Citation: Nosaka M, Ishida Y, Kimura A, Yamamoto H, Kuninaka Y, Shimada E, et al. Infuence of Circadian Rhythm on Thrombus Formation of Murine Deep Vein Thrombosis Model. Ann Hematol Oncol. 2017; 4(9): 1171. Ann Hematol Oncol - Volume 4 Issue 9 - 2017 ISSN : 2375-7965 | www.austinpublishinggroup.com Nosaka et al. © All rights are reserved Annals of Hematology & Oncology Open Access Abstract Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. In previous studies, how the time of therapy or drug delivery can impact treatment effect and outcomes. This study was to determine whether stasis-started time infuenced thrombus formation and resolution response. The mice ligated at 12:00 PM had decreased survival rate than the mice ligated at 7:00 AM. The thrombus size of at 12:00 PM-ligated mice were larger than thrombi of at 7:00-AM-ligated ones. In 12:00 PM-ligated mice, the Mmp9 gene expressions were lower than 7:00 AM-ligated ones. The treatment time may affect response in mice receiving stasis for formation of thrombi. Subsequent chronotherapy studies in anticoagulant therapy should investigate the time of therapy or medication. Keywords: Deep vein thrombosis; Pulmonary thromboembolism; Intravenous thrombus Introduction Deep vein thrombus (DVT) is a major problem as the cause of pulmonary thromboembolism (PTE) from both clinical and forensic aspects, and it is multifactorial and ofen results from a combination of risk factors such as genetic conditions, obesity, drugs, pregnancy, aging, trauma, and malignancy. DVT is frequently complicated with severe morbidity, leading sometimes to mortality. But the start time of the event that was a critical turning point and severity of thrombosis was not clear. Recently, in the researches were performed on circadian rhythm or chronotherapy. In this study, we found out the infuence of the time of surgical operation and stasis-state for thrombus formation using stasis-induced murine DVT model. Methods Mice Pathogen-free 8- to 10-week-old male BALB/c mice were obtained from SLC and designated as WT mice in this study. All mice were bred and housed at a constant temperature (23±2 ° C), with a 12-h light/dark cycle (light on at 8:00 AM and of at 8:00 PM). Tey were fed with standard feed and given water ad libitum. All animal experiments were approved by the Committee on Animal Care and Use at Wakayama Medical University. Stasis-induced DVT model Intravenous thrombus was induced as described previously. Briefy, afer deep anesthesia with intraperitoneal injection of 2,2,2-tribromoethanol (Avertin, 240 mg/kg body weight), a 1-cm incision was made along the abdominal midline, and the inferior vena cava (IVC) was exposed and ligated with 3-0 silk suture. Te abdominal wall was closed, and 1 ml of PBS was injected subcutaneously [1-3]. Te ligation time was at 7:00 AM and at 12:00 PM. Tis operation was performed within 7 minutes to exclude an efect of the ligation time lag. Special Article - Thrombosis Infuence of Circadian Rhythm on Thrombus Formation of Murine Deep Vein Thrombosis Model Nosaka M*, Ishida Y, Kimura A, Yamamoto H, Kuninaka Y, Shimada E, Okumoto K, Okada M, Hata S, and Kondo T Department of Forensic Medicine, Wakayama Medical University, Japan *Corresponding author: Nosaka M, Department of Forensic Medicine, Wakayama Medical University, Wakayama, Japan Received: August 22, 2017; Accepted: September 09, 2017; Published: October 12, 2017 Immunohistochemical staining Intravenous thrombi, obtained at the indicated time intervals afer IVC ligation, were fxed in 4 % formaldehyde bufered with PBS (pH 7.2) and transversely cut in the middle of the thrombus, and parafn- embedded sections (4-μm thick) were made. Immunostaining was automatically performed by Ventana Discovery ® XT (Ventana Medical Systems, Inc., AZ). Te primary Abs were diluted with the blocking bufer (PBS containing 1% normal serum corresponding to the secondary Abs and 1 % bovine serum albumin) to reduce nonspecifc reactions. Tereafer, the sections were reacted with anti- MPO mAbs at 37 ° C for 60 min. Afer incubation with biotinylated secondary Absat 37 ° C for 60 min, the DAB Map kit (Ventana Medical Systems, Inc., AZ) was used to visualize the antigens for all stains. Subsequently, all slides were counterstained with hematoxylin or lithium carbonate to visualize the nuclei [3-5]. Extraction of total RNAs and real-time RT-PCR Real-time RT-PCR was performed as described previously. Briefy, total RNA was extracted from thrombi samples using ISOGEN (Nippon Gene) according to the manufacturer’s instructions, and 1 μg of total RNA was reverse transcribed into cDNA. Tereafer, generated cDNA was subjected to real-time PCR analysis using SYBR Premix Ex Taq II kit (Takara Bio) with the specifc primers sets. Relative quantity of target gene expression to β-actin gene was measured by comparative Ct method [6]. Measurement of PT and APTT At 7:00 AM or at 12:00 PM, the blood was obtained from the hearts of mice. Tese blood samples were taken with 3.8% citrate solution and centrifuged to obtain plasma samples. PT and APTT of citrated plasma samples were measured by using COAGSEARCH (A&T) according to the manufacturer’s instructions [6]. Results We found out the mice reacted slowly to anesthesia at 7:00 AM,