Baytril

The fracture model was performed in 3 month-old male mice. Under isoflurane anesthesia a hemostat was used to make a closed fracture of the right tibia just distal to the middle of the tibia. Then the hindlimb was wrapped in casting tape (Delta-Lite) so the hip, knee and ankle were all fixed. The cast extended from the metatarsals of the hindpaw up to a spica formed around the abdomen. A window was left open over the dorsal paw and ankle to prevent constriction when post-fracture edema developed. After fracture and casting, the mice were given subcutaneously 2 days of Buprenorphine (0.05 mg/kg) and Baytril (5 mg/kg) as well as 1.5 ml of normal saline. At 3 weeks after surgery the mice were anesthetized with isoflurane and the cast removed. All mice had union at the fracture site by manual inspection.

All procedures on pigs were approved by the Johns Hopkins University Animal Care and Use Committee and by the Animal Care and Use Review Office of the US Army Medical Research and Materiel Command for Award Number W81XWH-19-C-0022 (Fort Detrick, MD). In conducting research using animals, the investigators adhered to the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and the principles set forth in the current version of the Guide for the Care and Use of Laboratory Animals, National Research Council.
Because there can be sex differences in the response to TBI (43 (link), 44 (link)) and TBI in the young and in military personnel is more prevalent in males (45 (link)), the study was conducted in male pigs. A total of 48 pigs weighing 28 ± 2 kg and approximately 3 months of age were used in the overall study. The experimental protocols for the TBI + HS experiment and the TBI alone experiment are delineated in Figure 1. The pigs were sedated with intramuscular injection of Telazol (50 mg/ml tiletamine and 50 mg/ml zolazepam, 4.4 mg/kg each component), ketamine 2.2 mg/kg and xylazine 2.2 mg/kg. Isoflurane (4% in 30% O₂) was administered via face mask to produce an anesthetic depth for oral intubation of the trachea. After a surgical plane of anesthesia was achieved, as assessed by the lack of limb withdrawal to hoof pinching and by looseness of muscle tone in the jaw, anesthesia was maintained with 2% isoflurane in approximately 30% O₂ with mechanical ventilation of the lungs. The antibiotic Baytril 10 mg/kg (100 mg/ml) was injected intramuscularly. Surgery was conducted using aseptic techniques. Through a 5-cm neck incision, an external jugular vein was isolated by blunt dissection. The vein was ligated and a catheter was advanced toward the heart and secured with another ligature. For arterial catheterization, we chose the axillary artery because occlusion of the carotid artery could limit cerebral blood flow after TBI and catheterization of the femoral artery can limit use of the hindlimb. An incision was made in the axilla, and the axillary artery was isolated, ligated, and cannulated with a flexible polyvinyl catheter that minimized kinking. The arterial and venous catheters were tunneled subcutaneously to the back of the neck, where they exited through a small incision. Pigs were able to bear weight on the forelimb and ambulate on the day after surgery.

Six days after infiltration, 18g of infiltrated leaves was harvested and homogenized in PBS as previously described. ND VLPs were partially purified using density gradient ultracentrifugation (70%/20% sucrose), whereafter the NDV F/HN VLPs in sucrose fractions 2 to 4 were pooled, dialyzed in 1 x PBS overnight using a 3500 MWCO Slide-A-Lyzer protein dialysis cassette (Thermo Fisher Scientific), and trehalose (15% [w/v]) was added as stabilising agent. Partially-purified NDV F/HN VLPs were tested by hemagglutination assay (see serological testing) and stored at 4°C until use. The vaccine dose of 25 µl partially-purified ND F/HN VLPs was calculated to correspond to an HA titre of 10 log₂ or 1024 HAU. On the day of vaccination, the partially purified plant extract containing the ND VLPs (25 µl) were either diluted in 1 x PBS to a final volume of 250 µl (non-adjuvanted treatment group; supplemented with 1% [v/v] enrofloxacin (Baytril^® 100, Bayer Animal Health, South Africa), or diluted in 1 x PBS and mixed with a commercial oil-in-water adjuvant Emulsigen^®-P (MVP adjuvants^®, Phibro Animal Health Corporation, United States; 20% [v/v]) plus 1% [v/v] enrofloxacin (Baytril^® 100, Bayer Animal Health, South Africa) to a final volume of 250 µl (adjuvanted treatment group). The Emulsigen-P commercial oil-in water adjuvant was selected due its effectiveness in a previous study conducted by the UP/CSIR research group involving the testing of a plant-produced VLP vaccine in poultry (Abolnik et al., 2022 (link)).

Rabbits were premedicated intramuscularly with a standard protocol that included 50 µg/kg dexmedetomidine (Dexdomitor; Orion Corporation, Espoo, Finland) and 0.6 mg/kg midazolam (Midazolam-hameln; Hospira Italia, Naples, Italy). All drugs were mixed in the same syringe and injected in the lumbar muscles. As rabbits lost the righting reflex, the abdomen in females and the inguinal and scrotal region in males were clipped; a 26-gauge (Delta Ven 1, Delta Med Spa, Viadana, Italy) over the needle catheter was inserted aseptically in the cephalic vein and buprenorphine (Buprenodale; Dechra Ltd., Skipton, UK) at the dose of 0.05 mg/kg was administered intravenously (IV). The effect of premedication was scored according to a previously described scale [6 (link)]. The descriptors of the scale were: posture from 0 (normal) to 5 (lying dorsally, not moving when stimulated); resistance to being rolled in dorsal recumbency from 0 (strong/normal resistance) to 3 (no resistance ); jaw muscle tone 0 (normal) or 1 (no resistance to mouth opened); palpebral reflex from 0 (normal) to 2 (absent). Oxygen at 1 L/min was administered via a Mapleson E breathing system placed close to the nose for at least 5 min and after, larynx was blindly sprayed with 0.2 mL of lidocaine (Lidocaina 2%; Ecuphar Italia S.r.l., Milan, Italy, diluted to 5 mg/mL) before the first attempt of insertion of the device. If the score was equal or higher than 9 out of 11, insertion of the LMA or the endotracheal tube was attempted, otherwise propofol (Vetofol; Norbrook Laboratories Ltd., Newry, Northern Ireland) was administered IV manually at 0.2 mg/kg every 10 seconds, until the score was considered adequate.
As the airways were secured, a pitot base spirometer connector (Pedi-Lite Spirometry Sensor; GE Healthcare, Helsinki, Finland) was connected to measure the tidal volume (Vt). After, the rabbits were placed in dorsal recumbency and connected to a circle breathing system with paediatric tubes; anaesthesia was maintained with isoflurane (Isoflo; Abbott Laboratories Ltd., Maidenhead, UK) carried in a mixture of oxygen and air (FiO₂ = 0.5); the surgical area was aseptically prepared. Initial flow rate was 1 L/min which was decreased to 0.5 L/min. End-tidal concentration of isoflurane was adjusted to maintain an adequate anaesthetic level minimizing the occurrence of surgery-induced cardiovascular and respiratory changes or spontaneous movement. Rabbits were maintained in spontaneous ventilation throughout anaesthesia, and manual ventilation at six breaths/min and a peak pressure of 10 cm H₂O was provided if apnoea was present or the respiratory rate was lower than < 6 breaths/min. A portable pulse oximeter (EDAN VE-H100B; Edan USA, San Diego, CA, USA), with a clip probe placed over the ear, measured the pulse rate (PR) and SpO₂, while the PEʹCO₂, the end-tidal concentration of isoflurane (FEʹIso) and the respiratory rate (RR) were obtained from a multiparameter monitor (Datex S/5; GE Healthcare, Helsinki, Finland). Non-invasive systemic arterial blood pressure was measured by an oscillometric device (PetTrust Blood Pressure Monitor; BioCare, Taoyuan City, Taiwan). The cuff was placed on the forelimb proximal to the carpus; the correct size of the cuff was obtained by a ruler provided by the manufacturer. A mixture of lactated Ringer’s solution and 5% dextrose (50:50) was infused at 5 mL/kg/h during anaesthesia. Before the beginning of the surgery the rabbits received meloxicam (1 mg/kg; Metacam; Boehringer Ingelheim, Milan, Italy) and enrofloxacin (5 mg/kg; Baytril 25 mg/mL injection solution; Bayer Spa, Milan, Italy) subcutaneously.