Abstract
Background/Aim: Ischemic stroke is a major health concern globally and developing reliable animal models is crucial for understanding its pathophysiology. This study evaluated the relationship between cerebral angiographic findings and neurologic dysfunction in an acute non-human primate thromboembolic stroke model and determined the minimum clot length for suitable middle cerebral artery (MCA) occlusion. Materials and Methods: A thromboembolic stroke model was developed by injecting autologous blood clots (length: 1, 2, 3, 4, 5, and 10 cm, n=1 to 3, 14 monkeys in total) into the internal carotid artery of male cynomolgus monkeys. Digital subtraction angiography (DSA) and neurologic deficit observation were performed pre-; immediately after (DSA only); and 1, 3, 6, and 24 h after embolization, and the relationship between clot length, neurologic deficits, and cerebral infarction was assessed. Results: DSA confirmed MCA occlusion in all animals after the clot injection. Recanalization of the MCA was observed within 6 h post-embolization in animals with shorter clots (≤3 cm). Neurologic deficits were evident in animals with MCA occlusion and correlated with the clot length. Larger clots (≥5 cm) led to permanent MCA occlusion, significant neurologic deficits, and extensive cerebral infarction. Histopathological examination revealed ischemic damage in brain regions corresponding to the infarcted areas. Conclusion: Clot length is critical in determining the extent of neurologic dysfunction and cerebral infarction, with larger clots producing more severe outcomes. Furthermore, the minimum clot length required for model creation is 5 cm.
- Experimental thromboembolic stroke model
- cynomolgus monkey
- digital subtraction angiography (DSA)
- neurologic deficits
Ischemic stroke remains a leading cause of death and severe neurologic dysfunctions worldwide (1, 2). Among the various experimental models employed, the middle cerebral artery (MCA) occlusion model in non-human primates (NHPs) emerges as a valuable tool for mimicking the complex pathophysiology of ischemic stroke. This monkey model allows for a closer approximation of human responses, providing a bridge between rodent studies (3, 4) and clinical observations (1).
The MCA occlusion model induces focal cerebral ischemia, leading to neurobehavioral changes that parallel those observed in human ischemic stroke (5, 6). NHPs, due to their structural and functional similarities to humans, offer a unique opportunity to unravel the intricacies of post-MCA occlusion neurobehavioral responses. However, despite the wealth of literature on MCA occlusion models and neurobehavioral assessments in NHPs, there exists a notable gap in our understanding of the integrated relationship between neurobehavioral changes and cerebral angiography findings in this specific context (2).
Angiography is a powerful tool for accurate identification of vessel occlusion, stenosis, and aneurysm sites and is used for planning thrombolytic therapy and mechanical thrombectomy in stroke patients. Currently, several modalities, such as computed tomography angiography (CTA) and magnetic resonance angiography (MRA), can be used for vessel imaging, in addition to digital subtraction angiography (DSA), which is considered to be the criterion standard for accurate identification of the occlusion site in acute stroke because it provides high-resolution images, shorter examination times, and real-time dynamic assessment, and can be combined with intervention techniques. The DSA technique has been used for the development of a stroke model (7-9); however, there are no reports examining the relationship between angiography and neurologic dysfunction in acute thromboembolic monkey stroke models.
In the present study, we evaluate the relationship between angiography and neurologic dysfunction in an acute NHP thromboembolic stroke model and determine the minimum clot length suitable for MCA occlusion model creation.
Materials and Methods
Animals and maintenance. Sixteen male cynomolgus monkeys weighing 3 to 6 kg and aged 4 to 6 years old (Source: Tian Hu Primate Animal Breeding Research Center Ltd., Phnom Penh, Cambodia) were used in this study. All procedures in animals were approved by the animal care and use committee of Shin Nippon Biomedical Laboratories, Ltd. (approval Nos. IACUC999591 and IACUC999626), a facility fully certified by AAALAC International, and the research was performed in accordance with the Institutional Guidelines for Animal Experiments and in compliance with the Japanese Law Concerning the Protection and Control of Animals (Law No. 105 and Notification No. 6), and under veterinary supervision. The principal investigator and the primate handling staff were present at all procedures. The animals were housed in cages 68 cm wide, 62 cm high, 77 cm deep [conforming to National Institutes of Health (NIH) requirements], and the room was maintained under the following condition: temperature: 26±3°C, humidity: 50%±20%, ventilation: 15 times/h, and a 12-h light/dark cycle (lighting from 07:00 a.m.).
Monkey thromboembolic stroke model. The procedure for development of the thromboembolic stroke model has been described previously (6, 10-12). A chronic catheter for the injection of a blood clot was implanted in the left internal carotid artery in each animal. Atropine sulfate hydrate, a preanesthetic medication, was administered intramuscularly to the animals. The animals were sedated by an intramuscular injection of ketamine hydrochloride (Ketalar for Intramuscular Injection 500 mg, Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan, 50 mg/ml), after which they underwent surgery while anesthetized by inhalation of isoflurane (0.5% to 2.0%, Isoflu, Zoetis Japan Inc., Tokyo, Japan). The catheter was filled with heparin saline solution (500 IU/ml) and tunneled subcutaneously into the neck toward the dorsal region. All animals received antibiotics [aqueous suspended injection of dihydrostreptomycin sulfate (250 mg potency/ml) and benzylpenicillin procaine (200,000 units/ml)], Meiji Seika Kaisha, Ltd., Tokyo, Japan, 0.05 ml/lg, intramuscularly) and were allowed to recover from the anesthesia. Two days after the operation, a single autologous blood clot was injected into the internal carotid artery through the carotid catheter under light sevoflurane (Maruishi Pharmaceutical Co., Ltd. Osaka, Japan) anesthesia.
Study design and animal assignment. A total of 16 monkey thromboembolic stroke model animals were used for the study, and 14 of 16 animals were assigned for evaluation of the relationship between the length of the blood clots and the embolisms, and the remaining 2 animals were assigned for evaluation of histopathological changes in the super-acute phase of the thromboembolic stroke model. For blood clot length/embolism evaluation, blood clots at lengths of 1, 2, 3, 4, 5, or 10 cm (diameter: approximately 1 mm, n=1, 2, or 3) were used (Table I). After injection of the blood clot (embolization), digital subtraction angiography (DSA) and neurologic evaluation were performed immediately (DSA only) and 3, 6, 18 (neurologic evaluation only), and 24 h after embolization, and cerebral infarct was measured 24 h after embolization. For histopathology evaluation, a 10 cm clot was injected, and the animal was euthanized 6 or 24 h after embolization (n=1 per timepoint).
Relationship between clot length and brain damage in cynomolgus monkey thromboembolic stroke model.
Neurologic evaluation. Neurologic deficits, according to a standardized score (1), were evaluated at 3, 6, and 24 h after embolization (Table I). Of the 100 points possible, 28 were assigned for consciousness, 22 for the sensory system, 32 for the motor system, and 18 for skeletal muscle coordination. In normal animals, the score for each category was 0 points. The total neurologic deficit score was the sum of the four category scores. Assessment of neurologic deficit was performed blindly by an experienced observer.
Digital subtraction angiography. DSA of the cerebral artery was performed using a C-arm machine (Infinix Celeve-i INFX-8000C, Canon Medical Systems Corporation, Tochigi, Japan). Animals were sedated by inhalation of isoflurane. DSA images were obtained after manual injection of nonionic contrast agent (Iopamidol Injection 300, Bayer Yakuhin, Ltd., Osaka, Japan) through the left internal carotid artery via the previously implanted catheter for clot injection at a frame rate of 30 f/s. DSA acquisition was performed before clot injection to obtain a baseline image.
Cerebral infarct volume analysis. After 24 h of embolization, 10 surviving animals assigned for evaluation of the relationship between blood clot length and embolism were sacrificed under pentobarbital anesthesia after the last neurologic scoring. The brain was then perfused with cold physiological saline and cut into 4 mm thick coronal sections. Brain slices were completely immersed in 1% 2,3,5-triphenyltetrazolium chloride (13, 14) (TTC, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) solution and were incubated at 37ºC for 40 min. The left hemispherical area and infarct area in each slice were measured from a photograph using a computerized image analytic system (analySIS, Soft Imaging System GmbH, Münster, Germany). The infarct size in each slice was expressed as a percentage of the total left hemispherical area in that slice. The infarct ratio was calculated from the sum of all 14 slices and represented as a percentage of the total area of all slices.
Histopathology. Six or twenty-four hours after embolization induced by the injection of the 10 cm clot, animals were euthanized under pentobarbital anesthesia. The brains were then perfused with cold saline from the right carotid artery and removed. Fourteen coronal brain slices (4 mm thickness) were prepared, stained with 1% TTC solution, and fixed in 10% phosphate-buffered formalin for 24 h. The paraffin-embedded tissue sections of 5 μm thickness were routinely processed and were stained with hematoxylin and eosin (H&E).
Results
Digital subtraction angiography. Before clot injection, the internal carotid artery (ICA), anterior carotid artery (ACA), and middle cerebral artery (MCA) were visualized clearly in the angiogram, and normal cerebral circulation was noted. Immediately after the blood clot injection, occlusion of the MCA was confirmed in all animals except for one (1 cm, No. 3); however, recanalization was observed in all animals injected with the 2 cm clot or shorter and in one of two animals injected with the 3 cm clot by 3 h after embolization. In the other animal injected with the 3 cm clot, both ACA and ICA occlusions in addition to MCA were observed immediately after embolization, and both MCA and ACA occlusions were still observed 3 h after embolization; however, recanalization was observed 6 h after embolization. In the 4 cm clot treated animals, incomplete MCA occlusion (partial recanalization observed in the territory supplied with blood from the MCA) was observed in all animals. In the animals with clots of 5 cm or longer, no complete recanalization was noted. However, one animal with a 4 cm clot, two animals with a 5 cm clot, and two animals with a 10 cm clot died within 24 h after embolization. These animals, except for the 4 cm clot animal, showed occlusion of ACA, MCA, and ICA immediately after clot injection. In the surviving animals with the 4 cm or 5 cm clot, only MCA was occluded immediately after clot injection.
At necropsy, the injected blood clot was not visually confirmed at the origin of MCA in the animals with a 3 cm or shorter clot, and ICA, MCA, and ACA were visualized in all these animals in DSA at 24 h after embolization. In the animals that were euthanized due to a humane endpoint, the injected clot was confirmed in both MCA and ACA at necropsy. In the surviving animals with a 4 cm or 5 cm clot, occlusion of the MCA was confirmed by DSA at 24 h after embolization, and the blood clot was located in the MCA (Figure 1, Table I).
Digital subtraction angiography (DSA) of the thromboembolic stroke monkey model. DSA of the internal carotid artery (ICA), anterior carotid artery (ACA), and middle cerebral artery (MCA) in the thromboembolic stroke monkey model is shown before; immediately after (i.a.); and 3, 6, and 24 h after embolization. After clot injection (embolization), the MCA was occluded (arrows); however, complete recanalization was noted with the 1 cm clot 3 h after embolization (arrowheads) and partial with the 4 cm clot 24 h after dosing (closed triangle). In the right panels, photographs of the origin of the MCA at necropsy are shown.
Relationship between clot length and neurologic deficits. As shown in Table I, 3 h after embolization all animals with MCA occlusion, except for one animal with a 4 cm clot (No. 8), showed decreased consciousness level (score: 8, conscious but clouded and accepting) in the consciousness category; absent or decreased in the contralateral facial sensation, pinna reflex, and pain response (score: 9 or 11) in the sensory system category; paralysis of contralateral hand, leg, and upper limb (score 16) in the motor system category; and unable to walk (score: 8, stands spontaneously, falls within a few steps or score: 12, sits, just able to circle) in skeletal muscle coordination category. The other animal with MCA occlusion (No. 9) showed similar neurologic deficits 6 h after embolization (total score: 45), but these changes recovered (total score: 12), and partial recanalization in the MCA territory was observed 24 h after embolization. In the other two animals that showed partial recanalization in the MCA territory, the neurologic score was less than the scores in animals with complete MCA occlusion. In the 3 cm clot animal (No. 5), neurologic deficit and MCA occlusion were observed 3 h after embolization; however, there was no MCA occlusion or neurologic deficits 6 h after embolization or later. In the animal (No. 2) with a 1 cm clot, even though there was no occlusion in MCA, ACA, or ICA in DSA, mild neurologic deficits (total scores: 23-27, slight decreases in conscious level and sensory and motor function) were observed from 3 to 24 h after embolization.
Relationship between clot length and cerebral infarction. As shown in Figure 2, all animals with a 3 cm clot or shorter, with the exception of two animals (Nos. 2 and 3) with a 1 cm clot, had no cerebral infarct 24 h after embolization, and the two animals with a 1 cm clot had a small cerebral infarct in the cerebral cortex (precentral gyrus, superior temporal gyrus, insula), cerebral white matter, and/or caudate nucleus of striatum in the left cerebral hemisphere (infarct ratio: less than 5%). One surviving animal (No. 7) with a 4 cm clot had a cerebral infarct in precentral gyrus and insula of the cerebral cortex and caudate nucleus and putamen of striatum (infarct ratio: 9.2%), but the other animal (No. 9) had a focal cerebral infarct located in the striatum only (infarct ratio: 30.4%). In the 5 cm clot animal, cerebral infarct was located in the cerebral cortex (front-orbital and precentral gyrus and insula), cerebral white matter, and striatum (putamen and caudate nucleus; No. 12, infarct ratio: 34.9%).
Cerebral infarct (TTC staining). TTC-stained coronal brain slices 24 h after embolization. Cerebral infarct was located in the precentral gyrus (Pr), superior temporal gyrus (St), frontal-orbital gyrus (Fo), and insula (Ins) in the cerebral cortex (ctx), cerebral white matter (cw), and caudate nucleus (CN) and putamen (Pu) of the striatum (str). The infarct ratio was calculated as the ratio of the infarct volume versus the left cerebral brain hemisphere volume. NC: Not calculated.
Histopathological changes in the super-acute phase of the monkey thromboembolic model. Six hours after embolization induction by injecting a 10 cm clot, assessment of cerebral infarct by TTC staining showed infarct mainly in the insula cortex and basal ganglia (caudate nucleus and putamen). Atrophy and necrosis of neurons and perivascular mononuclear cell infiltration, characteristic morphological features of brain ischemic damage, were observed in the caudate nucleus in the left hemisphere. In other regions, including the parietal and temporal cortices in the left hemisphere, no morphological changes were observed. Twenty-four hours after embolization by injecting a 10 cm clot, the infarct area extended to the parietal and temporal cortices, and the caudate nucleus, parietal, and temporal cortex neurons showed severe atrophy, vacuolation, perivascular mononuclear cell infiltration, and hemorrhage (Figure 3).
Histopathological changes in caudate nucleus (CN), and parietal (PC) and temporal cortices (TC) of the thromboembolic monkey stroke model. Brain section at 24 mm from the frontal pole was stained with TTC (A) and HE (B) 6 and 24 h after embolization. At 6 h after embolization, atrophy and necrosis of the neurons were observed in the CN (arrows). At 24 h after embolization, atrophy of neurons (arrows), vacuolation (arrowheads), and hemorrhage (open-arrow head) were observed in CN, PC, and TC. HE stains, (×400).
Discussion
In the present study, we demonstrated that creation of a permanent MCA occlusion cynomolgus monkey model induced by injecting an autologous blood clot into the internal carotid artery required a blood clot of at least 5 cm in length. With clots of 3 cm or shorter, although the MCA was confirmed to be occluded immediately after the clot injection (embolization), recanalization of the MCA was noted within 6 h after embolization, and most animals showed no neurologic deficits after recanalization. In the 4 cm clot animals, incomplete MCA occlusion (partial recanalization) was noted in all animals, the neurologic deficit score at partial recanalization of MCA was lower than that seen with complete MCA occlusion, and the partial recanalization, even 24 h after embolization, facilitated amelioration of the neurologic deficits. The cerebral infarction was observed mainly in the insula cortex and basal ganglia from 6 h after embolization and extended to the parietal and temporal cortex regions 24 h after embolization. The animals in which cerebral infarction in the parietal and temporal cortices were observed with TTC staining showed more severe neurologic deficits, but morphological histopathological changes, such as atrophy and necrosis of neurons, were not observed at 6 h after embolization.
Endogenous thrombolysis plays an important role as a biological defense mechanism that allows the breakdown of blood clots formed in the body. The fibrinolytic system is composed of the protease plasmin, its precursor, plasminogen, and their respective activators [tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)] and inhibitors [plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin] (15). Recanalization of the occluded cerebral arteries has been reported in stroke patients (16-18), and the presumptive mechanism through which partial recanalization facilitates fibrinolysis is through increased flow and arterial wall shear stress, which enhances plasminogen release and circulation throughout the clot in vitro (19), in vivo (20), and in healthy humans (21). In the present study, recanalization of MCA occlusion with blood clots of 3 cm or shorter was considered to be due to lysing of the thrombus by the animal’s endogenous fibrinolytic system, and the absence of severe neurological damage or large cerebral infarct volume was considered to reflect the symptoms of transient ischemic attack (TIA) in humans (22-24).
After the onset of ischemia, critical reduction of the cerebral blood flow (CBF) sequentially induces ischemic depolarization, excitatory cellular injury induced by Ca2+ and glutamate, oxidative injury induced by reactive oxygen species (including various free radicals), secondary microcirculatory disturbance, edema formation, apoptosis, inflammation, and brain injury, propagating from the ischemic core to the surrounding area (25, 26). In our NHP stroke model with a 10 cm blood clot, a marked decrease in the CBF region (<40% of the contralateral values) was noted in the temporal cortex and basal ganglia 1 h after embolization, and this region expanded 24 h after embolization (10). For acute stroke, there are two main methods of clinical treatment: drug-mediated thrombolysis and interventional surgery. Recombinant tissue plasminogen activator (rtPA), urokinase (known as uPA), recombinant pro-urokinase (27), and streptokinase (28) are commonly used as thrombolysis drugs in stroke patients within 4.5 h after onset (29-32). Endovascular thrombectomy has also emerged in the past decade as a major treatment for acute stroke when performed within 6 h after onset (33-35). We have previously reported that intra-arterial administration of urokinase from 1 h after embolization ameliorates neurologic deficits and decreases the cerebral infarct volume in the NHP thromboembolic stroke model (11). In the present study, the animals showed recanalization of the MCA within 6 h after embolization and had a smaller cerebral infarct (less than 10% of the infarct ratio), and amelioration of the neurologic deficits was noted, along with the absence of histopathological damage in the peripheral and temporal cortices, despite these areas being the sites of morphological brain injuries such as neuronal cell death in animals 24 h after embolization. These results suggest that the recanalization of the MCA within 6 h of embolization prevents brain ischemic neuronal cell death in the ischemic penumbra regions and immediately ameliorates neurologic deficits. This falls within the therapeutic time window of thrombolysis and thrombectomy.
In the present study, five animals (one with a 4 cm clot, two with a 5 cm clot, and two with a 10 cm clot) were euthanized within 24 h after embolization due to a humane endpoint, and the mortality ratio was higher than that in the previous report (less than 10% mortality) (6). Mortality in the monkey stroke model is considered to be related to frequent cerebral angiography for the following reasons: 1) iopamidol, the contrast agent for DSA, is known to induce renal dysfunction as a side effect (36, 37) and 2) injection into the cerebral blood vessels damaged by ischemic stroke enhances encephalopathy and brain edema (38).
In conclusion, we have shown that blood clots of 5 cm or longer are suitable for creation of a permanent MCA occlusion stroke monkey model that shows translatability of neurologic dysfunction and histopathological brain damage similar to that seen in human thromboembolic stroke. Furthermore, injection of clots of 4 cm or shorter into the MCA shows recanalization within 24 h after embolization, and this correlated with the amelioration of neurologic deficits.
Acknowledgements
The Authors thank the research members of the Laboratory Animal Management Department at Shin Nippon Biomedical Laboratories, Ltd. for providing expertise that greatly assisted the research.
Footnotes
Authors’ Contributions
T.Y. and Y.A. planned the study; T.Y., Y.A., K.M., K.N., and H.K. performed the experiments and data analysis; K.N. performed DSA measurement and analysis; H.K. performed histopathology review; T.Y., Y.A., and H.K. drafted the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received April 26, 2024.
- Revision received May 26, 2024.
- Accepted May 27, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
