CoQ10 fails to protect brain against focal and global ischemia in rats

Brain Research 877 (2000) 7–11 www.elsevier.com / locate / bres

Research report

CoQ10 fails to protect brain against focal and global ischemia in rats Hui Li a , Gary Klein a , Ping Sun a , Alastair M. Buchan b , * a

Alberta Stroke Program, Department of Clinical Neurosciences, Foothills Hospital, University of Calgary, Calgary, Alberta, Canada b Professor of Stroke Research, Rm 1162, Foothills Hospital, 1403 – 29 th St. NW, Calgary, Alberta T2 N 2 T9 Canada Accepted 20 June 2000

Abstract Objective: Release of oxygen free radicals occurs following cerebral ischemia. Studies show that oxygen free radicals mediate ischemic brain injury. CoQ10 is a potent free radical scavenger and may offset brain injury associated with reperfusion. We tested exogeneous CoQ10 as a neuroprotectant in rats following both global and focal ischemic insults. Methods: Rats were subjected to either 4-vessel occlusion ischemia (4-VO, 10 min occlusion, 7-day survival) or middle cerebral artery occlusion (MCAO, 120 min-occlusion, 22.5 h survival). Regional cerebral blood flows (rCBF) and physiological variables such as blood pressure, pO2, pCO2, plasma glucose and hematocrit were monitored and measured in focal ischemia. The animals were randomized to receive treatments of either phosphate buffered saline (PBS) vehicle or CoQ10 following global or focal ischemia. Injection times were at the end of ischemia and 3 h later for both models of ischemia. Histological outcomes are expressed as a percentage of hippocampal CA 1 cell injury in global ischemia or percentage of cortical infarct over that of non-ischemic hemisphere in focal ischemia. Results: In global ischemia, animals treated with PBS vehicle and CoQ10 had 8665% (n58) and 83610% (n58), respectively, of hippocampal CA 1 cell injury (P.0.05). The percentage of infarct volumes in animals following focal ischemia were 2369% (control, n510) and 2569% (CoQ10, n510). There were no temperature or physiological differences between the two treatment groups. Conclusion: Acute treatment with CoQ10 via intraperitoneal injection does not prevent neuronal injuries following global and focal ischemia.  2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Ischemia; Hippocampus; Infarct; Apoptosis; Inflammation; Free radical

1. Introduction Transient cerebral ischemia can trigger release of oxygen free radicals that may promote ischemic neuronal cell injury. Studies show that tissue reoxygenation following ischemia provides oxygen for a variety of enzymatic oxidation in ischemic tissues and leads to over-production of free radicals [7,38,8,16]. Contribution of free radicals to ischemic neuronal injury includes peroxidation of cellular lipid, protein and nucleic acid [7,29] as well as modulation of inflammation and tissue blood flow, microvascular permeability and disruption of blood brain barrier [4,32].

*Corresponding author. Tel.: 11-403-670-1581; fax: 11-403-6701602. E-mail address: [email protected] (A.M. Buchan).

The apparent role of free radicals in the development of neuronal cell death has stimulated interest in the use of antioxidants and free radical scavengers as potential therapeutic agents for ischemia. Many studies have shown that inhibition of free radicals, using scavengers or oxidative enzymes, results in significant neuroprotection in transient global ischemia [11,36,25]. Others report that infarcts in knock-out mutant mice [13] or in transgenic mice overexpressing superoxide dismutase [14] are smaller that that of wild-type normal control mice. CoQ10 is an essential co-factor of the electron transport chain but is also a potent free radical scavenger in lipid and mitochrondial membranes [23]. There is certainly some experimental evidence to suggest that CoQ10 can scavenge superoxide radicals in models of rat reperfusion injury [37]. CoQ10 may therefore be a logical choice as a therapy for cerebral ischemia. The purposes of our experi-

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02609-3

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H. Li et al. / Brain Research 877 (2000) 7 – 11

ments were to determine if CoQ10 is neuroprotective in rat models of global and focal ischemia.

2. Materials and methods

2.1. Animals Experiments using both global and focal ischemia were done with the approval of the Animal Care Committee of the University of Calgary. The animals were cared for according to guidelines published by the National Institutes of Health and the Canadian Council on Animal Care. All animals were obtained from Charles River (Montreal, Quebec, Canada).

temporal bone to expose the middle cerebral artery (MCA). A microclip was placed on the proximal end of the MCA. The ligature on the right common carotid artery was tightened, starting the period of ischemia. Surgical wounds were then closed with wound clips, and animals were disconnected from anesthesia and returned to cages. Physiological variables such as blood pressure, blood gases, plasma glucose and hematocrit were measured and recorded. Regional cerebral blood flow was measured using a laser Doppler flowmeter at locations of the core (R3) overlying the center of infarction, and the penumbra (R2) overlying the peripheral area of infarction at the onset of ischemia, the end of ischemia, the onset of reperfusion, and immediately prior to sacrifice of the animals. Rectal temperature of the animals was maintained at 378C during and following ischemia.

2.2. Global ischemia Male Wistar rats (150–175 g) underwent transient forebrain ischemia using four vessel occlusion [28]. In brief, the animals were anaesthetized with 2% halothane, 70% N 2 and 28% O 2 . Both common carotid arteries were isolated and a silk gently and loosely placed around each vessel. The vertebral arteries were electrocauterized and a ligature passed ventral to the cervical and paravertebral muscles but dorsal to the trachea, esophagus, external jugular veins, and common carotid arteries. The wound was closed with a wound clip and animals were allowed to recover overnight. On the following day, brief anesthesia was induced using halothane. Forebrain ischemia was induced by clamping both common carotid arteries with aneurysm clips and also tightening the collateral snare ligature to prevent the opening of collateral blood flow channels. Animals were observed during ischemia for unconsciousness, dilation of both pupils and pattern of respiration. Aneurysm clips were removed at the end of ischemia and the animals were returned to cages. Rectal temperature was maintained at 378C during and following ischemia.

2.4. Ischemic duration and drug treatment The animals were subjected to either 10 min of normothermic global ischemia or to 120 min of focal ischemia using middle cerebral occlusion, then divided randomly into two groups. Animals from group 1 were given Phosphate Buffered Saline vehicle as control and animals from group 2 were injected intraperitoneally (IP) with CoQ10 at 3 mg / kg / injection at time of reperfusion and 3 h after reperfusion. The dosage was chosen after evaluation of prior studies.

2.5. Neuropathological analysis

2.3. Focal ischemia

2.5.1. Global ischemia 7 days following the ischemia, animals were reanesthetized and perfusion-fixed with 4% buffered formalin. Serial coronal sections, 7 mm thick, of paraffin-embedded brains were cut and stained with hematoxylin and eosin. Both normal and injured neurons were counted in the hippocampal CA 1 area, and results expressed as a percentage of neurons injured, and presented as mean6S.D.%.

Male spontaneously hypertensive rats (200–250 g) underwent focal ischemia using middle cerebral artery occlusion. The surgical method has been described in detail [2]. In brief, animals were anesthetized with 70% N 2 , 28% O 2 , and 2% halothane. The tail artery was cannulated, connected to a BP monitor, and blood pressure was monitored during surgery. The right common carotid artery was isolated through a ventral midline incision and a ligature placed around it. A 1 cm incision was made perpendicular to and bisecting a line between the lateral canthus of the right eye and external auditory canal. The underlying temporalis muscle was retracted and a hole 1 mm in diameter drilled 2–3 mm rostral to the point of fusion of the zygomatic arch with the

2.5.2. Focal ischemia 22.5 h following ischemia, animals were reanesthetized, blood gases and rCBF were redetermined, and animals were decapitated. Brains were removed and frozen in isopentane, cooled on dry ice. 20 mm sections were cut, with 25 section intervals, and stained with hematoxylin and eosin. Areas of infarcted cortex, ipsilateral remaining regions and contralateral non-ischemic hemispheres were traced using an image-processing system (Image Pro II; Media Cybernetics, Silver Spring, MD, USA). The percentage of cortical infarction over that of normal hemisphere for each animal was calculated and presented as mean6S.D.%.

H. Li et al. / Brain Research 877 (2000) 7 – 11

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gases and rCBF. Comparisons between infarct sizes for treatment groups were by analysis of variance for focal ischemia. A P value of ,0.05 determined significance for both studies.

3. Results

3.1. Global ischemia Histological results are presented in Fig. 1. Animals treated with CoQ10 sustained 83610% (n58) of hippocampal CA 1 cell injuries, while the animals treated with vehicle had 8665% (n58) injuries. There were no significant differences between these groups (P.0.5).

3.2. Focal ischemia Fig. 1. Percentage of hippocampal CA 1 neuronal injury (mean6S.D.) measured in rats sacrificed after 7 days of survival following transient (10 min) forebrain ischemia using a four-vessel occlusion model.

2.6. Statistical analysis In global ischemia, differences in the percentage of injured hippocampal CA 1 neurons were analyzed by nonparametric statistics with a Mann–Whitney U test using a Bonferroni correction for global ischemia. In focal ischemia, student’s t-test was used to analyze differences in the physiological variables, including blood

Physiological variables are presented in Table 1 and rCBF in Table 2. Infarct volumes of both groups are shown in Fig. 2. In vehicle-treated animals the mean infarct volume was 2369% (n510), while in the CoQ10-treated group the mean volume was 2569% (n510) (P.0.5).

4. Discussion Transient cerebral ischemia can lead to either delayed selective neuronal death and / or apoptosis in the hippocam-

Table 1 Physiological Variables of CoQ10 (mean6S.D.)

Onset of Ischemia Control (n510) CoQ10 (n510) Reperfusion Control (n510) CoQ10 (n510) Decapitation Control (n510) CoQ10 (n510)

BP

pH

pO2 (mmHg)

pCO2 (mmHg)

Glu (mmol / l)

Hct

135617 140614

7.260 7.260

108633 115622

5664 6068

8.261 8.261

5263 5265

148618 164618

7.360 7.360

169640 175613

4363 4465

9.261 8.962

4964 4661

N /A N /A

7.360 7.360

125665 132653

4065 3966

6.662 6.861

4265 4463

Table 2 Regional Cerebral Blood Flows (Percentage of baseline; Mean6S.D.) Groups Core area (R3) Control (n510) CoQ10 (n510) Peripheral area (R2) Control (n510) CoQ10 (n510)

0 h of ischemia

End of ischemia

Reperfusion

Sacrifice

1064 862

1263 1062

106612 100619

97613 99615

864 1064

1064 1163

120630 125616

115638 106619

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H. Li et al. / Brain Research 877 (2000) 7 – 11

Administration of CoQ10 protects the myocardium from ischemic insult and enhances the recovery of myocardial tissue [21]. Studies also show that CoQ10 improves neurological function in Huntington’s disease [1] and decreases vasospasm [9]. It is impossible to directly compare the systemic benefits of any compound to those in the central nervous system, even if the underlying mechanism of damage is similar. The pharmacokinetics of CoQ10 are complex [34]. CoQ10 appears to cross the blood–brain barrier, with maximum concentrations being reached in the brain at 12 h post-ischemia [unpublished data, Astra Arcus]. It is possible that acute administration of CoQ10 is ineffective as a salvage maneuver, although long-term administration resulting in accumulation of CoQ10 in the brain may have potential for neuroprotection. Fig. 2. Percentage of cortical infarct volume (mean6S.D.) over that of normal hemispheric volume measured in rats sacrificed after 22.5 h of survival following 120 min of focal ischemia using a middle cerebral artery occlusion model.

pal CA 1 region [27,15,26,22,3], or infarction in ischemic cortex [5,18]. Interaction between extrinsic and intrinsic mechanisms such as glutamate release, free radical induced mitochondrial injury and inflammation may be responsible for the development of neuronal cell injury in ischemic neuronal tissues following global or focal ischemia. Our data fail to demonstrate that CoQ10 inhibits the effects of free radicals in either model of focal or global ischemia. There are, of course, factors other than the effects of free radicals which may mediate neuronal cell injury following cerebral ischemic insult. Caspase-mediated inflammation following both global and focal ischemia has attracted much attention as a possible cause of caspasemediated neuronal cell death. Cystein proteinases are activated and up-regulated following ischemia. Studies show that blocking activation of caspases using inhibitors such as z-VAD and z-DEVD can salvage brain tissues from ischemic insults following global [20] and focal ischemia [6]. Others show that mice with knock-out of caspase-1 [30] have less neuronal injury than that of wild-type control mice. Up-regulation of caspase activity is therefore likely a component of focal ischemia, especially in penumbral areas. Release of glutamate following ischemic insults also contributes to ischemic neuronal death. Studies have demonstrated that blocking the effects of glutamate, especially AMPA, can attenuate damage in ischemic neuronal tissues long after the global ischemic insult [17,35]. Although caspase inhibitors have been shown to attenuate cortical injury following focal ischemia, we were unable to duplicate the results following global ischemia [19]. This indicates that glutamate induced excitotoxicity and calcium mediated necrosis might account for selective neuronal necrosis and / or apoptosis in global ischemia.

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