Background
Currently, animal models of infection are considered essential for the study of physiopathology and pharmacology of infectious disease, being the best tool to bridge in vitro studies and clinical trials [
1]. The neutropenic mouse thigh infection model has been extensively used, and its impact on the current knowledge of antimicrobial pharmacology is undisputable [
2]. It allowed the quantitative determination of pharmacokinetic (PK) and pharmacodynamic (PD) indices (PK/PD) useful to predict antibiotic efficacy while minimizing selection of resistant mutants [
2]. This revolutionary advance in pharmacology was reached after characterizing the complex relationship between host, drug and pathogen, thanks to the unique opportunity offered by animal models for comparing the responses to infection and therapy under several immune statuses [
3].
Antibiotic activity can be positively influenced by the host immune system. For instance, the magnitude of the PK/PD index necessary for successful therapy is reduced in animal models by the presence of neutrophils [
3,
4]. Thus, in order to study in vivo the intrinsic activity of antibiotics without the influence of the immune system, animals are commonly rendered neutropenic with cytotoxic agents like 5-fluorouracil, methotrexate, and cyclophosphamide (CPM). In fact, the latter is the most frequently used immunosuppressive agent in experimental anti-infective pharmacology. CPM is a prodrug requiring metabolic transformation to generate active alkylating species. The initial activation is mediated by hepatic cytochrome P-450 enzymes and is the major pathway of elimination of the parent drug. Hydroxylation at the carbon-4 position of the oxazaphosphorine ring produces 4-hydroxycyclophosphamide in equilibrium with the tautomer aldophosphamide, which spontaneously degrades to phosphoramide mustard and acrolein [
5]. These alkylating species bind to DNA and induce strand breakage and cross-linking, killing actively replicating cells [
6].
Although the effects in mice of high doses of CPM (350–450 mg/kg) have already been described [
7] and some authors still use them [
8‐
10], a lower dose (250 mg/kg) is preferred nowadays [
11‐
13]. The low dose regimen is derived from a rat model [
14,
15], but the length and extent of the induced neutropenia in outbred mice have not been characterized in detail [
16]. At the same time, outbred mice are preferred for infection models because their genetic diversity allows determination of individual responses to infection and treatment. Such individuality constitutes another reason to study their response to the method of immunosupression.
The aim of this study was to evaluate the efficacy of a simplified 250 mg/kg regimen of CPM for murine immunosupression, characterizing its effects on white blood cells, including neutrophils, lymphocytes and monocytes, and its impact in three different animal models of infection with a variety of human pathogens.
Discussion
The results of this study provide the first comprehensive characterization of the effects of a commonly used, low-dose CPM regimen on peripheral blood leukocytes in female outbred mice. It allowed successful and reproducible induction of diverse models of infectious diseases, independently of the method of inoculation, the pathogen used, or its susceptibility pattern.
CPM, a chemotherapeutic agent, has several effects that can impair host resistance, but the decrease in the number of circulating granulocytes is probably one of the most important factors for its use in different animal models, because it promotes severe infection (e.g. sepsis) and allows close quantification of the intrinsic efficacy of antibiotics. Previous research on animal infection models using irradiated mice showed that bacterial growth is inversely related to the number of granulocytes in peripheral blood [
21,
22], and other authors demonstrated with the thigh infection model that there was a significant difference in CFU numbers at the site of infection between mice treated with CPM (250 mg/kg) and non-neutropenic mice [
23].
Previous works have also shown that, after profound neutropenia, mice recovered from CPM immunosupression with an increment in the number of blood leukocytes [
24‐
26]. We observed a similar phenomenon at day 11 when all mice, previously neutropenic, displayed relative neutrophilia compared with baseline values (Table
1 and Figure
1). It is important to bear this fact in mind when designing or modifying the duration of the experimental model, particularly when using survival as the end-point. The low-dose CPM regimen is best suited for animal models equal to or shorter than 4 days after infection, independent of the selected endpoint (number of remaining microorganisms or animal survival). Models longer than 5 days would have both of these endpoints affected by the incipient recovery of the immune system. If such a model was necessary, a third dose of CPM might be required.
Severe neutropenia is not necessarily defined by the same value in humans and mice. The lower limit of neutrophil count in the normal mouse is 110 cells/mm
3 [
20], while for normal humans it is 1800 cells/mm
3 [
27]. Although 100 neutrophils (5.56% of 1800) imply severe neutropenia for a patient, the same value is still very close to normal for the mouse (90.9% of 110). Based on this comparison, we preferred to use 10 neutrophils/mm
3 (9.1% of 110) to define severe neutropenia for the mouse.
We found that the first cell line affected by the low-dose CPM regimen was the granulocytic followed by the lymphocytic line. While neutropenia is obtained with the first dose (150 mg/kg), the second dose (100 mg/kg) must be administered to have lymphopenic mice. On the other hand, monocyte counts were reduced 96% by day 5 with our low-dose intraperitoneal CPM regimen, but the absolute cell number did not reach the inferior normal limit for the mouse (10 cells/mm
3). This is not surprising, since a previous work already showed that monocytopenia was induced only when the drug was administered by the subcutaneous route [
7].
The use of various animals as models for microbiological infections has been a fundamental part of infectious disease research for more than a century. The data from animal models provide means for screening potential treatments and eliminating undesirable ones, narrowing the successful candidates for use in human studies. Due to the ease of handling and reproducibility, the neutropenic murine thigh infection model has been extensively used, especially to evaluate the relationship between host, drug and microorganism in the absence of immunity, which often acts as a confounding variable [
16,
28‐
30]. While inbred and outbred stocks have been employed for this model, the latter are preferred in pharmacology and toxicology studies because they represent better the genetic variability observed in human populations. Although there is a brief description of the neutropenia process after injecting low-dose CPM to inbred mice [
14,
31], the response of outbred mice had not been thoroughly described, and some authors feel more comfortable using the high-dose regimen [
8‐
10]. The high-dose regimen requires greater CPM concentration per injection (250 mg/kg) or more than two injections, conditions implying unnecessary exposure of researchers and environment to this expensive carcinogenic agent [
32,
33], as well as greater risk of deadly peritoneal trauma and hemorrhagic cystitis for the animals [
34].
Our results demonstrate that a low-dose CPM regimen induces severe neutropenia (≤10 cell/mm3) in outbred mice, sustained enough to reliably execute animal models of infection with different endpoints up to 4 days long. Two doses simplify drug administration, and minimize the aforementioned problems associated with high-dose CPM, without compromising the model performance.
Conclusion
Previous results from a variety of papers suggest that intraperitoneal injections of CPM, administered 4 days (150) and 1 day (100 mg/kg) before experimental infection, induce neutropenia (≤100 neutrophils/mm3) in inbred mice and rats. Here, we showed that this low-dose regimen also induces profound and sustained neutropenia (<10 neutrophils/mm3) in outbred mice lasting at least 3 days. The use of a lower dose of CPM divided in two intraperitoneal injections is simpler, safer, and very effective to attain reproducible animal models of infection.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
OV conceived the study, directed its design and execution, obtained funding, reviewed, and re-wrote the final version of the manuscript. AFZ contributed in the study design, performed the analysis and interpretation of data, and prepared the manuscript with CAR. BES carried out the hematological analysis. CAR, MA and AXZ took care of the animals and carried out the experiments with assistance from the other authors.