Rompun

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.

Human Aβ_1-42 peptide was prepared as a stock solution at a concentration of 1 mg/mL in sterile saline solution, followed by aggregation via incubation at 37 °C for 4 days. The aggregated Aβ_1-42 peptide or vehicle (0.9% NaCl, 3 μL/ 5 min/mouse) was stereotaxically administered into the ventricle (i.c.v.) using a Hamilton microsyringe (−0.2 mm anterioposterior (AP), 1 mm mediolateral (ML), and −2.4 mm dorsoventral (DV) to Bregma) under anesthesia in combination with 0.05 mL/100 g body weight Rompun (Xylazine) and 0.1 mL/100 g body weight Zolitil (Ketamine). The rest of the protocol was the same as previously reported76 (link).
Twenty-four hours after Aβ_1-42 and vehicle i.c.v. injection, the mice were divided into the following groups: (1) control (C) mice injected i.c.v. with 0.9% saline as a vehicle or i.c.v. with Aβ_1-42 (Aβ_1-42 group), (2) mice injected with Aβ_1-42 and VA 30 mg/kg intraperitoneally (i.p.) for 3 wks (Aβ_1-42+ VA), and (3) mice treated with VA 30 mg/kg (i.p.) for 3 wks alone (VA). Twenty-four hours post i.c.v. Aβ_1-42 or vehicle injection, VA (30 mg/kg) was administered (i.p.) daily for 3 weeks.

Adult mice expressing GCaMP3 (6–12 weeks, male and female) were anesthetized using ketamine (KETAVET; Zoetis; 100 mg/kg), xylazine (Rompun; Bayer; 15 mg/kg), and acepromazine (Elanco; 2.5 mg/kg). Depth of anesthesia was confirmed by pedal reflex and breathing rate. Animals were maintained at a constant body temperature of 37°C using a heated mat (VetTech). Lateral laminectomy was performed at spinal level L3–5. In brief, the skin was incised longitudinally, and the paravertebral muscles were cut to expose the vertebral column. Transverse and superior articular processes of the vertebrae were removed using OmniDrill 35 (WPI) and microdissection scissors. To obtain a clear image of the sensory neuron cell bodies in the ipsilateral dorsal root ganglion (DRG), the dura mater and the arachnoid membranes were carefully opened using microdissection forceps. Artificial spinal fluid (values are in mm: 120 NaCl, 3 KCl, 1.1 CaCl₂, 10 glucose, 0.6 NaH₂PO₄, 0.8 MgSO₄, and 18 NaHCO₃, pH 7.4 with NaOH) was constantly perfused over the exposed DRG during the procedure to maintain tissue integrity. The animal was mounted onto a custom-made clamp attached to the vertebral column, rostral to the laminectomy. The trunk of the animal was slightly elevated to minimize interference caused by respiration. The DRG was isolated by coating with silicone elastomer.
Images were acquired using a Leica SP8 confocal microscope. A 10× dry, 0.4-N.A. objective with 2.2 mm working distanced was used, with image magnification of 0.75–3× optical zoom. GCaMP3 was excited using a 488 nm laser line (3–10% laser power). GCaMP was detected using a hybrid detector (60% gain). 512 × 512-pixel images were captured at a frame rate of 1.55 Hz, bidirectional scan speed of 800 Hz, and pixel dwell time of 2.44 μs.
Noxious and innocuous stimuli were applied to the left hindpaw, ipsilateral to the exposed DRG. For thermal stimuli, the ventral side of the paw was immersed with ice-water (nominally 0°C), acetone (100%) or water heated to 55°C using a Pasteur pipette. For delivery of precise temperature stimuli, a Peltier-controlled thermode (Medoc) was used. For mechanical stimuli, a pinch with serrated forceps was used. An interval of at least 30 s separated each stimulus application.

Approval for the study was granted by the Animal Research Ethics Committee of Health Sciences University Ankara Training and Research Hospital, Ankara, Turkey (meeting number: 66, decision no.: 666, dated: 26.07.2021). This experimental study was started in 2021 after the approval of the ethics committee and the study was carried out in the Animal Laboratory at Ankara Training and Research Hospital, Ankara, Turkey. The study sample comprised 21 male Wistar albino rats, each aging 8-9 weeks and weighting 250±25 grams. An environment was established before the experiment of room temperature 22-25°C, a 12-hour light/dark cycle (08:00-20:00 light; 20:00-08:00 dark), and humidity in the range of 55-60%. The rats had free access to standard rat food (23% protein, 5% fat, 15% fibre, and 50% carbohydrate) and tap water. The 21 rats were separated into 3 groups of 7. The anaesthesia procedure at the start of the study was applied to all the rats intraperitoneally with 90 mg/kg ketamine (Ketalar Flacon, 50 mg/ml Pfizer, Istanbul, Turkey) and xylazine 10 mg/kg intraperitoneal (Rompun 2% Flacon Bayer, Istanbul, Turkey). A blood sample was obtained from all the rats to determine the preoperative biochemical basal values. Laparotomy was applied to all the rats with a midline incision of approximately 3 cm.
The National Institutes of Health Guiding Principles has been taken as a guide regarding the care and use of animals used in this experimental study.¹²