scholarly journals Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

2020 ◽  
Vol 7 (01) ◽  
pp. 1 ◽  
Author(s):  
Smrithi Sunil ◽  
Sefik Evren Erdener ◽  
Blaire S. Lee ◽  
Dmitry Postnov ◽  
Jianbo Tang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Vessel Occlusion ◽  
Pial Vessel

scholarly journals Animal Models of Chronic Cerebral Hypoperfusion: From Mouse to Primate

2019 ◽  
Vol 20 (24) ◽  
pp. 6176 ◽  
Author(s):  
Kazuo Washida ◽  
Yorito Hattori ◽  
Masafumi Ihara
Keyword(s):  
White Matter ◽  
Animal Models ◽  
Mouse Model ◽  
Carotid Arteries ◽  
Amyloid Β ◽  
Chronic Cerebral Hypoperfusion ◽  
Motor Dysfunction ◽  
Cerebral Hypoperfusion ◽  
Left Vertebral Artery ◽  
Vessel Occlusion

Vascular cognitive impairment (VCI) or vascular dementia occurs as a result of brain ischemia and represents the second most common type of dementia after Alzheimer’s disease. To explore the underlying mechanisms of VCI, several animal models of chronic cerebral hypoperfusion have been developed in rats, mice, and primates. We established a mouse model of chronic cerebral hypoperfusion by narrowing the bilateral common carotid arteries with microcoils, eventually resulting in hippocampal atrophy. In addition, a mouse model of white matter infarct-related damage with cognitive and motor dysfunction has also been established by asymmetric common carotid artery surgery. Although most experiments studying chronic cerebral hypoperfusion have been performed in rodents because of the ease of handling and greater ethical acceptability, non-human primates appear to represent the best model for the study of VCI, due to their similarities in much larger white matter volume and amyloid β depositions like humans. Therefore, we also recently developed a baboon model of VCI through three-vessel occlusion (both the internal carotid arteries and the left vertebral artery). In this review, several animal models of chronic cerebral hypoperfusion, from mouse to primate, are extensively discussed to aid in better understanding of pathophysiology of VCI.

In Vitro and In Vivo Characterization of a Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FXRoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2697-2697
Author(s):  
Elise Roy ◽  
Paris Margaritis ◽  
Harre D. Downey ◽  
Katherine A. High
Keyword(s):  
Mouse Model ◽  
Thrombus Formation ◽  
Bleeding Tendency ◽  
Factor X ◽  
Wild Type ◽  
Intrinsic Pathway ◽  
Vessel Occlusion ◽  
Tail Clip

Abstract The complex and dynamic interplay between the intrinsic and extrinsic pathways of blood coagulation is incompletely understood. The mediator of prothrombin cleavage, Factor X (FX), plays a pivotal role as part of both the extrinsic and intrinsic tenase complexes. Moreover, the existence of naturally occurring Factor X mutations that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., 1988), is due to a missense mutation (Thr318←Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3% wild-type) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50% wild-type) in the extrinsic pathway as measured by PT. We chose to generate a mouse model of FX asymmetric activation to further probe the extrinsic-intrinsic pathway physiological relationship in hemostasis and thrombosis. For this, we used both an in vitro and an in vivo approach. We first constructed and purified the mouse homolog of FXRoma (mFXRoma) as well as wild-type mFX. Using a clotting-based assay, mFXRoma exhibited intrinsic and extrinsic activity comparable to that reported for the human mutation (5% and 18%, respectively). The reduced intrinsic and extrinsic activity of mFXRoma was not due to a secretion defect, based on Western blot analysis of supernatant and cell extracts from mFXRoma and mFX stably-transfected human embryonic kidney (HEK-293) cell lines. Mice homozygous for the analogous mutation (Thr315←Met) in exon 8 of the murine FX gene were generated by using a plug-and-socket approach. This resulted in the endogenous mFX exon 8 sequence being replaced with the mutated one, thus affording gene expression under the endogenous promoter. Analysis of mFXRoma homozygous mice showed a 6.4% and 19.2% intrinsic and extrinsic activity relative to wild-type littermates, respectively, confirming our in vitro data. The reduced activity in these mice resulted in a slight reduction in levels of the thrombin-antithrombin (TAT) complex. To determine any physiological defect of this mutation on the two pathways of coagulation, we performed two hemostatic challenges of the macrocirculation (tail clip and FeCl3-induced thrombus formation). In the tail-clip assay, blood loss showed no statistical difference between wild-type (n=5) and mFXRoma (n=6) mice. In contrast, following FeCl3-induced injury on the carotid artery (larger vessel diameter that in the tail), mFXRoma mice (3/3) failed to result in vessel occlusion (up to 30 min of observation), whereas wild-type littermates showed stable vessel occlusion (3/4) within ∼6 min of FeCl3 application. Although the type of injury was different, these data suggest that an impeded intrinsic activity of FX does not appear to affect hemostasis of the macrocirculation at relatively small diameter vessels but is essential for thrombus formation in large diameter vessels, and a relatively normal extrinsic activity does not compensate for this defect. This mouse model will aid in determining the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway of coagulation.

Antithrombotic Effects of a FXI Antisense Oligonucleotide in Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3076-3076
Author(s):  
Hong Zhang ◽  
Chenguang Zhao ◽  
Jeff Crosby ◽  
Curt Mazur ◽  
Susan M. Freier ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dose Range ◽  
Safety Margin ◽  
Mrna Levels ◽  
Vessel Occlusion ◽  
Antithrombotic Activity ◽  
Protein Levels ◽  
2Nd Generation ◽  
Vessel Injury ◽  
Antithrombotic Effects

Abstract FXI, a component of the intrinsic pathway of coagulation, contributes to the generation of thrombin and is also activated by thrombin on the surface of platelets, resulting in further amplification of the coagulation process. Reports have shown that FXI deficiency in mice or in humans is generally not associated with major bleeding yet is associated with reduced risk for thromboembolic disease. To determine whether targeting FXI with antisense oligonucleotides (ASOs) can produce antithrombotic activity without increasing risk of bleeding, a series of 2nd Generation 2′-O-methoxyethyl modified ASOs were identified that specifically reduce levels of FXI in mice and were evaluated for antithrombotic activity and bleeding potential. Mice were dosed with FXI ASO (ISIS 404071) subcutaneously for three weeks. FXI ASO treatment resulted in a dosedependent and specific reduction in FXI mRNA levels in liver and FXI protein levels in plasma with an ED50 of 3.2 mg/kg. These reductions in FXI levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXI ASO treatment also correlated well with antithrombotic activity in a 10% FeCl3 induced IVC thrombosis mouse model across a wide dose range. Furthermore, FXI ASO treatment was generally well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXI ASO targeting. Additional studies using intravital microscopy to study the effects of FXI ASO treatment on thrombosis formation in the mesenteric vein following 10% FeCl3 injury demonstrated that FXI ASO treatment significantly impeded platelet aggregation at the site of vessel injury, thrombus growth and vessel occlusion. Studies to further evaluate the safety of FXI targeting were conducted using a mouse model of intracerebral hemorrhage induced by intracranial injection of collagenase. In this model, FXI ASO treatment had no effect on hemorrhagic size, neurologic deficit score, or mortality at doses well above that needed to produce maximal antithrombotic activity. These results strongly suggest that FXI is an effective and safe target for antithrombotic therapy and that antisense drug technology could be an attractive approach to specifically target FXI for the treatment of thrombotic disorders.

Optimized mouse model of embolic MCAO: From cerebral blood flow to neurological outcomes

2020 ◽  
pp. 0271678X2091762 ◽  
Author(s):  
Rongrong Wang ◽  
Hailian Wang ◽  
Yaan Liu ◽  
Di Chen ◽  
Yangfan Wang ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Ischemic Stroke ◽  
Mouse Model ◽  
Infarct Volume ◽  
Laser Speckle ◽  
Core Area ◽  
Contrast Imaging ◽  
Vessel Occlusion ◽  
Doppler Flowmetry

The embolic middle cerebral artery occlusion (eMCAO) model mimics ischemic stroke due to large vessel occlusion in humans and is amenable to thrombolytic therapy with rtPA. However, two major obstacles, the difficulty of the eMCAO surgery and unpredictable occurrence of clot autolysis, had impeded its application in mice. In this study, we modified catheters to produce suitable fibrin-rich embolus and optimized the eMCAO model using cerebral blood flow (CBF) monitored by both laser Doppler flowmetry (LDF) and 2D laser speckle contrast imaging (LSCI) to confirm occlusion of MCA. The results showed that longer embolus resulted in higher mortality. There was a compensatory increase in MCA territory perfusion after eMCAO associated with decreased infarct volume; however, this was only partly dependent on recanalization as clot autolysis was only observed in ∼30% of mice. Cortical CBF monitoring with LSCI showed that the size of peri-core area at 3 h displayed the best correlation with infarct volume that is attributed to compensatory collateral blood flow. The peri-core area best predicted functional outcome after eMCAO. In summary, we developed a reliable eMCAO mouse model that better mimics embolic ischemic stroke in humans, which will increase the potential for successful translation of stroke neuroprotective therapies.

Estrogen restores postischemic pial microvascular dilation

2001 ◽  
Vol 281 (1) ◽  
pp. H155-H160 ◽  
Author(s):  
Y. Watanabe ◽  
M. T. Littleton-Kearney ◽  
R. J. Traystman ◽  
P. D. Hurn
Keyword(s):  
Vascular Endothelium ◽  
Vessel Diameter ◽  
Female Rats ◽  
Cellular Mechanisms ◽  
Vessel Occlusion ◽  
17Β Estradiol ◽  
Pial Vessel ◽  
The Brain ◽  
Experimental Cerebral Ischemia

Estrogen protects the brain from experimental cerebral ischemia, likely through both vascular and neuronal cellular mechanisms. The purpose of this study was to determine whether chronic estrogen treatment in males and repletion in ovariectomized (Ovx) females reverses abnormalities in pial arteriolar reactivity during reperfusion from global forebrain ischemia (4-vessel occlusion, 15 min) and whether the site of protection is vascular endothelium. Male and Ovx female rats were implanted with either placebo or a 25-μg 17β-estradiol pellet 10 days before ischemia. With the use of intravital microscopy, pial vessel dilation to ACh (10 μM) and S-nitroso- N-acetyl-penicillamine (SNAP; 1 μM) and vasoconstriction to serotonin (10 μM) was examined in situ at 30–60 min of reperfusion. Postischemic changes in vessel diameter were compared with preischemic values for each agent. Postischemic response to both ACh and SNAP was lost in males and Ovx females, but not in estrogen pellet-implanted males and estrogen-implanted Ovx females, suggesting that estrogen protects both endothelial and smooth muscle-mediated vasodilation. Ischemia blunted vessel constriction to serotonin regardless of treatment. These data demonstrate that estrogen acts as a vasoprotective agent within the cerebral circulation and can improve microvascular function under conditions of an acutely evolving ischemic pathology.

The Endothelial Protein C Receptor (EPCR) Regulates Endogenous Factor VII Levels in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3629-3629
Author(s):  
Juliana Small ◽  
Shannon Zintner ◽  
Lynn E Dankner ◽  
Paris Margaritis
Keyword(s):  
Mouse Model ◽  
Research Funding ◽  
Protein C ◽  
Binding Capacity ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Gain Of Function ◽  
Vessel Occlusion ◽  
Gla Domain

The endothelial protein C receptor (EPCR) has been demonstrated to bind activated FVII (FVIIa) through the Gla domain with equal affinity to Protein C (PC). Mouse studies suggest that EPCR is involved in the extravasation of infused human FVIIa, leading to an extended extravascular tissue persistence, longer than expected based on its circulating half-life. This provides a plausible explanation for the long-term benefits of hemophilic patients on human FVIIa prophylaxis. Collectively, these data suggest that EPCR sequesters administered FVIIa in tissues where it may have a hemostatic effect. However, the role of the endogenous FVII-EPCR interaction in normal conditions is largely unknown. For this, we have developed a mouse model to better understand this interaction in vivo. Endogenous mouse FVII and FVIIa (mFVII/FVIIa) do not bind mouse EPCR. However, our laboratory has demonstrated that L4F, L8M, and T9R substitutions in the Gla domain of mFVIIa enable its interaction with mouse EPCR while retaining full enzymatic activity in vitro. Based on that data, we utilized CRISPR/Cas9 technology to knock-in L4F, L8M, and T9R into the mFVII Gla domain in the mouse F7 locus (F7FMR), thereby developing mice with a chimeric endogenous FVII capable of binding EPCR. Founder animals were generated and capable of producing offspring, indicating that the gain-of-function in mFVII was compatible with life. Animals were subsequently backcrossed to wildtype C57BL/6 mice in order to remove potential off-target effects of the CRISPR/Cas9. Resultant heterozygous animals (F7FMR/WT) from the final cross were bred to generate F7FMR/FMR, F7FMR/WT, and F7WT/WTlittermates. We generated 59 male and 52 female animals and a binomial distribution test demonstrated that sex is equally distributed in the population. Moreover, the genotypes expected from the heterozygous crosses were inherited in a 1:2:1 ratio, further indicating that the gain-of-function in FVII is not lethal during development. As additional metrics of health, we measured weight longitudinally during weeks 1-10 of life and found no differences between the three genotypes for either gender. Complete blood counts (CBCs) revealed no differences between the F7FMR/FMR, F7FMR/WT, and F7WT/WTgenotypes, with the exception of a mild elevation in F7FMR/WTanimals compared to animals with wildtype FVII. Collectively, we found that the gain-of-function in EPCR binding by endogenous FVII is not detrimental to the overall health of the mice. Subsequently, we determined the mFVII levels in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals using an in-house ELISA. We observed that plasmatic mFVII levels were dependent on the EPCR-binding capacity of the endogenous mFVII. Specifically, F7WT/WTmice, whose mFVII does not bind EPCR, had a plasmatic mFVII concentration of ~690 ng/ml. In contrast, F7FMR/FMRhomozygote mice had ~350 ng/ml of mouse FVII, approximately half the plasma levels of the F7WT/WT. Heterozygote animals F7FMR/WThad an intermediate plasmatic mFVII level (~550 ng/ml), suggesting that EPCR may regulate plasmatic FVII levels in vivo. Lastly, we determined the hemostatic response to injury in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals. We did this in two ways, by measuring blood loss following tail clip assay and by determining time to vessel occlusion following ferric chloride injury of the carotid artery. We observed no differences between the three genotypes in response to either injury model. In conclusion, we have generated and characterized a novel mouse model in which endogenous FVII is capable of binding EPCR. Using this model, we demonstrated that EPCR can modulate plasmatic FVII levels in vivo but does not appear to affect hemostasis. Since this model mimics the FVII-EPCR interaction in humans, it can now be used to further investigate how this interaction participates in other normal or pathologic states that depend on FVII and/or EPCR. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol-Myers Squibb: Other: Salary (spouse); CSL Behring: Other: Salary (spouse); NovoNordisk A/S: Research Funding.

scholarly journals Antibody-mediated depletion of human CLEC-2 in a novel humanised mouse model

2021 ◽  
Author(s):  
Helena C Brown ◽  
Sarah Beck ◽  
Stefano Navarro ◽  
Ying Di ◽  
Eva M Soriano Jerez ◽  
...  
Keyword(s):  
Mouse Model ◽  
Thrombus Formation ◽  
Receptor Expression ◽  
Platelet Glycoprotein ◽  
Endogenous Ligand ◽  
Limited Effect ◽  
Vessel Occlusion ◽  
Novel Therapeutics ◽  
Tumour Metastasis

Platelet C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential anti-thrombotic target as genetic or antibody-mediated receptor deficiency prevents occlusive thrombus formation in mice. This occurs through interaction with an unknown ligand as the endogenous ligand podoplanin is not present in the vasculature. However, the CLEC-2-podoplanin interaction does have an important role in tumour metastasis. There are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo. We have therefore generated and characterised a humanised CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing. hCLEC-2KI mice were phenotypically normal and had comparable platelet glycoprotein receptor expression, activation and aggregation to wildtype platelets. hCLEC-2KI mice had both comparable bleeding and vessel occlusion times to WT mice. Challenging hCLEC-2KI mice with HEL1 or a second monoclonal anti-hCLEC-2 antibody, AYP1, resulted in transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks but had no effect on haemostasis. This illustrates the power of the humanised CLEC-2 mouse model in evaluating novel therapeutics in vivo, including antibodies that target CLEC-2, as well as the limited effect on haemostasis when targeting CLEC-2.

Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4870-4877 ◽  
Author(s):  
Julie Rayes ◽  
Martine J. Hollestelle ◽  
Paulette Legendre ◽  
Isabelle Marx ◽  
Philip G. de Groot ◽  
...  
Keyword(s):  
Mouse Model ◽  
Clinical Manifestations ◽  
Von Willebrand Disease ◽  
Bleeding Tendency ◽  
Vessel Occlusion ◽  
Giant Platelets ◽  
Circulating Platelet Aggregates ◽  
Platelet Aggregates ◽  
Von Willebrand

Abstract Von Willebrand disease (VWD)–type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

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