Exp. Anim. 58(1), 19–29, 2009

Direct Experimental Occlusion of the Distal Middle Cerebral Artery Induces High Reproducibility of

Brain Ischemia in Mice

Mutsuki KURAOKA1), Takahisa FURUTA2), Takashi MATSUWAKI3), Tsutomu OMATSU1), Yoshiyuki ISHII1), Shigeru KYUWA1), and Yasuhiro YOSHIKAWA1)

1)Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, 2)Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, 4–6–1 Shirokanedai, Minato-ku, Tokyo 108-8639, and

3)Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Abstract: Several investigators have used murine models to investigate the pathophysiology of brain ischemia. The focal ischemic model is a closer approximation to human stroke which includes a necrotic core, penumbra, and undamaged tissue. Occlusion of a unilateral artery, especially the middle cerebral artery (MCA), is performed in this model, but collateral circulation often induces variation of ischemic lesions both qualitatively and quantitatively. It is likely that the more proximal the artery which is unilaterally occluded is, the more inconsistent the outcomes. The present study was designed to examine the reproducibility of infarct lesion by distal or proximal artery occlusion. Direct occlusion of the distal MCA was performed and compared with unilateral common carotid artery occlusion (CCAO) in C57BL/6 mice. Direct MCA occlusion (MCAO) consistently induced ischemic lesions in cortical areas. All model animals (n=14) survived 24 h after occlusion, and exhibited a maximum infarct volume (20.0 ± 5.0%). In contrast, permanent and transient unilateral CCAO models had mortality rates of 62.5 and 25.0%, and showed severe to absent lesions with the infarct volumes of 29.0 ± 20.8 and 33.2 ± 24.2%, respectively. In conclusion, distal MCAO produces high reproducibility of ischemic insults and survivability compared to unilateral CCAO. Thus, distal MCAO is a useful method for the focal ischemic model.Key words: common carotid artery occlusion, middle cerebral artery occlusion, mouse

(Received 14 April 2008 / Accepted 4 September 2008)Address corresponding: M. Kuraoka, Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Introduction

Stroke is one of the most serious human disorders in countries with aging populations. The financial and social impact of stroke-related disorders cannot be un-derstated. Animal models of cerebral infarction are

crucial for understanding the mechanisms of neuronal death or survival following ischemic brain injury and for the development of therapeutic interventions for victims of all types of stroke. Many different species, especially rodents, and protocols have been used to ob-tain further knowledge in this research area.

20 M. KURAOKA, ET AL.

To induce hemispheric ischemia, occlusion of cerebral arteries distally straight from common carotid arteries (CCAs) has been performed in rodents [26]. According to the location of arterial occlusion, global or focal isch-emic models are presented. Bilateral CCAs branch rostrally with internal carotid arteries (ICAs) and exter-nal carotid arteries (ECAs). And bilateral ICAs link basilar arteries and form the Circle of Willis at the cere-bral base [30]. The Circle of Willis has a connection to cerebral arteries, such as the anterior cerebral artery (ACA), middle cerebral artery (MCA) and posterior cerebral artery (PCA). These arteries provide each ce-rebral region with blood supply. Bilateral CCA occlusion (CCAO) has been performed to induce global cerebral ischemia by reducing the blood flow in the Circle of Willis, whereas unilateral MCA occlusion (MCAO) in-duces focal ischemia [26].

The focal ischemic model is a closer approximation to human stroke and produces heterogenous pathological lesions which include a necrotic core and salvageable penumbra, as well as normal, undamaged tissue in both the ipsilateral and contralateral hemispheres [26]. Uni-lateral MCAO induces focal lesions in the cortex and caudate putamen, which are limited to MCA territory [12]. Recently, the MCAO model has mainly been in-duced by direct or indirect approach methods.

Direct MCAO is performed with the microsurgical techniques of coagulation [10, 12, 33] or ligation [4, 6, 32] on the distal MCA. The potential for invasion might increase in brain tissue, because craniectomy is also necessary for this method. There are only a small num-ber of reports of the craniectomized model in mice, as the operation is considered technically difficult [23]. On the other hand, the indirect MCAO method occludes the origin of MCA intraluminally with a monofilament thread through the unilateral CCA [3, 5, 16, 27, 29, 31]. The intraluminal thread model has the benefit of no brain invasion, so that a large number of studies can be per-formed under these conditions. In a previous study of direct or indirect MCAO rat models, however, the in-traluminal thread method was proposed as approximat-ing an ICA occlusion (ICAO) model rather than a simple MCAO model, because indirect MCAO induces isch-emic insult outside MCA territory [12]. In C57BL/6 mice, the intraluminal thread model occluded not only

MCA but also PCA, and caused severe hemispheric isch-emia [16]. Thus, direct MCAO may be better than in-direct MCAO to get a reproducible focal ischemia within MCA territory in mice models.

On unilateral artery occlusion, the effect of collateral circulation is indicated as the leading cause of variation of ischemic lesions [16, 18, 19]. The cerebrovascular anatomical issues have an advantage in the mouse mod-el because of the availability of strain-specific informa-tion. Development of posterior communicating arteries (PcomAs) leads to the lack of uniform architecture in the Circle of Willis [13, 24] and interferes with ischemic insults in the ipsilateral hemisphere. The more proximal the artery which is unilaterally occluded is, the more inconsistent the outcomes.

The present study is designed to examine the induction of infarct lesion by distal and proximal artery occlusion. Direct occlusion of the distal MCA was performed in C57BL/6 mice and was characterized by ischemic in-sults, involving neuronal death, glial cells reaction, and blood-brain barrier (BBB) damage. We compared the lesions of direct MCAO with unilateral CCAO models involving occlusion of a proximal artery. We demon-strate the efficiency of unilateral artery occlusion through the ischemic outcomes of distal and proximal artery oc-clusion.

Materials and Methods

AnimalsSpecific pathogen-free male C57BL/6 mice were pur-

chased from Japan SLC, Inc. (Shizuoka, Japan). They were acclimated for 1–2 weeks in a group of 3–4 indi-viduals per plastic cage (30 × 20 × 13 cm) filled with bedding of flaky spruce material (White Flake; Charles River Laboratories Japan Inc., Kanagawa, Japan). The mice were used at 10–13 weeks of age at weights of 25–30 g. All animals were housed on a 12 h light / dark cycle at a temperature of 22–24°C and humidity of 40–50%. They were fed on commercial pellets, (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. The number of animals used in this study was 78. All animal care and experimentation were conduct-ed in accordance with the guidelines of The University of Tokyo.

21BRAIN ISCHEMIA BY DIRECT MCAO IN MICE

Surgical preparation and direct MCAOFocal cerebral ischemia was induced by direct MCAO,

involving the craniectomy technique [7] with our modi-fi cation. Established intraluminal thread methods [3, 5, 12, 16, 29, 31] occlude the ICA at the origin of the MCA (Fig. 1A), whereas the direct MCAO occludes a more distal portion of the MCA (Fig. 1B). In detail, anesthe-sia was induced by intraperitoneal administration of chloral hydrate (400 mg/kg body weight in saline). Animals remained in anesthesia for 1.5–2 h before awak-ing. Temperature probes were inserted into the rectum and a heating pad was used to maintain rectal tempera-tures at 36.5 to 37.5°C. The right MCA was exposed via the transtemporal approach. The skin between the lat-eral part of the orbit and the external auditory meatus was incised, and the upper part of the temporalis muscle was pushed aside after partial resection. A 1- to 2-mm burr hole was drilled using a leutor (Mini Gold; Nihon Seimitsu Kikai Kosaku Co., Ltd., Hyogo, Japan) with a cemented carbide cutter (external diameter, 0.5 mm) (Nihon Seimitsu Kikai Kosaku Co., Ltd.), through the

frontal bone 1 mm rostral to the fusion of the zygoma and squamosal bone, and about 3.5 mm ventral to the dorsal surface of the brain (Fig. 1B). The MCA was exposed after the dura was opened and retracted. MCA was occluded by short coagulation with a general metal-lic heat applicator at a proximal location rather than in a branch (Fig. 1B), followed by transection of the vessel to ensure permanent disruption. The hole in the skull was sealed with dental cement (REPAIRSIN; GC Corp., Tokyo, Japan). After suturing, mice were returned to their cages in a controlled environment of 27–28°C, where they were kept until sacrifi ce. Postsurgical sur-vival was checked at 6, 12, and 24 h and 2, 4, and 8 day. Each experimental group consisted of four animals, ex-cept for the 24-h group. The 24-h group had fourteen animals for the comparison with CCAO, because we empirically observed the most developed lesions at this time point. Sham-operated animals (n=5) underwent the same procedure without occlusion and survived for 24 h. The operation was fi nished within 30 min.

Fig. 1. Schematic illustrations of the cerebral artery. (A) The Circle of Willis is formed by ACA, MCA, and PCA, and supplies blood fl ow to the brain. The portion of the ICA to the origin of the MCA (surrounded by a circle) is the target of occlusion in intraluminal thread models. (B) Presentation of the direct MCA occlu-sion (MCAO) method. A 1- to 2-mm burr hole is drilled in the frontal bone; MCA is exposed and coagu-lated. The burr hole is represented by a circle (an arrow). This circle is located 1 mm rostral to the fusion (an arrowhead) of the zygoma and squamosal bone on the zygoma arch, and about 3.5 mm ventral to the dorsal surface of brain. Abbreviations: anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA), internal carotid artery (ICA), posterior communicating artery (PcomA), superior cerebellar artery (SCA), and basilar artery (BA).

22 M. KURAOKA, ET AL.

Surgical preparation and CCAOUnilateral CCAO was performed permanently or tran-

siently to compare with direct MCAO. Anesthesia and maintenance of body temperature were performed as per the MCAO method. Through a small incision in the neck, the right common carotid artery was isolated from the vagal nerve and connective tissue by blunt dissection. Permanent CCAO (n=16) was performed by tightly knot-ting a 4–0 silk suture. Transient CCAO (n=8) was per-formed using an aneurysm clip applied unilaterally for 60 min, after which the clip was removed and blood flow through the vessel was confirmed. After occlusion, the neck incision was sutured. Postsurgical survival was checked at 24 h to compare with the peak lesions in the direct MCAO, then brain samples were obtained. Sham-operated mice (n=4) underwent the same procedure without CCAO and survived for 24 h.

Brain infarct lesion analysisAfter surgery, mice were sacrificed. Their brains were

then removed and sectioned coronally at 2-mm intervals. For vital staining [2], samples were incubated for 30 min in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; MP Biomedicals, OH, USA) at room temperature in the dark, fixed by immersion in 4% paraformaldehyde solution, and photographed. Image analysis was per-formed with the public domain Scion Image program (the U.S. National Institutes of Health; http://www.sci-oncorp.com/). Areas of infarction were plotted on trac-ings from projections of coronal sections, and infarct volume (InV), right hemisphere volume (RhV), and left hemisphere volume (LhV) were determined [25]. Infarct volume in each mouse was corrected for hemispheric edema using the following formula: corrected infarct volume (CIV, %) = [LhV – (RhV – InV)]/LhV × 100.

Histological and immunohistological assessmentAn additional two animals were perfusion fixed at 24

h, 4 and 8 day after direct MCAO and sham-operation, and postfixed with 4% paraformaldehyde in PBS (pH 7.4). Paraffin-embedded brains were sectioned at 10 mm. Hematoxylin and eosin (HE) staining was performed to identify morphologically normal and ischemic neurons as usual.

For immunohistological staining, the tissue specimens

were autoclaved (10 min at 121°C) as a pretreatment. After blocking with Block Ace (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) for 60 min at room temperature, the specimens were labeled with polyclonal primary antibody to glial fibrillary acidic protein for astrocytes (GFAP: dilution 1:1000; Dako A/S, Glostrup, Denmark) and Iba-1 for microglias/macrophages (dilu-tion 1:1000; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Managed specimens were incubated at 4°C overnight. The sections were then incubated with biotinylated goat anti-rabbit IgG (Dako A/S), which was followed by incubation with streptoavidin-biotin-horse-radish peroxidase complex (sABC kit; Dako A/S). For visualization, 3,3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Co., MO, USA) was used. Then, all sections were counterstained with hematoxylin.

In the analysis of BBB damage, we examined the ex-travasation of IgG in infarct lesions. Anti-mouse IgG immunohistological staining was performed [22]. With-out a primary antibody, we used purified biotinylated goat anti-mouse IgG antibody (Vector Labs Inc., CA, USA) in place of a secondary antibody in the protocol.

Statistical analysisAll parameters are presented as mean ± SD. Student’s

t-test was used for CIV in the comparison of the ischemic group vs the sham-operated group at 24 h after MCAO or CCAO. CIV values of each post-MCAO time group were analyzed by ANOVA followed by the post-hoc Tukey test. All statistical analyses used P<0.05 as the level of significance.

Results

Brain infarct lesions after direct MCAOAll mice treated with direct MCAO survived to each

time point of 6, 12, and 24 h and 2, 4, and 8 day. We checked neurological scores in all animals after each surgery, but notable neurological deficits (circling, sei-zure, and coma) were not observed. TTC staining was performed to measure infarct volumes of each post-MCAO time group. Figure 2 shows infarct lesions at 24 h after direct MCAO. Brain samples were sectioned at bregma levels +0.62, –1.58, and –2.54 mm correspond-ing to the mouse brain atlas [8]. The infarcted cortical

23BRAIN ISCHEMIA BY DIRECT MCAO IN MICE

area in the ipsilateral hemisphere was not stained with TTC. Sham-operated brain had no unstained areas, sug-gesting no visualized insult by craniectomy. To examine the potential hazards of the cauterization of the underly-ing brain tissue, we heated directly a part of cerebral cortex. At 24 h post-operation (the time of maximum lesions following direct MCAO), brain samples (n=3) were stained with TTC. Corresponding to the heated areas, the unstained areas had expanded to only 1 mm along the edges (data not shown). Therefore, the lesions following direct MCAO were demonstrated as being the result of ischemic insult, not heat insult.

To determine the temporal changes of lesions post-MCAO, the corrected infarct volume (CIV) was calcu-lated for each time point (Fig. 3). At 6 h, an unstained area was observed (8.35 ± 1.93%), which gradually in-creased with time. CIV was maximal at 24 h (20.0 ± 5.0%) but had decreased at 2 day. However, substantial CIV was still noted until 8 day after MCAO (11.1 ± 2.6%). Statistical signifi cance was observed between post-MCAO time groups in multiple comparisons. Histopathological and immunohistological features

Histopathological examinations were performed to observe the ischemic features. The brains 24 h after

Fig. 2. Triphenyltetrazolium chloride (TTC) staining of brain samples 24 h after direct MCAO (top) and sham operation (bottom) are shown. Brains were sectioned at the bregma level +0.62 (left), –1.58 (middle), and –2.54 mm (right). Dotted lines surround unstained parts indicating infarct areas. Note the infarct lesions in the cortex of the right hemisphere.

Fig. 3. Changes in corrected infarct volume (CIV) and post-MCAO time. Infarct lesions were visualized by TTC staining after direct MCAO. Each CIV value was calculated from sec-tions at 2-mm intervals. CIV gradually increased in size and peaked at 24 h. Each experimental group had four animals, except for the 24-h group (n=14). The post-hoc Tukey test was applied to the CIV value of each post-MCAO time group. Values with different letters (a, b, and c) are signifi cantly different (P<0.05) from each other.

24 M. KURAOKA, ET AL.

direct MCAO revealed zones of cortical infarct lesion in the ipsilateral hemisphere (Fig. 4A). Infarct lesion was characterized by vacuolation and pancellular necro-sis with dense eosinophilic areas, and shrunken neurons located along the edges of the infarct lesion (Fig. 4B). At 4 and 8 day, neuronal cells had decreased in the isch-emic core zone and penumbra, whereas glial cells were frequently observed (data not shown).

In the immunohistological assessment, GFAP-positive astrocytes were diffusely located in unimpaired regions at 24 h (Fig. 5A). At 4 day, GFAP-positive astrocytes had increased in the areas surrounding infarct lesions, and showed hypertrophy and hyperplasia as features of “reactive astrocytosis” (Fig. 5B). GFAP-positive pro-cesses were more developed at 8 day (Fig. 5C). On the other hand, Iba-1-positive cells, which are characterized as microglias and macrophages, infiltrated mildly into the ischemic core zone and penumbra at 24 h (Fig. 5D). At 4 day, Iba-1-positive cells had increased greatly in the penumbral region (Fig. 5E). Thereafter, the infarct lesion was completely inundated with considerable num-bers of Iba-1-positive cells (Fig. 5F). Sham-operated mice showed no features of activated glial cells (data not shown).

Anti-IgG immunohistological staining was performed for IgG extravasation following direct MCAO. At 24 h, an anti-IgG immunoreaction was not emphatically ob-served (Fig. 5G). At 4 day, however, IgG extravasation was found, moderately corresponding to infarct lesion (Fig. 5H), and a strong anti-IgG immunoreaction was shown at 8 day (Fig. 5I).

Comparative analysis of direct MCAO and unilateral CCAO

Permanent and transient CCAO was performed uni-laterally to compare with direct MCAO. We checked survival and infarct lesions at 24 h after each operation, because the maximum areas of lesions following direct MCAO were observed at this time point. All mice (n=14) subjected to MCAO survived, while more than half of animals (10/16) and 2 of 8 animals died in the permanent and transient CCAO groups, respectively. The mortal-ity rate was calculated by dividing the number of animals that died within 24 h after operation by the total number of animals in each group (Table 1).

The infarct occurrence rate was calculated by dividing the number of animals with ischemic insults by the num-ber of surviving animals (Table 1). MCAO reproducibly induced infarct lesion (100%), while permanent and transient CCAO groups showed lower rates, 50.0 and 66.7%, respectively.

Brain samples of the permanent CCAO model are shown at bregma levels –2.18 mm (Fig. 6). There was variation of cerebral infarcted areas, namely including intense lesions extending to the cortex, hippocampus, and nucleus ventralis thalami (Fig. 6A), mild lesions limited to the cortex (Fig. 6B), and absence of lesions (Fig. 6C). Based on sections at 2-mm intervals, CIV was calculated using only samples with infarct lesion (Table 1). The CIVs of MCAO, permanent and transient CCAO were 20.0 ± 5.0, 29.0 ± 28.8, and 33.2 ± 24.2%, respec-tively. Although both CCAO methods induced rela-tively large CIVs in comparison to MCAO, their SD was extremely high. Direct MCAO induced comparable infarct lesions, while large variation was observed in each CCAO group. CIV values of direct MCAO and transient CCAO were significantly higher than those of each sham-operated group (Table 1).

Discussion

The present study demonstrates the induction of in-farct lesion by distal and proximal artery occlusion. Direct occlusion of the distal MCA was performed in C57BL/6 mice and was characterized by ischemic in-sults. In comparative analysis with unilateral CCAO, the distal MCAO consistently induced focal ischemic lesions in the ipsilateral hemisphere, whereas the unilat-eral CCAO models showed large variations of infarction. We convincingly demonstrated the efficiency of the di-rect method of the distal MCAO for the focal cerebral ischemic model.

The direct MCAO models induced infarct lesions, which extended to cortical areas. From the results of TTC staining, the CIV value reached a peak at 24 h and gradually decreased thereafter. Histological ischemic features were observed as vacuolation and shrunken neurons in the ischemic core and penumbral region at 24 h. In the immunohistological assessment, at 4 and 8 day glial cell reactions were mainly observed. It is con-

25BRAIN ISCHEMIA BY DIRECT MCAO IN MICE

Fig. 4. Histopathological features of the brain 24 h after direct MCAO. (A and B) HE staining. Vacuola-tion and shrunken neurons were observed in cortical infarct lesions. The dotted line in (A) surrounds an infarct lesion. Arrows in (B) indicate shrunken neurons, which have eosinophilic cytoplasm. Scale bars: (A)=250 mm and (B)=25 mm.

Fig. 5. Immunohistological staining of cortical infarct lesions after direct MCAO. Glial cell reactions and IgG extravasation were examined by immunohistological staining with anti-GFAP (A–C), Iba-1 (D–F), and IgG (G–I) at 24 h (A, D, and G), 4 (B, E, and H), and 8 day (C, F, and I). Note that at 4 and 8 day hypertrophy and hyperplasia of GFAP-positive astrocytes were observed around infarct lesions, and that Iba-1-positive cells (microglias/macrophages) increase in the penumbral regions and ischemic core zones at 4 and 8 day, respectively. An anti-IgG immunoreaction was observed corresponding to infarct lesions at 4 and 8 day. The dotted lines in (G–I) surround infarct lesions. Scale bars: (A–F)=50 mm and (G–I)=100 mm.

26 M. KURAOKA, ET AL.

sidered that “reactive astrocytosis” for glial limitans and increase of Iba-1-positive cells (microglias/macrophag-es) for clearance of cellular debris gradually occurred. Accordingly these time points were on the transition from the acute to the chronic phase. Anti-IgG immuno-histological staining revealed that BBB damage was continually induced for as long as 8 day following direct MCAO, although ischemic lesions were most developed with edematous expansion at 24 h. Little is known about why BBB permeability changes maximally after isch-emic edema has peaked. Nonselective BBB permeabil-ity would be related to passive leakage in necrotic vessels

as previously indicated [10]. Similar to our results, Lambertsen et al. showed that infarct volume was max-imal in SJL mice at 24 h in the direct-permanent MCAO model [17]. On the other hand, the thread MCAO mod-el has a different CIV peak. The thread-permanent MCAO induces peak CIV at 18 h in C57BL/6 mice, and at 24 h in rats [31]. The difference in CIV peak time would be explained by differences of angioarchitectural anastomoses or speed of autolysis among rodent species [15, 26, 34]. Additionally, our study and previous reports suggest that the CIV peak might change with the type (direct or indirect) of MCAO model.

Fig. 6. TTC staining of brain samples 24 h after permanent unilateral common ca-rotid artery occlusion (CCAO). (A–C) Brain sections from the unilateral CCAO group and (D) sham-operation. Infarct lesions exhibit marked variation in the unilateral CCAO group. Sections are shown at bregma level –2.18 mm. Dot-ted lines surround unstained areas.

Table 1. Mortality rate, infarct occurrence rate, and CIV at 24 h after initiation of different types of unilateral artery occlusion

Occlusion type Mortality rate (%) Infarct occurrence rate (%) CIV (%)

MCAO 0 (0/14) 100 (14/14) 20.0 ± 5.0**

Permanent CCAO 62.5 (10/16) 50.0 (3/6) 29.0 ± 28.8Transient CCAO 25.0 (2/8) 66.7 (4/6) 33.2 ± 24.2#

Infarct occurence rate was calculated by dividing the number of animals with infarct detected by TTC staining by the number of surviving animals. CIV was analyzed from only brain samples with infarct lesion. Values are the mean ± SD. **P<0.01, #P<0.05, compared to MCAO sham-operated group and CCAO sham-operated group, respectively (Student’s t-test). CIV: corrected infarct volume. MCAO: middle cerebral artery occlusion. CCAO: common carotid artery occlusion.

27BRAIN ISCHEMIA BY DIRECT MCAO IN MICE

The direct MCAO method induced ischemic lesions only in the cortical areas, corresponding to the distal MCA territory. On the other hand, it is likely that the intraluminal thread insertion induces more severe hemi-spheric ischemia, because of occlusion at the origin of MCA. Xi et al. reported that the direct ligation of MCA induced less expanded infarct volumes than those of intraluminal thread models [32]. Recently, the intralu-minal thread model was proposed to be an ICAO model rather than a simple MCAO model [12]. Intraluminal occlusion in rats produced damage in the cortex and caudate putamen within MCA territory and also in the hippocampus and thalamus outside MCA territory [12]. In mice, the intraluminal thread occlusion leads to an occlusion duration-dependent [21] and filament thick-ness-dependent [24] increase in extension of the isch-emic lesion area outside MCA territory. Anomalies in the Circle of Willis are possibly a major cause for these ischemic lesions, and developed PcomAs lead to a lack of uniformity in the insulted regions [16]. Collateral circulation in cerebrovascular anatomy would relate to variability of ischemic insults [13, 19].

In the present study, we analyzed occlusion of the distal and proximal artery. The unilateral CCAO meth-od, which we performed for proximal artery occlusion, is used for Mongolian gerbils [11, 14, 20]. Mongolian gerbils lack PcomA necessary to complete the Circle of Willis, so this species has frequently been used to pro-duce ischemic models [26]. Similarly, C57BL/6 mice are most susceptible to global ischemia corresponding to poor development of PcomA [1, 13, 16, 34]. In our results, permanent and transient unilateral CCAO mod-els had mortality rates of 62.5 and 25.0%, respectively. Unilateral CCAO induced severe ischemic insults in this strain. Meanwhile, mice surviving at 24 h post-CCAO showed severe to absent lesions. The permanent and transient CCAO models showed variability of CIV, 29.0 ± 20.8 and 33.2 ± 24.2%, respectively. We consider that the ischemic outcomes of unilateral CCAO cannot be constantly induced in the ipsilateral hemisphere. Kita-gawa et al. reported that unilateral CCAO in C57BL/6 mice resulted in reduction of cortical microperfusion to 57.6 ± 7.9% of the baseline in the ipsilateral cerebral cortex, whereas bilateral CCAO reduced it to 4 to 7% [16]. It is likely that the blood supply from contralat-

eral CCA is partially redistributed to the ipsilateral ce-rebral circulation through the Circle of Willis, even in C57BL/6 mice. The possibilities of individual differ-ences in PcomA or leptomeningeal anastomoses on the cortical surface remain [1, 16, 18, 21]. It is necessary for more research into correlation of blood flow and angioarchitectural anastomoses in unilateral CCAO mouse models in order to define the influences exerted by collateral circulation.

Direct occlusion of the distal MCA consistently in-duced ischemic lesions. All model animals (n=14) sur-vived 24 h after MCAO and exhibited high reproduc-ibility of infarct lesions (CIV=20.0 ± 5.0%). Ischemic insults of distal MCAO are limited to small areas com-pared to other types of proximal artery occlusion. In addition, occlusion of the distal location would have had a lesser influence on the collateral circulation, including PcomA [1, 16, 18] and the pterygopalatine artery [28, 29]. This model could possibly be applied to the simple production of focal ischemia.

In the present study, we demonstrated the characters of the murine focal ischemic model by direct MCAO. With occlusion at the distal arterial portion, direct MCAO had high survivability and reproducibility of infarct le-sion within MCA territory. This method is more than adequate for the observation of ischemic insults, involv-ing neuronal cell death, glial cell reactions, and BBB damage. Moreover, direct MCAO can be performed without regard to variations in arterial augmentation or diameter that interfere with induction of consistent le-sions in the intraluminal thread MCAO model. We consider that the direct MCAO method is appropriate for the analysis of cerebral infarction in senescent mice, which have enlarged arterial augmentation [9]. In con-clusion, direct MCAO is a very useful method for the induction of focal ischemic lesion in mice.

Acknowledgment(s)

The authors thank Masaki Kurokawa and Makoto Hideshima for their skilled technical assistance and animal care.

28 M. KURAOKA, ET AL.

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