Figure 1.
Generation of bioluminescent T. brucei GVR35 for detection of trypanosomes in vivo.
(A) Live GVR35 WT and -LUC2 clones were assessed for luciferase activity in vitro after addition of D-luciferin. The in vitro detection limit was determined by imaging a dilution series of GVR35-LUC2 using IVIS. Data show, on a logarithmic scale, average total flux (in photons per second) ± SD of replicate wells containing a specified trypanosome number. The dotted line indicates background bioluminescence for GVR35 WT. (B) Brains of mice infected with WT or GVR35-LUC2 were compared for trypanosome DNA and neuropathology at different times after infection. Real-time qPCR of the trypanosome TbPFR2 gene was performed on brain homogenates. Neuropathology was scored on haematoxylin and eosin stained sections. Graphs show means and 95% confidence interval for each group (n = 11–12 per group). P value (Student's t test) compared to GVR35 WT is indicated *P<0.05; **P<0.01.
Table 1.
In vitro sensitivity of GVR35-LUC2 compared to WT for different trypanocidal compounds.
Figure 2.
In vivo imaging of bioluminescent T.
brucei GVR35 over the course of infection. GVR35 WT or -LUC2-infected mice were imaged at different times after infection. Total flux in photons per second (p/sec) for the mouse body (ventral view) or head region (dorsal view) is shown. D-luciferin (150 mg/kg) was injected intraperitoneally 10 minutes before imaging. ND indicates that trypanosomes were not detected in blood samples. Images show the same two representative mice over the full course of infection. Images of a GVR35 WT-infected mouse are shown to indicate the background bioluminescence. The colour scale indicates bioluminescent radiance in photons.second−1.centimeter−2.steradian−1.
Figure 3.
Ex vivo imaging of bioluminescent T.
brucei GVR35 over the course of infection. (A) Brains were removed from perfused GVR35-infected animals at indicated days after infection, soaked in D-luciferin and imaged ex vivo. A comparison between brains from perfused and non-perfused animals at day 14 is shown at the bottom. (B) Organs were harvested from perfused GVR35 WT or -LUC-infected mice at day 35, soaked in D-luciferin and imaged ex vivo. Colour scales indicate bioluminescent radiance. In A, different scales are used for weak (a) and stronger (b) bioluminescent radiance in photons.second−1.centimeter−2.steradian−1.
Figure 4.
brucei GVR35-LUC2- infected mice to assess in vivo trypanocidal activity. Mice were treated with (A) diminazene aceturate ((i) n = 11, (ii) n = 6), (B) melarsoprol (n = 12), (C) DB75 (n = 6) or (D) DB829 (n = 8) from day 21 and imaged on indicated days after infection. D-luciferin (150 mg/kg) was injected intraperitoneally 10 minutes before imaging. Bioluminescence from the heads of mice is shown as total flux in photons per second (p/sec). ND indicates that trypanosomes were not detected in blood samples. For each treatment images of the same two representative mice over the entire period are shown. In Aii brains were harvested from perfused GVR35-LUC2-infected mice at day 21 (untreated) or day 35 (diminazene-treated), soaked in luciferin and imaged ex vivo. Corresponding untreated heads and treated heads and brains are shown. The same two colour scales are used for all treatments for strong (a) and weaker (b) bioluminescent radiance in photons.second−1.centimeter−2.steradian−1. The colour scale used is indicated in the top right corner of each image.
Table 2.
In vivo antitrypanocidal activity in the GVR35-LUC2 mouse model.
Figure 5.
Trypanosomes invade the meninges.
(A) Lister 427-mCherry trypanosomes (red) in a meningeal blood vessel (outlined by dashed blue lines) at day 3 (Video S1). (B) Extravascular GVR35-mCherry trypanosomes (red) day 13. Blood and collagen appear green. A rapidly moving intravascular trypanosome is indicated by the arrow (Video S2). (C) 24 hr after intravenous (i.v.) injection of DB75 meningeal trypanosomes are labeled and motile (arrows), as are host nuclei (Video S5), day 28. Inset shows a different mouse at day 10, 25 min after i.v. injection of DB75, only the nucleus and kinetoplast are labeled (arrow heads, Video S3). (D) At day 25 numerous GVR35-LUC2 trypanosomes (moving dots in Video S4) are present in the meninges. Labeling by i.v. injection of DB75. (E) Numbers of meningeal trypanosomes plotted against days post-infection. Diminazene (40 mg/kg) at day 21 cleared the meninges of trypanosomes until about day 58 (crosses). (F) Treatment with diminazene at day 21 reduced luciferase emission but by day 44 there was strong emission from the head, despite zero blood parasitemia. Scale in photons.second−1.centimeter−2.steradian−1. (G) Subsequent multi-photon imaging in area circled in (F) showed no motile trypanosomes in the meninges. I.v. injection of DB75 labeled host nuclei. All scale bars 20 µm.