Tacrolimus,a Potential Neuroprotective Agent,Ameliorates Ischemic Brain Damage and Neurologic Deficits after Focal Cerebral Ischemia in Nonhuman Primates

Abstract

Tacrolimus (FK506), an immunosuppressive drug, is known to have potent neuroprotective activity and attenuate cerebral infarction in experimental models of stroke. Here we assess the neuroprotective efficacy of tacrolimus in a nonhuman primate model of stroke, photochemically induced thrombotic occlusion of the middle cerebral artery (MCA) in cynomolgus monkeys. In the first experiment, tacrolimus (0.01, 0.032, or 0.1 mg/kg) was intravenously administered immediately after MCA occlusion, and neurologic deficits and cerebral infarction volumes were assessed 24 hours after the ischemic insult. Tacrolimus dose-dependently reduced neurologic deficits and infarction volume in the cerebral cortex, with statistically significant amelioration of neurologic deficits at 0.032 and 0.1 mg/kg and significant reduction of infarction at 0.1 mg/kg. In the second experiment, the long-term efficacy of tacrolimus on neurologic deficits and cerebral infarction was assessed. Vehicle-treated monkeys exhibited persistent and severe deficits in motor and sensory function for up to 28 days. A single intravenous bolus injection of tacrolimus (0.1 or 0.2 mg/kg) produced long-lasting amelioration of neurologic deficits and significant reduction of infarction volume. In conclusion, we have provided compelling evidence that a single dose of tacrolimus not only reduces brain infarction but also ameliorates long-term neurologic deficits in a nonhuman primate model of stroke, strengthening the view that tacrolimus might be beneficial in treating stroke patients.

Keywords

Immunosuppressant FK506 Focal cerebral ischemia Nonhuman primate Neurologic deficits Long-term efficacy

Whereas a number of neuroprotective agents have reached phase 3 clinical trials for stroke, no promising results have been achieved (Clark et al., 2001; Diener et al., 2000; Lyden et al., 2002). The reasons for failure of neuroprotectants in clinical trials have been the subject of intense discussion (Gorelick, 2000; STAIR, 1999; STAIR-II, 2001). One reason for failure is the difficulty in translating the preclinical data obtained from rodents into a clinical setting. Whereas stroke models in rodents are easy to conduct and ischemic outcomes are generally uniform, allowing for robust detection of neuroprotective efficacy, human strokes exhibit considerable heterogeneity in both time-course and area of infarction. Furthermore, the obvious behavioral, anatomic, and circulatory differences between rodents and humans undermine the predictive value of these models.

Considering the failure of many clinical trials mentioned above, it is highly desirable to evaluate the neuroprotective efficacies of candidate compounds in larger animal models, which presumably mimic human stroke better and predict the clinical benefit, as recommended by STAIR-I (STAIR, 1999). Nonhuman primate stroke models offer distinct advantages to address the limitations of traditional rodent models. Nonhuman primates show striking similarity to humans in their microvascular collateral circulation and brain architecture, such as the ratio of white matter to gray matter (Gillilan, 1968). Occlusion of middle cerebral artery (MCA) in nonhuman primates produces tissue damage in the sylvian cortex, putamen, and subcortical white matter in the affected MCA territory, as commonly seen in human stroke patients (Darby et al., 1999; Watanabe et al., 1977). The damage in the white matter could be of particular importance because the ratio of the white matter to gray matter in monkeys is much closer to the ratio in humans than that in rodents. It is also desirable to establish a thrombotic MCA occlusion model in which the MCA is spontaneously recanalized. As many stroke patients show reperfusion of the MCA spontaneously or by thrombolytic therapeutic intervention (Ringelstein et al., 1992), these models would mirror human strokes more closely and allow better prediction of clinical outcomes. Furthermore, nonhuman primates exhibit more integrated behavioral changes following strokes, providing clinically relevant outcome measures generally lacking in rodent models, where assessment of infarct volume remains a common endpoint despite the unclear relationship between histopathologic changes and clinical outcomes. Thus, numerous neuroprotectants have produced robust reductions in infarct volume, only to generate disappointing outcomes in clinical trials. Assessing both behavioral and histopathologic changes in nonhuman primate would provide a vital platform for extrapolating to efficacy in humans. Such a platform also allows for the assessment of long-term behavioral changes, which more closely matches the later endpoints used in clinical trials.

Tacrolimus (FK506, Prograf) is an immunosuppressant used clinically to prevent allograft rejection and is known to inhibit calcineurin-mediated T-cell activation by forming a complex with FKBP12 (Liu et al., 1991). The drug has been well-characterized to have neuroprotective action on ischemic brain damage in rodents (Bochelen et al., 1999; Furuichi et al., 2003; Maeda et al., 2002; Sharkey and Butcher, 1994; Yagita et al., 1996). Because the neuroprotective effect of tacrolimus was first demonstrated to be mediated by inhibition of calcineurin activity (Sharkey and Butcher, 1994), the mechanism for antiischemic effects of tacrolimus have been studied, and antiinflammatory actions (Tsujikawa et al., 1998; Wakita et al., 1998) and antiapoptotic actions (Herr et al., 1999; Wang et al., 1999) have been proposed. The ischemic brain damage, including inflammatory reactions (Jean et al., 1998) and apoptotic cell death (Tsukada et al., 2001), similarly have been observed in primate stroke models. The aim of the present study was to evaluate the long-term neuroprotective efficacy of tacrolimus in a robust nonhuman primate stroke model, that is, MCA occlusion in cynomolgus monkeys. Using this model, we assessed neurologic deficits and infarction volumes following a single bolus dose of tacrolimus, both acutely (24 hours after the ischemic insult) and over a longer time course (28 days).

MATERIALS AND METHODS

Animals

Experiments were carried out using 3- to 5-year-old male cynomolgus monkeys weighing 3.1 to 5.5 kg purchased from Shin Nippon Biomedical Laboratories (Kagoshima, Japan). Animals were housed individually in cages 60 cm wide by 70 cm high by 70 cm deep (in accordance with the NIH requirement), at 23 ± 2°C, 55 ± 5% humidity, under 12-hour light/dark cycle (light on at 7:00 a.m.) for at least 1 week before experimental procedures. In the short-term study, 24 monkeys were used. There was no demise during the experimental period. In the long-term study, two monkeys died beyond 1 week after ischemia; one death was caused by severe brain edema, and the other monkey died possibly of infection. Thus, 24 monkeys were used for the long-term study, and 22 of them survived to 28 days. These studies were approved by the Ethics Committee for Laboratory Animal Experiments at Fujisawa Pharmaceutical Co., Ltd. The experimental protocol for the present study is schematically represented in Fig. 1.

FIG. 1.

Experimental protocols. A short-term study (A) and long-term study (B) were performed. Tacrolimus was administered intravenously immediately after MCA occlusion.

Surgical procedure

Surgery was carried out according to the method reported by Kaku et al. (1998). Cynomolgus monkeys were lightly anesthetized by intramuscular injection of ketamine hydrochloride (10 mg/kg), tracheostomized, and artificially ventilated immediately after intramuscular injection of atropine (0.05 mg/kg) and pancuronium bromide (0.05 mg/kg). Anesthesia was maintained with 0.6 to 1% isoflurane in a N₂O:O₂ gas mixture (N₂O:O₂ = 7:3), and immobilization was accomplished with 0.05 mg/kg of pancuronium bromide given intramuscularly every 2 hours for up to 6 hours after ischemia. The right femoral vein was cannulated with a polyethylene tube (PE50) for administration of rose bengal or test drug. The right femoral artery was also cannulated for measurement of blood pressure and heart rate, as well as blood sampling for blood gas analysis and determination of drug concentration. The right MCA was occluded by a transorbital approach. Under an operating microscope, the temporalis muscle was sectioned, and subtemporal craniotomy was performed without removing the zygomatic arch. The orbital content was dissected and excised. A window approximately 10 mm in diameter was opened just anterior to the foramen of the mandibular nerve at the skull base. The main trunk of the right MCA was visible through the window underneath the dura mater. After opening the dura mater, an optic fiber for green light irradiation was placed on the proximal site of the main MCA trunk, and the probe for a pulsed Doppler flowmeter (Model PDV-20, HHP-20, Crystal Biotech America, Northborough, MA, U.S.A.) for blood flow measurement was placed next to the optic fiber. Photoillumination with green light (wavelength, 540 nm; intensity, 4,000,000 lux) was achieved by using a xenon lamp (L-4887, Hamamatsu Photonics, Hamamatsu, Japan) with a heat-absorbing filter and a green filter. Irradiation was directed by the optic fiber mounted on the MCA trunk, and rose bengal (20 mg/kg) was infused intravenously for 6 minutes. Photoillumination was carried out for 20 minutes. After wound closure, buprenorphine hydrochloride (4 μg/kg) and cefazolin sodium hydrate (25 mg/kg) were intramuscularly administered to avoid pain and infection. Thereafter, anesthesia was stopped. After confirming good spontaneous breathing, each animal was returned to the home cage. Although most animals could feed themselves within 24 hours after surgery, some animals that could not sit upright and feed themselves were orally treated with eutrophic liquid, and their body temperature was maintained under a heating lamp. In the long-term study, analgesic and antibiotic were given daily for the first week. Rectal temperature was maintained at 37.5 ± 0.5 °C with a heating pad (TR-100, PS-100, Fine Science Tools, Canada) during surgery and regional cerebral blood flow (rCBF) measurement afterward.

Measurement of cerebral blood flow

Complete MCA photothrombotic occlusion was confirmed by the decrease of blood flow monitored for 6 hours after MCA occlusion by a pulsed Doppler flowmeter placed on the proximal MCA trunk. The time from rose-bengal injection to complete occlusion of the MCA and the time to first recanalization were measured. In the short-term study, rCBF at the surface of the cerebral cortex was also monitored for 6 hours after MCA occlusion. To perform continuous monitoring of cortical rCBF, the scalp and temporalis muscle were cut, and a small window was made 5 mm anterior and 35 mm lateral to the bregma. CBF was monitored by a laser-Doppler flowmeter (Omegaflow, FLO-N1, Neuroscience, Osaka, Japan), with the probe placed over the dura mater at the site. Change in rCBF is expressed as percentage of the baseline values.

Measurement of physiologic parameters

Arterial blood pressure, heart rate (AP-601G, AT-601G, Nihon Kohden, Tokyo, Japan), po₂, pco₂, pH, hematocrit, hemoglobin, and blood glucose (pH/blood gas analyzer i-STAT,i-STAT Corporation, Princeton, NJ, U.S.A.) were measured through a femoral arterial line at baseline and at 90, 180, 270, and 360 minutes after thrombotic occlusion for each group in the first acute experiment.

Assessment of neurologic deficits

Animals were subjected to a neurologic examination at 24 hours after MCA occlusion for the short-term study (Fig. 1A) and at day 1, day 2, and every 7 days for 28 days after MCA occlusion for the long-term study (Fig. 1B). Neurologic assessment was carried out in a blind fashion. Drugs were prepared and administered by the investigator who carried out surgery, and the scoring of neurologic deficits was carried out by another investigator who was a specialist for large animal care and was blinded to the treatment. The scoring method reported by Kito et al. (2001) was adopted as described in Table 1, where neurologic deficits were divided into the following categories: consciousness, motor system, muscle coordination, and sensory system. During the long-term study, animals were examined daily for obvious signs of pain or discomfort. Food intake and general health were also noted.

TABLE 1.

Neurologic scores for the monkey stroke model

Category	Score
Consciousness
Normal, consistently alert	0
Conscious and aggressive	4
Conscious and evasive	6
Conscious but clouded and tolerant	8
Drowsiness, aroused with stimulation	10
Lethargia, eyes open by intense stimulation	16
Stupor, aroused with persistent stimulation	20
Light coma, reflex movement only	24
Deep coma, no movement	28
Motor system	(ipsi-/contra)
Upper limb (strength and movement)
Normal	0/0
Reduced strength and skilled movement	2/2
Paralyzed and useless	4/4
Lower limb (strength and movement)
Normal	0/0
Raise with flexion of knee against gravity	2/2
Movement possible, but not against gravity	4/4
Paralyzed and useless	6/6
Upper limb tone
Normal	0/0
Overtly spastic or flaccid	3/3
Lower limb tone
Normal	0/0
Overtly spastic or flaccid	3/3
Musculoskeletal coordination
Normal, walks normally	0
Minimal incoordination, walks with some gait impairment	4
Incoordinated, but able to climb a wire net	6
Stands independently, falls within a few steps	10
Sit, only able to circle	12
Pose with lateral or dorsal recumbency	16
No movement	18
Sensory system	(ipsi-/contra)
Facial sensation
React consistently to touch in any area of face	0/0
Absent, does not react to touch in any area of face	3/3
Pinna reflex
Twitch ear in response to outer/inner hair stimulation	0/0
Absent, does not move ear in response to touch	3/3
Pain reflex
Strong, quick, complete withdrawal from toe pinch	0/0
Weak, slow, incomplete or inconsistent withdrawal from toe pinch	3/3
Absent, no withdrawal from toe pinch	5/5

“ipsi-” and “contra” indicate ipsilateral and contralateral to MCA occlusion.

Measurement of the volume of ischemic brain damage

For the short-term study, each monkey was deeply anesthetized with pentobarbital (50 mg/kg, i.p.) after neurologic examination, perfused transcardially with saline containing heparin (50 U/mL), and sacrificed. The brain was removed, and the cerebrum was coronally sectioned in 4-mm thickness using a brain slicer for cynomolgus money (Neuroscience, Osaka, Japan). The fourth coronal section was then sliced in 2-mm thickness to use the rostral slice for measurement of the drug concentration and the caudal slice for histologic assessment. The coronal sections for histologic assessment were immersed in neutrally buffered 10% formalin/PBS for at least 2 days. Each section was embedded in paraffin and cut into 3- to 4-μm thickness to make 21 consecutive slices and stained with hematoxylin-eosin. For the long-term study, the consecutive coronal slices were prepared in the same manner as for the short-term study and stained with hematoxylin-eosin. Using photographs of 22 consecutive slices, the area of ischemic brain damage and the whole area of the cerebrum were calculated using a computerized image analysis system. The area of infarction was calculated by subtracting the area of intact tissue in the ipsilateral brain region from the whole area of the contralateral brain region. The brain damage in each animal was expressed as the percentage of the sum of the area of damaged brain in comparison with the sum of the whole area of the cerebrum, and the volume of ischemic brain damage was calculated using the trapezoid method (Rosen, 1990).

Measurement of brain and blood concentration of tacrolimus

In the short-term study, arterial blood samples were collected from the cannulated femoral artery at 5, 15, 30, 90, 180, and 360 minutes and 24 hours after tacrolimus administration. Brain samples were taken at 24 hours after drug administration. Cortical and basal ganglia punches were made for measurement of drug concentration. Blood and brain samples were stored at −20°C until drug concentration was measured by enzyme immunoassay.

Drug

The injection formulation of tacrolimus prepared at Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan) was diluted with physiologic saline and administered intravenously at the doses of 0.01, 0.032, 0.1, and 0.2 mg/kg immediately after MCA occlusion. The vehicle ampoule prepared at Fujisawa Pharmaceutical Co., Ltd. was similarly diluted with physiologic saline and administered to monkeys for the vehicle-treated group. The injection volume was adjusted to 1 mL/kg throughout all experiments.

Statistical analyses

All values are mean ± SD. Statistical significance was evaluated using analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparison test for physiologic parameters, rCBF, and ischemic brain damage. Neurologic scores were analyzed with nonparametric Kruskal-Wallis test and post hoc Dunnett's multiple comparison test. P values less than 0.05 were considered significant.

RESULTS

Short-term efficacy of tacrolimus

In the first set of experiments, neurologic deficits and volumes of cerebral infarction were determined 24 hours after the ischemic insult to assess the dose-response effect for neuroprotective actions of tacrolimus. Physiologic variables, cerebral blood flow, and the concentration of tacrolimus in the blood and brain were also measured.

Cerebral blood flow

The MCA was occluded by thrombus formation, leading to a gradual decrease of cortical CBF. In most animals, cyclical reperfusion and reocclusion of the MCA were observed. The time from rose-bengal injection to complete occlusion of the MCA was 12.2 minutes for the vehicle-treated group and 13.8, 12.1, and 13.6 minutes for the groups treated with 0.01, 0.032, and 0.1 mg/kg of tacrolimus, respectively (Fig. 2A). The average time to first recanalization was 89.0 minutes for the vehicle-treated group and 66.8, 121.7, and 66.1 minutes for the groups treated with 0.01, 0.032, and 0.1 mg/kg of tacrolimus, respectively (Fig. 2B). There were no statistically significant differences in the time to occlusion and first recanalization among groups. Cortical CBF gradually decreased in most animals following MCA occlusion, although cortical CBF showed various patterns in individual animals. The average reduction of CBF at 360 minutes after MCA occlusion was 53.2% for the vehicle-treated group and 30.6%, 38.5%, and 34.8% for the 0.01, 0.032, and 0.1 mg/kg tacrolimus groups, respectively (Fig. 2C). There were no significant differences among groups.

FIG. 2.

Effects of tacrolimus on the time to complete occlusion of the middle cerebral artery (MCA) (A), time to first recanalization (B), and change in cerebral blood flow (C) induced by photochemical MCA occlusion in monkeys. Values are mean ± SD. Tacrolimus was administered intravenously immediately after MCA occlusion.

Physiologic parameters

Table 2 shows the physiologic parameters. In the vehicle-treated group, mean blood pressure (MBP) and heart rate (HR) gradually decreased and blood glucose increased after MCA occlusion. The value of pco₂ at 90 minutes after MCA occlusion significantly increased but was within physiologically normal range. There were no significant differences between vehicle- and tacrolimus-treated groups except for MBP, where the tacrolimus-treated groups showed significantly higher MBP at some time points compared with the vehicle-treated control group. However, these effects showed neither time dependency nor dose dependency. Other parameters, including HR, po₂, pco₂, pH, hematocrit, hemoglobin, and blood glucose, were not influenced by the administration of tacrolimus.

TABLE 2.

Effects of tacrolimus on physiological parameters after thrombotic MCA occlusion in monkeys

			Tacrolimus
	Time after MCA occlusion	Vehicle	0.01 mg/kg	0.032 mg/kg	0.1 mg/kg
MBP (mmHg)	Baseline	80 ± 12	84 ± 7	90 ± 22	86 ± 10
	90 min	74 ± 7	88 ± 22	95 ± 20	89 ± 10
	180 min	71 ± 7	91 ± 17^∗	83 ± 17	80 ± 7
	270 min	67 ± 2^##	80 ± 17	86 ± 20	77 ± 7
	360 min	66 ± 5^##	67 ± 7	75 ± 10	82 ± 10^∗∗
HR (beat/min)	Baseline	151 ± 17	142 ± 24	157 ± 22	154 ± 20
	90 min	141 ± 17^#	136 ± 29	148 ± 24	137 ± 22
	180 min	133 ± 17^##	137 ± 32	149 ± 24	133 ± 17
	270 min	129 ± 12^##	132 ± 24	149 ± 15	133 ± 15
	360 min	127 ± 12^##	131 ± 27	147 ± 22	134 ± 17
PO₂ (mmHg)	Baseline	173 ± 10	162 ± 24	175 ± 10	159 ± 2
	90 min	178 ± 15	167 ± 24	170 ± 10	174 ± 17
	180 min	172 ± 15	180 ± 12	173 ± 5	165 ± 15
	270 min	175 ± 10	164 ± 22	163 ± 17	158 ± 10
	360 min	175 ± 10	172 ± 10	174 ± 10	169 ± 12
PCO₂ (mmHg)	Baseline	34.8 ± 2.0	36.3 ± 2.4	37.3 ± 2.0	35.2 ± 1.7
	90 min	38.6 ± 3.4^#	40.3 ± 2.4	39.3 ± 5.1	38.6 ± 2.7
	180 min	37.0 ± 3.7	37.0 ± 2.0	37.3 ± 4.9	34.2 ± 1.7
	270 min	32.1 ± 2.2	31.7 ± 2.7	33.4 ± 3.7	33.5 ± 2.2
	360 min	35.4 ± 3.4	33.0 ± 2.9	35.9 ± 1.5	33.9 ± 2.4
PH	Baseline	7.479 ± 0.037	7.464 ± 0.027	7.453 ± 0.044	7.444 ± 0.034
	90 min	7.471 ± 0.039	7.472 ± 0.047	7.464 ± 0.061	7.462 ± 0.042
	180 min	7.464 ± 0.034	7.506 ± 0.042	7.481 ± 0.064	7.502 ± 0.027
	270 min	7.480 ± 0.032	7.484 ± 0.047	7.459 ± 0.017	7.471 ± 0.024
	360 min	7.469 ± 0.024	7.495 ± 0.059	7.471 ± 0.047	7.472 ± 0.032
Hematocrit (%)	Baseline	40 ± 2	37 ± 2	38 ± 7	37 ± 2
	90 min	41 ± 2	43 ± 0	42 ± 5	41 ± 2
	180 min	41 ± 2	44 ± 2	42 ± 5	42 ± 2
	270 min	40 ± 5	39 ± 2	41 ± 5	40 ± 2
	360 min	41 ± 2	42 ± 2	42 ± 5	41 ± 5
Hemoglobin (g/dL)	Baseline	14 ± 2	13 ± 0	13 ± 2	13 ± 0
	90 min	14 ± 0	15 ± 0	14 ± 2	14 ± 0
	180 min	14 ± 0	15 ± 0	14 ± 2	15 ± 0
	270 min	14 ± 2	13 ± 0	14 ± 2	14 ± 0
	360 min	14 ± 0	14 ± 0	14 ± 2	14 ± 2
Blood glucose (mg/dL)	Baseline	67 ± 15	56 ± 7	60 ± 5	65 ± 10
	90 min	75 ± 15	73 ± 5	85 ± 29	85 ± 20
	180 min	84 ± 12^##	83 ± 7	95 ± 29	100 ± 29
	270 min	90 ± 17^##	83 ± 10	96 ± 32	98 ± 20
	360 min	99 ± 15^##	98 ± 22	113 ± 27	107 ± 22

Tacrolimus was intravenously administered immediately after MCA occlusion. Values are mean ± SD obtained from 6 monkeys per group.

∗

P < 0.05

∗∗

P < 0.01, statistically significant compared to the vehicle-treated group

P < 0.05

P < 0.01, statistically significant compared to baseline in the vehicle-treated group (by one-way ANOVA followed by Dunnett's multiple comparison test). MBP, mean blood pressure; HR, heart rate.

Blood and brain concentration of tacrolimus

The blood concentrations of tacrolimus at a dose of 0.1 mg/kg declined with a half-life of 12.19 ± 1.45 hours. The brain concentrations of tacrolimus at 24 hours after administration in the ischemic and nonischemic cortices were similar. In the basal ganglia, the concentrations of tacrolimus were also comparable between ischemic and nonischemic sides (Table 3).

TABLE 3.

Blood and brain concentrations of tacrolimus in monkeys with middle cerebral artery occlusion

			Tacrolimus
		Time after administration	0.01 mg/kg	0.032 mg/kg	0.1 mg/kg
Blood concentration (ng/mL)		5 min	34.8 ± 3.8	120.3 ± 34.7	150.5 ± 18.1
		15 min	23.3 ± 4.6	72.2 ± 25.4	111.9 ± 21.8
		30 min	17.1 ± 1.8	44.2 ± 8.7	98.4 ± 27.4
		90 min	8.7 ± 1.0	20.9 ± 6.0	45.8 ± 10.5
		180 min	5.7 ± 0.5	13.2 ± 3.5	32.5 ± 9.2
		360 min	3.1 ± 0.8	9.0 ± 2.5	18.8 ± 4.1
		24 hour	0.2 ± 0.5	3.0 ± 1.5	8.4 ± 2.6
Brain concentration (ng/g tissue)	Ischemic cortex	24 hour	0.63 ± 0.09	1.74 ± 0.72	5.09 ± 1.57
	Non-ischemic cortex	24 hour	0.60 ± 0.13	1.84 ± 0.68	4.38 ± 1.38
	Ischemic basal ganglia	24 hour	0.26 ± 0.29	1.45 ± 0.91	2.63 ± 1.62
	Non-ischemic basal ganglia	24 hour	0.36 ± 0.29	1.33 ± 0.40	3.13 ± 1.04

Tacrolimus was intravenously administered immediately after MCA occlusion. Values are mean ± SD obtained from 6 monkeys per group.

Neurologic score

Vehicle-treated animals displayed severe neurologic deficits at 24 hours after MCA occlusion (Fig. 3). The common neurologic deficits were drowsiness, disappearance of pain reflex, paralysis of the upper and lower limbs, decrease of muscle tone, and incoordination. Although most animals showed neurologic signs only on the contralesional side, neurologic deficits in the sensory and motor system appeared on the ipsilesional side as well in some monkeys who showed severe ischemic damage. There were no deaths within 24 hours after MCA occlusion. The neurologic total score for the vehicle-treated group was 59 ± 19.6 points. Tacrolimus dose-dependently ameliorated the neurologic deficits in all subcategories of neurologic parameters. The effects of tacrolimus on the total neurologic score at 0.032 and 0.1 mg/kg were statistically significant.

FIG. 3.

Effects of tacrolimus on neurologic deficits induced by middle cerebral artery (MCA) occlusion in monkeys. Tacrolimus was administered intravenously immediately after MCA occlusion. Neurologic deficits were scored at 24 hours after occlusion. Values are mean ± SD. ∗ P < 0.05; statistically significant compared with vehicle-treated group (by Kruskal-Wallis followed by post hoc Dunnett's multiple comparison test).

Size of ischemic brain damage

Twenty-four hours after MCA occlusion, infarction was detected primarily in the cerebral cortex, including the precentral gyrus, superior temporal gyrus, insular cortex, and medial temporal gyrus. Basal ganglia and white matter, including the internal capsule, were also damaged extensively (Fig. 4A). Tacrolimus reduced the cortical infarction around the Sylvian fissure posteriorly (Fig. 4B and Fig. 4C). The infarct volume in the total area, cortex, basal ganglia, and white matter were 6256.7 ± 2186.5, 3618.1 ± 1788.5, 1494.8 ± 238.2, and 1143.9 ± 451.1 mm³, respectively. Tacrolimus reduced the size of cortical infarction by 58%, 43%, and 85% at doses of 0.01, 0.032, and 0.1 mg/kg, respectively, and a significant effect was obtained at 0.1 mg/kg. Although the neuroprotective action of tacrolimus was primarily observed in the cortex, tacrolimus also tended to reduce infarction in the basal ganglia by 10%, 15%, and 26% at doses of 0.01, 0.032, and 0.1 mg/kg, respectively (Fig. 5).

FIG. 4.

Neuroprotective effects of tacrolimus on brain damage induced by middle cerebral artery occlusion in monkeys. Tacrolimus was administered intravenously immediately after occlusion. Representative coronal sections of vehicle-treated (A) and tacrolimus-treated (B) animals as shown after staining with H&E. (C) Infarct areas in each coronal sections. Values are mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01; statistically significant compared with the vehicle-treated group (by one-way ANOVA followed by Dunnett's multiple comparison test).

FIG. 5.

Neuroprotective effects of tacrolimus on brain damage induced by middle cerebral artery (MCA) occlusion in monkeys 24 hours after ischemic insult. Tacrolimus was administered intravenously immediately after MCA occlusion. Values are mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01; statistically significant compared with the vehicle-treated group (one-way ANOVA followed by Dunnett's multiple comparison test).

Long-term efficacy of tacrolimus

Based upon the results of the short-term experiment, the study was extended to determination of the long-term efficacy of tacrolimus, and neurologic deficits were monitored for 28 days after focal ischemia in subsequent experiment. Brain damage was also measured at 28 days after MCA occlusion. Tacrolimus at 0.1 mg/kg, the dosage which was the most effective in the first study, was used in the second study, but 0.2 mg/kg was also added to verify the long-term neuroprotective action of tacrolimus.

Cerebral blood flow

Whereas the cortical rCBF was not monitored in the long-term study to minimize the insult to animals, blood flow at the MCA trunk was continuously monitored for 6 hours after MCA occlusion. As in the short-term study, tacrolimus (0.1 and 0.2 mg/kg) hardly altered the patency of MCA. The time to complete occlusion of the MCA was 9.9 minutes for the vehicle-treated group and 12.5 and 11.6 minutes for the groups treated with 0.1 and 0.2 mg/kg of tacrolimus, respectively, and the average time to first recanalization was 133.4 minutes for the vehicle-treated group and 122.2 and 125.8 minutes for the groups treated with 0.1 and 0.2 mg/kg of tacrolimus, respectively.

Physiologic parameters

As in the first study, occlusion of the MCA in vehicle-treated animals slightly decreased MBP and HR but increased blood glucose level, but it did not alter any of other physiologic parameters (data not shown). Tacrolimus at 0.2 mg/kg slightly but significantly elevated blood glucose compared with the vehicle-treated group at 270 minutes after MCA occlusion (data not shown). Otherwise, none of parameters, including MBP, HR, po₂, pco₂, pH, hematocrit, and hemoglobin, were significantly influenced by the administration of tacrolimus.

Neurologic score

Severe neurologic deficits were observed in vehicle-treated animals at 24 hours after MCA occlusion, which worsened at 48 hours after MCA occlusion (Fig. 6). The disturbance of consciousness and sensory dysfunction tended to show slight recovery 7 to 28 days after MCA occlusion. In contrast, motor system dysfunction did not show spontaneous recovery. Tacrolimus at 0.1 and 0.2 mg/kg administered as a single bolus injection immediately after MCA occlusion dose-dependently ameliorated the neurologic deficits in every category. Amelioration of neurologic deficits by tacrolimus lasted for 28 days, and improvement for muscle coordination was more pronounced.

FIG. 6.

Long-term effects of tacrolimus on neurologic deficits induced by middle cerebral artery occlusion in monkeys. Tacrolimus was administered intravenously immediately after occlusion. Neurologic deficits were scored at 1 day, 2 days, and then every 7 days for 28 days after occlusion. Values are mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01; statistically significant compared with the vehicle-treated group (by Kruskal-Wallis test followed by post hoc Dunnett's multiple comparison test).

Size of ischemic brain damage

Atrophy was observed in the cerebral cortex, basal ganglia, and white matter at 28 days after occlusion of MCA. The infarct volume was 5352.4 ± 1257.7, 2398.7 ± 714.9, 1341.7 ± 287.4, and 1612.0 ± 512.9 mm³ in the whole cerebrum, cortex, basal ganglia, and white matter, respectively. Tacrolimus at 0.1 and 0.2 mg/kg significantly reduced the infarct volume by 50% and 67%, respectively. In addition to the reduction of the cortical infarct, tacrolimus at 0.2 mg/kg significantly reduced infarction in the basal ganglia and white matter at 28 days after ischemia (Fig. 7).

FIG. 7.

Neuroprotective effects of tacrolimus on brain damage at 28 days after middle cerebral artery (MCA) occlusion in monkeys. Tacrolimus was administered intravenously immediately after MCA occlusion. Infarct volumes in different brain areas were measured at 28 days after MCA occlusion. Values are mean ± SD. ∗∗ P < 0.01; statistically significant compared with the vehicle-treated group (by one-way ANOVA followed by Dunnett's multiple comparison test).

DISCUSSION

Previous studies have shown that tacrolimus has potent neuroprotective effects in a variety of rodent models of stroke, focal ischemia in rats (Bochelen et al., 1999; Butcher et al., 1997; Furuichi et al., 2003; Sharkey and Butcher, 1994), thrombotic focal ischemia in rats (Maeda et al., 2002; Takamatsu et al., 1998), and transient global ischemia in gerbils (Furuichi et al., 2003; Yagita et al., 1996). However, the predictive value of these models is limited given the numerous neuroprotectants that have failed in clinical trials. Therefore, evaluating neuroprotective efficacy in larger animal models should permit a more robust prediction of clinical benefit. Although a recent report showed attenuation of early ischemic neuronal death by tacrolimus at 8 hours after MCA occlusion by MCA clipping in monkeys (Takamatsu et al., 2001), the study did not address dose-dependency of the effect on infarction and neurologic deficits at more extended time points. In the current study, we show that a single administration of tacrolimus dose-dependently and permanently reduced both ischemic brain damage and neurologic deficits in a nonhuman primate stroke model.

In nonhuman primates, the microvascular collateral circulation and the ratio of white matter to gray matter resemble those in humans. Thus primate stroke models should have better predictive value for clinical benefits in stroke patients. In cynomolgus monkeys, collateral circulation is sufficient to prevent formation of reproducible infarction after clipping of the proximal part of the MCA trunk (data not shown). However, we have successfully established a reproducible thrombotic occlusion model, where vascular endothelial injury is induced by a photochemical reaction between rose bengal and green light (Kawai et al., 1995), putatively causing blockage of collateral vessels by thrombus formation. The cyclical reperfusion and reocclusion of the MCA were observed in the majority of animals as previously observed with squirrel monkeys (Kaku et al., 1998). In control animals, the MCA was occluded approximately 12 minutes after rose bengal injection, but the MCA was recanalized at approximately 89 minutes after the occlusion. The cortical CBF measured by laser-Doppler flowmetry did not reduce rapidly after MCA occlusion but gradually decreased in all animals, and the reduction of cortical blood flow at 360 minutes after MCA occlusion was only 53.2%. In spite of the small reduction of cortical blood flow, the inner layer of the cerebral cortex was infarcted extensively in most animals. Therefore, we suspect that either microthrombi that are generated locally at the peripheral sites of the cortex or emboli dislodged from the MCA following repeated recanalization of MCA associated with cyclical flow reduction participated in the formation of stable infarction in the cortical area in the present model. Because we monitored blood flow of a limited cortical surface by the laser-Doppler probe, accurate cortical circulation could not be determined. Further studies using positron emission tomography or other imaging procedures will be required to measure cortical circulation accurately in this stroke model and to better understand the mechanism of the progression of cerebral infarction.

Tacrolimus administered immediately after MCA occlusion reduced ischemic brain damage without affecting the patency of MCA or cortical blood flow. This result is consistent with a lack of the effect of tacrolimus on rCBF or recanalization by t-PA in a rodent thrombotic MCA occlusion model (Maeda et al., 2002). Therefore, tacrolimus works through a neuroprotective mechanism rather than through a direct effect on cerebral circulation or hemodynamics. In fact, the neuroprotective effect of tacrolimus has been suggested to involve the antiapoptotic action (Wang et al., 1999), antiinflammatory effect (Tsujikawa et al., 1998), and the attenuation of free-radical production (Siesjo et al., 1999) in previous in vivo studies. In rodent MCA occlusion models, we have observed that tacrolimus attenuates both apoptotic and necrotic components of neuronal cell death and also inhibits the activation of inflammatory cells driven by activated microglia and infiltrated leukocytes (Furuichi et al., unpublished observation, 2003). There are reports suggesting that apoptotic neuronal death and reperfusion-driven inflammatory reactions are involved in ischemic brain injury in primates (Haring et al., 1996; Tsukada et al., 2001). It would be reasonable to assume that similar mechanisms are involved in the neuroprotective action of tacrolimus on cerebral infarction in the present monkey model, although further studies are required to elucidate the mode of neuroprotective actions of the drug.

Tacrolimus showed a similar pharmacokinetic profile in ischemic and normal monkeys, as well as similar concentrations in ischemic and normal brain tissue, suggesting that tacrolimus readily penetrates the ischemic brain area, in accordance with a previous study in rat focal ischemia (Butcher et al., 1997). Furthermore, the concentrations of tacrolimus in the blood and brain after administration of an effective dose at 0.1 mg/kg were comparable with those in rats given with effective doses of tacrolimus in focal ischemia models (Butcher et al., 1997; Furuichi et al., unpublished observations, 2002). Of interest, however, is the fact that the neuroprotective efficacy of tacrolimus in the present model ranks highly compared with other animal models (Furuichi et al., 2003; Maeda et al., 2002), with an 85% reduction of cortical infarction size at 24 hours and over a 50% reduction after 28 days. In addition to reducing infarct size, tacrolimus remarkably improved the motor system deficits and muscle coordination (Fig. 6 and Fig. 7), perhaps by ameliorating ischemic damage in the basal ganglia and white matter, in addition to the reduction of cortical infarction.

Another important finding was that the efficacy of tacrolimus in ameliorating neurologic deficits was long-lasting. Although the infarct volume has been used as an endpoint in animal models, endpoints for clinical trials are not infarct volumes but instead are neurologic and functional outcomes at 28 days or 90 days after stroke onset (STAIR-II, 2001). Thus, sophisticated behavioral assessments over an extended time period would allow better extrapolation to efficacy in humans. Furthermore, although tacrolimus has been shown to ameliorate skilled motor deficits for up to 2 weeks in a rodent stroke model (Sharkey and Butcher, 1994), functional deficits after ischemia in rodents could be different from those in humans. For example, impairment of consciousness, paralysis, or sensory deficits may develop and persist in stroke patients. Behavioral assessments relevant to these conditions are difficult in rodents. In our model, however, almost all animals in the vehicle-treated control group showed drowsiness, disturbance of muscle coordination and tone, paralysis of the upper and lower limbs, and disappearance of pain reflex. With the exception of consciousness and sensory function, which showed slight improvement, these neurologic deficits did not exhibit spontaneous recovery in vehicle-treated control animals. Thus our model more closely reflects the pattern and time course of deficits seen in stroke patients, and, importantly, provides sophisticated and extended behavioral assessments that confirm the neuroprotective potential of tacrolimus.

One reason for failure of many neuroprotectants in clinical trials is difficulty in translating the preclinical data obtained from animal studies into a clinical setting. In the case of tirilazad, it was proven to be effective in permanent focal ischemia in rodents but ineffective in transient focal ischemia in cats (Park and Hall, 1994; Takeshima et al., 1994). In a nonhuman primate stroke model, tirilazad was demonstrated to prevent edema 6 hours after focal ischemia (Boisvert and Hall, 1996) and reduced infarction 2 weeks after focal ischemia (Mori et al., 1995). Thus tirilazad was shown to reduce the edema formation in an appropriate ischemic condition and consequently reduce the infarction. In the case of clomethiazole, neuroprotective efficacy was demonstrated in transient global, permanent focal, and transient focal ischemia in rodents (Cross et al., 1995; Sydserff et al., 1995). Furthermore, it was shown to protect against hemineglect and to improve functional outcome in focal ischemia in the marmoset, a new world primate (Marshall et al., 1999, 2000). However, permanent MCA occlusion by elecrocoagulation in marmosets produced large infarction in the cortex presumably because of the poor collateral circulation, in contrast to that in cynomolgus monkeys and humans. Subsequent clinical trials demonstrated that both of these drugs did not work in stroke patients (Lyden et al., 2002; RANTTAS Investigators, 1996). These facts suggest that preclinical assessment should be performed in the setting close to the clinical situation as much as possible and the results should be carefully interpreted. On the other hand, it would also be important to make an appropriate clinical design based upon the information of the preclinical study. Attention should be drawn to construct a good preclinical/clinical design interfacing for the success of clinical trial.

In conclusion, we demonstrated that ischemic brain damage and long-lasting functional disability were dramatically ameliorated by a single administration of tacrolimus in a nonhuman primate model of stroke. The present results encourage a clinical trial with this drug in patients with acute stroke.

Footnotes

Acknowledgment

The authors thank Mr. Yuji Awaga for his care of animals and behavioral observations, Ms. Eriko Fujita for performing the drug concentration measure-ments, and Dr. Raymond D. Price for his critical review of the manuscript.

References

Bochelen

Rudin

Sauter

(1999) Calcineurin inhibitors FK506 and SDZ ASM 981 alleviate the outcome of focal cerebral ischemic/reperfusion injury. J Pharmacol Exp Ther 288:653–659

Boisvert

Hall

(1996) Tirilazad prevention of reperfusion edema after focal ischemia in cynomolgus monkeys. Can J Neurol Sci 23:46–52

Butcher

Henshall

Teramura

Iwasaki

Sharkey

(1997) Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism. J Neurosci 17:6939–6946

Clark

Wechsler

Sabounjian

Schwiderski

(2001) A phase III randomized efficacy trial of 2000 mg citicoline in acute ischemic stroke patients. Neurology 57:1595–1602

Cross

Jones

Snares

Jostell

Bredberg

Green

(1995) The protective action of chlormethiazole against ischemia-induced neurodegeneration in gerbils when infused at doses having little sedative or anticonvulsant activity. Br J Pharmacol 114:1625–1630

Darby

Barber

Gerraty

Desmond

Yang

Parsons

Tress

Davis

(1999) Pathophysiological topography of acute ischemia by combined diffusion-weighted and perfusion MRI. Stroke 30:2043–2052

Diener

Cortens

Ford

Grotta

Hacke

Kaste

Koudstaal

Wessel

(2000) Lubeluzole in acute ischemic stroke treatment: A double-blind study with an 8-hour inclusion window comparing a 10-mg daily dose of lubeluzole with placebo. Stroke 31:2543–2551

Furuichi

Katsuta

Maeda

Ueyama

Moriguchi

Matsuoka

Goto

Yanagihara

(2003) Neuroprotective action of tacrolimus (FK506) in focal and global cerebral ischemia in rodents: Dose dependency, therapeutic time window and long-term efficacy. Brain Res 965:137–145

Gillilan

(1968) The arterial and venous blood supplies to the forebrain (including the internal capsule) of primates. Neurology 18:653–670

10.

Gorelick

(2000) Neuroprotection in acute ischemic stroke: A tale of for whom the bell tolls? Lancet 355:1925–1926

11.

Haring

Berg

Tsurushita

Tagaya

del Zoppo

(1996) E-selectin appears in nonischemic tissue during experimental focal cerebral ischemia. Stroke 27:1386–1392

12.

Herr

Martin-Villalba

Kurz

Roncaioli

Schenkel

Cifone

Debatin

(1999) FK506 prevents stroke-induced generation of ceramide and apoptosis signaling. Brain Res 826:210–219

13.

Jean

Spellman

Nussbaum

Low

(1998) Reperfusion injury after focal cerebral ischemia: The role of inflammation and the therapeutic horizon. Neurosurgery 43:1382–1397

14.

Kaku

Umemura

Mizuno

Yano

Suzuki

Kawasaki

Nakashima

(1998) Evaluation of a GPIIb/IIIa antagonist YM337 in a primate model of middle cerebral artery thrombosis. Eur J Pharmacol 345:185–192

15.

Kawai

Umemura

Nakashima

(1995) Effect of argatroban on microthrombi formation and brain damage in the rat middle cerebral artery thrombosis model. Jpn J Pharmacol 69:143–148

16.

Kito

Nishimura

Susumu

Nagata

Kuge

Yokota

Minematsu

(2001) Experimental thromboembolic stroke in cynomolgus monkey. J Neurosci Methods 105:45–53

17.

Liu

Farmer

Jr. Lane

Friedman

Weissman

Schreiber

(1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP- FK506 complexes. Cell 66:807–815

18.

Lyden

Shuaib

Levin

Atkinson

Rajput

Wechsler

Ashwood

Claesson

Odergren

Salazar-Grueso

(2002) Clomethiazole Acute Stroke Study in ischemic stroke (CLASS-I): Final results. Stroke 33:122–129

19.

Maeda

Furuichi

Ueyama

Moriguchi

Satoh

Matsuoka

Goto

Yanagihara

(2002) A combined treatment with tacrolimus (FK506) and recombinant tissue plasminogen activator for thrombotic focal cerebral ischemia in rats: Increased neuroprotective efficacy and extended therapeutic time window. J Cereb Blood Flow Metab 22:1205–1211

20.

Marshall

JWB

Cross

Jackson

Green

Baker

Ridley

(2000) Clomethiazole protects against hemineglect in a primate model of stroke. Brain Res Bull 52:21–29

21.

Marshall

JWB

Cross

Ridley

(1999) Functional benefit from clomethiazole treatment after focal cerebral ischemia in a nonhuman primate species. Exp Neurol 156:121–129

22.

Mori

Ember

Copeland

Thomas

Koziol

del Zoppo

(1999) Effect of tirilazad mesylate on middle cerebral artery occlusion/reperfusion in non-human promates. Cerebrovas Dis 5:342–349

23.

Park

Hall

(1994) Dose-response analysis of the effect of 21-aminosteroid tirilazad mesylate (U-74006F) upon neurological outcome and ischemic brain damage in permanent focal cerebral ischemia. Brain Res 645:157–163

24.

RANTTAS Investigators (1996) A randomized trial of tirilazad mesylate in patients with acute stroke (RANTTAS). The RANTTAS Investigators. Stroke 27:1453–1458

25.

Ringelstein

Biniek

Weiller

Ammeling

Nolte

Thron

(1992) Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization. Neurology 42:289–298

26.

Rosen

Harry

(1990) Brain volume estimation from serial section measurements: A comparison of methodologies. J Neurosci Meth 35:115–124

27.

Sharkey

Butcher

(1994) Immunophilins mediate the neuroprotective effects of FK506 in focal cerebral ischemia. Nature 371:336–339

28.

Siesjo

Elmer

Janelidze

Keep

Kristian

Ouyang

Uchino

(1999) Role and mechanisms of secondary mitochondrial failure. Acta Neurochir Suppl 73:7–13

29.

(STAIR) STAIR (1999) Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 30:2752–2758

30.

(STAIR-II) STAIR (2001) Recommendations for clinical trial evaluation of acute stroke therapies. Stroke 32:1598–1606

31.

Sydserff

Cross

West

Green

(1995) The effect of chlormethiazole on neuronal damage in a model of transient focal ischemia. Br J Pharmacol 114:1631–1635

32.

Takamatsu

Kondo

Ikeda

Umemura

(1998) Neuroprotective effects depend on the model of focal ischemia following middle cerebral artery occlusion. Eur J Pharmacol 362:137–142

33.

Takamatsu

Tsukada

Noda

Kakiuchi

Nishiyama

Nishimura

Umemura

(2001) FK506 attenuates early ischemic neuronal death in a monkey model of stroke. J Nucl Med 42:1833–1840

34.

Takeshima

Kirsch

Koehler

Traystman

(1994) Tirilazad treatment does not decrease early brain injury after transient focal ischemia in cats. Stroke 25:670–676

35.

Tsujikawa

Ogura

Hiroshiba

Miyamoto

Kiryu

Honda

(1998) Tacrolimus (FK506) attenuates leukocyte accumulation after transient retinal ischemia. Stroke 29:1431–1438

36.

Tsukada

Watanabe

Yamashima

(2001) Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus. J Neurochem 79:1196–1206

37.

Wakita

Tomimoto

Akiguchi

Kimura

(1998) Dose-dependent, protective effect of FK506 against white matter changes in the rat brain after chronic cerebral ischemia. Brain Res 792:105–113

38.

Wang

Pathan

Ethell

Krajewski

Yamaguchi

Shibasaki

McKeon

Bobo

Franke

Reed

(1999) Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284:339–343

39.

Watanabe

West

Bremer

(1977) Experimental regional cerebral ischemia in the middle cerebral artery territory in primates. Part 2: Effects on brain water and electrolytes in the early phase of MCA stroke. Stroke 8:71–76

40.

Yagita

Kitagawa

Matsushita

Taguchi

Mabuchi

Ohtsuki

Yanagihara

Matsumoto

(1996) Effect of immunosuppressant FK506 on ischemia-induced degeneration of hippocampal neurons in gerbils. Life Sci 59:1643–1650