Hematologic Recovery and Survival of Irradiated Mice. By Shu Tanikawa ... (rhG-CSF) according to various administration schedules on the recovery of .... cology, National Institute of Radiological Sciences, Chiba: and. First Department of ...
RAPID COMMUNICATION
Effects of Recombinant Human Granulocyte Colony-Stimulating Factor on the Hematologic Recovery and Survival of Irradiated Mice By Shu Tanikawa, Masako Nose, Yoshiro Aoki, Kazuko Tsuneoka, Mikio Shikita, and Nobuo Nara We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony-forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not.
Twice-daily injections of rhG-CSF from day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twicedaily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-. and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays. 0 1990 by The American Society of Hematology.
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H E MAJOR CAUSE of death of patients exposed to lethal doses of whole body irradiation is hematopoietic injury. Thus far, bone marrow transplantation (BMT) has been considered the only approach to rescue these patients. However, BMT is not necessarily practical for the treatment of radiation accident victims. For example, the estimation of radiation doses and the measurement of patients’ HLA types are very difficult in these situations; and radiation exposures received as a result of accidents are not usually uniform in dose distribution. The developments of agents that improve the recovery of the surviving fraction of bone marrow cells are required for the treatment of radiation accident victims. We previously demonstrated that twice-daily i.p. injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from day 0 to day 6 enhanced the recovery of spleen colony-forming units (CFU-S), colony-forming units in culture (CFU-C), erythroid burst-forming units (BFU-E), and megakaryocyte colony-forming units (CFUMeg) on day 7 in the bone marrow of BDF, mice irradiated with 7.5-Gy x-rays.’ Schuening et aI2 also reported that twice-daily subcutaneous injections of rhG-CSF directly after a lethal dose of irradiation were effective in rescuing 4 of 5 dogs. On the other hand, all five dogs died with infections due to marrow aplasia when they delayed the start of treatment by 7 days after irradiation. In the present study, we show the importance of timing and duration of rhG-CSF treatment, and examine the effects of i.p. injections of rhG-CSF in various administration schedules on the hematologic recovery and survival of irradiated mice.
biologic responses obtained from in vivo experiments are not due to endotoxin contaminated in G-CSF. Assay of CFU-S. The CFU-S were assayed with the method of Till and M~Culloch.~ Briefly, pooled bone marrow cells were suspended in saline and IO5 cells were injected into each female recipient that had been irradiated with 8.5-Cy x-rays shortly before use. Ten days after the innoculation, the recipient mice were killed and their spleens were removed and fixed with Bouin’s solution. Colonies on the surface of the spleen were counted. Four or five mice were used for each assay point. Statistical analysis. The average numbers of CFU-S per femur, leukocytes, platelets, and values of hematocrit were analyzed by the Student’s t-test and reported as mean f SE. The differences in survival of irradiated mice were determined using the Cox-Mantel test.
MATERIALS AND METHODS
From the Divisions of Radiation Health and Chemical Pharmacology, National Institute of Radiological Sciences, Chiba: and First Department of Internal Medicine, Tokyo Medical and Dental University, Japan. Submitted April 4, 1990; accepted May 1, 1990. Address reprint requests to Shu Tanikawa. MD, First Department of Internal Medicine, Tokyo Medical and Dental University, 5-45. Yushima-I-chome, Bunkyo-ku, Tokyo, I 13, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7603-0030$3.00/0
Mice. Mice used were 12- to 16-week-old males of the strain BDF, (DBA/2 x C57BL), bred in our institute and matched for body weight before use. Ten days before starting experiments, all mice were fed with autoclaved food and water containing gentamicin sulfate and chlortetracycline hydrochloride at the concentration of 40 rg/mL and 110 fig/mL, respectively. Irradiation. Mice were irradiated with x-rays at a dose rate of 60 cGy/min. The physical factors used were 200 kilovolts (peak), 20 mA with 0.5-mm Cu and 0.5-mm AI filters. rhC-CSF. rhG-CSF was provided by Kirin-Brewery Co (Tokyo, Japan). The specific activity of rhG-CSF was approximately 10’ U/mg pure protein.’ A solution of 0.5 mg/mL of purified rhG-CSF contained less than 0.5 ng/mL endotoxin. The data ensure that the Blood, VOI 76,
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RESULTS
CFU-S in the femur of irradiated mice. According to the administration schedules illustrated in Fig 1, three mice of each group received twice-daily (for 7 days) injections of rhG-CSF or normal saline from about 2 hours after irradiation and were killed on day 7. Bone marrow cell suspensions obtained from the femurs of mice of each group were mixed and used for CFU-S assays. This experiment was repeated four times. The number of CFU-S per femur are illustrated in Fig 1. Twice-daily injections of rhG-CSF from day 0 to day 4 or day 6 significantly increased the number of CFU-S per femur, as compared with injections of saline alone. A single injection of rhG-CSF (2.0 rgldose) on day 0 and two injections of rhG-CSF (1.0 rgldose) on day 6 scarcely
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