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Table of Contents
Vol. 33, No. 4, 2012
Issue release date: April 2012
Cerebrovasc Dis 2012;33:329–339
(DOI:10.1159/000335309)

Thrombotic Stroke in the Anesthetized Monkey (Macaca mulatta): Characterization by MRI – A Pilot Study

Gauberti M.a · Obiang P.a · Guedin P.a, e · Balossier A.a, b · Gakuba C.a, c · Diependaele A.S.d · Chazalviel L.a · Vivien D.a · Young A.R.a · Agin V.a · Orset C.a
aINSERM U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, GIP Cyceron, Université de Caen-Basse Normandie, et bService de Neurochirurgie, cService d’anesthésie et réanimation, et dLaboratoire d’exploration fonctionnelle du système nerveux, CHU de Caen, Caen, et eService de Neuroradiologie, Hôpital Lariboisière, Paris, France
email Corresponding Author

Abstract

Background: The lack of a relevant stroke model in large nonhuman primates hinders the development of innova- tive diagnostic/therapeutic approaches concerned with this cerebrovascular disease. Our objective was to develop a novel and clinically relevant model of embolic stroke in the anesthetized monkey that incorporates readily available clinical imaging techniques and that would allow the possibility of drug delivery including strategies of reperfusion. Methods: Thrombin was injected into the lumen of the middle cere- bral artery (MCA) in 12 anesthetized (sevoflurane) male rhesus macaques (Macaca mulatta). Sequential MRI studies (including angiography, FLAIR, PWI, DWI, and gadolinium-enhanced T1W imaging) were performed in a 3T clinical MRI. Physiological and biochemical parameters were monitored throughout the investigations. Results: Once standardized, the surgical procedure induced transient occlusion of the middle cerebral artery in all operated animals. All animals studied showed spontaneous reperfusion, which occurred some time between 2 h and 7 days post-ictus. Eighty percent of the studied animals showed diffusion/perfusion mismatch. The ischemic lesions at 24 h spared both superficial and profound territories of the MCA. Some animals presented hemorrhagic transformation at 7 days post-ictus. Conclusion: In this study, we developed a pre-clinically relevant model of embolic stroke in the anesthetized nonhuman primate.


 Outline


 goto top of outline Key Words

  • Animal models
  • Embolic stroke
  • Focal ischemia
  • Middle cerebral artery occlusion
  • Methods
  • Monkey
  • MRI
  • Thrombin

 goto top of outline Abstract

Background: The lack of a relevant stroke model in large nonhuman primates hinders the development of innova- tive diagnostic/therapeutic approaches concerned with this cerebrovascular disease. Our objective was to develop a novel and clinically relevant model of embolic stroke in the anesthetized monkey that incorporates readily available clinical imaging techniques and that would allow the possibility of drug delivery including strategies of reperfusion. Methods: Thrombin was injected into the lumen of the middle cere- bral artery (MCA) in 12 anesthetized (sevoflurane) male rhesus macaques (Macaca mulatta). Sequential MRI studies (including angiography, FLAIR, PWI, DWI, and gadolinium-enhanced T1W imaging) were performed in a 3T clinical MRI. Physiological and biochemical parameters were monitored throughout the investigations. Results: Once standardized, the surgical procedure induced transient occlusion of the middle cerebral artery in all operated animals. All animals studied showed spontaneous reperfusion, which occurred some time between 2 h and 7 days post-ictus. Eighty percent of the studied animals showed diffusion/perfusion mismatch. The ischemic lesions at 24 h spared both superficial and profound territories of the MCA. Some animals presented hemorrhagic transformation at 7 days post-ictus. Conclusion: In this study, we developed a pre-clinically relevant model of embolic stroke in the anesthetized nonhuman primate.

Copyright © 2012 S. Karger AG, Basel


goto top of outline Introduction

To date, there is no consensus on the nonhuman stroke model that most acutely mimics the human pathology. Primate models of cerebral ischemia may offer many advantages over their rodent counterparts [1,2,3,4,5], but such investigations are time-consuming and rely on a multidisciplinary team, state-of-the-art imaging techniques, appropriate intensive care facilities, and a substantial financial commitment. Nonetheless, primate models of stroke offer full biochemical, physiological, and neurological assessment of the animal during a prolonged period of survival.

It is not surprising, therefore, that new experimental models of stroke in the primate are sparse as it may be just too difficult to induce a major stroke in healthy young adolescent nonhuman primates without elaborate manipulation of the cerebral vasculature. There are several models of stroke that have been used in nonhuman primates; some are more severe than others in terms of the degree of neuropathological changes. Experimental techniques to occlude the middle cerebral artery (MCA) involve mechanical occlusions by using either electrocoagulation [6,7], a filament model [8,9], an intravascular balloon or cuff [2,10,11,12], microvascular clips [13,14,15,16,17,18], or ligatures [19,20,21]. Other approaches use the intravascular route to inject autologous or heterologous preformed fibrin, blood clots [3,22,23], microemboli [24,25,26], or, in some cases, by in situ clot formation by photothrombosis [27,28,29]. However, it is important to establish which, if any, of these models of focal cerebral ischemia can be considered to be the most representative of an acute stroke in humans.

We believe that an animal model of embolic stroke would be the most appropriate manner in which to induce focal brain ischemia as it allows the possibility of thrombolytic and/or neuroprotector therapy. Furthermore, as the precise moment of MCA occlusion (MCAO) is known, the temporal evolution of the lesion and the time-to-treatment can be accurately determined through the use of MRI techniques, as in the present study.

The objective of the present study was to validate a model of thromboembolic stroke in anesthetized monkeys and to follow the time course of the resulting lesion through the use of clinically relevant imaging techniques and diagnostic procedures over a 3-month interval. This study consists of two parts: in the first one, which involved 6 animals, we tried to standardize the surgical procedure. In the second one, we used the standardized procedure to characterize and test the reproducibility of our model in 6 additional animals. The following is a result of our findings and our attempt to compare them with documented clinical studies.

 

goto top of outline Methods

Experiments were performed in 12 male rhesus macaques (Macaca mulatta) aged 5–6 years and with body weights ranging from 7 to 11 kg. An experimental protocol was submitted (A.R.Y.) to the Regional Ethics Committee for Animal Experimentation (Normandy) and approval was granted to conduct the study (referral No. N/02-03-08/03/02-11). Experiments were performed by licensed investigators (C.O., A.R.Y., L.C.) and in accordance with French ethical laws (act No. 87-848; Ministère de l’Agriculture et de la Forêt) and European Communities Council Directives (86/609/EEC and 2010/63/EU) guidelines for the care and use of laboratory animals. The animals were purchased directly from a French supplier (Station de primatologie – UPS 846, D56 – 13790 Rousset sur Arc). During the course of the present studies (2 years), the monkeys were housed at the Cyceron Research Centre (Establishment for Animal Experimentation, agreement No. B14118001) in individual cages maintained at 24°C with 50% relative humidity on a 12-hour/12-hour light/dark cycle and were fed commercial chow supplemented with fresh fruits and water ad libitum. Throughout the duration of these studies a veterinary surgeon was available to oversee the well-being of the animals.

goto top of outline Anesthesia

The monkeys were tranquilized with ketamine (Imogen®, 0.1 mg/kg, i.m.) and, subsequent to the placement of catheters in the external saphenous veins, anesthesia was induced by sevoflurane (Sevorane®, 2%) in 100% oxygen. Atracurium (Tracrium®, 0.5 mg/kg, i.v.) was used to achieve muscular relaxation and endotracheal intubation was performed. Monkeys were placed on intermittent positive pressure ventilation (Aestiva 5/MRI 7900; General Electric, France) with a fixed respiratory rate (22 breaths per min). The tidal volume (∼120 ml) was adjusted to maintain normocapnia (Pco2 38–42 mm Hg). Anesthesia was maintained with sevoflurane and 66% nitrous oxide in oxygen along with an intravenous perfusion of atracurium (0.75 mg/kg/h). During surgical interventions, the sevoflurane concentration was increased (to ∼3%, and sufentanyl 0.1 µg, i.v., was administered for further pain relief). Thereafter, and 30 min prior to the MRI studies, sufentanyl was discontinued and the concentration of sevoflurane was reduced to around 1.5–2.0%. Prior to the placement and removal of the stereotaxic ear bars, atropine sulphate (0.25 mg, i.v.) was administered. At the end of each study, the monkey was de-curarized with neostigmine (Prostigmine®, 0.5 mg, i.v.), extubated, and returned to its cage.

goto top of outline Post-Operative Recovery

Systemic antibiotic treatment with cephamandole was continued over 5 days (Kefandol®, 15 mg/kg, i.m., daily). A local antibiotic rifamycine (Rifocine®; Merrel Dow) was also applied into and around the orbit. In case of pain, an injection of the anti-inflammatory agent tolfedine 4% (Vétoquinol®, 4 mg/kg, i.m.) or the oripavine derivative buprenorphine (Buprecare®, 20 µg/kg, i.m.) was given as deemed necessary.

goto top of outline Surgical Procedures and Physiological Monitoring

The transorbital approach to the right MCA, originally described by Hudgins and Garcia [17], was employed as modified by Young et al. [30]. Following enucleation, a craniectomy was made using a high-speed saline-cooled dental drill to expose the right MCA. The dura was opened and the arachnoid dissected to allow placement of the micropipette. Two sutures were positioned to isolate the M1 branch into which the micropipette was inserted and thrombin injected (fig. 1a, b). During this procedure, warm saline was used to reduce the possibility of vascular spasm. Reconstruction of the orbit under aseptic conditions allowed complete post-operative recovery and permitted long-term survival in all animals. Arterial pressure and heart rate were measured continuously, as was the temperature, end-tidal CO2 levels, and Sao2 concentrations (In Vivo Magnitude 3150 MRI; Ademia, France). Following reconstructive surgery, the monkeys were immediately transferred to a 3T Philips MRI scanner (Philips Achieva) where physiological monitoring was continued. Basal physiological parameters were obtained from the basal imaging performed before the day of the surgery.

FIG01
Fig. 1.a Time line for the experimental protocol. b Schema of the surgical procedure showing the craniotomy, the isolated segment of the MCA between the two sutures and the pipette used to inject the thrombin to induce the cascade of clot formation (c).

goto top of outline Clot Formation and Injection

The hematological micropipette was made using an electrophysiology puller (PC-10; Narishige, USA) and calibrated (15 mm/µl; Assistent® ref. 555/5; Hoechst, Sondheim-Rhoen, Germany). Just before surgery, the micropipette was connected to a syringe filled with human thrombin (final concentration 1 UI/µl in PBS; Enzyme Research Laboratories, USA). Thrombin (total volume for the standardized procedure was 800 µl, 1 UI/µl) was injected into the lumen of the designated MCA segment in a step-by-step manner (firstly 6 × 50 µl at 2-min intervals). Thereafter, the proximal suture was removed and 2 × 100 µl were injected at 2-min intervals. Finally, the remaining thrombin (300 µl for the standardized procedure) was injected over 1–2 min after the distal suture was removed. The duration of the entire surgical procedure was approximately 3 h from endotracheal intubation to the end of reconstructive surgery.

The concentration of the thrombin solution was chosen according to the results obtained in rodent studies performed at our laboratory [31]. In the first set of monkeys operated, the total amount of thrombin injected was chosen to achieve a complete and stable arterial occlusion at the end of the thrombus formation procedure. Thereafter, we used this dose (800 UI) in the 6 other animals operated according to the standardized procedure.

goto top of outline In vivo MRI Acquisition

Monkeys were studied in a 3T clinical MRI equipped with two surface coils located on each side of the animal’s head (Philips Sense Flex M). Imaging was performed in the axial plane and included the following sequences (30 min total duration), 3D-time-of-flight angiography, T2-weighted, fluid attenuation inversion recovery (FLAIR), diffusion-weighted imaging (DWI), and pre- and post-contrast T1-weighted and perfusion-weighted imaging (PWI). This block of MRI sequences was repeated before and after surgery at 2 and 24 h, 7 and 21 days, and 3 months (fig. 1a). A bolus of 0.2 mmol/kg GdDOTA (Dotarem®) was injected i.v. for PWI imaging. Immediately before (pre-scan) and 15 min after GdDOTA injection (post-scan), we performed a T1-weighted scan. Subtractions of pre-scan from post-scan were visually inspected for BBB leakage at all imaging time points.

goto top of outline Image Analysis

Maps of relative hemodynamic indices from PWI data [time to peak (TTP), mean transit time, relative cerebral blood volume, and relative cerebral blood velocity] were calculated using the console software (Philips Achieva v1.5). The apparent diffusion coefficient (ADC) was calculated from the diffusion data using MedINRIA software (v1.9). Registrations of ADC maps were performed using the Fusion tool of the MedINRIA software. Maps of infarction probability were computed from manually outlined infarcted areas on registered DWI images using ImageJ software. For pixel-by-pixel analysis of perfusion/diffusion mismatch, we registered ADC maps (at 2 and 24 h) and TTP maps. Then, a region of interest was drawn in a nonischemic area allowing nonischemic ADC and TTP values to be measured. Subsequently, we thresholded the images to measure the volume of the brain regions showing low ADC (a threshold of 75% of the nonischemic region was chosen according to Røhl et al. [32]) and high TTP (a threshold of +3s compared to the nonischemic region was chosen because it provided the best estimate of the resulting ischemic lesion at 24 h post-occlusion with our data).

goto top of outline Neurological Score

Functional deficit was evaluated over the 3 months of the recovery period compared to the basal level before the MCA occlusion. The neurological score was obtained from observations of the animals’ behaviors (e.g. analysis of their spontaneous activities in the cages, hand use during feeding, interactions with animal caregivers) based on the following criteria: 0 = no apparent deficit; 1 = use of the left hand with noticeable deficits; 2 = no use of the left hand, and 3 = death. When the behaviors of the operated animals precluded accurate evaluation of the neurological score (because of altered consciousness, apathy, etc.), they were not tested. Evaluation of the neurological score was performed on videotapes in a blinded fashion by both basic scientists and clinicians.

goto top of outline Statistics

Statistical analyses of the data presented in table 1 were performed using the ANOVA test for multiple comparisons. p < 0.05 was considered statistically significant.

TAB01
Table 1. Physiological and biochemical parameters measured before and after controlled embolic stroke in anesthetized monkeys

 

goto top of outline Results

The schematic illustration of the surgical approach, placement of the micropipette, and the study protocol are illustrated in figure 1. The embolization procedure was difficult and evolved during the course of the study. The first 6 monkeys allowed us to standardize the procedure (determination of the volume of thrombin used and how the injection is performed). Once the procedure was established, we performed it on 6 additional animals to determine the reproducibility of the model.

goto top of outline Physiological and Biochemical Data

Anesthesia was regulated on an individual basis. The surgical intervention, the timing of the MRI examinations, and the survival period were the same for each monkey studied. No significant differences were noted in the physiological data measured with respect to time (n = 12). These data are presented in table 1.

goto top of outline Establishment of the Injection Procedure

Because of methodological issues (mainly due to dysfunctional injection systems leading to a low quantity of injected thrombin) the first two monkeys showed no significant parenchymal infarction on MRI during the course of the study. The next two monkeys received the thrombin injection into the M1 segment of the MCA while the artery was patent. The example of m576 (fig. 2a) showed that, due to the diameter of the artery and the perfusion pressure, the clot did not remain in place. Indeed, there was visual evidence [later confirmed by magnetic resonance angiography (MRA) at +2 h, fig. 2a] that, immediately following the injection, the clot was flushed through the circulation to lodge at the boundary zones of the vascular tree or in small arterioles, leading to a cortical hyperintense signal that covered the entire MCA territory (T2 image at +24 h, fig. 2a) with some involvement of the posterior aspect of the putamen. In the next monkey (m586, fig. 2b), a transient ligature of the distal part of the MCA was performed prior to the injection of thrombin into the MCA during blood flow interruption. This procedure resulted in an acute hyperintense signal on DWI that encompassed the putamen, the caudate nucleus, and the internal capsule (data not shown). The ischemic lesion was restricted to the basal ganglia region with sparing of the parasylvian cortex. The MRA showed a reduction in the apparent caliber of the MCA, but the MCA was by no means completely occluded (fig. 2b, MRA). Thus, the clot might have been flushed into lenticulostriate arteries. At +24 h, the T2 hyperintense signal (fig. 2b, T2) clearly showed the presence of large areas of edematous tissue in deep brain regions. Without proximal ligature the distal occlusion of the MCA preserves perfusion of the lenticulostriate arteries. This residual bloodstream drags the thrombin-induced clot from the proximal arteries to the deep brain. Then, occlusion of the striatal arteries leads to a persistent subcortical infarct. In the next monkey (m488, fig. 3), the injection of thrombin was performed in the isolated M1 segment of the MCA (with both proximal and distal ligatures). This procedure induced complete occlusion of the artery as illustrated in figure 3 (MRA at 2 h post-injection). Partial spontaneous reperfusion occurred some time between +2 h and 1 day post-ictus. The T2 image at +24 h (fig. 3) showed involvement of the pre-central gyrus with some preservation of the post-central gyrus which was in good agreement with the hypoperfused area detected at +2 h on the TTP image. Accordingly, MRI investigations highlighted a significant diffusion-perfusion mismatch (fig. 3). There was a clear difference 2 h post-ischemia between the area of reduced ADC (dark area) and the area of hypoperfusion (TTP; bright area) which may correspond to a brain zone in jeopardy. Twenty-four hours later, this area was recruited by the infarct core as illustrated by the size of the reduced ADC signal which matched perfectly with the TTP at 2 h post-ischemia. Cortical laminar necrosis was evident on the FLAIR image obtained at +3 months. Evidence of hemorrhagic transformation was not noted. When complete interruption of blood flow was observed, this situation led to an ischemic cortical injury in the territory of the MCA which displayed all of the characteristics of thromboembolic stroke in humans. Accordingly, this surgical procedure (as described in the Methods) was selected and reproduced in 6 additional animals.

FIG02
Fig. 2.a MRA (+2 h) and T2-weighted imaging (+24 h) after the MCAO approach in m576. b MRA (+2 h) and T2-weighted imaging (+24 h) after the MCAO approach in m586.

FIG03
Fig. 3. Complete occlusion of the proximal section of the MCA inducing cortical lesions. MRA at 2 and 24 h, and 3 months post-clot formation. T2 at 24 h post-clot formation. FLAIR at 3 months. ADC measured at 2 and 24 h post-clot formation. TTP at 2 h post-clot formation.

goto top of outline Reproducibility and Main Characteristics of the Model

No mortality was observed throughout the second part of the study (as for the first part). We performed MRI at +2 h in all of the animals (n = 6). MRI analyses at other time points (24 h, 7 and 21 days, and 3 months) as well as evaluation of the neurological deficits were performed in 3 randomized monkeys. The three other animals were included in a different protocol using a specific treatment and were thus not included in the present study. Maps of infarction probability at 2 h (n = 6) and 24 h (n = 3) are shown in figure 4a. Individual images of the 3 monkeys followed during 3 months are presented in figure 4b. Importantly, all animals showed complete occlusion of the MCA on the MRA performed +2 h after thrombin injection (fig. 5a). At +24 h, 66% of the animals showed arterial reperfusion and at +7 days, reperfusion had occurred in all animals (fig. 5a). Interestingly, pixel-by-pixel analysis revealed a perfusion mismatch at 2 h post-occlusion in 5 of 6 animals (not shown). Among the 3 animals which had MRI follow-up at +24 h and +7 days, 2 showed hemorrhagic transformation at +7 days (as illustrated in fig. 5e). The mean lesion volume on DWI at +2 h was 1.82 cm3 (±1.08 SD). As expected, the lesion volume (DWI) increased between +2 and +24 h (mean: 4.02 cm3 ± 1.38 SD at +24 h , fig. 5b). Pixel-by-pixel analysis revealed that most brain regions which infarcted between 2 and 24 h post-occlusion showed hemodynamic impairment on PWI imaging at 2 h (fig. 5c). No reversal of ADC was seen in our study using the 80% threshold. Ischemic lesions (as assessed by DWI-weighted imaging at +2 h) involved subcortical (1 of 6) or both cortical and subcortical (5 of 6) areas. BBB opening was evident at +24 h and +7 days post-ictus in all animals studied (fig. 5d).

FIG04
Fig. 4.a Probability infarction map calculated from DWI at 2 and 24 h post-occlusion. b Longitudinal MRI findings of the 3 monkeys with the 3-month follow-up including MRA at 2 h, T2-weighted imaging at 24 h, and FLAIR at 3 months post-occlusion.

FIG05
Fig. 5.a Percentage of animals with MCA reperfusion at 2 and 24 h, and 7 days post-clot formation. b Lesion volume measured on DWI images 2 and 24 h post-clot formation. c Representative images of the pixel-by-pixel analysis of the diffusion mismatch. d Representative image of BBB permeability to gadolinium at 2 and 24 h, and 7 days post-clot formation. e Representative image of hemorrhagic transformation (arrows).

MCA occlusion led to a measurable functional deficit in the 3 animals tested 21 days after MCAO, as expected after the MRI evaluation of the lesion (table 2). However, neurological deficit persisted at 3 months when the caudate nucleus and the putamen were encompassed by the infarct (m889, m907).

TAB02
Table 2. Neurological score performed at 7, 21, and 90 days post-stroke

 

goto top of outline Discussion

In accordance with the recommendations described in the updated STAIR report [33,34], we developed a novel model of embolic stroke in the anesthetized monkey. In this experimental model we observed many of the vascular abnormalities noted in humans during and after a stroke: the clot remains in place at least over several hours inducing downstream ischemic damages, thus allowing the timely administration of drugs including thrombolytic agents such as tissue-type plasminogen activator (tPA; with or without a neuroprotector agent).

MRI techniques were used to evaluate the consequences of ischemic stroke as this technology is readily available in most stroke centers. Although MRI technology identifies regions of mismatch, the hypothesis that DWI and PWI allows precise identification of the ischemic core and penumbra is still debated [35,36,37]. Nonetheless, most neurologists select their patients who are eligible for thrombolysis [38,39,40,41] based on, among other factors, PW- and DW-MRI. For this reason, we used the same diagnostic procedures as those performed in the stroke clinic to evaluate acute and chronic pathological changes associated with focal cerebral ischemia.

Among stroke models in nonhuman primates described so far, we consider that our present thrombotic model induced by local injection of thrombin displays most of the features of the clinical situation. For instance, in contrast to our present protocol, in models where autologous or heterologous preformed clots are used [22,23,42,43], the precise location of the arterial occlusion is not controlled (proximal or distal, middle or anterior MCA, involvement of the lenticulostriate arteries). In the photothrombotic model [27,28], although the location of the clot is also controlled, concerns have been recently raised about the mechanism involved in clot formation in such models and its relevance in what happens in the ‘real-life situation’ [44]. In contrast, in our present model, thrombus formation is triggered by a direct thrombin injection, thus mimicking the endogenous activation of thrombin mediated by the tissue factor, as could occur in humans during vascular thrombosis [45]. Thus, we believe that our model acutely mimics the human pathology and addresses some of the limitations of the previously described models.

This model could be relevant for future pre-clinical studies, either to validate strategies of brain protection, advance research on biomarkers, or for the development of new MRI sequences. In most nonhuman primate models of stroke, there are some caveats that must be taken into account. The various methodologies used to occlude a major cerebral artery, most often the MCA, are described above. All of these techniques are feasible for the study of therapeutic efficacy, but few, apart from the administration of autologous blood clots, provide the representative conditions of embolic stroke in humans. Notably, successful treatment of experimental embolic stroke has rarely been achieved in nonhuman primate models [42,43]. There is an urgent need for the development of new clinically relevant models of embolic stroke in the nonhuman primate. Our model of embolic stroke allows the direct induction of a single clot into the M1 segment of the MCA and permits localized occlusion of this major cerebral artery with, as in most humans experiencing ischemic stroke, spontaneous reperfusion in the following days.

It is difficult to accurately assess the time of onset of symptoms in a stroke patient. However, through the use of this novel experimental model we can accurately follow ischemic disturbances with respect to time and evaluate the efficacy of thrombolysis after administration of the drug even beyond the 4.5-hour time window as currently proposed [46,47].

One criterion of any model of focal cerebral ischemia is that it must have a reproducible lesion volume and thus a small number of ischemic animals could be compared to a treated group. We believe that variations in cerebrovascular architecture are an inherent factor of variability in man and, as was noted in the present study during the surgical preparation, in the nonhuman primate. Fortunately, the present model allows us to obtain a priori an individual MRA of each monkey prior to the surgical intervention and to assess the best possible approach for the injection site of thrombin. Furthermore, it is possible with this model to perform long-term evaluations using MRI, a technique rarely performed in the chronic stage of stroke in humans since the lesion is normally consolidated within 48–72 h.

Several key points remain, however, to be clarified and improved. In the present experimental stroke model, thrombus formation is triggered by a large amount of recombinant active thrombin. The injected thrombin could interact with the endothelium located downstream of the resulting thrombus and could trigger intracellular signaling unrelated to ischemia. However, in similar studies performed in rodents, intravascular thrombin did not produce any detectable side effects [48,49], suggesting that this mechanism is unlikely to significantly influence stroke outcome in the present model.

In contrast to other less invasive experimental stroke models, we performed enucleation to expose the MCA. Thus, ischemia-unrelated modifications of visual skills are expected and should be taken into account for neurological evaluation in enucleated animals. The craniotomy performed during the transorbital approach was as small as possible and reconstruction of the orbit avoided a significant effect on the post-stroke intracranial pressure. Nevertheless, this surgical procedure could trigger local inflammation and could be confounding in some cases (such as investigation of inflammatory-related biomarkers). Sham-operated animals would be needed as a control group in such cases.

Another critical issue is the number of animals needed to detect any significant effect of a neuroprotective treatment in this model. Since the lesion volume at 24 h is approximately twice the volume at 2 h, we can assume that neuroprotective treatments started 2 h after thrombus formation could reduce the ischemic lesion size by maximum 50% at 24 h. Sample size calculation [standardized difference = 0.5 ×(4.02/1.38) = 1.46] indicates that 8 animals per group are needed to detect the treatment effect on the lesion volume at 24 h with a power of 0.8 and a cutoff for statistical significance of 0.05 (t test). To detect a more reasonable difference of 25% in lesion size at 24 h, 15 animals per group would be needed. Thus, a study investigating a neuroprotective treatment in this model should involve 30 monkeys in order to be sufficiently powered.

Given that this model has most of the main characteristics of ischemic stroke in humans (i.e. arterial occlusion by a blood clot, diffusion/perfusion mismatch, and eventually hemorrhagic transformation), together with a very low mortality rate, we believe that it fulfills all of the criteria for assessing the pre-clinical efficacy of a treatment.

 

goto top of outline Acknowledgements

This work was supported by grants from the INSERM, the University of Caen-Basse Normandie, and the Regional Council of Lower Normandy.


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  18. Symon L: Regional cerebrovascular responses to acute ischaemia in normocapnia and hypercapnia: an experimental study in baboons. J Neurol Neurosurg Psychiatry 1970;33:756–762.
  19. Morawetz RB, DeGirolami U, Ojemann RG, Marcoux FW, Crowell RM: Cerebral blood flow determined by hydrogen clearance during middle cerebral artery occlusion in unanesthetized monkeys. Stroke 1978;9:143–149.
  20. Dodson RF, Aoyagi M, Chu LW: Ultrastructural changes in subacute cerebral infarction following middle cerebral artery occlusion in the baboon. Cytobios 1975;13:97–108.
  21. Ott EO, Abraham JA, Meyer JS, Tulleken CA, Mathew NT, Achari AN, Aoyagi M, Dodson RF: Regional cerebral blood flow measured by the gamma camera after direct injection of 133Xe into the distal stump of the occluded middle cerebral artery. Stroke 1975;6:376–381.
  22. Kuge Y, Yokota C, Tagaya M, Hasegawa Y, Nishimura A, Kito G, Tamaki N, Hashimoto N, Yamaguchi T, Minematsu K: Serial changes in cerebral blood flow and flow-metabolism uncoupling in primates with acute thromboembolic stroke. J Cereb Blood Flow Metab 2001;21:202–210.
  23. Xie Y, Munekata K, Seo K, Hossmann KA: Effect of autologous clot embolism on regional protein biosynthesis of monkey brain. Stroke 1988;19:750–757.
  24. Sato Y, Chin Y, Kato T, Tanaka Y, Tozuka Y, Mase M, Ageyama N, Ono F, Terao K, Yoshikawa Y, Hisatsune T: White matter activated glial cells produce BDNF in a stroke model of monkeys. Neurosci Res 2009;65:71–78.
  25. Watanabe O, Bremer AM, West CR: Experimental regional cerebral ischemia in the middle cerebral artery territory in primates. 1. Angio-anatomy and description of an experimental model with selective embolization of the internal carotid artery bifurcation. Stroke 1977;8:61–70.
  26. Bremer AM, Watanabe O, Bourke RS: Artificial embolization of the middle cerebral artery in primates: description of an experimental model with extracranial technique. Stroke 1975;6:387–390.
  27. Koketsu D, Furuichi Y, Maeda M, Matsuoka N, Miyamoto Y, Hisatsune T: Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia. Exp Neurol 2006;199:92–102.

    External Resources

  28. Furuichi Y, Maeda M, Moriguchi A, Sawamoto T, Kawamura A, Matsuoka N, Mutoh S, Yanagihara T: Tacrolimus, a potential neuroprotective agent, ameliorates ischemic brain damage and neurologic deficits after focal cerebral ischemia in nonhuman primates. J Cereb Blood Flow Metab 2003;23:1183–1194.
  29. Kaku S, Umemura K, Mizuno A, Yano S, Suzuki K, Kawasaki T, Nakashima M: Evaluation of a GPIIb/IIIa antagonist YM337 in a primate model of middle cerebral artery thrombosis. Eur J Pharmacol 1998;345:185–192.
  30. Young AR, Sette G, Touzani O, Rioux P, Derlon JM, MacKenzie ET, Baron JC: Relationships between high oxygen extraction fraction in the acute stage and final infarction in reversible middle cerebral artery occlusion: an investigation in anaesthetized baboons with positron emission tomography. J Cereb Blood Flow Metab 1996;16:1176–1188.
  31. Orset C, Macrez R, Young AR, Panthou D, Angles-Cano E, Maubert E, Agin V, Vivien D: Mouse model of in situ thromboembolic stroke and reperfusion. Stroke 2007;38:2771–2778.

    External Resources

  32. Røhl L, Ostergaard L, Simonsen CZ, Vestergaard-Poulsen P, Andersen G, Sakoh M, Le Bihan D, Gyldensted C: Viability thresholds of ischemic penumbra of hyperacute stroke defined by perfusion-weighted MRI and apparent diffusion coefficient. Stroke 2001;32:1140–1146.
  33. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH: Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009;40:2244–2250.
  34. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:2752–2758.
  35. Sobesky J, Weber OZ, Lehnhardt FG, Hesselmann V, Neveling M, Jacobs A, Heiss WD: Does the mismatch match the penumbra? Magnetic resonance imaging and positron emission tomography in early ischemic stroke. Stroke 2005;36:980–985.
  36. Kranz PG, Eastwood JD: Does diffusion-weighted imaging represent the ischemic core? An evidence-based systematic review. AJNR Am J Neuroradiol 2009;30:1206–1212.
  37. Kane I, Sandercock P, Wardlaw J: Magnetic resonance perfusion diffusion mismatch and thrombolysis in acute ischaemic stroke: a systematic review of the evidence to date. J Neurol Neurosurg Psychiatry 2007;78:485–491.
  38. Chemmanam T, Campbell BC, Christensen S, Nagakane Y, Desmond PM, Bladin CF, Parsons MW, Levi CR, Barber PA, Donnan GA, Davis SM, EPITHET Investigators: Ischemic diffusion lesion reversal is uncommon and rarely alters perfusion-diffusion mismatch. Neurology 2010;75:1040–1047.
  39. Donnan GA, Baron JC, Ma H, Davis SM: Penumbral selection of patients for trials of acute stroke therapy. Lancet Neurol 2009;8:261–269.
  40. Ebinger M, De Silva DA, Christensen S, Parsons MW, Markus R, Donnan GA, Davis SM: Imaging the penumbra – strategies to detect tissue at risk after ischemic stroke. J Clin Neurosci 2009;16:178–187.
  41. Solling C, Hjort N, Ashkanian M, Ostergaard L, Andersen G: Safety and efficacy of MRI-based selection for recombinant tissue plasminogen activator treatment: responder analysis of outcome in the 3-hour time window. Cerebrovasc Dis 2009;27:223–229.
  42. Susumu T, Yoshikawa T, Akiyoshi Y, Nagata R, Fujiwara M, Kito G: Effects of intra-arterial urokinase on a non-human primate thromboembolic stroke model. J Pharmacol Sci 2006;100:278–284.
  43. Omura T, Tanaka Y, Miyata N, Koizumi C, Sakurai T, Fukasawa M, Hachiuma K, Minagawa T, Susumo T, Yoshida S, Nakaike S, Okuyama S, Harder DR, Roman RJ: Effect of a new inhibitor of the synthesis of 20-HETE on cerebral ischemia reperfusion injury. Stroke 2006;37:1307–1313.
  44. Kleinschnitz C, Braeuninger S, Pham M, Austinat M, Nölte I, Renné T, Nieswandt B, Bendszus M, Stoll G: Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis. Stroke 2008;39:1262–1268.

    External Resources

  45. Owens AP 3rd, Mackman N: Tissue factor and thrombosis: the clot starts here. Thromb Haemost 2010;104:432–439.
  46. Carpenter CR, Keim SM, Milne WK, Meurer WJ, Barsan WG: Thrombolytic therapy for acute ischemic stroke beyond three hours. J Emerg Med 2011;40:82–92.

    External Resources

  47. del Zoppo GJ, Saver JL, Jauch EC, Adams HP Jr: Expansion of the time window for treatment of acute ischemic stroke with intravenous tissue plasminogen activator: a science advisory from the American Heart Association/American Stroke Association. Stroke 2009;40:2945–2948.
  48. García-Yébenes I, Sobrado M, Zarruk JG, Castellanos M, Pérez de la Ossa N, Dávalos A, Serena J, Lizasoain I, Moro MA: A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke 2011;42:196–203.
  49. Zhang Z, Zhang RL, Jiang Q, Raman SB, Cantwell L, Chopp M: A new rat model of thrombotic focal cerebral ischemia. J Cereb Blood Flow Metab 1997;17:123–135.

 goto top of outline Author Contacts

Prof. D. Vivien, PhD
INSERM U919 Serine Proteases and Pathophysiology of the Neurovascular Unit
GIP Cyceron, Bd H.-Becquerel, BP5229
FR–14074 Caen Cedex (France)
Tel. +33 2 31 47 01 60, E-Mail vivien@cyceron.fr


 goto top of outline Article Information

Maxime Gauberti, Pauline Obiang, Véronique Agin, and Cyrille Orset contributed equally to this work.

Received: June 14, 2011
Accepted: November 17, 2011
Published online: February 16, 2012
Number of Print Pages : 11
Number of Figures : 5, Number of Tables : 2, Number of References : 49


 goto top of outline Publication Details

Cerebrovascular Diseases

Vol. 33, No. 4, Year 2012 (Cover Date: April 2012)

Journal Editor: Hennerici M.G. (Mannheim)
ISSN: 1015-9770 (Print), eISSN: 1421-9786 (Online)

For additional information: http://www.karger.com/CED


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Background: The lack of a relevant stroke model in large nonhuman primates hinders the development of innova- tive diagnostic/therapeutic approaches concerned with this cerebrovascular disease. Our objective was to develop a novel and clinically relevant model of embolic stroke in the anesthetized monkey that incorporates readily available clinical imaging techniques and that would allow the possibility of drug delivery including strategies of reperfusion. Methods: Thrombin was injected into the lumen of the middle cere- bral artery (MCA) in 12 anesthetized (sevoflurane) male rhesus macaques (Macaca mulatta). Sequential MRI studies (including angiography, FLAIR, PWI, DWI, and gadolinium-enhanced T1W imaging) were performed in a 3T clinical MRI. Physiological and biochemical parameters were monitored throughout the investigations. Results: Once standardized, the surgical procedure induced transient occlusion of the middle cerebral artery in all operated animals. All animals studied showed spontaneous reperfusion, which occurred some time between 2 h and 7 days post-ictus. Eighty percent of the studied animals showed diffusion/perfusion mismatch. The ischemic lesions at 24 h spared both superficial and profound territories of the MCA. Some animals presented hemorrhagic transformation at 7 days post-ictus. Conclusion: In this study, we developed a pre-clinically relevant model of embolic stroke in the anesthetized nonhuman primate.



 goto top of outline Author Contacts

Prof. D. Vivien, PhD
INSERM U919 Serine Proteases and Pathophysiology of the Neurovascular Unit
GIP Cyceron, Bd H.-Becquerel, BP5229
FR–14074 Caen Cedex (France)
Tel. +33 2 31 47 01 60, E-Mail vivien@cyceron.fr


 goto top of outline Article Information

Maxime Gauberti, Pauline Obiang, Véronique Agin, and Cyrille Orset contributed equally to this work.

Received: June 14, 2011
Accepted: November 17, 2011
Published online: February 16, 2012
Number of Print Pages : 11
Number of Figures : 5, Number of Tables : 2, Number of References : 49


 goto top of outline Publication Details

Cerebrovascular Diseases

Vol. 33, No. 4, Year 2012 (Cover Date: April 2012)

Journal Editor: Hennerici M.G. (Mannheim)
ISSN: 1015-9770 (Print), eISSN: 1421-9786 (Online)

For additional information: http://www.karger.com/CED


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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    External Resources

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    External Resources

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  18. Symon L: Regional cerebrovascular responses to acute ischaemia in normocapnia and hypercapnia: an experimental study in baboons. J Neurol Neurosurg Psychiatry 1970;33:756–762.
  19. Morawetz RB, DeGirolami U, Ojemann RG, Marcoux FW, Crowell RM: Cerebral blood flow determined by hydrogen clearance during middle cerebral artery occlusion in unanesthetized monkeys. Stroke 1978;9:143–149.
  20. Dodson RF, Aoyagi M, Chu LW: Ultrastructural changes in subacute cerebral infarction following middle cerebral artery occlusion in the baboon. Cytobios 1975;13:97–108.
  21. Ott EO, Abraham JA, Meyer JS, Tulleken CA, Mathew NT, Achari AN, Aoyagi M, Dodson RF: Regional cerebral blood flow measured by the gamma camera after direct injection of 133Xe into the distal stump of the occluded middle cerebral artery. Stroke 1975;6:376–381.
  22. Kuge Y, Yokota C, Tagaya M, Hasegawa Y, Nishimura A, Kito G, Tamaki N, Hashimoto N, Yamaguchi T, Minematsu K: Serial changes in cerebral blood flow and flow-metabolism uncoupling in primates with acute thromboembolic stroke. J Cereb Blood Flow Metab 2001;21:202–210.
  23. Xie Y, Munekata K, Seo K, Hossmann KA: Effect of autologous clot embolism on regional protein biosynthesis of monkey brain. Stroke 1988;19:750–757.
  24. Sato Y, Chin Y, Kato T, Tanaka Y, Tozuka Y, Mase M, Ageyama N, Ono F, Terao K, Yoshikawa Y, Hisatsune T: White matter activated glial cells produce BDNF in a stroke model of monkeys. Neurosci Res 2009;65:71–78.
  25. Watanabe O, Bremer AM, West CR: Experimental regional cerebral ischemia in the middle cerebral artery territory in primates. 1. Angio-anatomy and description of an experimental model with selective embolization of the internal carotid artery bifurcation. Stroke 1977;8:61–70.
  26. Bremer AM, Watanabe O, Bourke RS: Artificial embolization of the middle cerebral artery in primates: description of an experimental model with extracranial technique. Stroke 1975;6:387–390.
  27. Koketsu D, Furuichi Y, Maeda M, Matsuoka N, Miyamoto Y, Hisatsune T: Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia. Exp Neurol 2006;199:92–102.

    External Resources

  28. Furuichi Y, Maeda M, Moriguchi A, Sawamoto T, Kawamura A, Matsuoka N, Mutoh S, Yanagihara T: Tacrolimus, a potential neuroprotective agent, ameliorates ischemic brain damage and neurologic deficits after focal cerebral ischemia in nonhuman primates. J Cereb Blood Flow Metab 2003;23:1183–1194.
  29. Kaku S, Umemura K, Mizuno A, Yano S, Suzuki K, Kawasaki T, Nakashima M: Evaluation of a GPIIb/IIIa antagonist YM337 in a primate model of middle cerebral artery thrombosis. Eur J Pharmacol 1998;345:185–192.
  30. Young AR, Sette G, Touzani O, Rioux P, Derlon JM, MacKenzie ET, Baron JC: Relationships between high oxygen extraction fraction in the acute stage and final infarction in reversible middle cerebral artery occlusion: an investigation in anaesthetized baboons with positron emission tomography. J Cereb Blood Flow Metab 1996;16:1176–1188.
  31. Orset C, Macrez R, Young AR, Panthou D, Angles-Cano E, Maubert E, Agin V, Vivien D: Mouse model of in situ thromboembolic stroke and reperfusion. Stroke 2007;38:2771–2778.

    External Resources

  32. Røhl L, Ostergaard L, Simonsen CZ, Vestergaard-Poulsen P, Andersen G, Sakoh M, Le Bihan D, Gyldensted C: Viability thresholds of ischemic penumbra of hyperacute stroke defined by perfusion-weighted MRI and apparent diffusion coefficient. Stroke 2001;32:1140–1146.
  33. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH: Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009;40:2244–2250.
  34. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:2752–2758.
  35. Sobesky J, Weber OZ, Lehnhardt FG, Hesselmann V, Neveling M, Jacobs A, Heiss WD: Does the mismatch match the penumbra? Magnetic resonance imaging and positron emission tomography in early ischemic stroke. Stroke 2005;36:980–985.
  36. Kranz PG, Eastwood JD: Does diffusion-weighted imaging represent the ischemic core? An evidence-based systematic review. AJNR Am J Neuroradiol 2009;30:1206–1212.
  37. Kane I, Sandercock P, Wardlaw J: Magnetic resonance perfusion diffusion mismatch and thrombolysis in acute ischaemic stroke: a systematic review of the evidence to date. J Neurol Neurosurg Psychiatry 2007;78:485–491.
  38. Chemmanam T, Campbell BC, Christensen S, Nagakane Y, Desmond PM, Bladin CF, Parsons MW, Levi CR, Barber PA, Donnan GA, Davis SM, EPITHET Investigators: Ischemic diffusion lesion reversal is uncommon and rarely alters perfusion-diffusion mismatch. Neurology 2010;75:1040–1047.
  39. Donnan GA, Baron JC, Ma H, Davis SM: Penumbral selection of patients for trials of acute stroke therapy. Lancet Neurol 2009;8:261–269.
  40. Ebinger M, De Silva DA, Christensen S, Parsons MW, Markus R, Donnan GA, Davis SM: Imaging the penumbra – strategies to detect tissue at risk after ischemic stroke. J Clin Neurosci 2009;16:178–187.
  41. Solling C, Hjort N, Ashkanian M, Ostergaard L, Andersen G: Safety and efficacy of MRI-based selection for recombinant tissue plasminogen activator treatment: responder analysis of outcome in the 3-hour time window. Cerebrovasc Dis 2009;27:223–229.
  42. Susumu T, Yoshikawa T, Akiyoshi Y, Nagata R, Fujiwara M, Kito G: Effects of intra-arterial urokinase on a non-human primate thromboembolic stroke model. J Pharmacol Sci 2006;100:278–284.
  43. Omura T, Tanaka Y, Miyata N, Koizumi C, Sakurai T, Fukasawa M, Hachiuma K, Minagawa T, Susumo T, Yoshida S, Nakaike S, Okuyama S, Harder DR, Roman RJ: Effect of a new inhibitor of the synthesis of 20-HETE on cerebral ischemia reperfusion injury. Stroke 2006;37:1307–1313.
  44. Kleinschnitz C, Braeuninger S, Pham M, Austinat M, Nölte I, Renné T, Nieswandt B, Bendszus M, Stoll G: Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis. Stroke 2008;39:1262–1268.

    External Resources

  45. Owens AP 3rd, Mackman N: Tissue factor and thrombosis: the clot starts here. Thromb Haemost 2010;104:432–439.
  46. Carpenter CR, Keim SM, Milne WK, Meurer WJ, Barsan WG: Thrombolytic therapy for acute ischemic stroke beyond three hours. J Emerg Med 2011;40:82–92.

    External Resources

  47. del Zoppo GJ, Saver JL, Jauch EC, Adams HP Jr: Expansion of the time window for treatment of acute ischemic stroke with intravenous tissue plasminogen activator: a science advisory from the American Heart Association/American Stroke Association. Stroke 2009;40:2945–2948.
  48. García-Yébenes I, Sobrado M, Zarruk JG, Castellanos M, Pérez de la Ossa N, Dávalos A, Serena J, Lizasoain I, Moro MA: A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke 2011;42:196–203.
  49. Zhang Z, Zhang RL, Jiang Q, Raman SB, Cantwell L, Chopp M: A new rat model of thrombotic focal cerebral ischemia. J Cereb Blood Flow Metab 1997;17:123–135.