Xylazine hydrochloride

Immunisation regime was based on similar studies performed investigating fractional IPV responses21 (link)32 (link)35 (link).Under ketamine hydrochloride (Ceva) and xylazine hydrochloride (Troy Laboratories) sedation, rats (n = 5) were immunised with 1 or 0.2 D-antigen units of IPV 2 by Nanopatch or IM injection, with an additional positive-control group receiving a full human dose of 8 D-antigen units IM. Two Nanopatches, each containing half the dose to be delivered were applied to each ventral ear pinnae using a proprietary applicator at a velocity 3.1 ms⁻¹ and kept in place for 2 minutes. IM injections were administered into the hind leg by a 29G needle. Rats received three doses by Nanopatch or IM injection, at 21-day intervals, with blood samples collected one day before vaccination and 21 days post final dose.

Thirteen 12–15 weeks old New Zealand female white rabbits weighing 3–4 kg were used for autologous implantation. The right eye of each rabbit underwent SMILE for tissue harvesting while the left eye underwent tissue implantation. The rabbits were randomly allocated into four groups: non-CXL inlays implanted at 90 μm depth (non-CXL-90 group, n = 3 eyes) and 120 μm depth (non-CXL-120 group, n = 3 eyes) and CXL treated inlays implanted at 90 μm depth (CXL-90 group, n = 3 eyes) and 120 μm depth (CXL-120 group, n = 3 eyes). One rabbit (n = 2 eyes) served as an untreated control.
The research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of SingHealth, Singapore, and conducted in accordance of the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. During pre- and postoperative examination and surgical intervention, the rabbits were anesthetized by an intramuscular injection of xylazine hydrochloride (5 mg/kg, Troy Laboratories, Smithfield, Australia) and ketamine hydrochloride (50 mg/kg, Parnell Laboratories, Alexandria, Australia). The rabbits were euthanized under anaesthesia by an overdose intracardiac injection of sodium pentobarbital (Jurox, Rutherford, Australia).

The immunization regime was based on similar studies performed investigating candidate dengue vaccines in mouse models. Under ketamine hydrochloride (Ceva Animal Health, Glenorie, Australia) and xylazine hydrochloride (Troy Laboratories, Gendenning, Australia) sedation, SV129 mice were immunized with 1 or 0.1 μg tetravalent sE (DENV1: 258848, DENV2: PR159, DENV3: CH53489, DENV4: H241 produced from S2 cells, donated by Prof Mathew Cooper, UQ) formulation by nanopatch or 1–10 μg tetravalent sE by intradermal (ID), subcutaneous (SC), or intra muscular (IM) injection, with or without 3 μg of the saponin adjuvant Quil-A (Brenntag, Essen, Germany). Control groups received PBS delivered via each investigated injection method, while nanopatch control groups received excipients only. Nanopatches containing the dose to be delivered were applied to each ventral ear pinnae using a proprietary applicator at a velocity of 3.1 ms⁻¹ and kept in place for 2 min. Mice received three doses by nanopatch or SC, ID, or IM injection at 28-day intervals, with blood samples collected one day before vaccination and 28 days after final dose.

Unless otherwise stated, all animal surgeries and follow-up evaluations were performed under general anesthesia achieved by intramuscular injections of xylazine hydrochloride (5 mg/kg; Troy Laboratories, Glendenning, NSW, Australia) and ketamine hydrochloride (50 mg/kg; Parnell Laboratories, Alexandria, NSW, Australia), together with a topical application of lignocaine hydrochloride (1%; Pfizer Laboratories, New York City, NY, USA).

New Zealand White rabbits (n = 20) were used for this study and all TE-EK surgeries and CE-CI procedures were performed by JSM. Their use, care and treatment strictly adhered to the regulation of the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, and all experimental procedures were approved by the Institutional Animal Care and Use Committee of SingHealth, Singapore (Ref: 2017/SHS/1292). All surgical procedures and follow-up evaluations were performed under general anesthesia achieved by intramuscular injections of 5 mg/kg xylazine hydrochloride (Troy Laboratories, New South Wales, Australia) and 50 mg/kg ketamine hydrochloride (Parnell Laboratories, New South Wales, Australia), along with topical application of lignocaine hydrochloride 1% (Pfizer Laboratories, New York, USA).

The procedure for IVT injection has been described previously [29 (link),75 (link),76 (link)]. Briefly, prior to injection animals were anesthetized with 150 mg/kg body weight (BW) ketamine hydrochloride (Parnell Laboratories, New South Wales, Australia) and 10 mg/kg BW xylazine hydrochloride (Troy Laboratories, New South Wales, Australia) and their pupils were dilated with a topical application of 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon Laboratories Inc., Fort Worth, TX, USA). For the injection, a 1 µL aliquot of solution containing each chemical was injected into the vitreous body of both eyes of each mouse using a Nanofil microsyringe with a 33G needle (World Precision Instruments Inc., Sarasota, FL, USA), under an ocular surgery microscope (Leica Microsystems GmbH, Wetzlar, Germany). A bilateral approach was taken to minimize the number of animals used in this study. All procedures were performed carefully to avoid injuring the lens or retina. Four or seven days following injections, the eyes were collected and subjected to further analysis as described below. Eyes with bleeding or inflammation caused by technical errors were excluded from analysis.

All pre and post procedures were carried out as previously described^{52 (link)}. Briefly, eight New Zealand White rabbits were used for this study. All CE-CI procedures were performed by JSM. The use of rabbits, their care and treatment adhered to the regulation of the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, and all experimental procedures were approved by the Institutional Animal Care and Use Committee of SingHealth, Singapore (Ref:2017/SHS/1292). All surgical procedures and follow-up evaluations were performed under general anesthesia achieved by intramuscular injections of 5 mg/kg xylazine hydrochloride (Troy Laboratories, New South Wales, Australia) and 50 mg/kg ketamine hydrochloride (Parnell Laboratories, New South Wales, Australia), along with topical application of lignocaine hydrochloride 1% (Pfizer Laboratories, New York, USA).