Ketaset

Experiments were performed in accordance with the Animals Scientific Procedures Act (1986) and were approved by the local Ethical Review Committee. Barrier-bred female 8–12 week old Balb/c mice (Charles River UK Ltd) were anaesthetised by intraperitoneal injection of 100 mg kg⁻¹ body weight ketamine (Ketaset; Fort Dodge Animal Health, Southampton, UK) and 10 mg kg⁻¹ body weight xylazine (Rompun; Bayer, Newbury, Berkshire, UK) and inoculated with M. smegmatis or M. tuberculosis by endotracheal aerosol application of a total volume of 25 µl using a Microsprayer® (PennCentury, Philadelphia, PA, USA) as previously described [74] (link).
Assessment of bioluminescence (photons s⁻¹cm⁻² steridian [sr]⁻¹) from living animals was performed using an IVIS® Spectrum system (Caliper Life Sciences, Alameda, USA) which consists of a cooled charge-coupled device camera mounted on a light-tight specimen chamber. Prior to bioluminescent imaging, mice were anaesthetised with 4% isoflurane. Luciferin dissolved in sterile D-PBS was then administered to animals inoculated with FFluc expressing strains [20 µl of 15 mg ml⁻¹ (47 µM) or 30 mg ml⁻¹ (94 µM) luciferin via the intranasal route, or 300 mg kg⁻¹ or 500 mg kg⁻¹ body weight by intraperitoneal injection]. To image mice infected with Gluc expressing M. smegmatis 50 µl of 0.48 mM or 0.96 mM coelenterazine (prepared by diluting the 10 mM stock in sterile D-PBS just before use) was intranasally administered (10 or 20 µg per mouse respectively), or 150 µl of 0.16 mM coelenterazine via the intraperitoneal route (10 µg). Mice were placed into the camera chamber of the IVIS® Spectrum imaging system where a controlled flow of 2.5% isoflurane in air was administered through a nose cone via the IXG8 gas anaesthesia system (Caliper Life Sciences). A grayscale reference image was taken under low illumination prior to quantification of emitted photons over 30 s to 5 min, depending on signal intensity, using the software program Living Image (Caliper Life Sciences) as an overlay on Igor (Wavemetrics, Seattle, WA). For anatomical localisation, a pseudocolour image representing light intensity (blue, least intense to red, most intense) was generated using the Living Image software and superimposed over the grayscale reference image. Bioluminescence within specific regions of individual mice was also quantified using the region of interest (ROI) tool in the Living Image software program (given as photons s⁻¹). Animals were imaged immediately after inoculation, to assess the success of the delivery, and 24 h post-infection. Animals inoculated with ffluc- or gluc- expressing M. smegmatis were imaged at different time points after substrate administration for up to 3 h.

Mice were anesthetized by intraperitoneal injection of a cocktail of 50 mg/kg of ketamine (Ketaset, Fort Dodge Animal Health, Overland Park, Kansas, United States), 10 mg/kg of xylazine (AnaSed, Ben Venue Laboratories, Bedford, Ohio), and 1.7 mg/kg of acepromazine (Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, United States). A portion of the right abdominal skin was removed and the peritoneal cavity opened along the rib cage. The mouse was then placed on the stage of an inverted Nikon (Tokyo, Japan) Diaphot microscope equipped with a Cooke SensiCam digital camera (Cooke, Romulus, Michigan, United States). The liver was immobilized with gauze and kept moist with warm PBS. Sporozoite infection was done on the microscope stage by bite of 100−200 infected mosquitoes. Images of sporozoites entering the liver were captured using either a fluorescein isothiocyanate long-pass filter combination or a GFP/DsRed dual band filter set (Chroma Technology, Rockingham, Vermont, United States) and imported into Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland, United States). Typical exposures times were 100 ms per image for gray tone and 300 ms for RGB images. Phototoxicity was limited by reducing light transmission to 20% with a neutral density filter.

Neonatal mice (PND2) were i.c.v. injected in the right hemisphere, between the confluence of sinuses with 100 µg of Ncald-ASO or CTRL-ASO using a glass needle [68 (link)]. ASOs concentration was determined photometrically (AD260) in order to administer 1.5 µL (1 µL ASO + 0.5 µL of 0.05% w/v trypan blue in PBS). For Ncald-ASO re-injections, i.c.v. bolus injection was performed at PND28. Animals were anesthetized using Ketamine (Ketaset 100 mg/mL, WDT Wirtschaftsgenossenschaft deutscher Tierärzte)/Xylazine (2%, Serumwerk Bernburg AG, Bernburg, Germany) and placed in a stereotaxic instrument (Bilaney, Cat# DKI940) with a mouse adaptor (Bilaney, Cat# DKI926) and a micropositioner (Bilaney, Cat#DK5000) using a thermostatic warming plate to maintain the body temperature during the procedure. Next, injection coordinates were determined from the mouse bregma (For HET: X = 1 mm, Y = 0.3 mm; for SMA X = 0.980 mm, Y = 0.250 mm; in all the Z = range between −1.6 mm and −1.7 mm) [69 (link)]. To drill a hole in the skull, we used a microdrill (burr size: 0.8 mm, KF Technology). A total of 5 µL of ASO was delivered at a rate of 1 µL/30 s.