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Restorative Neurology and Neuroscience 33 (2015) 131–142 DOI 10.3233/RNN-140440 IOS Press

An ischemic stroke model of nonhuman primates for remote lesion studies: A behavioral and neuroimaging investigation Xinran Chena , Ge Danga , Chao Danga , Gang Liua , Shihui Xinga , Yicong Chena , Qiang Xub and Jinsheng Zenga,∗ a Department

of Neurology and Stroke Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China b Guangdong Landau Biotechnology Co. Ltd, Guangzhou, Guangdong, China

Abstract. Purpose: We aimed to develop a nonhuman primate (NHP) stroke model for studies of secondary lesions in remote areas and to characterize its behavioral and neuroimaging features. Methods: Monkeys were either subjected to middle cerebral artery occlusion (MCAO) distal to the M1 branch (n = 17) or sham operation (n = 7). Neurological assessment and magnetic resonance imaging (MRI) were performed before and 1 week after operation. Results: After MCAO, six monkeys showed occlusion of the distal M1 segment and infarcts predominantly in the cortical and subcortical regions, without hippocampal and thalamic involvement. They had obvious neurological deficits. The other 11 monkeys showed blockage of the main trunk of the MCA, with infarcts extending into the hippocampus and thalamus, but no substantia nigra involvement. Their infarct volume were larger and neurological deficits were more severe than those after distal M1 occlusion. All sham-operated monkeys displayed normal behavior; however, MRI revealed small infarcts in three animals. Conclusions: MCAO or even sham operations might cause cerebral infarction in NHPs. Therefore, neurological assessment should be combined with MRI for screening candidate stroke models. Our model is suitable for studying secondary damage in remote regions, including the thalamus, hippocampus, and substantia nigra, after stroke. Keywords: Nonhuman primates, middle cerebral artery occlusion, remote lesions, model, magnetic resonance imaging

1. Introduction Cerebral infarction can lead to secondary neurodegeneration in structurally normal areas remote from, but synaptically connected to, the primary lesion site. Remote lesions of this type occur most ∗ Corresponding author: Jinsheng Zeng, Department of Neurology and Stroke Center, The First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, China. Tel.: +86 20 87755766; Fax: +86 20 87335935; E-mail: [email protected].

commonly in the ipsilateral thalamus, substantia nigra, and hippocampus several days or weeks after cerebral infarction in the middle cerebral artery (MCA) territory (Block et al., 2005; Zhang et al., 2012). Emerging evidence indicates that secondary damage in remote regions may hamper neurological recovery and can predict motor outcome after stroke (Devetten et al., 2010; Liang et al., 2007; Yu et al., 2009), providing a novel target for stroke management (Hiltunen and Jolkkonen, 2012; Zhang et al., 2012). The precise mechanisms underlying delayed neurodegeneration

0922-6028/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved

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secondary to cerebral infarction, however, remain unclear. In addition, treating remote lesions has clinical advantages, because, unlike primary insults, the therapeutic time window can extend up to several days/week after the initial insult. Thus, exploring the underlying mechanisms of remote lesions and evaluating therapies perturbing these targets would be of significant clinical feasibility and value. Animal stroke models are essential for elucidating stroke pathophysiology in humans (Qu et al., 2014; Sakakima et al., 2012). Our previous studies in rodents have demonstrated that a constant focal cortical infarction can be established, without primary ipsilateral thalamic lesions, by occluding the distal MCA in stroke-prone renovascular hypertensive rats. These animals are a representative model suitable for studying secondary cerebral infarction damage, and their underlying mechanisms in remote regions, including the thalamus and substantia nigra (Chen et al., 2014; Ling et al., 2009; Wang et al., 2012; Xing et al., 2012; Zhang et al., 2011, 2012). Similarly, several studies have assessed remote post-ischemic stroke lesions in stroke-prone spontaneously hypertensive rats and proposed some potential therapeutic targets (Onoue et al., 2005; Shima et al., 1996; Watanabe et al., 1997, 1998). However, the essential physiological conditions and anatomical structures of the rodent brain are dramatically different from those in humans. Therefore, rodent studies do not always reflect human situations, and the species-specific differences in stroke are considered as potential reasons for the failure of translation from rodent studies to clinical trials (Cook and Tymianski, 2011; Hoyte et al., 2004). To bridge this biological gap, the Stroke Therapy Academic Industry Roundtable (STAIR) committee suggested that nonhuman primates (NHPs) should be used for preclinical and translational stroke research (Fisher et al., 2009; Stroke therapy academic industry round table, 1999). NHP models offer distinct advantages regarding comparability because of their similarities to humans with regard to genetics, anatomy, pathophysiology, and behavior (Courtine et al., 2007; Enard et al., 2010). In addition, NHP models are highly suitable for evaluating the consequences of ischemic stroke with magnetic resonance imaging (MRI) techniques (D’Arceuil and de Crespigny, 2011). Over the last three decades, NHPs have been extensively used as experimental stroke models for different purposes. Two recent reviews have discussed their use in detail (Cook and Tymianski, 2012; Fukuda and Del, 2003); however, there is no

consensus about which NHP stroke model most closely models the human pathophysiologic condition. In addition, the use of different surgical approaches and vessel occlusion locations may be better suited to various research purposes or needs. Generally, the choice of an NHP stroke model depends on the study goal and should be in accordance with the STAIR recommendations (Fisher et al., 2009; Stroke therapy academic industry round table, 1999). To comply with the STAIR suggestion that it was reasonable to explore stroke recovery studies in primate models after sufficient evidence is gathered from rodent models, it is thus necessary to create a reliable NHP model suitable for studies designed to both elucidate the mechanisms of secondary damage in remote regions after cerebral infarction and to discover potential therapeutic strategies. However, a reliable and reproducible poststroke NHP model for the investigation of delayed degeneration in remote areas, including the thalamus, hippocampus, or substantia nigra, remains to be established. Therefore, the aim of the present study was to develop a cynomolgus macaque stroke model to study remote lesions after ischemic stroke and to characterize its behavioral and neuroimaging outcomes.

2. Material and methods 2.1. Animal preparation Twenty-four male cynomolgus monkeys (Guangdong Landau Biotechnology Co., Ltd, Guangzhou, China) ranging from 4 to 5 years old with body weights from 5.5 to 7.5 kg were used in the present study. Prior to operation, each monkey was randomly assigned to receive unilateral middle cerebral artery occlusion (MCAO) (n = 17) or sham procedures (n = 7). The animals were individually housed indoors at a constant temperature of 24–28◦ C and maintained on a 12-h light/12-h dark cycle (lights on at 08:00 A.M.). All animals received water ad libitum and were fed twice daily with monkey chow supplemented with fruits and vegetables. All experimental procedures were authorized by the Institutional Animal Ethical Committee of Sun Yat-Sen University, performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals, and were Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited.

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2.2. Surgical procedure

2.3. Post-operative recovery

The animals were fasted the evening prior to surgery. Anesthesia was induced with ketamine (10 mg/kg, intramuscular injection) and then maintained through intubation with isoflurane (1.5–3%) and vaporized in 100% oxygen. Heart rate, blood pressure, respiration, and rectal temperature were monitored, and intravenous fluids (0.9% saline with 5% glucose) were constantly administered throughout the surgery. The monkeys were fixed in a stereotaxic frame and placed on a warm heated surgical table with their heads turned to the right side. A left frontotemporal pterional craniotomy was performed under aseptic conditions. The dura mater was opened as a flap turned towards the greater sphenoidal wing, and the brain was gently retracted to expose the Sylvian fissure. After careful dissection of the arachnoid mater and trabecula of the related brain cisterns under an operative microscope, the left MCA was gradually exposed without disturbing the brain tissue. The left MCA was then occluded by bipolar electrocoagulation just distal to the M1 branch of the MCA (Fig. 1) and cut with microscissors. In order to avoid the induction of vasospasm or operative damage to the brain tissue as much as possible during the surgery, every step of the operation was carried out carefully and gently under a microscope, with suspension of the operation at regular intervals between each step. Monkeys that underwent all the procedures described above except MCAO served as the sham group. The operative field was washed with saline and covered with a flat piece of bone wax, and then the incision was sutured closed.

Within 1 week after operation, all monkeys were housed individually in single cages at constant room temperature, and their daily activity was observed. During the 3 days following operation, penicillin was administered by intramuscular injection twice a day to each animal at a dose of 0.4 million IU to prevent infection. Animals were observed continuously until they were able to self-care, eat, and drink. The wounds were examined for signs of infection. For monkeys that exhibited severe deficits in consciousness, enteral nutrition was provided via nasogastric gavage three times per day and 50 ml of 20% mannitol was administered intravenously twice a day; these monkeys also received intensive care, and were continuously monitored by 2 trained veterinarians. 2.4. MRI acquisition and analysis MRI scans were performed before and 1 week after operation on a Siemens’ 3.0-T Trio system (Siemens, Erlangen, Germany). Prior to the MRI scan, monkeys were anesthetized with ketamine (10 mg/kg, intramuscular injection). Imaging was performed in the axial plane and included the following sequences: T2weighted images (repetition time [TR] = 4130 ms, echo time [TE] = 100 ms, field of view [FOV] = 150 mm, flip angle = 150), fluid attenuation inversion recovery (FLAIR) images (TR = 9000 ms, TE = 117 ms, FOV = 150 mm, flip angle = 150), and magnetic resonance angiography (MRA) (TR = 21 ms, TE = 3.6 ms, FOV = 150 mm, flip angle = 18). The ischemic lesion

Fig. 1. Operative images before (A) and after (B) coagulation of the distal M1 segment of the middle cerebral artery (MCA). The arrow indicates the M1 segment of the MCA, and the arrowhead shows the orbitofrontal branch of the MCA.

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volume was measured on transverse slices of the T2-weighted image using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as described previously (West et al., 2009). For each slice, the regions where the signal intensity was 1.25 times higher than the counterpart in the contralateral hemisphere were defined as displaying hyperintensity (D’Arceuil et al., 2006; Sasaki et al., 2011). Infarct volume was expressed as a percentage of the ipsilateral hemisphere using the following formula: (volume of infarcted tissue of the ipsilateral hemisphere/total volume of the ipsilateral hemisphere) × 100%. 2.5. Neurological assessment Neurological assessments were performed before and 1 week after surgery by two observers who were blinded to the imaging data using a standardized neurological deficit score developed by Kito et al. (2001). Briefly, this scale consists of four categories, which are assigned to consciousness, the sensory system, the motor system, and skeletal muscle coordination; the points for each category are 28, 22, 32, and 18, respectively. Out of a total of 100 points, 0 corresponded to normal behavior, and 100 indicates severe bilateral neurological function impairment. 2.6. Statistical analysis All statistical analyses were performed in SPSS 13.0 for windows (SPSS Inc., Chicago, IL, USA). Results are expressed as the mean ± standard error of mean(SEM).Comparisonsofphysiologicalparameters between the MCAO and sham-operated groups were performed using Student’s t-tests. One-way analysis of variance followed by Bonferroni tests were conducted for multiple comparisons. Associations between neurological deficit scores and infarct volumes were analyzed by Spearman rank correlation analysis. Differences were considered statistically significant when P < 0.05. Power analyses were performed using the online toolkit provided by DSS Research (www.dssresearch.com) with an ␣ error of 5% and a ␤ error of 25% as described previously (West et al., 2009).

3. Results In all monkeys, blood pressure, heart rate, rectal temperature, and respiration were monitored before,

during, and after sham operation or MCAO under anesthesia. There were no significant differences in these physiological parameters between the sham-operated and MCAO groups at any time point (all P > 0.05, data not shown). All animals displayed normal behavior prior to surgery, corresponding to a neurological deficit score of 0. All animals manifested neurological impairment when awakened, except for the animals used as sham controls. The neurological symptoms of the animals became relatively stable 24 h after operation. The majority of the monkeys displayed constant deficits within 1 week after operation, whereas some exhibited improved behavior and consciousness at day 5. No intracerebral hemorrhages were detected on postoperative MR images. During the establishment of the stroke model, we observed two different outcomes in both the MCAO and sham-operated groups. 3.1. MCAO animals Monkeys subjected to the same MCAO surgery exhibited two different stroke manifestations. Six of the 17 animals had an occlusion of the distal M1 segment of the MCA, whereas occlusion of the main trunk of the MCA was confirmed on MRA 1 week after MCAO in the remaining 11 animals. All animals with occlusions of the distal M1 branch of the MCA were capable of drinking, eating, and self-grooming behaviors, without hand feeding and supplemental fluids within 24 h of recovery. Nevertheless, there was evidence of impaired activity due to the ischemic lesion. Representative MRI findings in those monkeys with a distal occlusion of the MCA M1 branch are shown in Fig. 2. The T2-weighted images demonstrated hyperintense signals in the territory of the left MCA 1 week after MCAO. These abnormal signals affected the majority of the subcortical white matter, temporal and parietal lobes, and insular cortex, but only a part of the basal ganglia was involved. Importantly, no apparent hyperintense signal was found in the hippocampus or thalamus in any of the T2-weighted images. The corresponding mean infarct volume was 24.7 ± 2.1% of the total ipsilateral hemisphere volume, corresponding to the sum of cortical including cortical and subcortical structures and basal ganglia infarct volumes (22.8 ± 1.9% and 1.9 ± 0.3%, respectively). The individual infarct volumes and neurological deficit scores are listed in Table 1. At 1 week after MCAO, the monkeys exhibited obvious neurological deficits, including drowsiness, spastic paralysis of the contralateral upper

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Fig. 2. MRA (A, C) and T2-weighted images (B, D) from a representative monkey before and 1 week after occlusion of the distal M1 segment of the middle cerebral artery (MCA). The arrowhead shows the occlusion of the MCA, and the dashed line indicates the partial involvement of the putamen. Table 1 Neurological deficit scores and infarct volumes in 6 monkeys with occlusion of the distal M1 of the MCA Mean ± SEM

Monkey Neurological Score Infarct Volume (%)

Before 1 week Before 1 week

1

2

3

4

5

6

0 41 0 26.1

0 45 0 30.4

0 43 0 24.5

0 37 0 19.8

0 33 0 17.7

0 45 0 29.5

0 41 ± 2 0 24.7 ± 2.1

Fig. 3. MRA (A, C) and T2-weighted images (B, D) from a representative monkey before and 1 week after occlusion of the main trunk of the middle cerebral artery (MCA). The arrowhead shows the occlusion of the MCA, and the arrow indicates the hyperintense area in the substantia nigra. The dashed lines show the partial involvement of the ipsilateral hippocampus and thalamus.

and lower limbs, disappearance of pain reflex, and musculoskeletal incoordination. Their average score was 41 ± 2. Moreover, the individual neurological deficit score correlated well with the infarct volume (r = 0.928, P = 0.008). A power calculation of these

monkeys revealed that an n of 6 would be required to determine statistical significance for a 25% reduction in infarct size. All monkeys with occlusions of the main trunk of the MCA survived. Seven of them exhibited severe

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3.2. Sham-operated animals

consciousness deficits, which required intensive care and continuous monitoring combined with enteral nutrition and mannitol therapy. After 1–3 days of treatment and care, all the monkeys self-recovered and were able to self-care, eat, and drink within 1 week, although functionality was obviously compromised due to neurological lesions. Representative MRI scans for monkeys with the main trunk of the MCA occluded are shown in Fig. 3. The results show that the infarct sizes were large, brain edema was common, and the infarcts were primarily located in the basal ganglia and subcortical regions. Cortical ischemia was variable, and the substantia nigra was not directly affected by ischemic areas. However, an area of high-signal intensity in the ipsilateral substantia nigra was observed on T2-weighed images 1 week after occlusion, which was limited to the ipsilateral substantia nigra and was not observed in adjacent structures. The hyperintensity was 2.36 times higher than counterparts in the contralateral brain area. Somewhat unexpectedly, we found that parts of the ipsilateral hippocampus and thalamus were involved to different extents in all of the monkeys; this was especially pronounced in the head of the hippocampus and the lateral portion of the thalamus. The extents of the cortical and basal ganglia infarct volumes were 22.4 ± 2.0% and 9.1 ± 0.4% of the ipsilateral hemisphere volume, respectively. Compared to monkeys with distal M1 branch occlusion, these monkeys showed a larger infarct volume and generally presented with more severe consciousness, motor system, sensory system, and skeletal muscle coordination impairment (all P < 0.05). These deficits corresponded to a mean infarct volume and average neurological deficit score of 31.5 ± 2.0% and 50 ± 2, respectively (Table 2). Notably, the neurological deficit score was significantly correlated with the infarct volume (r = 0.897, P = 0.000). Additionally, a power calculation revealed that 6 monkeys would be required to detect a 25% decrease in infarct size statistically significant.

All monkeys subjected to the sham operation awakened rapidly from anesthesia within 1 h, and manifested no neurological deficits. In the sham-operated group also, there were two different outcomes. T2weighted images obtained 1 week after operation revealed that three of the seven monkeys showed minimal but visible cortical infarction mainly involving the temporal and parietal lobes (Fig. 4, D and H). Nevertheless, the corresponding MRA findings demonstrated that the middle segment of the left MCA had disappeared in two monkeys (Fig. 4C), and the third had no postoperative change of the MCA (Fig. 4G). The other outcome was that no infarct was detected on any T2-weighted images and no evidence of left MCA occlusion was observed on MRA in the remaining monkeys (Fig. 5). Analyses of individual neurological deficit scores and infarct volumes in the seven monkeys of the sham-operated group are presented in Table 3. Results showed that the mean infarct volume was 2.1 ± 1.0% of the ipsilateral hemisphere. However, all animals appeared to display normal behavior 1 week after operation. 4. Discussion In accordance with the recommendations mentioned in the STAIR report, we developed an ischemic stroke model of cynomolgus monkeys suitable for studies investigating secondary damage in different remote brain regions, including the thalamus, hippocampus, and substantia nigra, after cerebral infarction. We also observed different outcomes in monkeys that underwent the same surgery in both the MCAO and sham-operated groups, and assessed the behavioral and neuroimaging consequences, during the establishment of the stroke model. Among NHP stroke models reported to date, our MCAO model exhibits several notable characteristics.

Table 2 Neurological deficit scores and infarct volumes in 11 monkeys with occlusion of the main trunk of the MCA Mean ± SEM

Monkey Neurological Score Infarct Volume (%)

Before 1 week Before 1 week

1

2

3

4

5

6

7

8

9

10

11

0 53 0 33.5

0 47 0 26.7

0 56 0 35.6

0 59 0 41.3

0 41 0 22.6

0 55 0 36.1

0 59 0 41.1

0 49 0 32.5

0 41 0 26.2

0 49 0 25.3

0 45 0 26.1

0 50 ± 2 0 31.5 ± 2.0

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Fig. 4. MRA (A, C, E, G) and T2-weighted images (B, D, F, H) from two representative monkeys before and 1 week after a sham operation that nevertheless showed cerebral infarction. A–D show a monkey with an infarction following middle cerebral artery occlusion (MCAO), and E–H depict a monkey with a small infarct but no MCA occlusion. The arrowhead shows an occlusion of the middle portion of the MCA, and the arrow indicates cortical infarction.

Fig. 5. MRA (A, C) and T2-weighted images (B, D) from a representative monkey without infarction before and 1 week after sham operation. A–D show that there was no infarction and no occlusion of the middle cerebral artery (MCA).

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Table 3 Neurological deficit scores and infarct volumes in 7 monkeys in the sham-operated group Mean ± SEM

Monkey Neurological Score Infarct Volume (%)

Before 1 week Before 1 week

1

2

3

4

5

6

7

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 6.7

0 0 0 3.9

0 0 0 4.1

For monkeys with an occlusion in the distal M1 branch, we observed consistently sized infarctions, primarily involving both the cortex and subcortical white matter in the MCA distribution. Remote regions, such as the thalamus and hippocampus, were not involved, which makes this a suitable model for studies investigating secondary damage to these areas after cerebral infarction. In monkeys with a blockage of the main trunk of the MCA, stable infarcts primarily located in the basal ganglia and subcortical regions were observed. The substantia nigra was not directly affected by ischemic lesions, making this a suitable model to study remote lesions in this region after ischemic stroke as well. The observation of consistently sized infarcts was restricted to monkeys with occlusions of the distal M1 branch and the main trunk of the MCA respectively, and did not apply to all the monkeys in the MCAO group. Moreover, the presence of stable infarcts would reduce the number of animals needed to test for the effectiveness of a given therapy (Huang et al., 2001). Both types of occlusion led to the development of clinical symptoms similar to those in humans with ischemic stroke, such as limb hemiplegia and hemiparesthesia. The consequences of our NHP stroke model could be assessed by both behavioral testing and MRI, which is in accordance with the updated STAIR recommendations (Fisher et al., 2009). In the present study, we found an area of highsignal intensity in the ipsilateral substantia nigra on T2-weighted images 1 week after MCAO in monkeys with the main trunk of the MCA occluded. The hyperintensity was limited to the ipsilateral substantia nigra and was not observed in the adjacent structures, indicating that the ipsilateral substantia nigra was not directly involved in, but was secondarily affected by, infarcts. Consistently, this phenomenon has been widely demonstrated to be a secondary degeneration in rodents, monkeys, and patients after ischemic stroke (Hirouchi et al., 2007; Nakane et al., 1992, 2001; Ohe et al., 2013). The potential mechanism is considered to be the result of disturbance of the

0 0 0 2.1 ± 1.0

nigrostriatal pathway caused by reduced inhibitory output of the neurotransmitter ␥-aminobutyric acid (GABA), which evoked the disinhibition mechanism leading to substantia nigra degeneration (Saji and Reis, 1987; Tamura et al., 1990). Consequently, there is no doubt that our “main trunk” model is suitable for studying remote degeneration in this region after cerebral infarction. In contrast, no high-signal intensity was observed in the thalamus and hippocampus of animals with distal occlusions of the M1 branch of the MCA on T2-weighted images 1 week after MCAO, which was consistent with previous studies that no T2 hyperintensity was found in the thalamus and hippocampus 1 week after stroke (Hirouchi et al., 2007; Justicia et al., 2008; Nakane et al., 2002; Xie et al., 2011). However, secondary degeneration of the ipsilateral thalamus and hippocampus could be detected on T2-weighted images after several weeks, which was confirmed by relevant histopathological studies. The underlying mechanism of the thalamic and hippocampal degeneration was considered to result from retrograde degeneration due to the destruction of the thalamocortical pathway (Iizuka et al., 1990; Kataoka et al., 1989). In addition, it is well known that the thalamus and hippocampus send synaptic projections to the ipsilateral frontoparietal cortex and entorhinal cortex, respectively (Block et al., 2005), while these projectile cortices were directly involved by infarcts, as assessed by T2-weighted images. Based on these observations, our “distal trunk” model was suitable for studying secondary thalamic and hippocampal damage after ischemic stroke. During the establishment of the stroke model, 11 monkeys had an occlusion of the main trunk of the MCA. This was an unexpected outcome because we selectively electrocoagulated the distal M1 segment of the MCA during surgery. We thought that our dissection of the arachnoid and pia mater to expose the MCA might result in vasospasm, as it was previously reported that microscopic surgery could cause cerebral vasospasm (Dodson et al., 1976). In addition,

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it is known that electrocoagulation can injure the MCA, which might provoke the vascular endothelial cells to release vasoactive substances. On the basis of this reaction, it could result in platelet aggregation and ultimately thrombosis (Dietrich et al., 1986; Wisniewski et al., 1995). Besides, perivascular edema after cerebral infarction could cause vascular compression, which is associated with thrombosis (Dietrich et al., 1986). On the other hand, our results from three infarcted monkeys in the sham-operated group showed that one monkey had a cortical infarction without a visible change in the MCA, which indicated that a vasospasm might have occurred during the operation and induced ischemia in distal areas (Fukuda and Del, 2003). Moreover, the other two monkeys had both cortical infarction and partial occlusion of the MCA, which suggested the potential of thrombosis. These results further confirmed the possibility of intraoperative vasospasm and thrombosis, which might partially account for occlusion of the proximal segment of the MCA and ischemia in the three monkeys that underwent sham operation. Nevertheless, viewed from another perspective, monkeys that underwent the same surgery ultimately had different outcomes in both the MCAO and sham-operated groups, which also indicated that there are variable vascular responses to ischemia and operation. Therefore, the application of MRI techniques to monitor the consequences of an NHP stroke model is very crucial. In 2007, Hirouchi et al. (2007) established a monkey model of delayed cerebral ischemia. This was the first demonstration of an NHP model that clarified delayed neurological disorder after MCAO. Consistent with their results, we also found an area of high-signal intensity in the ipsilateral substantia nigra on T2-weighted images 1 week after MCAO in monkeys with a blockage in the main trunk of the MCA. However, our results additionally revealed that parts of the ipsilateral hippocampus and thalamus were involved to different extents, which was not reported in their study. This could be due to anatomical reasons; the most lateral structures of the thalamus in macaque monkeys are consistently supplied by the ipsilateral internal carotid artery system (Coceani and Gloor, 1966). Occlusion of the main trunk of the MCA might therefore result in partial ipsilateral thalamic infarction. Additionally, a previous study revealed a fair amount of overlap of the carotid and posterior cerebral artery supplies within the thalamus (Coceani and Gloor, 1966), and brain swelling after cerebral infarction probably led

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to ischemia in a portion of the thalamus. With regard to the hippocampus, the infero-mesial portion of the macaque monkey temporal lobe has been reported to be entirely supplied with carotid blood (Kramer, 1912). Moreover, Coceani et al. (1966) demonstrated that the hippocampus is supplied with carotid blood to different degrees at various levels. Accordingly, these issues might account for the partial ipsilateral hippocampus infarction that occurred when the main trunk of the MCA was occluded. Besides, Nilges et al. (1944) demonstrated that the hippocampus is located on the watershed between the carotid and vertebrobasilar territories. Similarly, we could see that the proximal MCA was very close to the hippocampus on T2-weighted imaging before the occlusion. It was therefore not surprising that cerebral edema after MCAO would spread to the hippocampus and result in ischemia. Notably, West et al. (2009) produced a cortical NHP stroke model using a trans-orbital approach to occlude the MCA distal to the orbitofrontal branch and bilateral anterior cerebral arteries, which would be expected to result in secondary damage to remote regions after cerebral infarction. Conversely, we performed a microsurgical operation via pterional craniotomy and only occluded one vessel, which was technically less challenging and less stressful for the animals. The most significant limitation of the trans-orbital method is eyeball enucleation, which prevents behavioral testing requiring intact binocular vision and might affect studies elucidating changes in whole-brain structural and functional connectivity networks (Cook and Tymianski, 2012; Fukuda and Del, 2003). de Crespigny et al. (2005) induced focal cerebral ischemia in cynomolgus monkeys via an endovascular approach to achieve a more distal occlusion of the MCA M1 branch by wedging a microcatheter. Compared to the open vascular occlusion, the endovascular approach appeared to result in increased variability in stroke size and a higher mortality rate (D’Arceuil et al., 2006). However, the craniotomy approach also has some disadvantages; it leaves a significant residual dural defect, which might potentially lead to cerebrospinal fluid leakage. With protection from the temporal muscle, none of the animals developed this complication in our study. Another limitation is that it takes more time to produce the MCAO compared with other methods, which might increase the risk of complications and mortality. Despite these problems, procedures performed by skilled surgeons were generally well tolerated, and the animals could undergo a full battery of behavioral tests

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when they recovered. Moreover, longitudinal analyses of the neurological score and infarct volume were not carried out, which might impede a better understanding of the dynamic neuroimaging and behavioral features of our model. Finally, a correlative histopathological investigation was not performed in the present study, which is warranted in further studies to confirm the application of our model. In conclusion, our results suggest that MCAO or even a sham operation might result in cerebral infarction in NHPs. Thus, neurological assessment should be combined with MRI to select a qualified NHP stroke model. Our stroke model established in cynomolgus monkeys might be useful in elucidating the underlying mechanisms involved in the development of secondary damage in various remote areas, including the thalamus, hippocampus, and substantia nigra, after cerebral infarction. It could also be a valid model to test the efficacy of neuroprotective and regenerative therapies for ischemic stroke in preclinical settings.

Acknowledgments This study was supported by the National Basic Research Program of China (2011CB707804); grants from the Natural Science Foundation of China (39940012, 81000500, 81200903, 81200901 and 81371277); the Joint Funds of the Natural Science Foundation of China (U1032005); the Project of Science and Technology New Star of Pearl River (2012J2200089); and grants from the National Key Clinical Department, National Key Discipline, and Guangdong Key Laboratory for diagnosis and treatment of major neurological diseases.

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