Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

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Middle Cerebral Artery Blood Flows by Combining TCD Velocities and MRA Diameters: In Vitro and In Vivo Validations

Ultrasound in Medicine & Biology ◽

10.1016/j.ultrasmedbio.2014.05.022 ◽

2014 ◽

Vol 40 (11) ◽

pp. 2692-2699 ◽

Cited By ~ 7

Author(s):

K.A. Yonan ◽

E.R. Greene ◽

J.M. Sharrar ◽

A. Caprihan ◽

C. Qualls ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Cerebral Artery ◽

Blood Flows

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Neuroprotective and Angiogenesis Effects of Levetiracetam Following Ischemic Stroke in Rats

Frontiers in Pharmacology ◽

10.3389/fphar.2021.638209 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiang Yao ◽

Wenping Yang ◽

Zhendong Ren ◽

Haoran Zhang ◽

Dafa Shi ◽

...

Keyword(s):

Cerebral Cortex ◽

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Infarct Volume ◽

Hypoxia Inducible Factor ◽

Neuronal Cell ◽

Anti Inflammatory

Objective: The present study explored whether levetiracetam (LEV) could protect against experimental brain ischemia and enhance angiogenesis in rats, and investigated the potential mechanisms in vivo and in vitro.Methods: The middle cerebral artery was occluded for 60 min to induce middle cerebral artery occlusion (MCAO). The Morris water maze was used to measure cognitive ability. The rotation test was used to assess locomotor function. T2-weighted MRI was used to assess infarct volume. The neuronal cells in the cortex area were stained with cresyl purple. The anti-inflammatory effects of LEV on microglia were observed by immunohistochemistry. Enzyme-linked immunosorbent assays (ELISA) were used to measure the production of pro-inflammatory cytokines. Western blotting was used to detect the levels of heat shock protein 70 (HSP70), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF-1α) in extracts from the ischemic cortex. Flow cytometry was used to observe the effect of LEV on neuronal cell apoptosis.Results: LEV treatment significantly increased the density of the surviving neurons in the cerebral cortex and reduced the infarct size (17.8 ± 3.3% vs. 12.9 ± 1.4%, p < 0.01) after MCAO. Concurrently, the time required to reach the platform for LEV-treated rats was shorter than that in the saline group on day 11 after MCAO (p < 0.01). LEV treatment prolonged the rotarod retention time on day 14 after MCAO (84.5 ± 6.7 s vs. 59.1 ± 6.2 s on day 14 compared with the saline-treated groups, p < 0.01). It also suppressed the activation of microglia and inhibited TNF-α and Il-1β in the ischemic brain (135.6 ± 5.2 pg/ml vs. 255.3 ± 12.5 pg/ml, 18.5 ± 1.3 pg/ml vs. 38.9 ± 2.3 pg/ml on day 14 compared with the saline-treated groups, p < 0.01). LEV treatment resulted in a significant increase in HIF-1α, VEGF, and HSP70 levels in extracts from the ischemic cerebral cortex. At the same time, LEV reduced neuronal cell cytotoxicity and apoptosis induced by an ischemic stroke (p < 0.01).Conclusion: LEV treatment promoted angiogenesis and functional recovery after cerebral ischemia in rats. These effects seem to be mediated through anti-inflammatory and antiapoptotic activities, as well as inducing the expression of HSP70, VEGF, and HIF-1α.

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A review of experimental models of focal cerebral ischemia focusing on the middle cerebral artery occlusion model

F1000Research ◽

10.12688/f1000research.51752.2 ◽

2021 ◽

Vol 10 ◽

pp. 242

Author(s):

Melissa Trotman-Lucas ◽

Claire L. Gibson

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Cerebral Artery ◽

Artery Occlusion ◽

Lesion Volume ◽

Mechanistic Investigation ◽

Cerebral Artery Occlusion

Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.

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Effects of Cerebral Ischemia on Neuronal Hemoglobin

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2008.145 ◽

2008 ◽

Vol 29 (3) ◽

pp. 596-605 ◽

Cited By ~ 38

Author(s):

Yangdong He ◽

Ya Hua ◽

Wenquan Liu ◽

Haitao Hu ◽

Richard F Keep ◽

...

Keyword(s):

Cerebral Ischemia ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Ischemic Preconditioning ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Sprague Dawley ◽

Cerebral Neurons

This study examined whether neuronal hemoglobin (Hb) is present in rats. It then examined whether cerebral ischemia or ischemic preconditioning (IPC) affects neuronal Hb levels in vivo and in vitro. In vivo, male Sprague-Dawley rats were subjected to either 15 mins of transient middle cerebral artery occlusion (MCAO) with 24 h of reperfusion, an IPC stimulus, or 24 h of permanent MCAO (pMCAO), or IPC followed 3 days later by 24 h of pMCAO. In vitro, primary cultured neurons were exposed to 2 h of oxygen—glucose deprivation (OGD) with 22 h of reoxygenation. Results showed that Hb is widely expressed in rat cerebral neurons but not astrocytes. Hemoglobin expression was significantly upregulated in the ipsilateral caudate and the cortical core of the middle cerebral artery territory after IPC. Hemoglobin levels also increased more in the penumbral cortex and the contralateral hemisphere 24 h after pMCAO, but expressions in the ipsilateral caudate and the cortical core area were decreased. Ischemic preconditioning modified pMCAO-induced brain Hb changes. Neuronal Hb levels in vitro were increased by 2 h of OGD and 22 h of reoxygenation. These results indicate that Hb is synthesized in neurons and can be upregulated by ischemia.

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A review of experimental models of focal cerebral ischemia focusing on the middle cerebral artery occlusion model

F1000Research ◽

10.12688/f1000research.51752.1 ◽

2021 ◽

Vol 10 ◽

pp. 242

Author(s):

Melissa Trotman-Lucas ◽

Claire L. Gibson

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Cerebral Artery ◽

Artery Occlusion ◽

Lesion Volume ◽

Mechanistic Investigation ◽

Cerebral Artery Occlusion

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Adamts-18 Is a Novel Candidate Gene Of Vascular Development That Is Related To Aggravated Thrombosis: Evidence From a Adamts-18 Knock Out Mice

Blood ◽

10.1182/blood.v122.21.31.31 ◽

2013 ◽

Vol 122 (21) ◽

pp. 31-31

Author(s):

Wei Zhang ◽

Dawei Bu ◽

Suying Dang ◽

Tao Hong ◽

Thomas Wisniewski

Keyword(s):

Blood Flow ◽

Carotid Artery ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Common Carotid Artery ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Surface Display

Abstract HIV-ITP patients have a unique antibody (Ab) against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). The search for a physiologic ligand that could induce this reaction was undertaken by panning the GPIIIa49-66 peptide with a phage surface display 7-mer peptide library. From 20 positive clones, 1 had 70% identity with a C-terminal region of ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin (TSR)-like motif 18), which is secreted by endothelial cell (EC). The recombinant C-terminal fragment of ADAMTS-18 can completely dissolve platelet aggregates formed in vitro. Moreover, this fragment lyses thrombi formed in the cerebral artery of mice and reduces infarction and neurologic impairment in murine ischemic stroke model (Blood 113: 6051, 2009). However, whether ADAMTS-18 represents the dominant physiologic mechanism controlling thrombus dissolution in vivo remains to be clarified. Here, we used ADAMTS-18-deficient (ADAMTS-18-/-) mice to study the contributions of ADAMTS-18 to thrombus formation in vivo. To investigate possible functional differences between WT and ADAMTS-18-/- platelets, we tested WT and ADAMTS-18-/- platelets in a model of pulmonary thromboembolism induced by infusion of a mixture of platelet agonist collagen (25 µg per mouse) and epinephrine (1 µg per mouse). In lung tissue Hematoxylin and eosin-stained (HE) slides, the mean number of thrombi per lung was same in the ADAMTS-18-/- group compared with WT group (163.7 ±14.38 vs 174.9 ±11.73, n=30/group, P=0.5480). In vitro, there is no difference between WT and ADAMTS-18-/- platelet aggregation trace and activation initiated by various platelet agonists ADP (10 µM) or collagen (2 µg/mL). No difference was noted on WT and ADAMTS-18-/- platelet adhesion on immobilized ligand (fibrinogen). These results indicated ADAMTS-18 had no effect on platelet function. We next evaluate the effect of ADAMTS-18 on thrombus formation in a second well-established carotid artery thrombosis model, which is induced by 10% FeCl3 patch. In the process of surgical operation, we unexpectedly observed that all ADAMTS-18-/- mice have premature common carotid artery bifurcation compared with WT mice. A Doppler flow monitor showed ADAMTS-18-/- mice exhibited significantly reduced carotid artery blood flow than WT mice (ADAMTS-18-/- vs WT, 0.5 ± 0.11 vs 0.75 ± 0.21 mL/min, n=7/group, P=0.0298), which results in shortened time of thrombus formation (ADAMTS-18-/- vs WT, 452.17 ± 68.88 vs 611.43 ± 92.02 sec, n=7/group, P=0.0005 ). Immunohistochemistry staining showed that the common carotid artery of ADAMTS-18-/- mice had increased adventitial collagen deposition compared with WT mice. In vivo matrigel plug assay demonstrated that ADAMTS-18-/- mice had significantly lower density of blood vessels compared to the WT mice. Since the middle cerebral artery arises from the internal carotid artery, we conjecture that ADAMTS-18-/- mice would have aggravated brain infarction for the less cerebral blood flow supplying. This proved to be true. In transient middle cerebral artery occlusion (tMCAO) model, the infarction size in ADAMTS-18-/- mice was significantly larger than in WT mice (mean infarction %, 25.68 ± 4.13 vs 17.41 ± 3.24, n=8, P=0.0012). Taken together, these observations suggest vasculature is the potential site of action of ADAMTS-18. To our knowledge, this is the first validation study of linkage and association of ADAMTS-18 as a pro-vasculature gene that is related to aggravated thrombosis. Disclosures: No relevant conflicts of interest to declare.

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The Cortical Ischaemic Penumbra Associated with Occlusion of the Middle Cerebral Artery in the Cat: 2. Studies of Histopathology, Water Content, and in vitro Neurotransmitter Uptake

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1983.12 ◽

1983 ◽

Vol 3 (1) ◽

pp. 97-108 ◽

Cited By ~ 89

Author(s):

A. J. Strong ◽

B. E. Tomlinson ◽

G. S. Venables ◽

G. Gibson ◽

J. A. Hardy

Keyword(s):

Specific Gravity ◽

Middle Cerebral Artery ◽

Water Content ◽

Cerebral Artery ◽

Ultrastructural Changes ◽

Neurotransmitter Uptake ◽

Critical Ischaemia ◽

Ischaemic Penumbra

The nature of the ischaemic penumbra, as defined by suppression of electroencephalogram amplitude in the absence of increase in steady state pial surface potassium activity in excess of 13 m M, was examined in the marginal gyrus of cats subjected to middle cerebral artery occlusion. In vitro synaptosomal neurotransmitter uptake, water content (specific gravity), and histopathology at the light and electron microscopic level were studied and the results compared with those obtained at deeper, critical levels of ischaemia (less than 15 ml 100 g−1 min−1). [3H]4-Aminobutyric acid uptake was 104% of control in the marginal gyrus (NS), and 61 and 48% (p < 0.05) in critical ischaemia. It is concluded that impairment of in vivo synaptosomal uptake is a marker of simultaneous widespread damage to neurones, rather than of a change restricted to the synaptic compartment, although the present findings cannot exclude reversible, substrate-limited impairment of uptake in vivo. Reductions in specific gravity were seen only with critical ischaemia. In 5 of 6 experiments, early or classical ischaemic neuronal cell changes and reactive glia were seen on light microscopy in restricted areas in the marginal gyrus, either in microfoci or scattered more diffusely. Ultrastructural changes were more frequent but were considered to affect only a minority of neurones. Hypotheses for selective electrophysiological suppression in penumbra are discussed.

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Abstract 334: An Echogenic Clot Method for Thrombolysis Monitoring of Directed Lytics in Thrombotic Stroke

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.334 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Dalton E Carter ◽

Tao Peng ◽

Melanie R Moody ◽

Shao-Ling Huang ◽

David D McPherson ◽

...

Keyword(s):

Mass Loss ◽

Dissolution Rate ◽

Microcrystalline Cellulose ◽

Quantitative Assessment ◽

Imaging Techniques ◽

Ultrasound Contrast Agent ◽

Rabbit Blood ◽

Blood Clots

Background: We have previously demonstrated thrombolytic efficacy of tissue plasminogen activator (tPA)-loaded echogenic liposomes (TELIP), a directed therapeutic construct that features exposed tPA fibrin-binding sites, in a rabbit abdominal aorta thrombosis model. The most relevant animal model for evaluation of directed thrombolytic therapy for ischemic stroke is the rabbit thrombotic stroke model previously used to validate recombinant tPA (rtPA; Alteplase). The ability to precisely monitor tPA-mediated thrombus dissolution in vivo is important for quantitative assessment of directed TELIP efficacy. Hypothesis: A gas-free ultrasound contrast agent can be incorporated into blood clots at a concentration that does not affect the tPA-mediated clot dissolution rate, while enabling quantitative assessment of the clot dissolution rate. Methods: In 1.5-ml Eppendorf tubes, 0.2-ml clots were formed from a mixture of whole rabbit blood, 1 M calcium chloride, human thrombin and various amounts of microcrystalline cellulose. At 0-time, 50 μg rtPA in 0.5 ml porcine plasma was added to each tube. Washed clots in tubes were weighed with an analytical balance at 30, 60 and 90 minutes. Clot echogenicity at each time point was assessed with a Philips HDI 5000 ultrasound system using an L12-5 linear array probe positioned laterally to tubes placed in an anechoic chamber. Recorded Images were subjected to videodensitometric analysis that converted image reflectivity to mean gray scale values (MGSV). Results: We found that 1.12 mg/ml of microcrystalline cellulose in rabbit blood clots (0.2 ml) provided optimal echogenicity without affecting the clot dissolution rates (0.3-0.6 mg/min.) caused by 100 μg/ml of rtPA. The clot dissolution rate measured by videodensitometric analysis of serial sonograms of the echogenic clots agreed well with that determined by mass loss measurements (0.28% 0-time value/minute). Conclusions: We have developed a method for monitoring rtPA-mediated clot mass loss by ultrasound imaging techniques and validated it in vitro. This method will be important for demonstrating the enhanced efficacy and decreased hemorrhagic effects provided by directed tPA vehicles relative to systemic administration of the free thrombolytic.

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Abstract W MP89: Silencing VEGF-B Diminishes the Neuroprotective Effect of Candesartan Treatment after Experimental Focal Cerebral Ischemia

Stroke ◽

10.1161/str.46.suppl_1.wmp89 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Sahar Soliman ◽

Tauheed Ishrat ◽

Bindu Pillai ◽

Adviye Ergul ◽

Susan C Fagan

Keyword(s):

Cerebral Ischemia ◽

Infarct Size ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Focal Cerebral Ischemia ◽

Neuroprotective Effect ◽

Critical Role ◽

Hemoglobin Content

Background & Objective: The pro-survival effect of VEGF-B has been documented in different in vivo and in vitro models. We have previously shown an enhanced VEGF-B expression in response to candesartan treatment after focal cerebral ischemia. In this study, we tested the hypothesis that VEGF-B expression contributes to candesartan-mediated recovery. Methods: Silencing VEGF-B expression was achieved by bilateral intracerebroventricular injection of lentiviral particles containing short hairpin RNA against VEGF-B (KD) or vehicle (NTC). Middle cerebral artery (MCA) was occluded for 90 minutes. At reperfusion, animals received either intravenous saline or candesartan. Neurobehavioral outcome was assessed 24, 48 and 72 hours after the insult and infarct size was measured at 72 hours. In an additional set of experiment, middle cerebral artery was occluded for 3h followed by 21h reperfusion. Rats were sacrificed at 24h post-MCAO and brains perfused for evaluation of vascular markers (edema and hemoglobin content). Results (Table): Candesartan-treated animals showed a significant reduction in the infarct size and edema accompanied by functional recovery in Bederson, beam walk, paw grasp and grip strength performance only in the presence of VEGF-B. In addition, candesartan-treated animals showed significantly reduction of hemoglobin content, a marker for hemorrhage and edema at 24 h after MCAO. Conclusion: Our results suggest VEGF-B plays a critical role in mediating candesartan’s neuronal and vascular protective effect after stroke. Identifying growth factors that mediate recovery after ischemic stroke presents possible targets for stroke therapeutics.

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Detecting apoptosis as a clinical endpoint for proof of a clinical principle

Ophthalmologica ◽

10.1159/000513584 ◽

2020 ◽

Author(s):

Piero Zollet ◽

Timothy E.Yap ◽

M Francesca Cordeiro

Keyword(s):

Drug Development ◽

Therapeutic Response ◽

Imaging Techniques ◽

Brain Diseases ◽

Promising Strategy ◽

Sight Loss ◽

Apoptosis Detection ◽

Novel Therapeutic Strategies

The transparent eye media represent a window through which to observe changes occurring in the retina during pathological processes. In contrast to visualising the extent of neurodegenerative damage that has already occurred, imaging an active process such as apoptosis has the potential to report on disease progression and therefore the threat of irreversible functional loss in various eye and brain diseases. Early diagnosis in these conditions is an important unmet clinical need to avoid or delay irreversible sight loss. In this setting, apoptosis detection is a promising strategy with which to diagnose, provide prognosis, and monitor therapeutic response. Additionally, monitoring apoptosis in vitro and in vivo has been shown to be valuable for drug development in order to assess the efficacy of novel therapeutic strategies both in the pre-clinical and clinical setting. Detection of Apoptosing Retinal Cells (DARC) technology is to date the only tool of its kind to have been tested in clinical trials, with other new imaging techniques under investigation in the fields of neuroscience, ophthalmology and drug development. We summarize the transitioning of techniques detecting apoptosis from bench to bedside, along with the future possibilities they encase.

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