Each mouse was pre-medicated subcutaneously with 0.1 mg/kg buprenorphine (Vetergesic; Animalcare, York, UK) and anaesthesia was induced and maintained with isoflurane (Isoforine®-Cristália) diluted in 100% oxygen delivered by mask. Sciatic neurectomy (SN) of the right limb of each mouse was accomplished as previously described and the contralateral left limb served as an internal contralateral control (contralateral) (De Souza et al., 2017 (link); de Souza et al., 2005 (link)). Briefly, an incision was made caudal to the right hip joint and the biceps femoris muscle elevated to expose the nerve. SN was achieved by resecting a 3-4 mm segment of the sciatic nerve posterior to the hip joint. The neurectomised mice were able to move around in the cage and gained access to food and water without difficulties. The mice were sacrificed through cervical dislocation at one of four different time points: either 5 days (n = 5), 35 days (n = 4), 65 days (n = 5) or 95 days (n = 3) after right SN. The tibiae, both left and right, were subsequently dissected, fixed in neutral-buffered formaldehyde for 24 h before washing and storage in 70% alcohol prior to microCT scanning.
Isoforine
Manufactured by Cristália
Sourced in United Kingdom, Brazil
Isoforine is a laboratory equipment product manufactured by Cristália. It is a specialized device designed for scientific and research applications.
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6 protocols using isoforine
1
Sciatic Nerve Neurectomy in Mice
2
Pharmacological Modulation of Angiotensin Receptors
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Losartan potassium (selective AT1 receptor antagonist) (Tocris, Westwoods Business Park, Ellisville, Missouri, USA), A-779 (selective MAS receptor antagonist) (Tocris), PD123319 ditrifluoroacetate (selective AT2 receptor antagonist) (Tocris) and urethane (Sigma-Aldrich, St Louis, Missouri, USA) were dissolved in saline (NaCl 0.9%). The poly-antibiotic formulation (Pentabiotico; Fontoura-Wyeth, Campinas, São Paulo, Brazil), isoflurane (Isoforine; Cristália, Itapira, São Paulo, Brazil), lidocaine (Harvey Química Farmacêutica Ind. e Comércio Ltd.a., Catanduva, São Paulo, Brazil), and flunixin meglumine (Banamine, Schering Plough, Cotia, São Paulo, Brazil) were used as provided.
3
Bacterial Strain Instillation in Wistar Rats
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In the early morning (8:00 a.m.), 28 male Wistar rats (mean body weight ± SD, 333±78g) were anesthetized under spontaneous breathing with 1.5–2.0% isoflurane (Isoforine®; Cristália, Itapira, SP, Brazil) and underwent intratracheal instillation of the above-mentioned bacterial cultures. This bacterial strain is deposited in the Culture Collection of Hospital-Acquired Bacteria (CCBH) located at the Hospital Infection Research Laboratory at Oswaldo Cruz Institute, FIOCRUZ, Brazil.
4
Stem Cell Therapy for Spinal Cord Injury
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Prior to anesthesia, the animals were injected with 0.05 mg/kg acepram (acepromazine®1) associated with 4 mg/kg dolosal (Meperidine®2), both intramuscularly. After 15 minutes anaesthesia was induced with 5 mg/kg propofol (Propovan®3 - Cristália) intravenously and maintained by inhalational anaesthesia with isoflurane (Isoforine®4 - Cristália). During surgery the animals underwent dorsolateral hemilaminectomy in the injured spinal cord. The incision was made lateral to the midline and dorsal to the lesion. After incision of the skin and muscles, the fascia was incised lumbodorsal alongside the corresponding spinous processes of the vertebrae, and, with the aid of a periosteum elevator, the spinal processes were isolated. By exposing the dorsal surface of the articular process, a window was opened to provide access to the spinal canal. After which, the medullary mass was compressed and damaged tissue was removed. After durotomia, intramedullary injection of 1×106 of stem cells derived from foetal canine bone marrow
[31 (link)] was completed cranial to the injury, at the focal point of the lesion, and caudal to the lesion. The postoperative therapy consisted of 30 mg/kg Kefazol ® (cefazolin sodium) 30 mg/kg, 0.1 mg/kg meloxicam (Generic Biosintética) and 2 mg/kg Tramal ® (tramadol hydrochloride) for seven days.
[31 (link)] was completed cranial to the injury, at the focal point of the lesion, and caudal to the lesion. The postoperative therapy consisted of 30 mg/kg Kefazol ® (cefazolin sodium) 30 mg/kg, 0.1 mg/kg meloxicam (Generic Biosintética) and 2 mg/kg Tramal ® (tramadol hydrochloride) for seven days.
5
Feline Ovariohysterectomy Procedural Protocol
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To perform the ovariohysterectomy, acepromazine (0.05 mg/kg IM) (acepromazine 2%; Syntec) and morphine (0.3 mg/kg IM) (morphine sulfate 10 mg/ml; Hipolabor + Sanval) were used as pre-anesthetic medication for healthy cats in diestrus, while meperidine (3 mg/kg IM) (Petidina; Uniao Chemical Laboratory) was used for cats with pyometra. For anesthetic induction, propofol (6 mg/kg IV) (Propotil; Biochimico) was used, with subsequent continuous infusion of Ringer’s lactate (6 ml/kg/h). The cats were intubated and anesthetic maintenance was performed with isoflurane (Isoforine; Cristália) in 100% oxygen (300 ml/kg/min) in an open circuit (Baraka) using an anesthesia machine (WATO EX-65; Mindray). Before the surgical procedure, the animals received sodium cephalothin (30 mg/kg IV) as prophylactic antibiotic therapy, even with all the proper aseptic conditions. Ovariohysterectomy was performed using the three-clamp technique after laparotomy through a midline incision.
6
Animal Euthanasia and Tissue Collection
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As previously described [25 (link)], at the end of the experimental period, the animals were anesthetized by inhalation (Isoforine, Cristália®, São Paulo, Brazil), and euthanized by exsanguination by cardiac puncture. The tissues were immediately collected, weighted, and frozen in liquid nitrogen, and stored at −80 °C for further analysis. Feces were collected from the cecum, weighted, and stored at −80 °C for future analyses. Colon fragments were collected and fixed in formaldehyde (10% v/v) for the first 24 h and then stored in ethanol (70% v/v) and embedded in paraffin for histological analysis.
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