓). The median lesion score for the left and right hippocampi was 3 in the control group and 0.5 in the treated group (Fig 2⇓). As a result of treatment, there was a median score decrease of −2 (95% CI, −3 to −1) in the left hippocampus (P=.002) and of −1.5 (95% CI, −2.5 to −0.5) in the right hippocampus (P=.011).
These results were confirmed by quantitative data. Ischemic injury in the 2-μm sections was restricted to the pyramidal cell layer of the CA1 subfield of the two hippocampi and characterized by irreversible coagulative necrosis of pyramidal neurons and pronounced gliosis (Fig 1⇑). Statistical analysis on counts of viable neurons showed a significant neuroprotection in both hippocampi after lubeluzole treatment. The median number of viable neurons per millimeter in the left hippocampus was 42 in the control group and 99 in the lubeluzole group. In the right hippocampus, the values were 69 and 113 for the control and the treated group, respectively (Fig 2⇑). The median treatment effect was +64 cells (95% CI, +26 to +82) for the left (P=.002) and +34 cells (95% CI, +20 to +86) for the right hemisphere (P=.013).
A highly significant linear relationship between histological scores and quantitative results was found for both hemispheres (Spearman rank correlation, r=−.827 and r=−.797 for the left and the right hemispheres, respectively).
As can be derived from rectal temperature registration in awake rats up to 48 hours after ischemia, there was no indication that lubeluzole induced delayed hypothermia (Table 2⇓).
Posttreatment with lubeluzole after an incomplete cerebral ischemic insult resulted in marked reduction of delayed neuronal cell death, as evidenced by the significantly higher number of viable neurons in CA1 hippocampus. This neuroprotective effect is probably also reflected in the improved survival rate of the treated animals. Apart from one rat that died from tracheal tube obstruction, no mortality was observed in the lubeluzole group. The cause of death in four rats in the control group is unknown, but cerebral complications cannot be excluded. One hour after recirculation, Paco2 and actual base excess remained significantly lower in the solvent group than in the drug-treated group. The extent to which a more rapid return to the normal range of one or both blood parameters can be held responsible for the beneficial effect of lubeluzole on mortality and/or delayed neuronal death is not known.
The morphological aspect and degree of ischemic cell change, resulting from combined carotid artery occlusion and severe hypotension, were comparable to those observed in previous studies.20 21 The CA1 hippocampal subfield in unchallenged Wistar rats of comparable weight contains 149 viable neurons per millimeter.20 Damage in this subfield consisted of coagulative neuronal cell change, characterized by shrinkage of the cell body, nuclear pyknosis, and fragmentation of the apical dendrite. These necrotic neurons could hardly be distinguished from proliferating microglia. Therefore, only surviving pyramidal neurons of the CA1 subfield were counted. These were easily identifiable on the basis of a fairly extended perikaryon and a centrally located round nucleus. Moreover, viable cells were clearly delineated from the surrounding neuropil. Since numerical counts might have been influenced by edematous swelling of the tissue, lesion scores based on staining properties of CA1 cells were also included. A clear linear relationship was present between scores and counts, suggesting the unlikelihood that eventual edema formation would influence the results.
There is no doubt that ischemic and postischemic brain temperatures may influence structural outcome of hippocampal neurons after global ischemia and that certain pharmacological agents exert their protective effect by interaction with brain temperature.22 23 The present results show a median reduction of brain temperature of −0.45°C at the end of 1 hour of lubeluzole infusion. This is much below the protective shifts reported with an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist.22 Moreover, when body temperature was followed in a limited number of ischemic animals treated with vehicle or with lubeluzole, no temperature difference could be detected up to 48 hours after ischemic challenge. Even higher doses of lubeluzole did not affect body temperature up to 4 hours after photochemical stroke in rats.11 Therefore, there is no indication that drug-induced hypothermia might have been responsible for the observed beneficial effects of lubeluzole.
Although the mechanism of action of lubeluzole is not fully understood, recent experimental evidence shows that the drug may exert its effect at the neuronal cell level. In this study, the structural outcome of brain cells after global cerebral ischemia was improved by lubeluzole. A similar effect occurred after systemic hypoxia in juvenile rats (J. Van R., unpublished data, 1991). Functional outcome was improved after photothrombotic infarction of sensorimotor cortex in rats,11 and neuronal excitability was normalized in ipsilateral and contralateral hemispheres after unilateral photothrombotic infarction in rats.13 These beneficial effects on the structural and functional preservation of neurons compromised by oxygen shortage may be accounted for by an attenuation of excessive extracellular glutamate levels that are observed subsequent to ischemia, as shown by Scheller et al.12 Glutamate plays a crucial role in the occurrence of ischemic damage because of its excitotoxic potency. Prevention of its postischemic increase might preserve neuronal integrity and function. Reduction of glutamate by lubeluzole can be explained by improved clearance from the extracellular space by glial cells but also by the ability of lubeluzole to prevent the pathological consequences of sodium-dependent calcium overload, as shown by Ashton et al.15 In this manner, not only could excessive excitatory transmitter release be avoided, but cell destruction triggered by pathological amounts of calcium might also be prevented.
Although the exact mechanism of action remains to be elucidated, the neuroprotective effects of lubeluzole already justify its clinical use in stroke.
The authors are grateful to Raymond Broeckx for technical advice and equipment support over the years; to Lambert Leijssen, who prepared the illustrations; and to Drs Marc De Ryck and Koen van Rossem for their critical scientific comments.
- Received July 9, 1996.
- Revision received October 22, 1996.
- Accepted October 24, 1996.
- Copyright © 1997 by American Heart Association
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Haseldonckx et al demonstrated that lubeluzole was neuroprotective in a rat model of global incomplete ischemia. In this study the authors took care to monitor and/or control the important physiological variables. Lubeluzole was administered 5 minutes after ischemia which, in the case of global ischemia, is a clinically relevant protocol. In phase I and II stroke clinical trials, lubeluzole has demonstrated safety and preliminary efficacy1R and has advanced into phase III trials. In addition, focal ischemia studies in rats demonstrated that lubeluzole was neuroprotective alone2R or in combination with other agents.3R
Although lubeluzole was shown to have neuroprotective effects in this and in several other studies, the mechanism(s) through which these beneficial effects are produced is not well defined. It will be important to further clarify the mechanisms of lubeluzole's neuroprotective effect. The new data presented in this study suggest that, in addition to potential therapeutic efficacy in focal stroke, lubeluzole may be useful in reducing brain damage after global ischemia.
Diener HC, Hacke W, Hennerici M, Radberg J, Hantson L, DeKeyser J. Lubeluzole in acute ischemia stroke: a double-blind, placebo-controlled phase II trial. Stroke.. 1996;27:76-81.
Aronowski J, Strong R, Grotta JC. Combined neuroprotection and reperfusion therapy for stroke: effect of lubeluzole and diaspirin cross-linked hemoglobin in experimental focal ischemia. Stroke.. 1996;27:1571-1577.