brain research 1599 (2015) 85–92
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Research Report
Preischemic neuroprotective effect of minocycline and sodium ozagrel on transient cerebral ischemic rat model Sang In Parka, Sang Kyu Parkb,n, Kyoung Sool Jangb, Yong Min Hanb, Choong Hyeon Kimc, Seok Jeon Ohc a
Institute of Catholic Integrative Medicine (ICIM), Incheon St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea b Department of Neurosurgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea c Department of Neurosurgery, Hanyang University Hospital, College of Medicine, Hanyang University, Seoul, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
We investigated the neuroprotective properties of single doses of minocycline and ozagrel
Accepted 25 December 2014
when administered prior to stroke. Male Sprague-Dawley rats were assigned randomly to
Available online 31 December 2014
one of the following groups: (1) control (Con) group (n ¼10), (2) minocycline (Mino) group
Keywords:
(n ¼10), (3) sodium ozagrel (SO) group (n ¼10). Rats were treated with a single dose of
Minocycline
minocycline or ozagrel at 30 min before stroke. A middle cerebral artery occlusion (MCAO)
Sodium ozagrel
was made at 30 min after drug administration and reperfusion was done. The rats were
Neuroprotection
subjected to a neurobehavioral test at days 1, 3 and 7 after MCAO. The cerebral ischemic
Stroke prevention
volume was quantified by MetaMorph imaging software after TTC staining. The neuronal
Reperfusion
cell survival and astrocytes expansion were assessed by the NeuN and GFAP immunohistofluorescence staining. Apoptosis was detected by the TUNEL assay. We statistically analyzed and compared the results with each other. Mino and SO groups had neuroprotective effect and showed a better behavioral performance of adhesive removal and treadmill test at 7 days after stroke. Mino and SO groups also showed a smaller infarct volume than control group at 7 days after stroke. Immunohistofluorescence staining showed a higher number of NeuN positive cells, lower activated astrocytes in GFAP and a lower apoptosis in TUNEL staining. This study showed that single doses of minocycline and ozagrel prior to stroke had neuroprotective effects. These agents will be useful not only in post-stroke therapy but also in stroke prevention in several cerebrovascular procedures like carotid endarterectomy, bypass procedure, endovascular angioplasty, thromboembolectomy or thrombolysis. & 2014 Elsevier B.V. All rights reserved.
n Correspondence to: Department of Neurosurgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea. Fax: þ82 32 280 5991. E-mail address: [email protected] (S.K. Park).
http://dx.doi.org/10.1016/j.brainres.2014.12.051 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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1.
brain research 1599 (2015) 85–92
Introduction
Stroke is one of the most common causes of death worldwide and the most frequent cause of permanent disability. The poor prognosis of stroke is due to irreversible loss of brain cells before the patient receives medical attention, which is exacerbated by the void in delayed treatment options to protect against secondary injury. Cell death associated with the initial blood flow interruption and the immediately ensuing excitotoxicity are abrupt, while inflammation occurs over a long period of time from stroke onset. Accordingly, anti-inflammatory treatment is likely to extend the therapeutic window allowing improved intervention in stroke (Zhang et al., 2004). Within several hours after stroke onset, ischemia induces inflammation that causes simultaneous alterations in the cerebrovasculatures including apoptosis and activation of microglia (Fawcett and Asher, 1999; Silver and Miller, 2004). Activated microglia and proliferated astrocytes release toxic substances such as nitric oxide (NO) and free radicals that can damage healthy neurons (Giulian, 1993) although some reports showed the beneficial role of microglia (Neumann et al., 2006, 2008). Free radicals generated during ischemia and reperfusion also play an important role in the development of brain damage via obstruction of cell membranes and formation of edema (Matsuo et al., 1995; Simonian and Coyle, 1996; Takamatsu et al., 1998). Platelet aggregation was also reported as one of the aggravating factors of cerebral ischemia (Suzuki et al., 1989). Thromboxane A2 (TXA2), a strong vasoconstrictor and platelet aggregator, is increased, and aggravates brain damage after cerebral ischemia–reperfusion (Chen et al., 1986; Petroni et al., 1989). Minocycline is a semi-synthetic tetracycline with independent anti-inflammatory effects that is neuroprotective in models of brain injury including stroke (Domercq and Matute, 2004; Yrjanheikki et al., 1999). Neuroprotective effects of Minocycline include inhibition of gliosis, apoptosis, free radical formation, and peripheral inflammation (Fox et al.,
2005; Tomas-Camardiel et al., 2004). Specifically, minocycline inhibits the post-ischemic induction of nitric oxide synthase (NOS) and reduces the release of cytochrome c by directly conferring stability to the mitochondrial membrane (Wang et al., 1998). Sodium ozagrel (ozagrel) is a selective TXA2 synthase inhibitor. It ameliorates platelet aggregation, vasoconstriction and brain edema in acute cerebral ischemia (Chen et al., 1986). Most of studies of minocycline and ozagrel are about the post-stroke therapeutic effect. However, many cerebrovascular procedures, like conventional cerebral angiography, carotid endarterectomy (CEA), balloon angioplasty or stenting, temporary block of cerebral circulation during cerebral aneurysm clipping or external carotid–internal carotid (EC–IC) bypass, often cause ischemic stroke (Bendszus et al., 1999; Britt et al., 2000; Enevoldsen et al., 1999; Kato et al., 2003; Soeda et al., 2003). Arterial vasospasm after subarachnoid hemorrhage can cause large cerebral infarct. The aim of this study is to assess the neuroprotective effect of minocycline and ozagrel when administered prior to these several neurovascular procedures which have a risk of ischemic stroke.
2.
Results
2.1.
Behavior evaluation
Fig. 1 shows behavioral results. Before ischemia, no rats showed a neurobehavioral deficit. Mino group or SO group showed more improved performance than control group in the adhesive removal test at 7 days after MCAO. In the adhesive removal test, there was a significant difference in the scores at 7 days in the Mino and SO group compared to the control group (Con: 129.90717.00, Mino: 56.33720.47, SO: 73.80720.47 s, po0.05) although there was no significant difference at day 1 (Con: 154.2873.86, Mino: 159.00715.32, SO: 152.40715.20 s). Mino group or SO group also showed significant improvement in treadmill test at day 1 (Con: 29.4776.95, Mino: 48.5077.48,
Fig. 1 – Behavior evaluations after middle cerebral artery occlusion (MCAO). Rats in Mino and SO group remove adhesive paper more quickly and more tolerable in treadmill test. The Mino group shows a slightly improved performance than the SO group although there is no statistical significance. npo0.05, nnpo0.01.
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SO: 50.1774.93 m/min, po0.05) and at day 7 (Con: 44.7176.72, Mino: 68.4274.50, SO: 60.1272.81 m/min, po0.05) after MCAO.
2.2.
Infarct volume
A representative photograph of brain slices obtained at 7 days after MCAO is shown in Fig. 2. Both non-stained (white) parts and weakly stained (pale-red) parts can be seen adjacent to the non-stained parts. The brains of rats were stained with TTC to obtain the infarct volume and calculated by measuring the area of infarction area (Fig. 2B). Both the area and volume of the non- or weakly-stained parts in Mino and SO groups were significantly decreased on TCC stained slices. There were significant differences in infarct volume between control groups and Mino group or SO group, and also between Mino and SO group (Con: 394.31786.85, Mino: 213.58752.94, SO: 255.10763.14 mm3, po0.05).
2.3.
Immunohistofluorescence staining
The number of astrocytes, neurons and apoptotic cells in the peri-infarct region was observed at 7 days after MCAO. All drug-treated groups showed a significant decrease in the density of GFAP positive cells compared to the control group in the analysis of GFAP positive cells (Fig. 3). There was no statistical difference between Mino and SO group although the GFAP expression was slightly lower in Mino group (Con: 24097300, Mino: 18007250, SO: 20277311 optical density, po0.05). This means that Mino and SO group may protect against the delayed expansion of the infarct through secondary oxidative injury caused by activated astrocytes and reactive gliosis. The cell survival of neurons was represented by NeuN positive cells. All drug-treated groups showed a higher number of NeuN positive cells compared to the control group (Fig. 4). Especially, Mino group showed a significantly higher
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number of NeuN positive cells compared to the SO group (Con: 3473, Mino: 73715, SO: 62713 cells/mm2, po0.05). Apoptotic cells were recognized by identification of DNA fragmentation using TUNEL method (Fig. 5). All treated groups had reduced TUNEL-positive cells compared to control group (Con: 148721, Mino: 96720, SO: 118714 cell/mm2, po0.05). Mino group showed a significantly decreased number of TUNEL positive cells compared to SO group.
3.
Discussion
In ischemic brains, an increased chemokine mRNA expression displays a biphasic profile, initially being found in neurons and then subsequently found in astrocytes (Wang et al., 1998). Accumulation of macrophage/microglia is found in areas of gliosis surrounding recent infarcts (Flynn et al., 2003). Reperfusion activates the arachidonic cascade, leading to the production of excessive amounts of TXA2, which is a metabolite of arachidonic acid (Gaudet et al., 1980; Petroni et al., 1989). The arachidonic cascade also is one of the production pathways for free radicals (Simonian and Coyle, 1996). There are several studies that minocycline or ozagrel are effective post-ischemic stroke treatments. Minocycline is reported to have neuroprotective effects after cerebral ischemia or intracerebral hemorrhage in animal models of stroke (Fox et al., 2005; Yrjanheikki et al., 1999). In addition to their known antibiotic properties, tetracyclines also have been shown to have anti-inflammatory properties in a variety of cells and tissues. Minocycline treatment reduces the amount of cyclooxygenase-2, inducible NO synthase, prostaglandin E2 and interleukin-1β in models of neuronal damage (Chen et al., 2000; Du et al., 2001). This effect may be caused by actions of minocycline on suppression of microglial activation (Du et al., 2001). Cerebral ischemia results in a localized secondary
Fig. 2 – Comparison of infarct volume by 2% 2-3-5-triphenylterazolium staining (TTC) among three groups. The infarct volumes of Mino and SO group grossly decreased compared with the control group. The infarct volumes are more decreased in the Mino group than in the SO group. npo0.05, nnpo0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3 – Glial fibrillary acidic protein (GFAP) positive cells in the peri-infarct area of precentral cortex at 7 days after MCAO. 40 ,6-Diamidino-2-phenylindole (DAPI) shows gross coronal section of peri-infarct area. The optical density is most prominent in the control group (A). The SO group (C) shows less prominent than the control group, although it shows more prominent optical density than the Mino group (B). npo0.05.
inflammation that can significantly contribute to the final outcome. Reduction of this ischemia-induced inflammation by minocycline may contribute to damage reduction and functional recovery enhancement. Furthermore, the results of the current study demonstrate the promising therapeutic potential of minocycline in stroke. Ozagrel has been widely used for the treatment of thrombotic or lacunar stroke over the last few years. It is a TXA2 synthase inhibitor, modulates the arachidonic acid cascade thereby reducing TXA2 and increases PGI2 (Chen et al., 1986). TXA2 is a potent inducer of platelet aggregation and a vasoconstrictor and PGI2 is a strong inhibitor of platelet aggregation and a vasodilator. Ozagrel administration improves the balance of PGI2/TXA2 during the acute phase of cerebral ischemia. The present study demonstrates that a single dose of minocycline or ozagrel prior to MCAO exerted the direct protection against ischemic stroke in neurons. Previous articles reported that the risk of high intensity lesion on diffusion-weighted imaging (DWI) after cerebral angiography
ranges from 9% to 29% (Kato et al., 2003). Temporary cerebral blood flow occlusion is needed in several cerebrovascular procedures like CEA, balloon angioplasty, stenting, or bypass surgery. Sometimes these procedures cause cerebral ischemia or stroke. In case of CEA or carotid stenting, perioperative ischemic complication rates are reported as 3–24% (Asai et al., 2013). Previous articles have reported that patients who underwent neuro-interventional procedures carry a greater risk of presentation of high-intensity lesion on DWI, which ranges from 51.3% to 61% (Soeda et al., 2003). Preventive administration of neuroprotective agent in high risk patients can minimize the damage by stroke. Several agents were introduced for an intraoperative cerebral protection like barbiturate, calcium channel blocker (CCB) and NO synthase inhibitor. However, barbiturate frequently causes respiratory depression on locally anesthetized patients and CCB cause hypotension with weak neuroprotective effect. The prevention of a reperfusion injury is an important therapeutic target for stroke. Moreover, intracerebral hemorrhage of basal ganglia and hyperperfusion brain
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Fig. 4 – NeuN positive cells in the peri-infarct area of precentral cortex at 7 days after MCAO. There is a significant larger number of NeuN positive cells in the Mino and SO group than in the control group. NeuN positive cells are more prominent in the Mino group than in the SO group. npo0.05, nnpo0.01.
Fig. 5 – Terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end (TUNEL) staining in the peri-infarct area of precentral cortex at 7 days after MCAO. Apoptotic cells are significantly decreased in the Mino and SO groups, especially in the Mino group. npo0.05, nnpo0.01.
swelling are not rare after reperfusion of MCAO. Blood brain barrier (BBB) breakdown may play an important role in these reperfusion injuries. Minocycline or ozagrel can be another
intraoperative cerebral protection agent although these agents have minor side effects such as headache, nausea, elevated liver enzyme and others.
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In case of endovascular thromboembolectomy or thrombolysis for acute stroke caused by major artery occlusion, occluded vessel selection with microcatheter is a first step for the recanalization procedure. It takes a great deal of time from the selection of the occluded vessel to the successful recanalization. An intra-arterial injection of minocycline or ozagrel in the occluded vessel would be helpful to prevent the progression of an infarct core until recanalization and reperfusion injury. Minocycline and ozagrel decreased the total volume of infarction. We think that minocycline or ozagrel target the ischemic rim and prevent microthrombi formation and further aggravation in the rim by suppressing the platelet activity and inflammatory enzymes along with edema and vasoconstriction, ultimately resulting in a smaller infarct volume. TCC staining is a simple and widely used method for the detection of infarction, with the non-stained part indicating the infarction. The weakly stained area, which situated adjacent to the non-stained part in each slice, seemed to include both surviving cells and dead cells. Thus, this part seemed likely to be the penumbra. The above results suggest that minocycline or ozagrel prevented cell death in the penumbra and suppressed an enlargement of infarct core. A smaller margin of improvement with diminished drug delivery could be the reason for ineffectiveness in the delayed administration. Thus, early administration before temporary block of cerebral circulation would be helpful to prevent an unexpected ischemic injury. There were some differences in the effectiveness between minocycline and ozagrel and these differences seem likely to be related to their differing in the diversity of effects. Minocycline have been reported to suppress free radicals in the ischemic period and to have more multiple effects than ozagrel (Tomas-Camardiel et al., 2004). Ozagrel has been reported to suppress the decrease of cerebral blood flow but not to have an effect on free radicals (Pettigrew et al., 1989).
4.
Conclusions
In the present study, both minocycline and ozagrel showed smaller infarct volume, more neuronal survival, fewer astrocyte activation and apoptosis in addition to more improvement of behavioral disability than the control group when administered prior to ischemia. It suggests that minocycline and ozagrel attribute to neuroprotective effects in MCAO rat models. Additional to post-ischemic therapy, it will be useful to prevent cerebral ischemia in multiple cerebrovascular procedures.
housed in temperature–humidity and light–dark controlled rooms, and allowed food and tap water ad libitum. Animals were assigned randomly to one of the following three groups: (1) the first group of rats was treated with saline as the control group (n ¼10, Con group), (2) the second group was injected with minocycline (n¼ 10, Mino group), and (3) the third group was injected with sodium ozagrel (n¼ 10, SO group).
5.2.
Minocycline (Sigma, Inc. St. Louis, MO, USA) was prepared by dissolving in 0.9% saline as 4 mg/ml. Ozagrel (Kissei Pharmaceutical Co. Ltd., Matsumoto, Japan) was prepared by dissolving in 0.9% saline as 5 mg/ml. Minocycline (45 mg/kg) and ozagrel (5 mg/kg) were intravenously administrated to the tail vein at 30 min before performing the middle cerebral artery occlusion (MCAO) in the rat models.
5.3.
Experimental procedures
5.1.
Animals
All animal procedures were carried out in accordance with the animal experimental guideline established by the institutional Animal Care and Use Committee of Catholic University Medical School (CIMH 2012-005). Male Sprague-Dawley rats (270–300 g) aged 10–12 weeks old were used. The rats were
MCAO model
Transient MCAO was induced as described by Longa et al. (1989) with a minor modification. Under inhalation anesthesia with isoflurane, the right common carotid artery (CCA) and the external carotid artery (ECA) were exposed through a midline incision. The occipital artery and the superior thyroid artery were dissected and coagulated. The right internal carotid artery (ICA) was exposed and the pterygopalatine artery was ligated. Two loose ties (5–0 silk suture) were made around the ECA stump and tightened the distal tie. A small puncture opening was made in the ECA and 3–0 monofilament (intraluminal suture with a rounded tip) was inserted through the opening and the silk suture was tightened around the lumen containing the filament. The monofilament was gently advanced into the ICA for a distance of 19 to 20 mm beyond bifurcation until it blocked the bifurcating origin of the middle cerebral artery (MCA). The body temperature was maintained at 37 1C using heating pad during the procedure. The animal was allowed to regain consciousness and checked to determine whether the MCA was successfully occluded by placing the animal on a flat surface, lifting it by the base of the tail 2 in. off the surface and observing its behavior. When the MCA is successfully occluded, rats exhibit marked thorax twisting when suspended by its tail. Contralateral forepaw flexure should also be observed. Ninety minutes after the occlusion, animals were reanesthetized and reperfused by removing the suture and the ECA was ligated.
5.4.
5.
Drug administration
Behavior evaluation
Behavioral functions were evaluated with adhesive removal and treadmill test at 1, 3 and 7 days after MCAO. All animals were trained three times for a week before MCAO. A square dot of adhesive-backed paper (12Ø) was used in the adhesive removal test as bilateral tactile stimuli occupying the distal radial region on the wrist of each forelimb. Animals were given three trials with a cut-off time of 180 s. The data are presented as mean time to remove the left dot. The treadmill
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was accelerated from 20 to 80 m/min with a cut-off time of 300 s.
5.5.
Infarct volume
At 7 days after MCAO, all rats (n¼ 10, for each group) were sacrificed under deep anesthesia with 5% halothane. The brain was dissected out and five 2 mm thick coronal slices were prepared using a rodent brain matrix. The slices were stained a 2% solution of 2-3-5-triphenylterazolium chloride (TCC; Sigma, St. Louis, MO, USA) at 37 1C for 15 min. The infarct area was measured using image analyzing software (MetaMorph, Molecular Devices Inc., Downingtown, PA, USA), determined by using the method of subtracting the area of the non-infarct ipsilateral hemisphere from that of the contralateral tissue to reduce errors that might occur due to cerebral edema.
5.6.
Immunohistofluorescence staining
The tissue was coronally cut at a thickness of 12 mm with 0.1 mm of boundary area of cerebral infarct in the precentral gyrus, located 24 mm posterior to the frontal pole from each rat for 20 min and washed with 0.01 M phosphate-buffered solution (PBS) for 10 min. Sections were blocked in normal goat serum for 1 h at room temperature. The sections were incubated with mouse antibody to glial fibrillary acidic protein (GFAP, Millipore, Inc., Billerica, MA, USA, diluted at 1:200) and neuronal nuclei (NeuN, Millipore, Inc., Billerica, MA, USA, diluted at 1:100) overnight at 4 1C. Subsequently, sections were incubated for 1 h at room temperature with Alexa 488-conjugated goat antimouse IgG (Vector Laboratories, Southfield, MI, USA, diluted at 1:200). After washing, the sections were counterstained and stored at 20 1C and then observed under both a fluorescence microscope equipped with a spot digital camera and a confocal scanning laser microscope (LSM 510, Zeiss, Jena, Germany). Apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by means of the in situ cell death detection kit (Roche, Indianapolis, IN, USA) developed using the Cy2-conjugated streptavidin observed by confocal scanning laser microscope. The ischemia penumbra was determined by optical density of GFAP, NeuN and TUNEL positive cells using the MetaMorph imaging program (Molecular Device Inc., Downingtown, PA, USA).
5.7.
Statistical analysis
Data are expressed as mean7SD. Statistical analyses were performed using analysis of variance (ANOVA) with post-hoc analysis. A value of po0.05 was considered statistically significant.
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www.elsevier.com/locate/brainres
Research Report
Preischemic neuroprotective effect of minocycline and sodium ozagrel on transient cerebral ischemic rat model Sang In Parka, Sang Kyu Parkb,n, Kyoung Sool Jangb, Yong Min Hanb, Choong Hyeon Kimc, Seok Jeon Ohc a
Institute of Catholic Integrative Medicine (ICIM), Incheon St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea b Department of Neurosurgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea c Department of Neurosurgery, Hanyang University Hospital, College of Medicine, Hanyang University, Seoul, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
We investigated the neuroprotective properties of single doses of minocycline and ozagrel
Accepted 25 December 2014
when administered prior to stroke. Male Sprague-Dawley rats were assigned randomly to
Available online 31 December 2014
one of the following groups: (1) control (Con) group (n ¼10), (2) minocycline (Mino) group
Keywords:
(n ¼10), (3) sodium ozagrel (SO) group (n ¼10). Rats were treated with a single dose of
Minocycline
minocycline or ozagrel at 30 min before stroke. A middle cerebral artery occlusion (MCAO)
Sodium ozagrel
was made at 30 min after drug administration and reperfusion was done. The rats were
Neuroprotection
subjected to a neurobehavioral test at days 1, 3 and 7 after MCAO. The cerebral ischemic
Stroke prevention
volume was quantified by MetaMorph imaging software after TTC staining. The neuronal
Reperfusion
cell survival and astrocytes expansion were assessed by the NeuN and GFAP immunohistofluorescence staining. Apoptosis was detected by the TUNEL assay. We statistically analyzed and compared the results with each other. Mino and SO groups had neuroprotective effect and showed a better behavioral performance of adhesive removal and treadmill test at 7 days after stroke. Mino and SO groups also showed a smaller infarct volume than control group at 7 days after stroke. Immunohistofluorescence staining showed a higher number of NeuN positive cells, lower activated astrocytes in GFAP and a lower apoptosis in TUNEL staining. This study showed that single doses of minocycline and ozagrel prior to stroke had neuroprotective effects. These agents will be useful not only in post-stroke therapy but also in stroke prevention in several cerebrovascular procedures like carotid endarterectomy, bypass procedure, endovascular angioplasty, thromboembolectomy or thrombolysis. & 2014 Elsevier B.V. All rights reserved.
n Correspondence to: Department of Neurosurgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea. Fax: þ82 32 280 5991. E-mail address: [email protected] (S.K. Park).
http://dx.doi.org/10.1016/j.brainres.2014.12.051 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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1.
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Introduction
Stroke is one of the most common causes of death worldwide and the most frequent cause of permanent disability. The poor prognosis of stroke is due to irreversible loss of brain cells before the patient receives medical attention, which is exacerbated by the void in delayed treatment options to protect against secondary injury. Cell death associated with the initial blood flow interruption and the immediately ensuing excitotoxicity are abrupt, while inflammation occurs over a long period of time from stroke onset. Accordingly, anti-inflammatory treatment is likely to extend the therapeutic window allowing improved intervention in stroke (Zhang et al., 2004). Within several hours after stroke onset, ischemia induces inflammation that causes simultaneous alterations in the cerebrovasculatures including apoptosis and activation of microglia (Fawcett and Asher, 1999; Silver and Miller, 2004). Activated microglia and proliferated astrocytes release toxic substances such as nitric oxide (NO) and free radicals that can damage healthy neurons (Giulian, 1993) although some reports showed the beneficial role of microglia (Neumann et al., 2006, 2008). Free radicals generated during ischemia and reperfusion also play an important role in the development of brain damage via obstruction of cell membranes and formation of edema (Matsuo et al., 1995; Simonian and Coyle, 1996; Takamatsu et al., 1998). Platelet aggregation was also reported as one of the aggravating factors of cerebral ischemia (Suzuki et al., 1989). Thromboxane A2 (TXA2), a strong vasoconstrictor and platelet aggregator, is increased, and aggravates brain damage after cerebral ischemia–reperfusion (Chen et al., 1986; Petroni et al., 1989). Minocycline is a semi-synthetic tetracycline with independent anti-inflammatory effects that is neuroprotective in models of brain injury including stroke (Domercq and Matute, 2004; Yrjanheikki et al., 1999). Neuroprotective effects of Minocycline include inhibition of gliosis, apoptosis, free radical formation, and peripheral inflammation (Fox et al.,
2005; Tomas-Camardiel et al., 2004). Specifically, minocycline inhibits the post-ischemic induction of nitric oxide synthase (NOS) and reduces the release of cytochrome c by directly conferring stability to the mitochondrial membrane (Wang et al., 1998). Sodium ozagrel (ozagrel) is a selective TXA2 synthase inhibitor. It ameliorates platelet aggregation, vasoconstriction and brain edema in acute cerebral ischemia (Chen et al., 1986). Most of studies of minocycline and ozagrel are about the post-stroke therapeutic effect. However, many cerebrovascular procedures, like conventional cerebral angiography, carotid endarterectomy (CEA), balloon angioplasty or stenting, temporary block of cerebral circulation during cerebral aneurysm clipping or external carotid–internal carotid (EC–IC) bypass, often cause ischemic stroke (Bendszus et al., 1999; Britt et al., 2000; Enevoldsen et al., 1999; Kato et al., 2003; Soeda et al., 2003). Arterial vasospasm after subarachnoid hemorrhage can cause large cerebral infarct. The aim of this study is to assess the neuroprotective effect of minocycline and ozagrel when administered prior to these several neurovascular procedures which have a risk of ischemic stroke.
2.
Results
2.1.
Behavior evaluation
Fig. 1 shows behavioral results. Before ischemia, no rats showed a neurobehavioral deficit. Mino group or SO group showed more improved performance than control group in the adhesive removal test at 7 days after MCAO. In the adhesive removal test, there was a significant difference in the scores at 7 days in the Mino and SO group compared to the control group (Con: 129.90717.00, Mino: 56.33720.47, SO: 73.80720.47 s, po0.05) although there was no significant difference at day 1 (Con: 154.2873.86, Mino: 159.00715.32, SO: 152.40715.20 s). Mino group or SO group also showed significant improvement in treadmill test at day 1 (Con: 29.4776.95, Mino: 48.5077.48,
Fig. 1 – Behavior evaluations after middle cerebral artery occlusion (MCAO). Rats in Mino and SO group remove adhesive paper more quickly and more tolerable in treadmill test. The Mino group shows a slightly improved performance than the SO group although there is no statistical significance. npo0.05, nnpo0.01.
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SO: 50.1774.93 m/min, po0.05) and at day 7 (Con: 44.7176.72, Mino: 68.4274.50, SO: 60.1272.81 m/min, po0.05) after MCAO.
2.2.
Infarct volume
A representative photograph of brain slices obtained at 7 days after MCAO is shown in Fig. 2. Both non-stained (white) parts and weakly stained (pale-red) parts can be seen adjacent to the non-stained parts. The brains of rats were stained with TTC to obtain the infarct volume and calculated by measuring the area of infarction area (Fig. 2B). Both the area and volume of the non- or weakly-stained parts in Mino and SO groups were significantly decreased on TCC stained slices. There were significant differences in infarct volume between control groups and Mino group or SO group, and also between Mino and SO group (Con: 394.31786.85, Mino: 213.58752.94, SO: 255.10763.14 mm3, po0.05).
2.3.
Immunohistofluorescence staining
The number of astrocytes, neurons and apoptotic cells in the peri-infarct region was observed at 7 days after MCAO. All drug-treated groups showed a significant decrease in the density of GFAP positive cells compared to the control group in the analysis of GFAP positive cells (Fig. 3). There was no statistical difference between Mino and SO group although the GFAP expression was slightly lower in Mino group (Con: 24097300, Mino: 18007250, SO: 20277311 optical density, po0.05). This means that Mino and SO group may protect against the delayed expansion of the infarct through secondary oxidative injury caused by activated astrocytes and reactive gliosis. The cell survival of neurons was represented by NeuN positive cells. All drug-treated groups showed a higher number of NeuN positive cells compared to the control group (Fig. 4). Especially, Mino group showed a significantly higher
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number of NeuN positive cells compared to the SO group (Con: 3473, Mino: 73715, SO: 62713 cells/mm2, po0.05). Apoptotic cells were recognized by identification of DNA fragmentation using TUNEL method (Fig. 5). All treated groups had reduced TUNEL-positive cells compared to control group (Con: 148721, Mino: 96720, SO: 118714 cell/mm2, po0.05). Mino group showed a significantly decreased number of TUNEL positive cells compared to SO group.
3.
Discussion
In ischemic brains, an increased chemokine mRNA expression displays a biphasic profile, initially being found in neurons and then subsequently found in astrocytes (Wang et al., 1998). Accumulation of macrophage/microglia is found in areas of gliosis surrounding recent infarcts (Flynn et al., 2003). Reperfusion activates the arachidonic cascade, leading to the production of excessive amounts of TXA2, which is a metabolite of arachidonic acid (Gaudet et al., 1980; Petroni et al., 1989). The arachidonic cascade also is one of the production pathways for free radicals (Simonian and Coyle, 1996). There are several studies that minocycline or ozagrel are effective post-ischemic stroke treatments. Minocycline is reported to have neuroprotective effects after cerebral ischemia or intracerebral hemorrhage in animal models of stroke (Fox et al., 2005; Yrjanheikki et al., 1999). In addition to their known antibiotic properties, tetracyclines also have been shown to have anti-inflammatory properties in a variety of cells and tissues. Minocycline treatment reduces the amount of cyclooxygenase-2, inducible NO synthase, prostaglandin E2 and interleukin-1β in models of neuronal damage (Chen et al., 2000; Du et al., 2001). This effect may be caused by actions of minocycline on suppression of microglial activation (Du et al., 2001). Cerebral ischemia results in a localized secondary
Fig. 2 – Comparison of infarct volume by 2% 2-3-5-triphenylterazolium staining (TTC) among three groups. The infarct volumes of Mino and SO group grossly decreased compared with the control group. The infarct volumes are more decreased in the Mino group than in the SO group. npo0.05, nnpo0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3 – Glial fibrillary acidic protein (GFAP) positive cells in the peri-infarct area of precentral cortex at 7 days after MCAO. 40 ,6-Diamidino-2-phenylindole (DAPI) shows gross coronal section of peri-infarct area. The optical density is most prominent in the control group (A). The SO group (C) shows less prominent than the control group, although it shows more prominent optical density than the Mino group (B). npo0.05.
inflammation that can significantly contribute to the final outcome. Reduction of this ischemia-induced inflammation by minocycline may contribute to damage reduction and functional recovery enhancement. Furthermore, the results of the current study demonstrate the promising therapeutic potential of minocycline in stroke. Ozagrel has been widely used for the treatment of thrombotic or lacunar stroke over the last few years. It is a TXA2 synthase inhibitor, modulates the arachidonic acid cascade thereby reducing TXA2 and increases PGI2 (Chen et al., 1986). TXA2 is a potent inducer of platelet aggregation and a vasoconstrictor and PGI2 is a strong inhibitor of platelet aggregation and a vasodilator. Ozagrel administration improves the balance of PGI2/TXA2 during the acute phase of cerebral ischemia. The present study demonstrates that a single dose of minocycline or ozagrel prior to MCAO exerted the direct protection against ischemic stroke in neurons. Previous articles reported that the risk of high intensity lesion on diffusion-weighted imaging (DWI) after cerebral angiography
ranges from 9% to 29% (Kato et al., 2003). Temporary cerebral blood flow occlusion is needed in several cerebrovascular procedures like CEA, balloon angioplasty, stenting, or bypass surgery. Sometimes these procedures cause cerebral ischemia or stroke. In case of CEA or carotid stenting, perioperative ischemic complication rates are reported as 3–24% (Asai et al., 2013). Previous articles have reported that patients who underwent neuro-interventional procedures carry a greater risk of presentation of high-intensity lesion on DWI, which ranges from 51.3% to 61% (Soeda et al., 2003). Preventive administration of neuroprotective agent in high risk patients can minimize the damage by stroke. Several agents were introduced for an intraoperative cerebral protection like barbiturate, calcium channel blocker (CCB) and NO synthase inhibitor. However, barbiturate frequently causes respiratory depression on locally anesthetized patients and CCB cause hypotension with weak neuroprotective effect. The prevention of a reperfusion injury is an important therapeutic target for stroke. Moreover, intracerebral hemorrhage of basal ganglia and hyperperfusion brain
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Fig. 4 – NeuN positive cells in the peri-infarct area of precentral cortex at 7 days after MCAO. There is a significant larger number of NeuN positive cells in the Mino and SO group than in the control group. NeuN positive cells are more prominent in the Mino group than in the SO group. npo0.05, nnpo0.01.
Fig. 5 – Terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end (TUNEL) staining in the peri-infarct area of precentral cortex at 7 days after MCAO. Apoptotic cells are significantly decreased in the Mino and SO groups, especially in the Mino group. npo0.05, nnpo0.01.
swelling are not rare after reperfusion of MCAO. Blood brain barrier (BBB) breakdown may play an important role in these reperfusion injuries. Minocycline or ozagrel can be another
intraoperative cerebral protection agent although these agents have minor side effects such as headache, nausea, elevated liver enzyme and others.
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In case of endovascular thromboembolectomy or thrombolysis for acute stroke caused by major artery occlusion, occluded vessel selection with microcatheter is a first step for the recanalization procedure. It takes a great deal of time from the selection of the occluded vessel to the successful recanalization. An intra-arterial injection of minocycline or ozagrel in the occluded vessel would be helpful to prevent the progression of an infarct core until recanalization and reperfusion injury. Minocycline and ozagrel decreased the total volume of infarction. We think that minocycline or ozagrel target the ischemic rim and prevent microthrombi formation and further aggravation in the rim by suppressing the platelet activity and inflammatory enzymes along with edema and vasoconstriction, ultimately resulting in a smaller infarct volume. TCC staining is a simple and widely used method for the detection of infarction, with the non-stained part indicating the infarction. The weakly stained area, which situated adjacent to the non-stained part in each slice, seemed to include both surviving cells and dead cells. Thus, this part seemed likely to be the penumbra. The above results suggest that minocycline or ozagrel prevented cell death in the penumbra and suppressed an enlargement of infarct core. A smaller margin of improvement with diminished drug delivery could be the reason for ineffectiveness in the delayed administration. Thus, early administration before temporary block of cerebral circulation would be helpful to prevent an unexpected ischemic injury. There were some differences in the effectiveness between minocycline and ozagrel and these differences seem likely to be related to their differing in the diversity of effects. Minocycline have been reported to suppress free radicals in the ischemic period and to have more multiple effects than ozagrel (Tomas-Camardiel et al., 2004). Ozagrel has been reported to suppress the decrease of cerebral blood flow but not to have an effect on free radicals (Pettigrew et al., 1989).
4.
Conclusions
In the present study, both minocycline and ozagrel showed smaller infarct volume, more neuronal survival, fewer astrocyte activation and apoptosis in addition to more improvement of behavioral disability than the control group when administered prior to ischemia. It suggests that minocycline and ozagrel attribute to neuroprotective effects in MCAO rat models. Additional to post-ischemic therapy, it will be useful to prevent cerebral ischemia in multiple cerebrovascular procedures.
housed in temperature–humidity and light–dark controlled rooms, and allowed food and tap water ad libitum. Animals were assigned randomly to one of the following three groups: (1) the first group of rats was treated with saline as the control group (n ¼10, Con group), (2) the second group was injected with minocycline (n¼ 10, Mino group), and (3) the third group was injected with sodium ozagrel (n¼ 10, SO group).
5.2.
Minocycline (Sigma, Inc. St. Louis, MO, USA) was prepared by dissolving in 0.9% saline as 4 mg/ml. Ozagrel (Kissei Pharmaceutical Co. Ltd., Matsumoto, Japan) was prepared by dissolving in 0.9% saline as 5 mg/ml. Minocycline (45 mg/kg) and ozagrel (5 mg/kg) were intravenously administrated to the tail vein at 30 min before performing the middle cerebral artery occlusion (MCAO) in the rat models.
5.3.
Experimental procedures
5.1.
Animals
All animal procedures were carried out in accordance with the animal experimental guideline established by the institutional Animal Care and Use Committee of Catholic University Medical School (CIMH 2012-005). Male Sprague-Dawley rats (270–300 g) aged 10–12 weeks old were used. The rats were
MCAO model
Transient MCAO was induced as described by Longa et al. (1989) with a minor modification. Under inhalation anesthesia with isoflurane, the right common carotid artery (CCA) and the external carotid artery (ECA) were exposed through a midline incision. The occipital artery and the superior thyroid artery were dissected and coagulated. The right internal carotid artery (ICA) was exposed and the pterygopalatine artery was ligated. Two loose ties (5–0 silk suture) were made around the ECA stump and tightened the distal tie. A small puncture opening was made in the ECA and 3–0 monofilament (intraluminal suture with a rounded tip) was inserted through the opening and the silk suture was tightened around the lumen containing the filament. The monofilament was gently advanced into the ICA for a distance of 19 to 20 mm beyond bifurcation until it blocked the bifurcating origin of the middle cerebral artery (MCA). The body temperature was maintained at 37 1C using heating pad during the procedure. The animal was allowed to regain consciousness and checked to determine whether the MCA was successfully occluded by placing the animal on a flat surface, lifting it by the base of the tail 2 in. off the surface and observing its behavior. When the MCA is successfully occluded, rats exhibit marked thorax twisting when suspended by its tail. Contralateral forepaw flexure should also be observed. Ninety minutes after the occlusion, animals were reanesthetized and reperfused by removing the suture and the ECA was ligated.
5.4.
5.
Drug administration
Behavior evaluation
Behavioral functions were evaluated with adhesive removal and treadmill test at 1, 3 and 7 days after MCAO. All animals were trained three times for a week before MCAO. A square dot of adhesive-backed paper (12Ø) was used in the adhesive removal test as bilateral tactile stimuli occupying the distal radial region on the wrist of each forelimb. Animals were given three trials with a cut-off time of 180 s. The data are presented as mean time to remove the left dot. The treadmill
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was accelerated from 20 to 80 m/min with a cut-off time of 300 s.
5.5.
Infarct volume
At 7 days after MCAO, all rats (n¼ 10, for each group) were sacrificed under deep anesthesia with 5% halothane. The brain was dissected out and five 2 mm thick coronal slices were prepared using a rodent brain matrix. The slices were stained a 2% solution of 2-3-5-triphenylterazolium chloride (TCC; Sigma, St. Louis, MO, USA) at 37 1C for 15 min. The infarct area was measured using image analyzing software (MetaMorph, Molecular Devices Inc., Downingtown, PA, USA), determined by using the method of subtracting the area of the non-infarct ipsilateral hemisphere from that of the contralateral tissue to reduce errors that might occur due to cerebral edema.
5.6.
Immunohistofluorescence staining
The tissue was coronally cut at a thickness of 12 mm with 0.1 mm of boundary area of cerebral infarct in the precentral gyrus, located 24 mm posterior to the frontal pole from each rat for 20 min and washed with 0.01 M phosphate-buffered solution (PBS) for 10 min. Sections were blocked in normal goat serum for 1 h at room temperature. The sections were incubated with mouse antibody to glial fibrillary acidic protein (GFAP, Millipore, Inc., Billerica, MA, USA, diluted at 1:200) and neuronal nuclei (NeuN, Millipore, Inc., Billerica, MA, USA, diluted at 1:100) overnight at 4 1C. Subsequently, sections were incubated for 1 h at room temperature with Alexa 488-conjugated goat antimouse IgG (Vector Laboratories, Southfield, MI, USA, diluted at 1:200). After washing, the sections were counterstained and stored at 20 1C and then observed under both a fluorescence microscope equipped with a spot digital camera and a confocal scanning laser microscope (LSM 510, Zeiss, Jena, Germany). Apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by means of the in situ cell death detection kit (Roche, Indianapolis, IN, USA) developed using the Cy2-conjugated streptavidin observed by confocal scanning laser microscope. The ischemia penumbra was determined by optical density of GFAP, NeuN and TUNEL positive cells using the MetaMorph imaging program (Molecular Device Inc., Downingtown, PA, USA).
5.7.
Statistical analysis
Data are expressed as mean7SD. Statistical analyses were performed using analysis of variance (ANOVA) with post-hoc analysis. A value of po0.05 was considered statistically significant.
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