- 1.
All animal procedures described in this chapter are in accordance with the guidelines of the UK Home Office under authorized Home Office licenses and approved by the relevant Animal Welfare and Ethics Committees. Its implementation in other laboratories will require approval from relevant government and institutional committees before commencing work.
- 2.
T.b.b. GVR35-VSL2 lines can be obtained from the corresponding author as culture stabilates or as blood stabilates for infection of donor mice.
- 3.
Outbred CD1 (ICR) mice tolerate infection with T. brucei GVR35 well. Pilot studies with inbred BALB/c mice indicated that this strain is more susceptible to secondary infections when infected with T. brucei.
- 4.
The GVR35 imaging model described here makes use of bioluminescence imaging (Subheading 3.2.2) with the IVIS spectrum imaging system (PerkinElmer) and Living Image analysis software (Subheading 3.3); the model can also be adapted for use with other in vivo small animal imaging systems and analysis software.
- 5.
In vitro culturing will affect the virulence of T. brucei GVR35; use of F1 blood stabilates for the generation of new blood stabilate working stocks is recommended.
- 6.
Cells may grow very slowly after the first passage. Some cells will die while others continue to divide resulting in no net increase in cells in the first couple of days. The culture will recover and grow normally (~three fold overnight) between 1 and 2 weeks after thawing of the stabilate. Cells will start growing faster (growth of fivefold overnight) over time but may also become more virulent so this should be avoided to ensure reproducibility of the model.
- 7.
The cells will start dying from 1.5–2 × 106 cells/mL so are best kept at a density below 1 × 106 cells/mL.
- 8.
Mice will develop parasitemia at different times and parasites harvested from blood may vary in their infectivity. It is therefore advised to infect two mice for harvesting of parasites and to make two sets of stabilates for later selection of the set which results in the most reliable infections (Subheadings 3.1.2 and 3.1.3).
- 9.
Single-use needles and lancets should be used to avoid contamination and additional injury to mice.
- 10.
It is important to harvest parasites from mice while parasitemia is increasing. Once parasitemia has reached its peak and decreases again the parasites will be less infective. Harvest parasites when parasitemia is in the range of 5 × 106–1.5 × 107 parasites/mL of blood.
- 11.
Parasites will be harvested from donor mice during their first peak of parasitemia and then used to infect experimental mice (designated as day 0). Imaging and treatment of mice is usually done from day 7 (stage 1 model) or day 21 (stage 2 model) after infection and should ideally fall on a convenient day that will also allow for preparation of compounds one day in advance. For example, our blood straws produce an infection in donor mice with the first peak of parasitemia developing 4–5 days postinfection. By infecting donor mice on a Thursday blood and parasites can usually be harvested on the following Tuesday for infection of experimental mice. This allows for preparation of compounds on Monday and imaging as well as start of compound treatment on Tuesday.
- 12.
We routinely set mice up in groups of six per treatment with three diminazene aceturate-treated controls and three untreated controls. These animal numbers have been determined using a power analysis with variables estimated from previous experiments. The variables and calculated group sizes may change depending on the mice used, the bioluminescence of the parasites and sensitivity of the imaging systems. Pilot studies should be performed when setting up a new model to estimate the variables for power analysis.
- 13.
The GVR35 mouse model mimics stage 1 disease at day 7 postinfection. By day 21 the parasites become established in the CNS and infections can only be cured by compounds active in the brain [16, 17]. For screening in stage 2, mice should always be treated after 21 days of infection. Treatment can also be delayed by 1 or 2 days if required for logistical reasons (e.g., day 8 or 9 for stage 1 and day 22 or 23 for stage 2) with subsequent delay in the posttreatment imaging time points.
- 14.
The imaging schedule should be determined in advance and will depend on what is feasible and what analysis is to be done. To analyze in vivo kill kinetics daily imaging after treatment may be needed. For stage 1, imaging usually starts on day 7 and can be done daily afterward up to day 14 (e.g., day 7, 8, 9, 10, 14). For stage 2, imaging usually starts at day 21 and is done weekly, or later (usually after day 63), 2-weekly intervals following start of treatment (e.g., day 21, 28, 35, 42, 49, etc.).
- 15.
The number of animals to be imaged will depend on the IVIS imaging system being used. We prefer to image three mice at a time to allow space between mice.
- 16.
It is preferable to inject luciferin subcutaneously or intraperitoneally. While subcutaneous injection results in a slightly lower signal compared with intraperitoneal injection it has a longer and flatter signal kinetics making it more suitable for taking multiple images of the whole body or head. Furthermore, there is a higher risk of failure for intraperitoneal injection (where injection is placed into a site other than the peritoneal cavity, such as the lumen of the stomach or bowel, or injection is subcutaneous, retroperitoneal or intravascular), particularly in trypanosome-infected mice where spleens are enlarged. A failed injection may reduce the luciferin concentration in tissues resulting in lower bioluminescence or result in death.
- 17.
Luciferin can be injected once the animal is anesthetized; however, this will alter the kinetics of the signal compared with an animal that is awake. This should be taken into account when establishing acquisition timings and setting.
- 18.
Imaging acquisition settings and the timing of imaging after luciferin injection need to be determined in advance. This will depend on the site of luciferin injection, strength of the signal, and location of the signal. To establish the imaging start time calculate a kinetic curve during a pilot study—this is done by taking multiple images over time and determining when the signal peaks and falls.
- 19.
The mouse to be imaged is moved to the middle nose cone while the other two remain on outer nose cones. For head images the “Field of view (FOV)” on the IVIS acquisition panel should be set on A—this increases resolution by moving the stage upward but will only allow imaging of the middle mouse (e.g., for the IVIS spectrum FOV A is 3.9 × 3.9 cm). Once head images of the middle mouse are completed it is transferred to a holding box and another mouse is moved to the middle for imaging of head (any open nose cones should be blocked with black plugs as mice are transferred from the imaging chamber).
- 20.
Mice may have low bioluminescence signal after treatment with a trypanocidal compound; to detect these low signals increase the exposure time and the binning. In untreated mice, or mice with relapsed parasitemia, signal may be saturated at high exposure and binning; in these instances the exposure time and binning should be decreased or the F/stop increased.
- 21.
Transparent nose cones ensure gas delivery to individual mice from the anesthesia manifold. Black rubber plugs seal unused ports of the anesthesia manifold. Nose cones and plugs should be provided with the IVIS gas anesthesia system.
- 22.
XMD-2 manifold dividers are available in different heights and can be purchased from PerkinElmer or may have been provided with the IVIS system. We cut dividers from black Lexan plastic sheets. In cases where high signal from a mouse still spills over to other mice with low signal it may be necessary to re-image mice separately (e.g., this may occur when parasites have recrudesced in one mouse while others remain clear of parasite signal).
- 23.
The vehicle and dosing regimen for compounds should be determined in advance and tested for toxicity in uninfected mice before used for treatment in the T. b. brucei infection model.
- 24.
Compound administration should be done in volumes as specified in the appropriate animal project license or as recommended by animal research advisory boards. For example for oral dosing it is considered good practice to administer compounds in 10 mL vehicle/kg of mouse [18].
- 25.
CD1 mice infected with T. b. brucei
GVR35 may develop symptoms related to infection of the CNS such as hind-leg paralysis and paresis. This usually occurs from day 28–35 postinfection. To avoid these adverse effects from developing untreated mice should be culled at day 28–30 postinfection (e.g., after the imaging session, 7 days after start of treatment) or when symptoms become visible.