Towards a standardization of the murine tail bleeding model

Greene, Teshell K.; Schiviz, Alexandra; Hoellriegl, Werner; Poncz, Mortimer; Muchitsch, Eva-Maria

doi:10.1111/j.1538-7836.2010.04084.x

Cited by 76 publications

(89 citation statements)

References 14 publications

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“…However, no significant differences were observed between the TULA-2 −/− and TULA-2 +/+ groups ( Figure Under normal physiological wound healing process, platelets gradually retract and form dense fibrin-platelet plugs to prevent further blood loss and protect deeper tissue for healing. 34,35 Therefore, we studied clot retraction, another readout for platelet outside-in signaling. Platelet-rich plasma isolated from TULA-2 −/− or TULA-2 +/+ mice were stimulated with 1 U/mL thrombin, and retraction was measured.…”

Section: Knockout Of Tula-2 May Not Affect the Platelet Outside-in Simentioning

confidence: 99%

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice

Zhou

Abraham

Renna

et al. 2016

ATVB

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Objective-The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). Approach and Results-HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2 −/− mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2 −/− murine platelets. Compared with wild-type mice, TULA-2 −/− mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2 −/− and TULA-2 +/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2 +/− mice, whose platelets contained 50% as much protein as the TULA-2 +/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2 +/+ mice. Conclusions-Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

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Section: Knockout Of Tula-2 May Not Affect the Platelet Outside-in Simentioning

confidence: 99%

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice

Zhou

Abraham

Renna

et al. 2016

ATVB

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“…The mice were anesthetized with 100 mg kg -1 ketamine and 10 mg kg -1 xylazine and placed in a warming chamber. Two millimeters of the tail tip were clipped with a guillotine [4], the tails were immersed in warm saline (2 mL, 35 AE 2°C), and blood was collected over a period of 60 min; anesthesia was maintained as required by subcutaneous supplementation with one-third of the starting dose. Blood loss was determined gravimetrically.…”

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confidence: 99%

Influence of genetic background on bleeding phenotype in the tail‐tip bleeding model and recommendations for standardization: communication from the SSC of the ISTH

Schiviz

Magirr

Leidenmühler

et al. 2014

Journal of Thrombosis and Haemostasis

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The murine tail-tip bleeding model is widely used to measure bleeding time, blood loss, and survival after treatment [1][2][3][4]. Because of the considerable variation in data from these studies and thus the need for large sample sizes per group, and the lack of comparability of results from different laboratories, research groups have tried to standardize the model. In 2010, the SSC of the ISTH proposed standardized methods for determining tail bleeding time in mice. The SSC report standardized the tail clipping by endorsing the use of a warming chamber to evenly warm the anesthetized mouse, and assessing the influence of anesthesia and systolic blood pressure on blood loss [5].We sought to test the ability of the standardized method to detect differences in bleeding phenotypes of wild-type mouse strains, which may show different bleeding phenotypes [6]. The relevance of the genetic background of inbred mouse strains or transgenic mice on phenotype has been described in various research fields. Mice may differ in physiologic parameters [7] or in response to triggers. Polymorphisms exist for substrains of the same inbred strains [8]. Genetic background also influences pathways that regulate thrombosis and hemostasis: differences were observed in global bleeding and thrombosis assays [9], coagulation and fibrinolytic factors [10], and susceptibility to treatment [11].To investigate the ability of the tail-tip bleeding assay to detect the influence of genetic background on bleeding phenotype, we used hemophilic mice on different backgrounds, i.e. coagulation factor VIII (exon 17) knockout (FVIII À/À ) [12] mice on a BALB/c, C57BL/6, or mixed background, along with different substrains of wild-type C57BL/6 or BALB/c mice ( Table 1). The mice were anesthetized with 100 mg kg -1 ketamine and 10 mg kg -1 xylazine and placed in a warming chamber. Two millimeters of the tail tip were clipped with a guillotine [4], the tails were immersed in warm saline (2 mL, 35 AE 2°C), and blood was collected over a period of 60 min; anesthesia was maintained as required by subcutaneous supplementation with one-third of the starting dose. Blood loss was determined gravimetrically. At the end of this observation period, the mice were removed from the chamber and humanely killed by cervical dislocation. Pairwise tests of equality of variance were performed with a bootstrap approach; the level of statistical significance was set to P = 0.05.C.FVIII À/À mice showed markedly lower median blood loss (365.7 mg) than B6.FVIII À/À (965.2 mg) and B6; FVIII À/À (890.4 mg) mice. For all three strains, median blood loss was lower in females than in males, with the largest sex difference (634 mg vs. 954 mg for females vs. males) and the highest interindividual variation being observed in B6;FVIII À/À mice (Fig. 1). In 129S1/SvmJ mice and the four substrains of BALB/c mice, median blood loss (23.5 mg and 10.0-19.0 mg, respectively) and interanimal variability were low. In contrast, median blood loss in C57BL/6 substrains varied from 36.0 mg (C57BL/...

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“…The results of this study are consistent with the research conducted by Greene, Schivis, Hoellriegl, Poncz, and Muchitsch (2010). Greene et al (2010) stated that the bleeding time of normal mice is 51-62 seconds. Heparin which injected intravenously to provided enough hemorrhage effect demonstrated with average bleeding time is six times greater than normal bleeding time.…”

Section: Resultsmentioning

confidence: 99%

“…Heparin which injected intravenously to provided enough hemorrhage effect demonstrated with average bleeding time is six times greater than normal bleeding time. The aim of heparin's injection in this study was to eliminate the bias of clotting factors in the body of mice (Greene et al, 2010;Gray et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

Hemostatic Effect of Ethanol Extract of Piper betle, Linn Leaves to Male Mice

Sinulingga

Subandrate²,

Kesumaputri³

et al. 2017

Molekul

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Hemorrhage occurs in most of the dental care. Untreated hemorrhage could cause excessive blood loss, hypotension, and cyanosis. A Natural resource that reported has an hemostatic effect is ethanol extract of betel leaves (Piper betel, Linn).The aim of this study is to find the minimum concentration of ethanol extract of betel leaves which capable of shortening the bleeding time in mice. The experimental study used pretestposttest with control group design was conducted on 35 mice that divided into 7 group which are negative control, positive control (feracrylum 1%), the ethanol extract of betel leaves 1%, 5%, 10%, 15%, and 20%. All mice were injected heparin intravenously. Mice's tail was cut at diameter 3 mm and pretest bleeding time was counted. Mice's tail was recut at diameter 4 mm, given treatment for 5 seconds and posttest bleeding time was counted. Results of paired t-test showed that reduction of bleeding time between pretest and posttest was significant (p<0,050). The enhancement of ethanol extract of betel leaves concentration leads to better hemostatic effect. Results of ANOVA test showed that comparison of posttest bleeding time among groups was significant (p<0,050). The minimum concentration of ethanol extract of betel leaves which capable of shortening the bleeding time in mice is 5%.Keywords: bleeding, bleeding time, dental care, the ethanol extract of betel leaves. ABSTRAKPerdarahan terjadi pada sebagian besar perawatan gigi. Perdarahan yang tidak ditangani dapat menyebabkan terjadinya kehilangan darah yang berlebihan, hipotensi, dan sianosis.Bahan alami yang dilaporkan memiliki efek hemostatik adalah ekstrak etanol daun sirih hijau (Piper betle, Linn).Penelitian ini bertujuan mengetahui konsentrasi minimal ekstrak etanol daun sirih hijau yang dapat memperpendek waktu perdarahan mencit.Penelitian ini bersifat eksperimental dengan pretest-posttest control group designmenggunakan 35 ekor mencit yang dibagi dalam 7 kelompok, yaitu kelompok kontrol negatif, kontrol positif (feracrylum 1%), ekstrak etanol daun sirih hijau 1%, 5%, 10%, 15%, dan 20%. Semua mencit diinjeksi heparin intravena.Kemudian ekor mencit dipotong pada diameter 3 mm dan dihitung waktu perdarahan sebelum perlakuan. Ekor mencit dipotong kembali pada diameter 4 mm, diberikan perlakuan selama 5 detik, dan dihitung waktu perdarahan setelah perlakuan menggunakan stopwatch.Hasil uji t berpasangan waktu perdarahan sebelum dan sesudah perlakuan menunjukkan penurunan signifikan (p<0,05).Semakin tinggi konsentrasi ekstrak etanol daun sirih hijau, maka efek hemostatik semakin baik.Hasil uji ANOVA menunjukkan bahwa perbandingan rata-rata waktu perdarahan antar kelompok setelah perlakuan signifikan (p<0,05). Konsentrasi minimal ekstrak etanol daun sirih hijauyang dapat memperpendek waktu perdarahan mencit adalah 5%.Kata Kunci: ekstrak etanol daun sirih hijau, perawatan gigi, perdarahan, waktu perdarahan.

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Towards a standardization of the murine tail bleeding model

Cited by 76 publications

References 14 publications

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice

Influence of genetic background on bleeding phenotype in the tail‐tip bleeding model and recommendations for standardization: communication from the SSC of the ISTH

Hemostatic Effect of Ethanol Extract of Piper betle, Linn Leaves to Male Mice

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