Rodent Leukocyte Isolation and Radiolabeling for Inflammation Imaging Study

김은미,오필선,BOUD FATIMA MUNEIM HAMAD,정환정,임석태,손명희 대한핵의학회 2020 핵의학 분자영상 Vol.54 No.3

Purpose The objective of this study was to describe to develop methods of rodent leukocyte isolation and radiolabeling for in vivo inflammation imaging. Methods Thigh muscle inflammation was induced by injection of collagenase. Blood was collected from the jugular vein and separated by Histopaque. The collected cells were incubated in a 37 °C CO2 incubator for 1~2 h. After incubation, 99mTc- HMPAO and 18F-FDG were used to treat leukocytes followed by incubation for 30 min. 99mTc-HMPAO and 18F-FDG labeled autologous leukocytes were injected into the tail veins of rats. The images were then acquired at various time points. Image-based lesion to normal muscle ratio was compared. Results After Histopaque separation, the proportion of lymphocytes was higher than that of other cell types. After CO2 incubation, the collected leukocytes were viable, while room temperature exposed leukocytes without CO2 incubation were non-viable. Granulocytes, especially, were more quickly influenced by various conditions than the mononuclear cells. Labeling efficiencies of 99mTc-HMPAO and 18F-FDG were 4.00 ± 2.06 and 1.8%, respectively. 99mTc-HMPAO- and 18F-FDG-labeled leukocytes targeted well the inflamed lesion. 99mTc-HMPAO-labeled leukocytes, but not 18F-FDG-labeled leukocytes, were found in the abdomen activity. Conclusion Inflamed lesions of rats were well visualized using autologous radiolabeled leukocytes. This method might provide good information for understanding inflammatory diseases.

허혈-재관류에 의해 유도된 백혈구-혈관내피세포 유착에 대한 Videomicroscopy 영상소견

이영배,강한석,박신병,Lee, Young Bae,Kang, Han Sug,Park, Shin Byung 대한신경외과학회 2000 Journal of Korean neurosurgical society Vol.29 No.10

Purpose : Recent evidence suggests a possible role for leukocytes in brain injury following ischemia and reperfusion. This study examined the temporal profile of ischemic tissue damage and leukocyte response after transient middle cerebral artery occlusion(MCAO) with reperfusion in the mouse. Methods : Focal cerebral ischemia was made by temporary occluding of the stem of the proximal MCA. Two groups of the mouse were investigated : (1) sham operation(n=10), and (2)those having the arterial occlusion released after 90 minute(n=20). By 4 hours(n=10) and 24 hours(n=10) after the onset of ischemia-reperfusion, fluorescein videoimages were under-taken in the pial venules of the mouse using a closed cranial window technique. Rhodamine 6G was administered as a $80-100{\mu}l/min$ i.v. loading dose and a $30-40{\mu}l/min$ i.v. maintenance dose in saline to selectively label circulating leukocytes. Neuropathologic evaluation for brain injury was accomplished using the histochemical stain 2,3,5-triphen-yltetrazolium chloride(TTC) and hematoxylin and eosin(H & E) stain. Results : The mean number of adherent leukocytes to cerebral venules in the 90 minutes MCAO and 24 hours reperfusion group were $306{\pm}24$ compared with $72{\pm}8$ in the sham operation group. In the TTC staining method, the cortical infarct affecting 34.8% of hemispheric volume were created in all of animals (n=10) undergoing 90 minute MCAO with 24 hours reperfusion, but the infarcted area were not found in the other(sham operation and 90 minute MCAO with 4 hours reperfusion)groups. In the H & E stain, the brain tissue following 90 minute MCAO with 4 hours reperfusion revealed only a pyknosis of the nuclei with shrunken cytoplasm, but infiltrated leukocytes were not observed. After 24 hours of reperfusion, a many leukocytes were infiltrated within parenchyma and blood vessles. Conclusions : These findings demonstrate the feasiblity of continous in vivo monitoring of leukocyte adherence in cerebral venules and suggest that reperfusion induced leukocyte adherence to venular endothelium may contribute to tissue injury following focal cerebral ischemia.

자동혈구분석기 Hemavet 950FS를 이용한 돼지 혈액 내 혈구분석의 유효시간 평가

김기현(Ki-Hyun Kim),김광식(Kwang-Sik Kim),김두완(Doo-Wan Kim),사수진(Soo-Jin Sa),김영화(Young-Hwa Kim) 한국산학기술학회 2017 한국산학기술학회논문지 Vol.18 No.1

본 연구는 자동혈구분석기 Hemavet 950FS를 이용한 돼지 혈액 내 혈구분석의 유효성을 검증하고, 정확한 분석을 위한 유효시간을 결정하기 위하여 실시하였다. 분석시료는 체중 60 kg (±3.5)의 Duroc 종 34두로부터 채취하였으며, 이들 중에서 분석치가 정상범위를 벗어나거나 용혈이 발견된 시료는 연구에서 제외하고 최종적으로 10개의 시료를 본 연구에 이용하였다. 임의의 하나의 시료에 대해 10회 연속분석을 통하여 일내변동을 조사한 결과, leukocytes계 혈구들 중에서 백혈구, 호중구, 림프구에서는 낮은 일내변동으로 분석의 유효성이 인정되었으나, 단핵구, 호산구, 호염구에 있어서는 큰 변동을 나타내었다. 이와 반면에 erythrocytes 및 thrombocytes계 혈구들의 모든 지표들은 안정적인 일내변동이 관찰되었다. 채혈 후 시간경과에 따른 혈구변화를 분석한 결과 leukocytes계 혈구 및 thrombocytes계 혈구는 시간경과에 따라 검출농도가 유의적으로 감소되는 것으로 관찰되었다. 반면, erythrocytes계 혈구들은 채혈 후 120시간까지는 안정적으로 분석이 가능한 것으로 나타났다. 본 연구의 결과를 종합하면, Hemavet 950FS는 돼지의 혈액 내에서 단핵구, 호산구, 호염구를 제외하고 일반 혈구분석에 유용한 것으로 판단된다. 또한, leukocytes 및 thrombocytes계 혈구들에 대한 정확한 분석을 위해서는 채혈 후 4시간 이내에 분석이 이루어져야하며, erythrocytes계 혈구들은 채혈 후 120시간까지는 안정적으로 검출이 가능한 것으로 사료된다. This study was conducted to determine the valid time for accurate detection of complete blood cell count (CBC) in pig blood using an automatic blood corpuscle analyzer (Hemavet 950FS). Blood samples were collected from 34 pigs (Duroc) with a 60 kg (±3.5) body weight. Ten specimens with CBC parameters in normal range and with no hemolysis were selected among 34 samples and used in this study. Regarding leukocytes parameters, white blood cell (WBC), neutrophil (NE), and lymphocyte (LY) counts showed a low daily variation (coefficient of variation, CV), whereas monocyte (MO), eosinophil (EO), and basophil (BA) CVs were significantly high (19.7, 56.9, and 53.3%, respectively). On the other hand, all parameters of erythrocytes and thrombocytes showed stable daily variation. All parameters of leukocytes and thrombocytes were significantly reduced as storage time passed (P<0.01 or 0.001), except for lymphocytes (P=0.535). However, no significant differences were observed in parameters of erythrocytes from blood up to 120 hours. From above results, we assert that Hemavet 950FS is useful in analyzing CBC, except for MO, EO, and BA. For accurate detection of leukocyte and thrombocyte parameters, analysis should be performed within 4 hours after blood collection when using Hemavet 950FS. On the other hand, parameters of erythrocytes could be stably detected for at least 120 hours after blood collection.

Changes in muscle damage markers and circulating Leukocytes following Local muscle eccentric contractions of the elbow flexors

이호성 ( H. S. Lee ),박우영 ( W. Y. Park ) 한국운동생리학회(구 한국운동과학회) 2011 운동과학 Vol.20 No.3

The purpose of this study were to measure changes in markers of muscle damage and circulating leukocytes after eccentric contractions of elbow flexors and to determine whether these changes are influenced by a repeated bout of the same exercise. All subjects (n=10) performed two bouts of eccentric exercise of the elbow flexors (10 sets of 6 repetitions) using the non-dominant separated by 4 weeks. Changes in circulating leukocyte numbers (neutrophils, lymphocytes, monocytes and eosinophils) in the blood and muscle damage indicators (MVC, ROM, CIR, VAS and CK) were taken immediately before exercise, immediately after exercise, 24, 48, 72, and 96 hours following each exercise bout. changes in MVC, ROM, CIR, and VAS were significantly larger following the first bout compared to the second bout, CK increased significantly only after the first bout. However, no significant differences between the two bouts were evident for the changes in any of the circulating leukocytes. Therefore, it seems reasonable to state that severe muscle damage by eccentric exercise does not induce significant alterations in circulating leukocytes.

Combined Genotoxic Effects of Aflatoxin B1, Ochratoxin A and Zearalenone in Rat Bone Marrow and Blood Leukocytes

( Harutyunyan Tigran ),( Karapetyan Anna ),( Hovhannisyan Galina ),( Aroutiounian Rouben ) 한국환경생물학회 2013 환경생물 : 환경생물학회지 Vol.31 No.3

Mycotoxins such as aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEA) are widespread contaminants of food and feedstuffs. It is very likely, that humans and animals are always exposed to mixtures of mycotoxins rather than to individual compounds. Therefore, risk assessments should consider mixture toxicity data. In the present study the combination of AFB1, OTA and ZEA was tested for genotoxicity in rat bone marrow and blood leukocytes after 15, 30 and 60 days treatment. The level of DNA damage was determined by the comet assay. The tail intensity and Olive tail moment in leukocytes and bone marrow cells were significantly higher than in controls. At the same time, the level of DNA damage in bone marrow cells was higher than in leukocytes. The data suggests that prolonged exposure to mycotoxins combination through food consumption can induce DNA damage contributing to the harmful effects in vivo.

운지버섯 열수 추출액이 Sarcoma-180 복강암세포에 대한 NK 활성도 및 백혈구의 Chemotaxis에 미치는 영향

고순자,정규선,이연태 숙명여자대학교 약학연구소 2001 약학논문집-숙명여자대학교 Vol.17 No.-

This experiment was conducted to evaluate the inhibitory effect of Water-Soluble Extraction from Coriolus versicolor on the growth of tumor cells through the activation of immunocytes in vivo. The lymphocytes were seperated from the spleen of tumor-bearing mice administrated orally Coriolus versicolor extract. The cytotoxicity of NK cells was observed by using the method of four-hours ^(51)Cr-release. Chemotaxis of leukocytes seperated from peripheral blood was measured using Modified Boyden chamber. The results obtained were summarized as following. 1. The cytotoxicity of NK cells from normal saline treated tumor-bearing mice was not significantly increased, but the cytotoxicity of NK cells from Coriolus versicolor treated tumor-bearing mice was significantly increased (P<0.025). 2. The chemotaxis of leukocytes from normal saline treated tumor-bearing mice was not significantly increased, but the chemotaxis of leukocytes from Coriolus versicolor treated tumor-bearing mice was significantly increased (P<0.025). According to these results, the Water-Soluble Extraction from Coriolus versicolor could inhibit the growth of mouse tumor cells through the activation of cytotoxicity of NK cells and the activation of chemotactic leukocytes.

뇌허혈-재관류 손상실험의 Ideal Model과 뇌허혈-재관류 손상시 뇌세정맥내 백혈구의 유착의 증가

박종운 대한신경외과학회 1996 Journal of Korean neurosurgical society Vol.25 No.3

We developed an in vivo model of cerebral ischemia-reperfusion wherein on-line videoimages of fluorescently-labelled leukocytes flowing within pial venules of anesthetized newborn pigs can be obtained. A closed cranial window was implanted over the parietal cortex, and venules were imaged with an fluorescence microscope(Olympus BHMJ) outfitted with a 100 watt mercury lamp, rhodamine filter cube, 10×immersion lens(Olympus : 0.4NA), and newvicon tube campera. Final image magnification captured to Super VHS videotape was 470×. Rhodamine 6G was administered as a 0.5㎎/㎏ i.v. loading dose and a 0.5㎎/㎏/h i.v. maintenance dose in saline to selectively label circulating leukocytes. We made an ideal model with 6 piglets and under basaline conditions in piglets, few labelled leukocytes were observed adhering to the endothelium of 50-100㎛ diameter venules. However, following 10 min, of global cerebral ischemia induced by reversible subclavian and branchiocephalic occlusion, a dramatic increase in rolling and adherent leukocytes was observed at 30, 60, and 90 min of reperfusion. At these tims, cortical blood flow, measured by both laser doppler flowmetry and hydrogen clearance, was decreased by 25∼40%(p<0.05). These findings demonstrate the feasibility of continuous in vivo monitoring of leukocyte adherence in cerebral venules, and suggest that reperfusion-induced adherence to venular endothelium may contribute to hypoperfusion following global cerebral ischemia.

Leukocyte-Stimulating Effect and Phytochemical Screening of Trichilia hirta Extracts Edgar Hernandez Sosa, Yulianis Martín Castejón, Alexander Batista Duharte, Deivys

Edgar Hernandez Sosa,Yulianis Martín Castejón,Alexander Batista Duharte,Deivys Portuondo,Vivian Tamayo,Humberto J. Morris Quevedo,Clara Esther Martínez Manrique 한국식품영양과학회 2011 Journal of medicinal food Vol.14 No.9

Trichilia hirta (Family Meliaceae) is a tree traditionally used in the folk medicine of Cuba to treat asthma, cancer, and ulcers. The objective of this study was to determine the phytochemical composition of ethanol extracts obtained from leaves, roots, and stem bark and to evaluate the leukocyte-stimulating effect of T. hirta root extracts on BALB/c mice. The chemical composition of the extracts was determined by phytochemical screening. Saponins, tannins, flavonoids, and coumarins were detected in extracts of T. hirta. The leukocyte-stimulating effect was evaluated by oral application of ethanol extracts (81.8 and 976 mg/kg) in BALB/c mice for 7 days. The application of 976 mg of extract/kg increased the total leukocyte count up to 15–33%; this effect was significant for neutrophil counts compared with control animals (P<.05). In addition, a dose of 82 mg/kg significantly increased total leukocytes at day 4 of the study (P<.05). The results indicated that T. hirta extracts contain phytochemicals reported as immunostimulants. The administration of these extracts to BALB/c mice indicated that ethanol extract could exhibit leukocyte-stimulating properties and makes it a promising alternative for the development of an immunoprotective agent.

덱스트란-단백질 복합체의 제조 및 이의 혈구 거동

윤취임,김진일,김승수 한국생체재료학회 2009 생체재료학회지 Vol.13 No.2

Dextran-albumin and dextran-gelatin complexes were prepared and evaluated for coating at a leukocyte removal filter to improve a removal rate of leukocytes. Dextran aldehyde was prepared to make cross-linked polymer complexes. The chemical characteristics of the complexes were carried out with Fourier transform-infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Water contact angle and water absorbancy of the polymer complexes were measured to evaluate hydrophilicity. The recovery rate of red blood cells was above 97% and was not dependent on the polymer complexes. The removal rate of leukocyte by dextran-albumin complexes was higher than that by dextran- gelatin complexes. The removal rate of leukocytes by dextran-albumin complexes improved with increased the amount of dextran in the complex. It was 82.9 ± 3.4% when the complex contained 75% of dextran.

한국 성인의 비만과 과체중에 따른 혈청 페리틴과 백혈구의 차이

이혜순(Lee Hea Shoon) 한국기초간호학회 2019 Journal of korean biological nursing science Vol.21 No.2

Purpose: The purpose of this study was to investigate the difference in serum ferritin and leukocyte regarding overweight and obese South Korean adults. Methods: This study was conducted on 5,281 subjects older than 19, according to data from the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-3), 2015. Data were analyzed using descriptive statistics, t-test, ANOVA, Scheffe s test, Pearson’s correlation coefficient, and stepwise multiple regression analysis (SPSS 24.0). Results: First, serum ferritin and leukocyte were higher regardubg obesity, followed by being overweight and within normal weight. Second, body mass index (BMI) was positively correlated with serum ferritin and leukocyte. Third, factors affecting serum ferritin were gender, and being obese and overweight. Explanatory power of the model was 26.2%. Factors affecting leukocyte were gender, obesity, being overweight, and weight change over the past year (weight gain). Explanatory power of the model was 10.2%. Conclusion: Obesity and being overweight were factors affecting serum ferritin and leukocyte, and obesity was more affected than being overweight in Koreans older than 19. In conclusion, serum ferritin was a marker of inflammation, rather than iron status, in overweight and obese Korean adults.