Combined Neuroprotection and Reperfusion Therapy for Stroke
Effect of Lubeluzole and Diaspirin Cross-Linked Hemoglobin in Experimental Focal Ischemia
Background and Purpose In search of a better treatment for acute ischemic stroke, we evaluated the use of lubeluzole and hemodilution with diaspirin cross-linked hemoglobin (DCLHb) therapy to test whether treatment with two complementary acting compounds provides more potent protection than either treatment alone.
Methods We used unilateral reversible middle cerebral artery (MCA) and common carotid artery (CCA) occlusion of various durations in Long-Evans rats to produce ischemic cortical lesions. We calculated the average maximal lesion volume (Volmax) and the time required to produce half maximal lesion size (T50) in control animals (n=31) and evaluated the effects on cerebral perfusion and infarct size of treatment with lubeluzole (n=23), hemodilution (to 30% hematocrit) with albumin (n=17) or DCLHb (n=23), and combined lubeluzole+DCLHb therapy initiated 15 minutes after MCA/CCA occlusion.
Results The Volmax produced by MCA/CCA occlusion in control animals was 138.5±7.7 mm3, and T50 was 98.5±10.2 minutes. Lubeluzole alone reduced Volmax by 53% with no significant effect on T50. In contrast to lubeluzole, DCLHb hemodilution prolonged T50 by 68% with no significant effect on Volmax. Prolongation of T50 by DCLHb was not due to hemodilution itself, since a similar degree of hemodilution with albumin had no effect. Finally, combined lubeluzole+DCLHb rescued 72% of the tissue and augmented the effect of lubeluzole alone by 40% (Volmax, 66.3±13.0 versus 39.4±12.2 mm3) while prolonging T50 by 31%.
Conclusions Combination therapy for acute stroke using compounds with complementary action can result in more complete attenuation of neuronal damage and demonstrates the possible clinical utility of combined neuroprotective and reperfusion therapies.
Outcome from ischemic stroke reflects a summation of early (ischemia-related) and late (reperfusion-related) pathological vascular and cellular processes that ultimately combine to produce neuronal death. Because of this complexity, it is unlikely that optimal protection of the brain from stroke can be achieved through a single therapy, suggesting that an alternative approach based on combination therapy be considered.
There are two major strategies currently being evaluated for ischemic stroke. One is to restore and/or improve cerebral blood flow through the use of thrombolysis, anticoagulation, antiplatelet drugs, and/or hemodynamic manipulation. A second is to block the damaging cascade of biochemical events that occurs as a result of an imbalance between supply and demand of oxygen caused by decreased cerebral blood flow.
Blood flow in vessels is dependent on principles of tubular flow and hemorheology. Blood is a non-newtonian fluid: its viscosity increases at the lower flow rates that probably exist in ischemic regions at the time of occlusion and postischemic hypoperfusion. The force required to restore flow after it is interrupted (the yield stress) is proportional to the third power of the hematocrit level, which is the major determinant of blood viscosity.1 2 3 4 Lowering hematocrit level by hemodilution can improve brain perfusion beyond an obstructed artery. Although hemodilution may improve CP, its benefit is offset by a simultaneous decrease in the blood oxygen-carrying capacity because of lowered erythrocyte count. Therefore, the ideal substance for hemodilution should be a low-viscosity fluid that also carries oxygen.
Recently, a blood substitute consisting of stable, nontoxic DCLHb was developed and characterized.5 6 Hemodilution with DCLHb was shown to be effective in ameliorating ischemic damage in both rat and rabbit stroke models5 7 8 and is currently under evaluation for use in stroke treatment in preliminary clinical trials.
Alternative strategies to reduce ischemic brain damage include neuroprotective agents that interact with ion fluxes, neurotransmitter receptors and release, or signal transduction. Many of these strategies have been tested and found effective in animal models of ischemia and are undergoing clinical trials. One substance that has shown promise in preclinical evaluation and initial clinical trials is lubeluzole.9 10 Lubeluzole exhibits diverse mechanisms of action, including inhibition of glutamate release, prevention of glutamate-activated nitric oxide synthase–induced neurotoxicity, and blockage of voltage-gated Na+ and Ca2+ ion channels.9 11
To evaluate whether a multistrategy approach to treatment of ischemic stroke can provide more protection than a single-strategy approach, we analyzed the effect of lubeluzole and DCLHb alone and in combination in a well-described, reproducible rat model of acute focal ischemia. Results showed that each strategy alone either delayed and/or reduced neuronal damage and that combined therapy had an additive effect greater than either of the drugs alone.
Materials and Methods
Production of Ischemia
Focal ischemia in male Long-Evans rats (324 to 389 g) was induced by tandem occlusion of the left MCA and left CCA as previously described.12 13 The animals were fasted overnight with free access to water and then anesthetized, first with ether for baseline blood pressure measurement and then an intraperitoneal injection of chloral hydrate (a single 0.5-g/kg IP bolus in 1 mL of saline provided surgical anesthesia for at least 2 hours). The femoral artery was cannulated for measurement of blood pressure. Temperature of the right temporalis muscle was monitored by means of a digital needle microprobe (Omega Engineering model 410B-T with 0.1°C resolution) and was maintained at a constant value during ischemia and during the first hour of reperfusion with a heating lamp and a warming blanket. The left CCA was isolated through a midline incision and tagged with a suture. An incision was made through the left temporalis muscle perpendicular to a line between the external auditory canal and the lateral canthus of the left eye. Under direct visualization with the surgical microscope, two burr holes were made with a hand-held drill: a 1×3-mm rectangular burr hole, situated 2 to 3 mm rostral to the fusion of the zygomatic arch with the squamosal bone to expose the left MCA rostral to the rhinal fissure, and a 1-mm round burr hole for CP measurement 3 mm dorsal to the MCA exposure, which is the locus that we find to be the core of the infarction. The underlying brain was covered with sterile cotton saturated with normal saline. A laser-Doppler flow probe (0.8-mm diameter; Vasamedic) was placed over the saline-wet intact dura to measure baseline CP. The beveled edge of a 23-gauge hypodermic needle was used to pierce and open the dura along the entire length of the rectangular burr hole. A 0.005-in diameter stainless steel wire (Small Parts Inc) was placed underneath the left MCA rostral to the rhinal fissure, proximal to the major bifurcation of the MCA, and distal to the lenticulostriate arteries. The artery was then lifted, and the wire was rotated clockwise. The left CCA was then occluded with two atraumatic Heifetz aneurysm clips. Interruption of flow through the MCA was inspected under the microscope and verified by CP measurement from the laser-Doppler flowmeter. After a predetermined period of MCA/CCA occlusion ranging from 0 to 240 minutes, reperfusion was established by first removing the aneurysm clips from the CCA and then rotating the wire counterclockwise and removing it from beneath the MCA. This model of MCA/CCA occlusion produces exclusively cortical infarction. Infarct size was measured approximately 24 hours later.
DCLHb (cross-linked between the α-chains) is a cell-free pasteurized hemoglobin, purified from outdated human blood and solubilized in buffered electrolytes.5 It is similar to whole blood hemoglobin with regard to oxygen-carrying capacity, and a 10% solution has 290 mOsm/L osmolarity, 1.3 centistokes viscosity (similar to serum albumin), and 43 mm Hg oncotic pressure (hyperoncotic with respect to plasma). Human serum albumin solution in the same buffer was used for comparison. Both albumin and DCLHb solutions were provided by Baxter Healthcare Corporation.
Lubeluzole, (+)-(S)-4-(2-benzothiazolylmethylamino)-alfa-[(3,4-difluorophenoxy)methyl]-1-piperidineethanol, is a benzothiazole derivative formulated for intravenous injection and was provided by Janssen Research Foundation, Beerse, Belgium.
The objective of this study was to evaluate the effect of neuroprotective therapy with lubeluzole, hemodilution with DCLHb, and combined neuroprotective and hemodilution therapies on histopathologic outcome after acute ischemic stroke of variable duration ranging from 0 to 240 minutes and followed by reperfusion lasting 20 to 24 hours. Animals were randomly allocated to one of the following groups (the total number of animals per group before exclusion due to mortality is indicated): (1) untreated control (n=31), (2) lubeluzole alone (n=25; treatment with lubeluzole was initiated 15 minutes after onset of ischemia administered as a 0.31-mg/kg IV bolus in 0.5 mL followed by a 2-hour IV infusion of 0.31 mg/kg per hour [0.42 mL/h]), (3) albumin hemodilution (n=18), (4) DCLHb alone (n=34), and (5) DCLHb+lubeluzole (n=30). Groups 3, 4, and 5 had hemodilution that led to reduction of hematocrit to 30%. For hemodilution, 5% albumin solution (group 3) or 10% DCLHb (groups 4 and 5) was used. A two-step protocol was performed to reduce hematocrit. First, isovolemic hemodilution was carried out by withdrawal of arterial blood through the femoral artery and the simultaneous infusion of the same volume of either DCLHb or albumin through the femoral vein, beginning 15 minutes after induction of ischemia and lasting 12 to 15 minutes. Second, a top load of either of the hemodiluents (over 7 to 10 minutes) was administered to achieve a hematocrit of 30%. The volumes of hemodiluent used to reach a hematocrit of 30% for each group are indicated in Table 1⇓. Rats in group 5 (DCLHb+lubeluzole) received the same hemodilution protocol as group 4 (DCLHb alone). Lubeluzole was given as for group 2 (lubeluzole alone) except that the start of lubeluzole was delayed to 30 minutes after induction of ischemia, together with the DCLHb top load, to avoid any decrease in plasma concentration of drug due to blood exchange.
Infarct Volume Measurement
Infarct volume was measured 24 hours after induction of ischemia as previously described.13 Rats were killed under deep chloral hydrate anesthesia with intracardiac perfusion of 50 mL of 0.9% saline delivered under a constant pressure of 120 to 140 mm Hg.
The dissected brains were cooled immediately in ice-cold PBS, and 2-mm coronal sections were made using a Jacobowitz brain slicer. The infarcted regions of each of the sections were visualized by 30-minute staining at room temperature in 2% TTC in PBS. TTC-stained sections were transferred to phosphate-buffered 10% formalin before measurement of infarct volume.
Morphometric determination of the manually outlined infarcted surface (in square millimeters) of each section was performed using a computer-based Drexel University image analyzer (DUMAS) operated by the “Brain” version 1.2 for Macintosh software (Drexel University). The direct total infarct volume (in cubic millimeters) was calculated by summing the infarct area of sequential sections and multiplying by the interval thickness between sections.
Correlation Between Duration of Ischemia and Infarct Volume
The correlation between duration of ischemia and infarct size in terms of basal and maximal responses, ED50, and curve shape and steepness were computed as previously described.13 14 The computer program (Allfit) used to perform this analysis uses the logistic function y=(a−d)/[1+(x/c)b]+d, where y is the infarct volume, x is the duration of ischemia, a is the response when x=0, d is the maximal infarct volume (hereafter labeled Volmax), b is a slope “factor” that determines the steepness of the curve, and c is the ED50 (the duration of ischemia resulting in a half maximal infarct volume, hereafter labeled T50). This program was developed for the simultaneous fitting of families of sigmoidal dose-response curves and was obtained from the Laboratory of Theoretical and Physical Biology at the National Institutes of Health. The statistical difference between the groups with regards to T50 and Volmax was calculated by means of Student's t test, using log of the mean and log of the standard error of compared values provided by Allfit.
The temperature of the right temporalis muscle during the entire duration of ischemia and first hour of reperfusion was 36.5±0.32°C, 36.2±0.26°C, 36.5±0.27°C, 36.3±0.29°C, and 36.7±0.44°C for the control, lubeluzole, albumin, DCLHb, and DCLHb+lubeluzole groups, respectively.
Baseline, anesthesia, and occlusion MAPs were not significantly different among the five groups (Table 2⇓). Chloral hydrate anesthesia, in agreement with previous reports,13 lowered MAP by 17% to 20% of its baseline value in all groups (P<.05). Occlusion of MCA/CCA resulted in a moderate 6% to 9% elevation (P<.05) of the blood pressure that presumably reflects the effect of ischemia on insular cortex and/or cingulate gyrus.15 16 17 Finally, lubeluzole and albumin did not affect MAP, whereas infusion of DCLHb elevated MAP by 17% above baseline. DCLHb+lubeluzole also resulted in increased MAP. MAP during reperfusion (30 minutes after reversal of ischemia) remained elevated in animals treated with DCLHb alone and DCLHb+lubeluzole. Lubeluzole alone or albumin did not affect MAP during reperfusion.
CP in the core of the infarct was measured with a laser-Doppler flowmeter. Although the values obtained are in units that do not represent the actual flow in milliliters per 100 g of brain tissue, they are an accurate reflection of relative perfusion in the core of the infarct. Baseline (ether anesthesia) values of CP were not significantly different among the groups and were 122.7±17.2, 107.3±21.9, 120.0±23.1, 115.0±12.5, and 118.7±15.6 for the control, lubeluzole, albumin, DCLHb, and DCLHb+lubeluzole rats, respectively (Table 3⇓). Similar to MAP, CP was decreased 26% to 37% after chloral hydrate anesthesia. Occlusion of the MCA/CCA dramatically reduced CP to 4.6% to 6.5% of baseline (not significantly different among the groups). After treatment and 30 minutes of reperfusion, CP returned to approximately 50% of baseline value in all but the DCLHb-alone group. DCLHb alone (but not with lubeluzole) further improved CP by 20% (relative increase, 44%) compared with the control group.
Levels of Pco2, Po2, and pH at baseline and after treatment (during ischemia) did not differ among the groups except for a slightly higher pH (7.38±0.03) in the DCLHb-alone group compared with control (7.32±0.04) during ischemia.
Mortality among the groups varied significantly. In the control group, 6% of animals died because of the surgery and/or ischemia. Neither albumin hemodilution (5%) nor lubeluzole (8%) significantly affected mortality rate. However, mortality rates in the DCLHb-alone and DCLHb+lubeluzole groups were 33% and 16%, respectively.
Effect of MCA/CCA Occlusion on Infarct Volume
Twenty-nine Long-Evans rats were subjected to variable durations of ischemia ranging from 45 to 240 minutes, as illustrated in Fig 1A⇓. Duration of MCA/CCA occlusion lasting up to 90 minutes did not produce substantial brain damage. Four of 5 rats with 90 minutes of ischemia developed infarct volumes less than 20 mm3. Extending the duration of ischemia to 120 minutes produced a dramatic increase in lesion volume in all 5 animals. Prolonging MCA/CCA occlusion for up to 240 minutes did not result in further enlargement of lesion size. Predicted Volmax computed by the Allfit computer program (see “Materials and Methods” for details) was 138.5±7.7 mm3, whereas the duration of MCA/CCA occlusion that produced the T50 amounted to 98.5±10.2 minutes.
Effects of Lubeluzole and DCLHb Hemodilution Alone on Infarct Volume
Lubeluzole administered 15 minutes after induction of ischemia of variable duration potently alleviated neuronal damage. Its major benefit was to reduce average Volmax from 138.5 to 65.3 mm3 (approximately 50% reduction, Fig 1B⇑). However, T50 was not significantly prolonged by treatment with lubeluzole and was 111.7±15.0 minutes.
Hemodilution with 10% DCLHb to a hematocrit of 30% was also effective in alleviating ischemic damage. However, the component of observed benefit was distinct from that provided by lubeluzole (Figs 1D and 2⇑⇓). Whereas lubeluzole predominantly affected Volmax, DCLHb hemodilution mostly affected T50, extending the duration of MCA/CCA occlusion that produced half maximal damage from 98.5 minutes in controls to 170.3 minutes. By contrast, DCLHb did not affect Volmax. The effect of DCLHb hemodilution presumably was not due to hemodilution itself, since albumin, substituted for DCLHb, did not have a significant effect on T50 (98.5±10.2 versus 93.9±19.5 minutes for control and albumin hemodilution, respectively) (Figs 1C and 2⇑⇓).
Effect of Combined Treatment With Lubeluzole and DCLHb Hemodilution on Infarct Volume
The distinct profiles of benefit provided by lubeluzole (decrease in Volmax) and DCLHb hemodilution (prolongation of T50) suggested the possible additive effect of therapy with combined lubeluzole and DCLHb. As seen in Figs 1E and 2⇑⇑, combination therapy provided protection superior to either treatment alone. Lubeluzole coadministered with DCLHb hemodilution further reduced by approximately 40% the Volmax observed after treatment with lubeluzole alone (66.3±13.0 versus 39.4±12.2 mm3). This translates into a greater than 70% reduction of control infarct volume. The 70% reduction of Volmax was associated with simultaneous prolongation of T50. Combined therapy extended the T50 by approximately 30 minutes more than that of controls. It is likely that the lower steepness of the curve for DCLHb+lubeluzole (−9.1) versus DCLHb alone (−25.7), due to reduction of Volmax in the combined therapy group, accounts for the longer T50 calculated by Allfit for DCLHb alone compared with the combined therapy group.
Over the last decade, there has been dramatic progress in understanding the basic mechanisms of brain pathology after ischemia. This has led to the development of a variety of experimental strategies aimed at the treatment of stroke. Numerous treatments have shown variable degrees of efficacy in reducing brain damage using various animal models of ischemia. None, however, except very recently TPA,18 have produced full protection in the laboratory or proven beneficial in clinical trials in humans. To provide the most effective treatment for ischemic stroke, it will be necessary to both optimize treatment strategy and shorten the time between the onset of ischemia and the start of treatment.
While the search for an ideal monotherapy will continue, an alternative approach to augment protection is to combine the most promising known therapies, which complement each other on the basis of their mechanisms of action.
Two treatments, lubeluzole and DCLHb hemodilution, were selected on the basis of their distinct modes of action to test the usefulness of the combined-therapy approach in treating experimental ischemia using our model of unilateral MCA/CCA occlusion in rats. Initially, each treatment was tested separately. Distinct profiles of protection by lubeluzole and DCLHb hemodilution, with the first mostly affecting Volmax and the second having a predominant effect on T50, indicated that these drugs might complement each other in their anti-ischemic effect when administered together (Fig 2⇑).
The region of ischemic brain most amenable to rescue by antistroke therapy surrounds the core of the infarct. This peri-ischemic or penumbral region is characterized by intermediate impairment of blood perfusion and preserved cerebral metabolism. Excessive glutamate release and abnormal Na+ and Ca2+ ion homeostasis are characteristic of the penumbra.19 Reduction of the Volmax by lubeluzole, which has been shown to block glutamate release and Na+ and Ca2+ ion channels, may indicate rescue of this penumbral region. The core of the infarct appeared to be equally damaged by the same duration of ischemia in both control and lubeluzole-treated animals. On the other hand, hemodilution with DCLHb might temporarily provide improved oxygenation throughout the entire ischemic region, thereby delaying formation of the infarct (prolonging the T50). One explanation for the additive effect of DCLHb and lubeluzole is that DCLHb temporarily maintains perfusion and oxygenation in what would be the “core,” thereby transiently maintaining it in a “penumbral” state. When DCLHb is combined with lubeluzole, lubeluzole can reach an area that otherwise would have no perfusion but that still suffers from excessive glutamate release and ionic imbalance amenable to the presumed mechanism of action of lubeluzole.
The mechanism of benefit provided by DCLHb is not entirely clear. We demonstrated improved CP during the reperfusion phase in DCLHb-treated animals. We measured CP in the region just distal to the MCA occlusion to ensure similar ischemic insults in all animals; although we were unable to demonstrate improved perfusion during ischemia in this region with DCLHb, it is likely (but unproven) that perfusion was improved in other more peripheral areas of the ischemic core and penumbra where collateral flow was more possible.
It is unlikely that hemodilution and reduced viscosity by themselves were responsible for the positive results with DCLHb in our experiments, since hemodilution with albumin did not affect blood flow during reperfusion. DCLHb as opposed to albumin can compensate for loss of hemoglobin due to blood exchange and thereby improve oxygen delivery. DCLHb, like whole blood hemoglobin, can bind and quench nitric oxide.20 21 Nitric oxide is toxic to neuronal tissue22 ; therefore, inhibition of excessive nitric oxide concentration by DCLHb may be responsible for its observed neuroprotection. Furthermore, DCLHb has a pressor effect that may account for the improved reperfusion found with DCLHb and not albumin. Although a negative effect of inhibition of endothelial nitric oxide on ischemic outcome has been postulated,23 this may be counteracted by the effect of DCLHb on endothelin and adrenergic mechanisms.21 24
The cause of death in the DCLHb-treated groups is unknown. However, preliminary results indicate that some delayed interaction between chloral hydrate anesthesia and DCLHb might play a critical role. Normal animals given chloral hydrate plus DCLHb also died (J. Aronowski, unpublished observations, 1995), and previous studies using general anesthesia did not report increased mortality with DCLHb.5 7 In most cases in our study, death occurred several hours after ischemia, phlebotomy, and hemodilution and after animals had already recovered from surgery. DCLHb-treated rats demonstrated no obvious behavioral or physiological abnormalities coinciding with mortality, and postmortem examination in 2 animals showed only pulmonary congestion, which was thought to be agonal in nature. There was also no correlation (r=.01; P=.976) between duration of ischemia and mortality, suggesting that ischemic brain injury did not affect mortality rate. Consequently, it is unlikely that animals treated with DCLHb with larger lesions were eliminated from analysis because of death, thereby causing artifactual lowering of Volmax or prolongation of T50 in groups treated with DCLHb.
In conclusion, we have shown that hemodilution with the blood substitute DCLHb can delay the onset of ischemic damage and that the neuroprotective compound lubeluzole can reduce the size of the infarcted region. The complementary action of these compounds when administered in combination results in more complete neuronal protection and demonstrates the possible clinical utility of combined reperfusion and neuroprotection therapies.
Selected Abbreviations and Acronyms
|CCA||=||common carotid artery|
|DCLHb||=||diaspirin cross-linked hemoglobin|
|MAP||=||mean arterial blood pressure|
|MCA||=||middle cerebral artery|
|T50||=||half maximal lesion size|
|Volmax||=||average maximal infarct volume|
Support for this study was provided by the National Institutes of Health National Institute of Neurological Disorders and Stroke (grant NS-23979), Janssen Research Foundation, and Baxter Healthcare Corporation.
- Received December 18, 1995.
- Revision received March 19, 1996.
- Accepted May 24, 1996.
- Copyright © 1996 by American Heart Association
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The number of agents with established effectiveness in reducing ischemic injury of the brain in experimental animals is increasing rapidly. This development has led to interest in finding out whether the simultaneous administration of two or more agents might be more effective than the benefit from their individual action. Such a potentiation might occur because of a number of mechanisms. First, since the mechanisms of ischemic cellular damage are complex, it is possible that two agents that have independent actions might be more effective in interrupting the cytotoxic mechanisms that lead to neuronal death when such agents are given together. Second, the administration of agents that increase blood flow together with agents that act on the cellular ischemic processes might be more effective not only because the increased blood flow relieves ischemia but also because it allows the delivery of the agent that acts on cellular processes to a larger volume of ischemic tissue. Finally, the combination of procedures or agents that increase blood flow may be coupled with increased delivery of oxygen by chemical means, as well as with the administration of an agent that acts on the cellular ischemic processes.
In the article above, Aronowski and his colleagues confirm that in an experimental ischemic model, combination therapy is more effective than the administration of two agents separately. The authors found that the simultaneous administration of lubeluzole and DCLHb reduced infarct volume from vascular occlusion to a greater extent than the two agents administered separately. The precise mechanism of action and the reasons for the greater effectiveness of combination therapy were not entirely clear. Because they found that hemodilution comparable with that achieved by the DCLHb, induced by administration of albumin, was not effective, simple hemodilution and consequent increase in blood flow does not appear to be the mechanism involved. It is possible that the DCLHb might not only have increased blood flow but also delivered more oxygen, which is not the case with the albumin-induced hemodilution, or that the DCLHb might have scavenged damaging agents, such as nitric oxide, released during ischemia. Irrespective of the mechanism involved, it is of great importance to have experimental confirmation of the concept that combination therapy might be more effective than would be expected from the administration of agents alone.
One reservation needs to be mentioned: the administration of lubeluzole and DCLHb in this study was initiated 15 minutes after the onset of vascular occlusion. This is obviously too short a time for therapeutic intervention in most patients with stroke. Therefore, additional studies will be needed to show whether combination therapy given at a later time is as effective as when given at the earlier stages of ischemia.
Selected Abbreviations and Acronyms
|CCA||=||common carotid artery|
|DCLHb||=||diaspirin cross-linked hemoglobin|
|MAP||=||mean arterial blood pressure|
|MCA||=||middle cerebral artery|
|T50||=||half maximal lesion size|
|Volmax||=||average maximal infarct volume|