Zuluaga Andres F, Salazar Beatriz E, Rodriguez Carlos A, Zapata Ana X, Agudelo Maria, Vesga Omar
GRIPE: Infectious Disease Problems Research Group, University of Antioquia Medical School, Medellín, Colombia.
BMC Infect Dis. 2006 Mar 17;6:55. doi: 10.1186/1471-2334-6-55.
For its low cost and ease of handling, the mouse remains the preferred experimental animal for preclinical tests. To avoid the interaction of the animal immune system, in vivo antibiotic pharmacodynamic studies often employ cyclophosphamide (CPM) to induce neutropenia. Although high doses (350-450 mg/kg) are still used and their effects on mouse leukocytes have been described, a lower dose (250 mg/kg) is widely preferred today, but the characteristics and applicability of this approach in outbred mice have not been determined.
Fifteen female ICR mice were injected intraperitoneally with 150 and 100 mg/kg of CPM on days 1 and 4, respectively. Blood samples (approximately 160 microL) were drawn from the retro-orbital sinus of each mouse on days 1, 4, 5, 6, 7 and 11. Leukocytes were counted manually and the number of granulocytes was based on microscopic examination of Wright-stained smears. The impact of neutropenia induced by this method was then determined with a variety of pathogens in three different murine models of human infections: pneumonia (Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus), meningoencephalitis (S. pneumoniae), and the thigh model (S. aureus, Escherichia coli, Bacteroides fragilis).
The basal count of leukocytes was within the normal range for outbred mice. On day 4, there was an 84% reduction in total white blood cells, and by day 5 the leukopenia reached its nadir (370 +/- 84 cells/mm3). Profound neutropenia (< or =10 neutrophils/mm3) was demonstrated at day 4 and persisted through days 5 and 6. Lymphocytes and monocytes had a 92% and 96% decline between days 1 and 5, respectively. Leukocytes recovered completely by day 11. Mice immunosupressed under this protocol displayed clinical and microbiological patterns of progressive and lethal infectious diseases after inoculation in different organs with diverse human pathogens.
A CPM total dose of 250 mg/kg is sufficient to induce profound and sustained neutropenia (<10 neutrophils/mm3) at least during 3 days in outbred mice, is simpler than previously described methods, and allows successful induction of infection in a variety of experimental models.
由于成本低且易于操作，小鼠仍然是临床前试验首选的实验动物。为避免动物免疫系统的相互作用，体内抗生素药效学研究通常采用环磷酰胺（CPM）诱导中性粒细胞减少。尽管仍使用高剂量（350 - 450mg/kg），且其对小鼠白细胞的影响已有描述，但如今较低剂量（250mg/kg）更受广泛青睐，不过这种方法在远交系小鼠中的特性和适用性尚未确定。
15只雌性ICR小鼠分别在第1天和第4天腹腔注射150mg/kg和100mg/kg的CPM。在第1、4、5、6、7和11天从每只小鼠的眶后窦采集血样（约160μL）。手动计数白细胞，并通过对瑞氏染色涂片的显微镜检查确定粒细胞数量。然后在三种不同的人类感染小鼠模型中，用多种病原体确定这种方法诱导的中性粒细胞减少的影响：肺炎（肺炎克雷伯菌、肺炎链球菌、金黄色葡萄球菌）、脑膜脑炎（肺炎链球菌）和大腿模型（金黄色葡萄球菌、大肠杆菌、脆弱拟杆菌）。
远交系小鼠白细胞的基础计数在正常范围内。在第4天，白细胞总数减少了84%，到第5天白细胞减少达到最低点（370±84个细胞/mm³）。在第4天出现严重中性粒细胞减少（≤10个中性粒细胞/mm³），并持续到第5天和第6天。淋巴细胞和单核细胞在第1天和第5天之间分别下降了92%和96%。白细胞在第11天完全恢复。按照该方案免疫抑制的小鼠在接种不同人类病原体至不同器官后，表现出进行性和致命性传染病的临床和微生物学模式。
25mg/kg的CPM总剂量足以在远交系小鼠中至少3天诱导严重且持续的中性粒细胞减少（<10个中性粒细胞/mm³），比先前描述的方法更简单，并能在多种实验模型中成功诱导感染。
