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Therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h in rats
European Journal of Pharmacology 666 (2011) 105–110
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h in rats Jing Sun a, Yunman Li a,⁎, Weirong Fang a, Lishun Mao b a b
Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China Jiangsu Kefeiping Pharmaceutical Company Limited, Nanjing 210016, PR China
a r t i c l e
i n f o
Article history: Received 15 December 2010 Received in revised form 11 May 2011 Accepted 17 May 2011 Available online 1 June 2011 Keywords: XQ-1h Therapeutic time window Focal cerebral ischemia reperfusion injury Middle cerebral artery occlusion (MCAO)
a b s t r a c t Pervious experimental studies have shown that XQ-1h has beneficial neuroprotective effect in the cerebral ischemia reperfusion injury. However, the therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h is not clear. Under chloral hydrate anesthesia, transient focal cerebral ischemia was induced in rats by 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h of reperfusion. Saline as vehicle or XQ-1h at the doses of 31.2, 15.6 and 7.8 mg/kg i.v. was administered at 0.5, 1, 2, 3 h after induction of ischemia. Subsequently, 24 h after MCAO brain edema, infarct volume, neurological deficits and cerebral blood flow were evaluated. Administrations of XQ-1h at the doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly reduced infarct rate (%) by 75.6% (5.2 ± 1.7), 66.2% (7.2 ± 1.9), and 47.9% (11.1 ± 1.2), respectively. XQ-1h (31.2 mg/kg) treatment, 0.5, 1, and 2 h after reperfusion produced significant improvement in neurological score compared to vehicle-treated group (P b 0.01). Administrations of XQ-1h at the doses of 31.2 mg/kg and 15.6 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly increased cerebral blood flow (mv) by 16.9 ± 1.9, 11.7 ± 1.3, 9.5 ± 1.0, respectively (P b 0.01). In conclusion the therapeutic time window of XQ-1h for cerebral ischemia reperfusion injury is within 2 h. Interestingly, we also discovered that the therapeutic time window of XQ-1h is deeply related with the activity of scavenging oxidative stress products. Further studies need to be conducted more drug combination therapy programs in order to assess the potential clinical application of XQ-1h. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Stroke is one of the most frequent causes of death and disability worldwide, and has significant clinical and socioeconomic impact. The direct pathological consequence of the stroke is the cerebral ischemicreperfusion injury a complex interplay of multiple pathways, which includes: depletion of adenosine-triphosphate (ATP), excitotoxic glutamate efflux, ionic imbalance, loss of metabolic function with increased acidosis, oxidative stress, and activation of inflammatory processes (Kristian et al., 2008). Ultimately, it involves the destruction and/or dysfunction of brain cells, for which a very few available drugs have proved to be partially effective. Over the last decade, substantial scientific evidence has accumulated to suggest that ginkgolide-B, a major constituent of Ginkgo biloba extract, is a Platelet Activating Factor (PAF) highly selective and competitive receptor antagonist, which is exert beneficial effects in animal models of acute neurodegeneration (Ahlemeyer and Krieglstein, 2003; Bate et al., 2004). PAF is an important modifiable factor for
⁎ Corresponding author. Tel./fax: + 86 25 83271173. E-mail address: [email protected] (Y. Li). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.05.048
cerebral ischemic-reperfusion injury, which regulates the Nitric Oxide Synthase (NOS) and deal with the inflammatory response of the leukocytes (Park et al., 1999). Previous studies from our laboratory have shown that Ginkgolide B at dose of 16 and 8 mg/kg produced significant reduction in infarct volume, edema volume and neurological deficits when treatment was initiated within 4 h after the initiation of focal cerebral ischemia by MCAO, i.e. 2 h after reperfusion. (Weirong and Yan, 2010). XQ-1h is a novel ginkgolide B derivative; its structure is shown in Fig. 1. From Fig. 1 we can see that the structural difference between them is that XQ-1h has a dimethylamino-ethoxy group combining with methane sulfonic acid. The possible mechanism of the protective effect of XQ-1h on the blood–brain barrier is that XQ-1h antagonizes the PAF receptor and thus inhibits PAF-induced calcium overload and up-regulation of iNOS (Yan and Weirong, 2009). The previous study from our laboratory has shown that XQ-1h at doses of 15.6 and 7.8 mg/kg produced neuroprotective effects in pretreatment of cerebral ischemic-reperfusion injury in vivo (data not shown), but there has not yet been any detailed investigation of the therapeutic time window associated with the use of XQ-1h in the focal stroke model. Therefore it is very important to evaluate the therapeutic time window and its possible mechanisms by which XQ-1h exhibit potential for the treatment for stroke.
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The following exclusion criteria were applied during the experiment. Exclusion criteria: – Mortality animals; – No stroke: after MCAO surgery the awaken rats' neurological deficits has been tested. The rat which neurological deficits score (Longa et al., 1989) is 0 points will be excluded; – Problems during induction of MCAO (excessive bleeding, prolonged operation time ≥ 15 min, thread placement). The rats in the different groups were sacrificed 24 h after MCAO operation and the brains were removed respectively. 2.4. Middle cerebral artery occlusion (MCAO) Fig. 1. Chemical structure of XQ-1h.
In this study, we delineated the window of opportunity for treatment of focal cerebral ischemic damage using XQ-1h after middle cerebral artery occlusion (MCAO). Infarct volume, water content, cerebral blood flow (CBF), and neurologic deficit scores were used to evaluate the efficacy of XQ-1h administered at different times after MCAO. We also study the production of oxidative stress in homogenates from the perifocal brains. 2. Materials and methods 2.1. Chemicals and reagents XQ-1h was kindly provided by Jiangsu Kefeiping Pharmaceutical Company Limited. Ozagrel was bought from Haerbing Sanlian Pharmaceutical Company Limited. Superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit and Na +/K+ ATPase kit were obtained from Nanjing Jiancheng Bioengineering Institute. All other reagents used were of analytical grade and commercially available. 2.2. Animals Male Wistar rats weighing 250–300 g were obtained from Zhejiang Laboratory Animals Center, Zhejiang Academy of Medical Science (Hangzhou, China). Rats were maintained in a clean room (Animal Center for Pharmaceutical Research, China Pharmaceutical University, Nanjing, China) at a temperature between 20 and 23 °C, with a 12 h light–dark cycle and a relative humidity of 50%. Rats were housed in metabolic cages under the supply of filtered pathogen-free air with access to food and water ad libitum. The experimental protocols used in this study were approved by our ethics committees for animal experiment. 2.3. Grouping Three hundred and sixty healthy Wistar male rats were randomly divided into 24 groups (n= 15) : 0.5 h sham-operation group, 0.5 h vehicle group, 0.5 h XQ-1h 31.2 mg/kg, 0.5 h XQ-1h 15.6 mg/kg, 0.5 h XQ-1h 7.8 mg/kg and 0.5 h Ozagrel 12 mg/kg group after reperfusion; 1 h sham-operation group, 1 h vehicle group, 1 h XQ-1h 31.2 mg/kg, 1 h XQ-1h 15.6 mg/kg, 1 h XQ-1h 7.8 mg/kg and 1 h Ozagrel 12 mg/kg group after reperfusion; 2 h sham-operation group, 2 h vehicle group, 2 h XQ-1h 31.2 mg/kg, 2 h XQ-1h 15.6 mg/kg, 2 h XQ-1h 7.8 mg/kg and 2 h Ozagrel 12 mg/kg group after reperfusion; 3 h sham-operation group, 3 h vehicle group, 3 h XQ-1h 31.2 mg/kg, 3 h XQ-1h 15.6 mg/kg, 3 h XQ-1h 7.8 mg/kg and 3 h Ozagrel 12 mg/kg group after reperfusion. 2.3.1. Model assessment (Ulrich, 2009) Because the success rate of MCAO model is about 60%, the experimental design is calculated at 15 rats in each group to ensure that each group has 8 valid data.
The right middle cerebral artery (MCA) was occluded using the intraluminal suture technique described by (Longa et al., 1989), with minor modification. Male rats weighing 260–300 g were anesthetized with 300 mg/kg chloral hydrate i.p. The right carotid region was exposed through a midline cervical incision. In order to block the origin of the MCA, a monofilament nylon suture (diameter about 0.26 mm) was prepared by rounding its tip by heating and coating with poly-L-lysine (Sigma). The nylon suture was introduced through the right external carotid artery into the internal carotid artery and advanced approximately 18–20 mm intracranially from the common carotid artery bifurcation. Body temperature was maintained within the normal physiological range with a heating lamp during the operation. 2.5. Neurological deficits 2 h after MCAO, the animals were anesthetized with aether and the MCA was reopened by withdrawing the inserted suture. 0.5, 1, 2, and 3 h after reperfusion of MCAO, XQ-1h (31.2, 15.6, 7.8 mg/kg), ozagrel 12 mg/kg and vehicle were administrated i.v. respectively via vena caudalis (Fig. 2). Neurological deficits in the vehicle-treated group and XQ-1h-treated group were measured according to the method of Longa et al. (Longa et al., 1989) at 24 h after reperfusion. • • • •
Score 0: no apparent neurological deficits Score 1: contra lateral forelimb flexion Score 2: decreased resistance to lateral push Score 3: spontaneous movement in all directions and contra lateral circling when pulled by tail • Score 4: did not walk spontaneously and had depressed levels of consciousness. 2.6. Cerebral blood flow (CBF) CBF was continuously monitored using a laser-Doppler flow meter (AD Instruments, Australia) on the base of the skull at the level of the fronto parietal cortex. Rats were placed in a stereotaxic frame (Kopf)
Fig. 2. Design for therapeutic time window study of XQ-1h in ischemia and reperfusion injured rats.
J. Sun et al. / European Journal of Pharmacology 666 (2011) 105–110
and maintained anesthetized with an adapted facial mask under 1.5–2 s halothane. A 2-mm diameter burr hole was drilled on the skull at 1.3 mm posterior and 4 mm lateral to Bregma on the brain side ipsilateral to further MCA occlusion, and was frequently moistened with saline solution. The duramater was exposed but was maintained intact to prevent any cortical injury. A 2-cm-long plastic guide tube was positioned inside the hole with a micromanipulator (avoiding large blood vessels) and fixed on the skull with dental cement. Relative CBF was monitored with a laser-Doppler probe (AD Instruments, Australia) 5 min prior to the first artery occlusion to establish the baseline. Values used for graphical representation of CBF time course were the mean of 5-min window record laserDoppler flowmeter data using an appropriate software (AD Instruments, Australia) (Robert, 1995). CBF was measured 23 hrs after cerebral ischemic- reperfusion in each group. 2.7. Infarct volume rate Then animals were deeply anesthetized and killed by cervical dislocation. The brains were quickly removed and cut into 5 slices of 2 mm thickness. Then, the brain slice were stained with 2% 2,3,5triphenyl-tetrazolium chloride (TTC, Sigma) at 37 °C in a water bath for 15 min and separately determined the infarcted and total areas of each hemisphere (Wang et al., 2007). TTC stains viable brain tissue dark red based on intact mitochondrial function whereas infarcted tissue areas remain unstained (white). Infarct rate was calculated as following: (weight of total infarction area / weight of brain) × 100% (Bouley et al., 2007). 2.8. Water content To determine brain water content, brain hemispheres were superficially dried, transferred to aluminum foil, weighed (“wet weight”) and dried overnight at 105 °C in a desiccating oven. The dried slices were weighed again (“dry weight”), and total brain water was calculated according to [(wet weight− dry weight) / wet weight] × 100% (Bouley et al., 2007). 2.9. SOD, MDA and Na +/K + ATPase detection In rats with the same operation and administration methods followed, we investigated the SOD, MDA and Na +/K + ATPase. After 24 h reperfusion rats were sacrificed by decapitation, and the brain was removed and dissected on ice. The hippocampus was weighed and homogenized (50 mg tissue/ml buffer) in ice-cold Locke's buffer using 15 strokes in a Teflon/glass homogenizer. The level of SOD, MDA and Na +/K +ATPase were determined in chromometry with spectrophotometer at 550 nm (SOD), 532 nm (MDA) and 636 nm (Na +/K + ATPase) wave length (Dezhi and Renata, 2008).
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administration after 3 h reperfusion did show a trend in the neurological improvement, statistical significance was not observed. 3.2. Effect of post-treatment of XQ-1h on cerebral blood flow The CBF (mv) in vehicle-treated animals was 4.6 ± 1.1, and in sham animals was 23.5 ± 1.8 at 0.5 h after reperfusion following MCAO. XQ-1h at the doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly increased CBF by 16.9 ± 1.9, 11.7 ± 1.3, 9.5 ± 1.0, respectively (P b 0.01), shown in Fig. 4. There was no significant decrease in XQ-1h 7.8 mg/kg group when administration time was longer than 1 h after reperfusion. 3.3. Effect of post-treatment of XQ-1h on cerebral edema To measure brain edema formation, we determined brain water content (%) as described above. As shown in Table 1, at 0.5 h brain water content in the sham operation group was 77.9 ± 1.2. MCAO induced an increase in water content to 80.7 ± 1.6 indicating a significant post-stroke edema formation (P b 0.01). No significant alteration in cerebral edema was observed (P ≥ 0.05). 3.4. Effect of post-treatment of XQ-1h on cerebral infarction The infarct rate (%) in vehicle-treated animals was 21.3 ± 2.8 i.v. administrations of XQ-1h at doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly reduced infarct rate by 75.6% (5.2 ± 1.7), 66.2% (7.2 ± 1.9), 47.9% (11.1 ± 1.2), respectively (P b 0.05, P b 0.01, shown in Table 1). There was no significant decrease in infarct rate if administration time was longer than 2 h after reperfusion. 3.5. Effect of post-treatment of XQ-1h on SOD, Na +/K + ATPase activity and MDA content As compared with the sham operation group and the vehicletreated group, SOD activity significantly decreased and MDA content increased (P b 0.01). As shown in Table 2, SOD activity (U/mg prot) in vehicle-treated group was 40.91 ± 10.50. Post-treatment with XQ-1h 31.2 mg/kg i.v. significantly increasing SOD activity at 0.5 h and 1 h after reperfusion (P b 0.01). MDA content (nmol/mg prot) in vehicletreated group was 12.35 ± 3.08. Post-treatment with XQ-1h 31.2 mg/ kg i.v. significantly decreased MDA content at 0.5 h and 1 h after reperfusion (P b 0.01). As compared with the sham operation group and the vehicle-treated group, Na +/K +ATPase (U) significantly decreased (P b 0.01). Na +/K +ATPase in vehicle-treated group were 1.64 ± 0.71. Post-treatment with XQ-1h 31.2 mg/kg i.v. significantly increased Na +/K +ATPase at 0.5 h after reperfusion (P b 0.01). 4. Discussion
2.10. Statistics All values are expressed as the mean ± S.D. except neurological deficit scores; all other statistical significance of difference between groups was determined by one-way analysis of variance (ANOVA) test. Results were considered to be statistically significant when P b 0.05. 3. Results 3.1. Effect of post-treatment of XQ-1h on neurological deficits Vehicle-treated rats showed prominent neurological deficits in Table 1. XQ-1h (31.2 mg/kg) treatment, 0.5, 1, and 2 h after reperfusion produced significant improvement in neurological score compared to vehicle-treated group (P b 0.01). Although groups of
XQ-1h is a novel ginkgolide B derivative. From Fig. 1 we can see that the structural difference between them is that XQ-1h has a dimethylamino-ethoxy group combining with methane sulfonic acid, which enables XQ-1h, has better water solubility. Thus we may conclude that the bioactivity of XQ-1h is similar to that of ginkgolide B and exhibits its effects by antagonizing the PAF receptor. Previous researches done by our laboratory suggested that XQ-1h can effectively permeate the blood brain barrier, dilate blood vessels, improve microcirculation, inhibit platelet aggregation and protect Rat brain microvessel endothelial cells against Na2S2O4-induced hypoxia and re-oxygenation insult (Yan and Weirong, 2009). Platelet aggregation is reported as one of the aggravating factors of cerebral ischemia. The role of thromboxane A2 (TXA2), a strong vasoconstrictor and platelet aggregator, is well established by several studies (Arii et al., 2002; Imamura et al., 2003). Ozagrel, a selective
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Table 1 The effect of XQ-1h post-treatment on neurological deficits, cerebral edema (%) and cerebral infarction (%) in MCAO rats (n = 8). Groups
Neurological deficits
Dose (mg/kg) – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12
Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel
Cerebral edema
Cerebral Infarction
Administration time after reperfusion (h) 0.5
1
2
3
2.9 ± 0.5 1.6 ± 0.4a 1.8 ± 0.3a 2.6 ± 0.5 1.6 ± 0.6a 77.9 ± 1.2a 80.7 ± 1.6 79.4 ± 1.6 80.1 ± 1.0 81.8 ± 1.4 79.8 ± 1.1
3.1 ± 0.8 2.2 ± 0.5a 2.4 ± 0.6 2.9 ± 0.4 1.9 ± 0.4a 78.3 ± 1.6a 81.1 ± 1.8 80.9 ± 0.7 81.5 ± 1.2 83.2 ± 1.0 80.9 ± 1.2
2.9 ± 0.7 2.6 ± 0.5 2.8 ± 0.2 2.9 ± 0.7 2.8 ± 0.6 78.7 ± 1.4a 81.3 ± 1.7 81.8 ± 1.4 81.3 ± 1.2 82.1 ± 0.7 81.6 ± 1.1
24.3 ± 2.8 5.2 ± 1.7a 9.3 ± 2.3a 20.5 ± 3.5 5.5 ± 1.5a
25.7 ± 1.9 7.2 ± 1.9a 16.0 ± 4.6 24.8 ± 1.5 10.9 ± 1.6a
3.0 ± 0.7 2.0 ± 0.3a 2.6 ± 0.3 2.7 ± 0.3 2.8 ± 0.5 78.5 ± 1.7a 80.9 ± 1.9 79.6 ± 0.7 81.1 ± 1.5 82.2 ± 1.5 79.5 ± 0.4 0a 28.7 ± 2.3 11.1 ± 1.2a 18.3 ± 4.4 27.8 ± 4.0 10.0 ± 2.2a
28.5 ± 3.1 20.4 ± 1.9 23.7 ± 2.5 27.2 ± 3.7 20.7 ± 2.6
Values are the mean ± S.D. a P b 0.01 as compared to the vehicle-treated group.
TXA2 synthetase inhibitor, shows a suppressive effect on vasospasm and platelet aggregation in animal and human experiments (Arii et al., 2002; Imamura et al., 2003). As ozagrel has a similar mechanism of anti-cerebral ischemia–reperfusion injury, we chose ozagrel as the experimental positive control drug. After cerebral ischemic injury, two types of neuronal cell death have been described: necrosis and apoptosis. Those apoptotic neurons can survive for some time but will continue to die for many hours or even days after ischemia in the ischemic penumbra (Charriaut et al., 1996; Fisher, 1997; Yan et al., 1995). This delayed evolution of neuronal death provides a potential window of opportunity for neuronal rescue with effective. Many neuroprotective agents have been found to produce considerable neuroprotection in pre-treatment of ischemia–reperfusion insult, but they often fail at clinical trials. There are many reasons for the failures, the short therapeutic window included. Although XQ-1h at dose of 15.6 and 7.8 mg/kg shows neuroprotective effects in pretreatment of cerebral ischemic-reperfusion injury in vivo (data not shown), little work is done to illustrate its effects on post-treatment of cerebral ischemic-reperfusion injury and its therapeutic time window remains unknown. According to the doses of pre-treatment of cerebral ischemicreperfusion we designed the doses of three groups in this research as 31.2 mg/kg, 15.6 mg/kg and 7.8 mg/kg respectively. Before starting the formal experiment, we did a bioequivalence experiment between ozagrel and XQ-1h, indicating that ozagrel 12 mg/kg i.v was equal to XQ-1h 31.2 mg/kg i.v. (data not shown). Consequently, we chose 12 mg/kg of ozagrel as the experimental positive control dose.
0.5h
Sham
1h
2h
3h
In this research, we evaluated four indexes, neurological deficits, cerebral edema, cerebral infarction and cerebral blood flow (CBF), in MCAO rats after the administration of XQ-1h to identify its therapeutic window. After cerebral ischemic-reperfusion injury, the core of the ischemic lesion necrosis of neurons and axons, axoplasm transport collapse, which will lead to the appearance of a large area of infarction and Cerebral edema (Kristian et al., 2008). Cerebral edema and associated increased intracranial pressure are the major immediate consequences of cerebral ischemic-reperfusion injury that contribute to most early deaths (Kristian et al., 2008). Secondary damage in cerebral ischemic-reperfusion injury is influenced by changes in cerebral blood flow (CBF), cerebral metabolic dysfunction and inadequate cerebral oxygenation (Graham and Chen, 2001). Therefore, the measurement of these four indexes can directly reflect the effects of XQ-1h in posttreatment of cerebral ischemic-reperfusion injury. From Table 1 and Fig. 3 we can see XQ-1h at dose of 31.2 and 15.6 mg/kg produced significant reduction in infarct volume and neurological deficits when treatment was initiated within 2 h after the initiation of focal cerebral ischemia by MCAO, i.e. 2 h after reperfusion. However, treatment at 3 h did not improve cerebral ischemia injury evidently in rats. Therefore, we could conclude that the most perfect therapeutic time of XQ-1h was within 2 h after ischemic stroke in rats. It has been widely accepted that the brain requires this large amount of oxygen to generate sufficient ATP by oxidative phosphorylation to maintain and restore ionic gradients. Na ±/K ±ATPase found on the plasma membrane of neurons, consumes 70% of the energy supplied to the brain (Kristian et al., 2008). This ion pump maintains
Ozagrel XQ-1h (H)XQ-1h(M) XQ-1h(L)
Ozagrel XQ-1h(H) XQ-1h(M) XQ-1h(L)
0.5h
2h
1h
3h
Vehicle
Ozagrel (12mg/kg), XQ-1h (H) (31.2mg/kg), XQ-1h (M) (15.6mg/kg), XQ-1h (L) (7.8mg/kg) Fig. 3. Effect of post-treatment of XQ-1h on cerebral infarction and cerebral edema. Infarct volumes at 24 h after reperfusion in the rats with 120 min of MCAO (n = 8).
J. Sun et al. / European Journal of Pharmacology 666 (2011) 105–110
Fig. 4. Effect of post-treatment of XQ-1h on cerebral blood flow. Cerebral blood flow at 24 h after reperfusion in the rats with 120 min of MCAO (n = 8). Ozagrel (12 mg/kg), XQ-1h (H) (31.2 mg/kg), XQ-1h (M) (15.6 mg/kg), XQ-1h (L) (7.8 mg/kg).
the high intracellular K + concentration and the low intracellular Na + concentration necessary for the propagation of action potentials. After ischemia, mitochondrial inhibition of ATP synthesis leads to ATP being consumed within two minutes, which will cause neuronal plasma membrane depolarization, release of potassium into the extracellular space and entry of sodium into cells (Kristian et al., 2008). ATP is an important neuromodulators in the hippocampus. Endogenous ATP is released frequency-dependently from in vitro hippocampal slices upon low and high frequency electrical stimulation (Cunha et al., 1996; Wieraszko et al., 1989). Therefore, we detected the activity of the Na ±/K ±ATPase in hippocampal to evaluate the protective effect of XQ-1h on the acute path physiological changes of cerebral ischemicreperfusion injury. From Table 2, we can see that XQ-1h had beneficial effect on protecting Na +/K +ATPase and the optimal effect was obtained when XQ-1h was injected at 0.5 h after the reperfusion and no effect 1 h later. The results probably indicated that at 1 h after the reperfusion the activity of Na +/K +ATPase was irreversibly damaged.
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To further validate our conclusion, it is necessary to study on possible mechanism of XQ-1h on post-treatment of cerebral ischemicreperfusion injury in vivo. XQ-1h shares a chemical structure similar to that of ginkgolide B which has been shown to scavenge free radicals and non-specifically inhibit seryl and aspartyl proteases (Ahlemeyer and Krieglstein, 2003; Bate et al., 2004). Therefore, we may conclude that the mechanism of XQ-1h's effects on ischemia is similar to that of ginkgolide B. During acute phage of ischemia, excitotoxity, oxidative stress, calcium overload lead to the damage of DNA, the deposition of protein and phosphoric acid and the accumulation of intracellular free radicals (Kristian et al., 2008). N-methyl-D-aspartate-receptor subtype of glutamate, which is coupled with NO synthesis by activation of Nitric oxide synthases (NOS), plays a crucial role in the production of kindling seizures (Bruno and Battaglia, 2001). Moreover, the accumulation of free radicals, including the accumulation of NO, will result in lipid peroxidation. Malondialdehyde (MDA) is the last product of lipid peroxidation which is toxic to cells and cell membranes (Crack and Taylor, 2001). Therefore, the amount of MDA can indirectly reflect the degree of lipid peroxidation and the accumulation of free radicals. It is accepted that SOD (superoxidase dismutase) is one of the most important physiological antioxidants against free radicals and that they prevent subsequent lipid peroxidation (Bordet and Deplanque, 2000). Many experiments have proved that cerebral ischemia/reperfusion of the rat at the acute stage can increase the content of MDA and consume SOD to decrease its activity (Crack and Taylor, 2001). Free radicals are thought to cause behavioral deficits in experimental animals. Fukui and Stadaman suggest that balanced antioxidants are required to control the cognitive and motor functions of the cerebral cortex and the hippocampus. Fukui accounts that oxidative stress and aging impair memory functions (Fukui et al., 2001; Stadtman, 1992). The hippocampus, in contrast, while showing similar changes as the striatum in the first 20– 30 min past ischemia, seemed to recover thereafter and evidently represents penumbral tissue which is the major target for neuroprotective drugs (Cornelia and Alexander, 2010). Therefore, we chose SOD and MDA in hippocampus as indexes to evaluate XQ-1h's ability of eliminating free radicals which are excessively generated during the process of cerebral ischemic-reperfusion injury. From Table 2, we can see the activity of SOD in MCAO rats were ameliorated after the 2 h administration of XQ-1h, and posttreatment with XQ-1h 31.2 mg/kg i.v. significantly decreased MDA
Table 2 The effect of XQ-1h post-treatment on SOD (U/mg prot), Na+/K+ ATPase activity (U) and MDA content (nmol/mg prot) in MCAO rats (n = 8).
SOD Activity
MDA content
Na+/K+ ATPase activity
Groups
Dose (mg/kg)
Administration time after reperfusion (h) 0.5 h
1h
2h
3h
Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel
– – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12
70.83 ± 8.73a 40.91 ± 10.5 62.05 ± 8.87a 57.79 ± 17.07b 38.77 ± 5.38 58.95 ± 2.66a 3.44 ± 0.44a 12.35 ± 3.08 4.11 ± 0.71a 5.77 ± 1.86a 7.1 ± 3.01a 4.67 ± 0.45b 3.57 ± 0.48a 1.64 ± 0.71 3.33 ± 0.84a 2.9 ± 0.97b 2.05 ± 0.43 2.93 ± 0.64a
69.89 ± 9.21a 41.2 ± 0.88 56.57 ± 6.81b 49.67 ± 7.62 40.33 ± 8.05 57.3 ± 19.37b 3.78 ± 0.65a 12.42 ± 2.8 7.17 ± 2.79a 9.58 ± 1.53b 10.19 ± 3.59 5.97 ± 2.5b 3.35 ± 0.34a 1.41 ± 0.69 1.63 ± 0.83 0.8 ± 0.54 0.72 ± 0.42 1.27 ± 0.61
66.54 ± 7.65a 40.68 ± 0.79 48.58 ± 10.99 42.93 ± 14.29 40.1 ± 5.58 50.82 ± 8.4 4.21 ± 1.32a 12.89 ± 1.12 8.44 ± 3.72b 10.23 ± 2.38 11.32 ± 2.06 9.18 ± 2.87a 2.86 ± 0.52a 1.44 ± 0.39 1.1 ± 1.61 0.93 ± 0.72 0.49 ± 0.41 1.1 ± 0.62
70.22 ± 6.76a 39.31 ± 3.92 45.22 ± 1.28 40.68 ± 1.98 39.89 ± 1.77 44.81 ± 0.45 5.11 ± 0.4a 13.11 ± 1.45 10.49 ± 1.42 12.02 ± 1.43 12.33 ± 1.47 10.21 ± 1.16 2.69 ± 0.49a 1.28 ± 0.31 1.04 ± 1.92 0.87 ± 1.51 0.72 ± 1.29 0.98 ± 1.23
Values are mean ± S.D. a P b 0.01 as compared to the vehicle-treated group. b P b 0.05 as compared to the vehicle-treated group.
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content at 2 h after reperfusion (P b 0.05); however, treatment at 3 h did not improve cerebral ischemia injury evidently in rats. The mechanism of antioxidative activity of XQ-1h might be XQ-1h antagonizes the PAF receptor and inhibits PAF-induced iNOS over expression (Yan and Weirong, 2009), leading to the decreased release of NO and the low accumulation of free radical further easing the lipid peroxidation, decreasing the amount of MDA and protecting the ability of SOD. This result further verified that the therapeutic time window of XQ-1h is 2 h, and it suggested that XQ-1h is a potent antioxidant, somehow reduced neurobehavioral deficits by scavenging free radicals. It also indicated that the therapeutic time window of XQ-1h is deeply related with the activity of scavenging oxidative stress products. In conclusion, the most perfect therapeutic time of XQ-1h was within 2 h after ischemic reperfusion in MCAO rats, XQ-1h had protective effect on Na ±/K ±ATPase within 0.5 h after the reperfusion, and the beneficial effects on ischemic injury is related with the activity of scavenging oxidative stress products. Further studies can be focused on looking for more drug combination therapy programs in order to assess the potential clinical application of XQ-1h. References Ahlemeyer, B., Krieglstein, J., 2003. Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci 60, 1779–1792. Arii, K., Igarashi, H., Arii, T., Katayama, Y., 2002. The effect of ozagrel sodium on photochemical thrombosis in rat: therapeutic window and combined therapy with heparin sodium. Life Sci 71, 2983–2994. Bate, C., Salmona, M., Williams, A., 2004. Ginkgolide B inhibits the neurotoxicity of prions or amyloid-beta1-42. J Neuroinflamm 36, 1–4. Bordet, R., Deplanque, D., 2000. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J Cereb Blood Flow Metab 20, 1190. Bouley, J., Fisher, M., Henninger, N., 2007. Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging. Neurosci. Lett. 412, 185–190. Bruno, V., Battaglia, G., 2001. Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab 21, 1013–1033. Charriaut, C., Margaill, I., Represa, A., 1996. Apoptosis and necrosis following reversible focal ischemia: an in situ DNA fragmentation analysis. J Cereb Blood Flow Metab 16, 186–194.
Cornelia, K., Alexander, M., 2010. Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice. Brain Res 1312, 101–107. Crack, P., Taylor, J., 2001. Reactive oxygen species and the modulation of stroke. Free Radical Biol Med 38, 1433–1444. Cunha, R., Correia, P., Sebastiao, A., Ribeiro, J., 1996. Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides. Br. J. Pharmacol. 119, 253–260. Dezhi, T., Renata, I., 2008. Protein kinase M zeta regulation of Na+/K+ATPase: a persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures. Brain Res 1213, 127–139. Fisher, M., 1997. Characterizing the target of acute stroke therapy. Stroke 28, 866–872. Fukui, K., Onodera, K., Shinkai, T., Suzuki, S., Urano, S., 2001. Impairment of learning and memory in rats caused by oxidative stress and aging, and changes in antioxidative defense systems. Ann. N. Y. Acad. Sci. 928, 168–175. Graham, S., Chen, J., 2001. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 21, 99–109. Imamura, T., Kiguchi, S., Kobayashi, K., Ichikawa, K., Yama, Y., Kojima, M., 2003. Effect of ozagrel, a selective thromboxane A2-synthetase inhibitor, on cerebral infarction in rats, comparative study with norphenazone, a free-radical scavenger. Arzneimittel Forsch 53, 688–694. Kristian, P., Roger, P., Mary, P., 2008. Neuropharmacology-special issue on cerebral ischemia mechanisms of ischemic brain damage. Neuropharmacol 55, 310–318. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Park, T.S., Gonzales, E.R., Gidday, J.M., 1999. Platelet-activating factor mediates ischemia-induced leukocyte-endothelial adherence in newborn pig brain. J Cereb Blood Flow Metab 19, 417–424. Robert, B., 1995. Decreased cerebral blood flow during acute hyperglycemia. Brain Res 703, 145–150. Stadtman, E., 1992. Free radicals in the genesis of Alzheimer's disease. Ann. N. Y. Acad. Sci. 695, 73–76. Ulrich, D., 2009. Standard operating procedures (SOP) in experimental stroke research: SOP for middle cerebral artery occlusion in the mouse. Nat Preced 3492, 1. Wang, C., Zhang, D., Ma, H., Liu, J., 2007. Neuroprotective effects of emddin-8-O-beta-dglucoside in vivo and in vitro. Eur. J. Pharmacol. 577, 58–63. Weirong, F., Yan, D., 2010. Blood brain barrier permeability and therapeutic time window of Ginkgolide B in ischemia–reperfusion injury. Eur. J. Pharmacol. 39, 8–14. Wieraszko, A., Goldsmith, G., Seyfried, T.N., 1989. Stimulation-dependent release of adenosine triphosphate from hippocampal slices. Brain Res 485, 244–250. Yan, D., Weirong, F., 2009. Blood-brain barrier breakdown by PAF and protection by XQ1H due to antagonism of PAF effects. Eur. J. Pharmacol. 616, 43–47. Yan, L., Chopp, M., Jiang, N., 1995. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 15, 389–397.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h in rats Jing Sun a, Yunman Li a,⁎, Weirong Fang a, Lishun Mao b a b
Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China Jiangsu Kefeiping Pharmaceutical Company Limited, Nanjing 210016, PR China
a r t i c l e
i n f o
Article history: Received 15 December 2010 Received in revised form 11 May 2011 Accepted 17 May 2011 Available online 1 June 2011 Keywords: XQ-1h Therapeutic time window Focal cerebral ischemia reperfusion injury Middle cerebral artery occlusion (MCAO)
a b s t r a c t Pervious experimental studies have shown that XQ-1h has beneficial neuroprotective effect in the cerebral ischemia reperfusion injury. However, the therapeutic time window for treatment of focal cerebral ischemia reperfusion injury with XQ-1h is not clear. Under chloral hydrate anesthesia, transient focal cerebral ischemia was induced in rats by 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h of reperfusion. Saline as vehicle or XQ-1h at the doses of 31.2, 15.6 and 7.8 mg/kg i.v. was administered at 0.5, 1, 2, 3 h after induction of ischemia. Subsequently, 24 h after MCAO brain edema, infarct volume, neurological deficits and cerebral blood flow were evaluated. Administrations of XQ-1h at the doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly reduced infarct rate (%) by 75.6% (5.2 ± 1.7), 66.2% (7.2 ± 1.9), and 47.9% (11.1 ± 1.2), respectively. XQ-1h (31.2 mg/kg) treatment, 0.5, 1, and 2 h after reperfusion produced significant improvement in neurological score compared to vehicle-treated group (P b 0.01). Administrations of XQ-1h at the doses of 31.2 mg/kg and 15.6 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly increased cerebral blood flow (mv) by 16.9 ± 1.9, 11.7 ± 1.3, 9.5 ± 1.0, respectively (P b 0.01). In conclusion the therapeutic time window of XQ-1h for cerebral ischemia reperfusion injury is within 2 h. Interestingly, we also discovered that the therapeutic time window of XQ-1h is deeply related with the activity of scavenging oxidative stress products. Further studies need to be conducted more drug combination therapy programs in order to assess the potential clinical application of XQ-1h. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Stroke is one of the most frequent causes of death and disability worldwide, and has significant clinical and socioeconomic impact. The direct pathological consequence of the stroke is the cerebral ischemicreperfusion injury a complex interplay of multiple pathways, which includes: depletion of adenosine-triphosphate (ATP), excitotoxic glutamate efflux, ionic imbalance, loss of metabolic function with increased acidosis, oxidative stress, and activation of inflammatory processes (Kristian et al., 2008). Ultimately, it involves the destruction and/or dysfunction of brain cells, for which a very few available drugs have proved to be partially effective. Over the last decade, substantial scientific evidence has accumulated to suggest that ginkgolide-B, a major constituent of Ginkgo biloba extract, is a Platelet Activating Factor (PAF) highly selective and competitive receptor antagonist, which is exert beneficial effects in animal models of acute neurodegeneration (Ahlemeyer and Krieglstein, 2003; Bate et al., 2004). PAF is an important modifiable factor for
⁎ Corresponding author. Tel./fax: + 86 25 83271173. E-mail address: [email protected] (Y. Li). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.05.048
cerebral ischemic-reperfusion injury, which regulates the Nitric Oxide Synthase (NOS) and deal with the inflammatory response of the leukocytes (Park et al., 1999). Previous studies from our laboratory have shown that Ginkgolide B at dose of 16 and 8 mg/kg produced significant reduction in infarct volume, edema volume and neurological deficits when treatment was initiated within 4 h after the initiation of focal cerebral ischemia by MCAO, i.e. 2 h after reperfusion. (Weirong and Yan, 2010). XQ-1h is a novel ginkgolide B derivative; its structure is shown in Fig. 1. From Fig. 1 we can see that the structural difference between them is that XQ-1h has a dimethylamino-ethoxy group combining with methane sulfonic acid. The possible mechanism of the protective effect of XQ-1h on the blood–brain barrier is that XQ-1h antagonizes the PAF receptor and thus inhibits PAF-induced calcium overload and up-regulation of iNOS (Yan and Weirong, 2009). The previous study from our laboratory has shown that XQ-1h at doses of 15.6 and 7.8 mg/kg produced neuroprotective effects in pretreatment of cerebral ischemic-reperfusion injury in vivo (data not shown), but there has not yet been any detailed investigation of the therapeutic time window associated with the use of XQ-1h in the focal stroke model. Therefore it is very important to evaluate the therapeutic time window and its possible mechanisms by which XQ-1h exhibit potential for the treatment for stroke.
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The following exclusion criteria were applied during the experiment. Exclusion criteria: – Mortality animals; – No stroke: after MCAO surgery the awaken rats' neurological deficits has been tested. The rat which neurological deficits score (Longa et al., 1989) is 0 points will be excluded; – Problems during induction of MCAO (excessive bleeding, prolonged operation time ≥ 15 min, thread placement). The rats in the different groups were sacrificed 24 h after MCAO operation and the brains were removed respectively. 2.4. Middle cerebral artery occlusion (MCAO) Fig. 1. Chemical structure of XQ-1h.
In this study, we delineated the window of opportunity for treatment of focal cerebral ischemic damage using XQ-1h after middle cerebral artery occlusion (MCAO). Infarct volume, water content, cerebral blood flow (CBF), and neurologic deficit scores were used to evaluate the efficacy of XQ-1h administered at different times after MCAO. We also study the production of oxidative stress in homogenates from the perifocal brains. 2. Materials and methods 2.1. Chemicals and reagents XQ-1h was kindly provided by Jiangsu Kefeiping Pharmaceutical Company Limited. Ozagrel was bought from Haerbing Sanlian Pharmaceutical Company Limited. Superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit and Na +/K+ ATPase kit were obtained from Nanjing Jiancheng Bioengineering Institute. All other reagents used were of analytical grade and commercially available. 2.2. Animals Male Wistar rats weighing 250–300 g were obtained from Zhejiang Laboratory Animals Center, Zhejiang Academy of Medical Science (Hangzhou, China). Rats were maintained in a clean room (Animal Center for Pharmaceutical Research, China Pharmaceutical University, Nanjing, China) at a temperature between 20 and 23 °C, with a 12 h light–dark cycle and a relative humidity of 50%. Rats were housed in metabolic cages under the supply of filtered pathogen-free air with access to food and water ad libitum. The experimental protocols used in this study were approved by our ethics committees for animal experiment. 2.3. Grouping Three hundred and sixty healthy Wistar male rats were randomly divided into 24 groups (n= 15) : 0.5 h sham-operation group, 0.5 h vehicle group, 0.5 h XQ-1h 31.2 mg/kg, 0.5 h XQ-1h 15.6 mg/kg, 0.5 h XQ-1h 7.8 mg/kg and 0.5 h Ozagrel 12 mg/kg group after reperfusion; 1 h sham-operation group, 1 h vehicle group, 1 h XQ-1h 31.2 mg/kg, 1 h XQ-1h 15.6 mg/kg, 1 h XQ-1h 7.8 mg/kg and 1 h Ozagrel 12 mg/kg group after reperfusion; 2 h sham-operation group, 2 h vehicle group, 2 h XQ-1h 31.2 mg/kg, 2 h XQ-1h 15.6 mg/kg, 2 h XQ-1h 7.8 mg/kg and 2 h Ozagrel 12 mg/kg group after reperfusion; 3 h sham-operation group, 3 h vehicle group, 3 h XQ-1h 31.2 mg/kg, 3 h XQ-1h 15.6 mg/kg, 3 h XQ-1h 7.8 mg/kg and 3 h Ozagrel 12 mg/kg group after reperfusion. 2.3.1. Model assessment (Ulrich, 2009) Because the success rate of MCAO model is about 60%, the experimental design is calculated at 15 rats in each group to ensure that each group has 8 valid data.
The right middle cerebral artery (MCA) was occluded using the intraluminal suture technique described by (Longa et al., 1989), with minor modification. Male rats weighing 260–300 g were anesthetized with 300 mg/kg chloral hydrate i.p. The right carotid region was exposed through a midline cervical incision. In order to block the origin of the MCA, a monofilament nylon suture (diameter about 0.26 mm) was prepared by rounding its tip by heating and coating with poly-L-lysine (Sigma). The nylon suture was introduced through the right external carotid artery into the internal carotid artery and advanced approximately 18–20 mm intracranially from the common carotid artery bifurcation. Body temperature was maintained within the normal physiological range with a heating lamp during the operation. 2.5. Neurological deficits 2 h after MCAO, the animals were anesthetized with aether and the MCA was reopened by withdrawing the inserted suture. 0.5, 1, 2, and 3 h after reperfusion of MCAO, XQ-1h (31.2, 15.6, 7.8 mg/kg), ozagrel 12 mg/kg and vehicle were administrated i.v. respectively via vena caudalis (Fig. 2). Neurological deficits in the vehicle-treated group and XQ-1h-treated group were measured according to the method of Longa et al. (Longa et al., 1989) at 24 h after reperfusion. • • • •
Score 0: no apparent neurological deficits Score 1: contra lateral forelimb flexion Score 2: decreased resistance to lateral push Score 3: spontaneous movement in all directions and contra lateral circling when pulled by tail • Score 4: did not walk spontaneously and had depressed levels of consciousness. 2.6. Cerebral blood flow (CBF) CBF was continuously monitored using a laser-Doppler flow meter (AD Instruments, Australia) on the base of the skull at the level of the fronto parietal cortex. Rats were placed in a stereotaxic frame (Kopf)
Fig. 2. Design for therapeutic time window study of XQ-1h in ischemia and reperfusion injured rats.
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and maintained anesthetized with an adapted facial mask under 1.5–2 s halothane. A 2-mm diameter burr hole was drilled on the skull at 1.3 mm posterior and 4 mm lateral to Bregma on the brain side ipsilateral to further MCA occlusion, and was frequently moistened with saline solution. The duramater was exposed but was maintained intact to prevent any cortical injury. A 2-cm-long plastic guide tube was positioned inside the hole with a micromanipulator (avoiding large blood vessels) and fixed on the skull with dental cement. Relative CBF was monitored with a laser-Doppler probe (AD Instruments, Australia) 5 min prior to the first artery occlusion to establish the baseline. Values used for graphical representation of CBF time course were the mean of 5-min window record laserDoppler flowmeter data using an appropriate software (AD Instruments, Australia) (Robert, 1995). CBF was measured 23 hrs after cerebral ischemic- reperfusion in each group. 2.7. Infarct volume rate Then animals were deeply anesthetized and killed by cervical dislocation. The brains were quickly removed and cut into 5 slices of 2 mm thickness. Then, the brain slice were stained with 2% 2,3,5triphenyl-tetrazolium chloride (TTC, Sigma) at 37 °C in a water bath for 15 min and separately determined the infarcted and total areas of each hemisphere (Wang et al., 2007). TTC stains viable brain tissue dark red based on intact mitochondrial function whereas infarcted tissue areas remain unstained (white). Infarct rate was calculated as following: (weight of total infarction area / weight of brain) × 100% (Bouley et al., 2007). 2.8. Water content To determine brain water content, brain hemispheres were superficially dried, transferred to aluminum foil, weighed (“wet weight”) and dried overnight at 105 °C in a desiccating oven. The dried slices were weighed again (“dry weight”), and total brain water was calculated according to [(wet weight− dry weight) / wet weight] × 100% (Bouley et al., 2007). 2.9. SOD, MDA and Na +/K + ATPase detection In rats with the same operation and administration methods followed, we investigated the SOD, MDA and Na +/K + ATPase. After 24 h reperfusion rats were sacrificed by decapitation, and the brain was removed and dissected on ice. The hippocampus was weighed and homogenized (50 mg tissue/ml buffer) in ice-cold Locke's buffer using 15 strokes in a Teflon/glass homogenizer. The level of SOD, MDA and Na +/K +ATPase were determined in chromometry with spectrophotometer at 550 nm (SOD), 532 nm (MDA) and 636 nm (Na +/K + ATPase) wave length (Dezhi and Renata, 2008).
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administration after 3 h reperfusion did show a trend in the neurological improvement, statistical significance was not observed. 3.2. Effect of post-treatment of XQ-1h on cerebral blood flow The CBF (mv) in vehicle-treated animals was 4.6 ± 1.1, and in sham animals was 23.5 ± 1.8 at 0.5 h after reperfusion following MCAO. XQ-1h at the doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly increased CBF by 16.9 ± 1.9, 11.7 ± 1.3, 9.5 ± 1.0, respectively (P b 0.01), shown in Fig. 4. There was no significant decrease in XQ-1h 7.8 mg/kg group when administration time was longer than 1 h after reperfusion. 3.3. Effect of post-treatment of XQ-1h on cerebral edema To measure brain edema formation, we determined brain water content (%) as described above. As shown in Table 1, at 0.5 h brain water content in the sham operation group was 77.9 ± 1.2. MCAO induced an increase in water content to 80.7 ± 1.6 indicating a significant post-stroke edema formation (P b 0.01). No significant alteration in cerebral edema was observed (P ≥ 0.05). 3.4. Effect of post-treatment of XQ-1h on cerebral infarction The infarct rate (%) in vehicle-treated animals was 21.3 ± 2.8 i.v. administrations of XQ-1h at doses of 31.2 mg/kg at 0.5, 1, and 2 h after reperfusion of MCAO significantly reduced infarct rate by 75.6% (5.2 ± 1.7), 66.2% (7.2 ± 1.9), 47.9% (11.1 ± 1.2), respectively (P b 0.05, P b 0.01, shown in Table 1). There was no significant decrease in infarct rate if administration time was longer than 2 h after reperfusion. 3.5. Effect of post-treatment of XQ-1h on SOD, Na +/K + ATPase activity and MDA content As compared with the sham operation group and the vehicletreated group, SOD activity significantly decreased and MDA content increased (P b 0.01). As shown in Table 2, SOD activity (U/mg prot) in vehicle-treated group was 40.91 ± 10.50. Post-treatment with XQ-1h 31.2 mg/kg i.v. significantly increasing SOD activity at 0.5 h and 1 h after reperfusion (P b 0.01). MDA content (nmol/mg prot) in vehicletreated group was 12.35 ± 3.08. Post-treatment with XQ-1h 31.2 mg/ kg i.v. significantly decreased MDA content at 0.5 h and 1 h after reperfusion (P b 0.01). As compared with the sham operation group and the vehicle-treated group, Na +/K +ATPase (U) significantly decreased (P b 0.01). Na +/K +ATPase in vehicle-treated group were 1.64 ± 0.71. Post-treatment with XQ-1h 31.2 mg/kg i.v. significantly increased Na +/K +ATPase at 0.5 h after reperfusion (P b 0.01). 4. Discussion
2.10. Statistics All values are expressed as the mean ± S.D. except neurological deficit scores; all other statistical significance of difference between groups was determined by one-way analysis of variance (ANOVA) test. Results were considered to be statistically significant when P b 0.05. 3. Results 3.1. Effect of post-treatment of XQ-1h on neurological deficits Vehicle-treated rats showed prominent neurological deficits in Table 1. XQ-1h (31.2 mg/kg) treatment, 0.5, 1, and 2 h after reperfusion produced significant improvement in neurological score compared to vehicle-treated group (P b 0.01). Although groups of
XQ-1h is a novel ginkgolide B derivative. From Fig. 1 we can see that the structural difference between them is that XQ-1h has a dimethylamino-ethoxy group combining with methane sulfonic acid, which enables XQ-1h, has better water solubility. Thus we may conclude that the bioactivity of XQ-1h is similar to that of ginkgolide B and exhibits its effects by antagonizing the PAF receptor. Previous researches done by our laboratory suggested that XQ-1h can effectively permeate the blood brain barrier, dilate blood vessels, improve microcirculation, inhibit platelet aggregation and protect Rat brain microvessel endothelial cells against Na2S2O4-induced hypoxia and re-oxygenation insult (Yan and Weirong, 2009). Platelet aggregation is reported as one of the aggravating factors of cerebral ischemia. The role of thromboxane A2 (TXA2), a strong vasoconstrictor and platelet aggregator, is well established by several studies (Arii et al., 2002; Imamura et al., 2003). Ozagrel, a selective
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Table 1 The effect of XQ-1h post-treatment on neurological deficits, cerebral edema (%) and cerebral infarction (%) in MCAO rats (n = 8). Groups
Neurological deficits
Dose (mg/kg) – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12
Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel
Cerebral edema
Cerebral Infarction
Administration time after reperfusion (h) 0.5
1
2
3
2.9 ± 0.5 1.6 ± 0.4a 1.8 ± 0.3a 2.6 ± 0.5 1.6 ± 0.6a 77.9 ± 1.2a 80.7 ± 1.6 79.4 ± 1.6 80.1 ± 1.0 81.8 ± 1.4 79.8 ± 1.1
3.1 ± 0.8 2.2 ± 0.5a 2.4 ± 0.6 2.9 ± 0.4 1.9 ± 0.4a 78.3 ± 1.6a 81.1 ± 1.8 80.9 ± 0.7 81.5 ± 1.2 83.2 ± 1.0 80.9 ± 1.2
2.9 ± 0.7 2.6 ± 0.5 2.8 ± 0.2 2.9 ± 0.7 2.8 ± 0.6 78.7 ± 1.4a 81.3 ± 1.7 81.8 ± 1.4 81.3 ± 1.2 82.1 ± 0.7 81.6 ± 1.1
24.3 ± 2.8 5.2 ± 1.7a 9.3 ± 2.3a 20.5 ± 3.5 5.5 ± 1.5a
25.7 ± 1.9 7.2 ± 1.9a 16.0 ± 4.6 24.8 ± 1.5 10.9 ± 1.6a
3.0 ± 0.7 2.0 ± 0.3a 2.6 ± 0.3 2.7 ± 0.3 2.8 ± 0.5 78.5 ± 1.7a 80.9 ± 1.9 79.6 ± 0.7 81.1 ± 1.5 82.2 ± 1.5 79.5 ± 0.4 0a 28.7 ± 2.3 11.1 ± 1.2a 18.3 ± 4.4 27.8 ± 4.0 10.0 ± 2.2a
28.5 ± 3.1 20.4 ± 1.9 23.7 ± 2.5 27.2 ± 3.7 20.7 ± 2.6
Values are the mean ± S.D. a P b 0.01 as compared to the vehicle-treated group.
TXA2 synthetase inhibitor, shows a suppressive effect on vasospasm and platelet aggregation in animal and human experiments (Arii et al., 2002; Imamura et al., 2003). As ozagrel has a similar mechanism of anti-cerebral ischemia–reperfusion injury, we chose ozagrel as the experimental positive control drug. After cerebral ischemic injury, two types of neuronal cell death have been described: necrosis and apoptosis. Those apoptotic neurons can survive for some time but will continue to die for many hours or even days after ischemia in the ischemic penumbra (Charriaut et al., 1996; Fisher, 1997; Yan et al., 1995). This delayed evolution of neuronal death provides a potential window of opportunity for neuronal rescue with effective. Many neuroprotective agents have been found to produce considerable neuroprotection in pre-treatment of ischemia–reperfusion insult, but they often fail at clinical trials. There are many reasons for the failures, the short therapeutic window included. Although XQ-1h at dose of 15.6 and 7.8 mg/kg shows neuroprotective effects in pretreatment of cerebral ischemic-reperfusion injury in vivo (data not shown), little work is done to illustrate its effects on post-treatment of cerebral ischemic-reperfusion injury and its therapeutic time window remains unknown. According to the doses of pre-treatment of cerebral ischemicreperfusion we designed the doses of three groups in this research as 31.2 mg/kg, 15.6 mg/kg and 7.8 mg/kg respectively. Before starting the formal experiment, we did a bioequivalence experiment between ozagrel and XQ-1h, indicating that ozagrel 12 mg/kg i.v was equal to XQ-1h 31.2 mg/kg i.v. (data not shown). Consequently, we chose 12 mg/kg of ozagrel as the experimental positive control dose.
0.5h
Sham
1h
2h
3h
In this research, we evaluated four indexes, neurological deficits, cerebral edema, cerebral infarction and cerebral blood flow (CBF), in MCAO rats after the administration of XQ-1h to identify its therapeutic window. After cerebral ischemic-reperfusion injury, the core of the ischemic lesion necrosis of neurons and axons, axoplasm transport collapse, which will lead to the appearance of a large area of infarction and Cerebral edema (Kristian et al., 2008). Cerebral edema and associated increased intracranial pressure are the major immediate consequences of cerebral ischemic-reperfusion injury that contribute to most early deaths (Kristian et al., 2008). Secondary damage in cerebral ischemic-reperfusion injury is influenced by changes in cerebral blood flow (CBF), cerebral metabolic dysfunction and inadequate cerebral oxygenation (Graham and Chen, 2001). Therefore, the measurement of these four indexes can directly reflect the effects of XQ-1h in posttreatment of cerebral ischemic-reperfusion injury. From Table 1 and Fig. 3 we can see XQ-1h at dose of 31.2 and 15.6 mg/kg produced significant reduction in infarct volume and neurological deficits when treatment was initiated within 2 h after the initiation of focal cerebral ischemia by MCAO, i.e. 2 h after reperfusion. However, treatment at 3 h did not improve cerebral ischemia injury evidently in rats. Therefore, we could conclude that the most perfect therapeutic time of XQ-1h was within 2 h after ischemic stroke in rats. It has been widely accepted that the brain requires this large amount of oxygen to generate sufficient ATP by oxidative phosphorylation to maintain and restore ionic gradients. Na ±/K ±ATPase found on the plasma membrane of neurons, consumes 70% of the energy supplied to the brain (Kristian et al., 2008). This ion pump maintains
Ozagrel XQ-1h (H)XQ-1h(M) XQ-1h(L)
Ozagrel XQ-1h(H) XQ-1h(M) XQ-1h(L)
0.5h
2h
1h
3h
Vehicle
Ozagrel (12mg/kg), XQ-1h (H) (31.2mg/kg), XQ-1h (M) (15.6mg/kg), XQ-1h (L) (7.8mg/kg) Fig. 3. Effect of post-treatment of XQ-1h on cerebral infarction and cerebral edema. Infarct volumes at 24 h after reperfusion in the rats with 120 min of MCAO (n = 8).
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Fig. 4. Effect of post-treatment of XQ-1h on cerebral blood flow. Cerebral blood flow at 24 h after reperfusion in the rats with 120 min of MCAO (n = 8). Ozagrel (12 mg/kg), XQ-1h (H) (31.2 mg/kg), XQ-1h (M) (15.6 mg/kg), XQ-1h (L) (7.8 mg/kg).
the high intracellular K + concentration and the low intracellular Na + concentration necessary for the propagation of action potentials. After ischemia, mitochondrial inhibition of ATP synthesis leads to ATP being consumed within two minutes, which will cause neuronal plasma membrane depolarization, release of potassium into the extracellular space and entry of sodium into cells (Kristian et al., 2008). ATP is an important neuromodulators in the hippocampus. Endogenous ATP is released frequency-dependently from in vitro hippocampal slices upon low and high frequency electrical stimulation (Cunha et al., 1996; Wieraszko et al., 1989). Therefore, we detected the activity of the Na ±/K ±ATPase in hippocampal to evaluate the protective effect of XQ-1h on the acute path physiological changes of cerebral ischemicreperfusion injury. From Table 2, we can see that XQ-1h had beneficial effect on protecting Na +/K +ATPase and the optimal effect was obtained when XQ-1h was injected at 0.5 h after the reperfusion and no effect 1 h later. The results probably indicated that at 1 h after the reperfusion the activity of Na +/K +ATPase was irreversibly damaged.
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To further validate our conclusion, it is necessary to study on possible mechanism of XQ-1h on post-treatment of cerebral ischemicreperfusion injury in vivo. XQ-1h shares a chemical structure similar to that of ginkgolide B which has been shown to scavenge free radicals and non-specifically inhibit seryl and aspartyl proteases (Ahlemeyer and Krieglstein, 2003; Bate et al., 2004). Therefore, we may conclude that the mechanism of XQ-1h's effects on ischemia is similar to that of ginkgolide B. During acute phage of ischemia, excitotoxity, oxidative stress, calcium overload lead to the damage of DNA, the deposition of protein and phosphoric acid and the accumulation of intracellular free radicals (Kristian et al., 2008). N-methyl-D-aspartate-receptor subtype of glutamate, which is coupled with NO synthesis by activation of Nitric oxide synthases (NOS), plays a crucial role in the production of kindling seizures (Bruno and Battaglia, 2001). Moreover, the accumulation of free radicals, including the accumulation of NO, will result in lipid peroxidation. Malondialdehyde (MDA) is the last product of lipid peroxidation which is toxic to cells and cell membranes (Crack and Taylor, 2001). Therefore, the amount of MDA can indirectly reflect the degree of lipid peroxidation and the accumulation of free radicals. It is accepted that SOD (superoxidase dismutase) is one of the most important physiological antioxidants against free radicals and that they prevent subsequent lipid peroxidation (Bordet and Deplanque, 2000). Many experiments have proved that cerebral ischemia/reperfusion of the rat at the acute stage can increase the content of MDA and consume SOD to decrease its activity (Crack and Taylor, 2001). Free radicals are thought to cause behavioral deficits in experimental animals. Fukui and Stadaman suggest that balanced antioxidants are required to control the cognitive and motor functions of the cerebral cortex and the hippocampus. Fukui accounts that oxidative stress and aging impair memory functions (Fukui et al., 2001; Stadtman, 1992). The hippocampus, in contrast, while showing similar changes as the striatum in the first 20– 30 min past ischemia, seemed to recover thereafter and evidently represents penumbral tissue which is the major target for neuroprotective drugs (Cornelia and Alexander, 2010). Therefore, we chose SOD and MDA in hippocampus as indexes to evaluate XQ-1h's ability of eliminating free radicals which are excessively generated during the process of cerebral ischemic-reperfusion injury. From Table 2, we can see the activity of SOD in MCAO rats were ameliorated after the 2 h administration of XQ-1h, and posttreatment with XQ-1h 31.2 mg/kg i.v. significantly decreased MDA
Table 2 The effect of XQ-1h post-treatment on SOD (U/mg prot), Na+/K+ ATPase activity (U) and MDA content (nmol/mg prot) in MCAO rats (n = 8).
SOD Activity
MDA content
Na+/K+ ATPase activity
Groups
Dose (mg/kg)
Administration time after reperfusion (h) 0.5 h
1h
2h
3h
Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel Sham Vehicle XQ-1h XQ-1h XQ-1h Ozagrel
– – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12 – – 31.2 15.6 7.8 12
70.83 ± 8.73a 40.91 ± 10.5 62.05 ± 8.87a 57.79 ± 17.07b 38.77 ± 5.38 58.95 ± 2.66a 3.44 ± 0.44a 12.35 ± 3.08 4.11 ± 0.71a 5.77 ± 1.86a 7.1 ± 3.01a 4.67 ± 0.45b 3.57 ± 0.48a 1.64 ± 0.71 3.33 ± 0.84a 2.9 ± 0.97b 2.05 ± 0.43 2.93 ± 0.64a
69.89 ± 9.21a 41.2 ± 0.88 56.57 ± 6.81b 49.67 ± 7.62 40.33 ± 8.05 57.3 ± 19.37b 3.78 ± 0.65a 12.42 ± 2.8 7.17 ± 2.79a 9.58 ± 1.53b 10.19 ± 3.59 5.97 ± 2.5b 3.35 ± 0.34a 1.41 ± 0.69 1.63 ± 0.83 0.8 ± 0.54 0.72 ± 0.42 1.27 ± 0.61
66.54 ± 7.65a 40.68 ± 0.79 48.58 ± 10.99 42.93 ± 14.29 40.1 ± 5.58 50.82 ± 8.4 4.21 ± 1.32a 12.89 ± 1.12 8.44 ± 3.72b 10.23 ± 2.38 11.32 ± 2.06 9.18 ± 2.87a 2.86 ± 0.52a 1.44 ± 0.39 1.1 ± 1.61 0.93 ± 0.72 0.49 ± 0.41 1.1 ± 0.62
70.22 ± 6.76a 39.31 ± 3.92 45.22 ± 1.28 40.68 ± 1.98 39.89 ± 1.77 44.81 ± 0.45 5.11 ± 0.4a 13.11 ± 1.45 10.49 ± 1.42 12.02 ± 1.43 12.33 ± 1.47 10.21 ± 1.16 2.69 ± 0.49a 1.28 ± 0.31 1.04 ± 1.92 0.87 ± 1.51 0.72 ± 1.29 0.98 ± 1.23
Values are mean ± S.D. a P b 0.01 as compared to the vehicle-treated group. b P b 0.05 as compared to the vehicle-treated group.
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content at 2 h after reperfusion (P b 0.05); however, treatment at 3 h did not improve cerebral ischemia injury evidently in rats. The mechanism of antioxidative activity of XQ-1h might be XQ-1h antagonizes the PAF receptor and inhibits PAF-induced iNOS over expression (Yan and Weirong, 2009), leading to the decreased release of NO and the low accumulation of free radical further easing the lipid peroxidation, decreasing the amount of MDA and protecting the ability of SOD. This result further verified that the therapeutic time window of XQ-1h is 2 h, and it suggested that XQ-1h is a potent antioxidant, somehow reduced neurobehavioral deficits by scavenging free radicals. It also indicated that the therapeutic time window of XQ-1h is deeply related with the activity of scavenging oxidative stress products. In conclusion, the most perfect therapeutic time of XQ-1h was within 2 h after ischemic reperfusion in MCAO rats, XQ-1h had protective effect on Na ±/K ±ATPase within 0.5 h after the reperfusion, and the beneficial effects on ischemic injury is related with the activity of scavenging oxidative stress products. Further studies can be focused on looking for more drug combination therapy programs in order to assess the potential clinical application of XQ-1h. References Ahlemeyer, B., Krieglstein, J., 2003. Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci 60, 1779–1792. Arii, K., Igarashi, H., Arii, T., Katayama, Y., 2002. The effect of ozagrel sodium on photochemical thrombosis in rat: therapeutic window and combined therapy with heparin sodium. Life Sci 71, 2983–2994. Bate, C., Salmona, M., Williams, A., 2004. Ginkgolide B inhibits the neurotoxicity of prions or amyloid-beta1-42. J Neuroinflamm 36, 1–4. Bordet, R., Deplanque, D., 2000. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J Cereb Blood Flow Metab 20, 1190. Bouley, J., Fisher, M., Henninger, N., 2007. Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging. Neurosci. Lett. 412, 185–190. Bruno, V., Battaglia, G., 2001. Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab 21, 1013–1033. Charriaut, C., Margaill, I., Represa, A., 1996. Apoptosis and necrosis following reversible focal ischemia: an in situ DNA fragmentation analysis. J Cereb Blood Flow Metab 16, 186–194.
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