Ketamine hydrochloride

Forty Long-Evans male rats (1–3 months, 200–250 g) purchased from Envigo Bioproducts Incorporated (Madison, WI) were used to induce the ocular infection model. The animals were handled and cared for according to the IIT Institutional Animal Care and Use Committee (IACUC) protocol with the principles embodied in the Statement for the Use of Animals in Ophthalmic and Vision Research adopted by the Association for Research in Vision and Ophthalmology. Animals were fed ad libitum. Animals were anesthetized using 0.8 mg/kg body weight of 100 mg/mL ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, IA) and 0.1 mg/kg body weight of 100 mg/mL xylazine (AnaSed Injection, Akron, Inc., Decatur, IL) via intraperitoneal (IP) injection. Proparacaine drops (Bausch and Lomb, Rochester, NY) were used to anesthetize the corneas, and pupils were dilated using phenylephrine (Bausch and Lomb) and atropine drops (Bausch and Lomb). Heart rate and blood oxygen saturation were monitored with a Pulse Oximeter (8500AV; Nonin Medical, Inc., Plymouth, MN). Animals were placed on a custom-built heated stage and monitored to maintain a core body temperature of 37°C.

Monkeys were implanted with catheters to the lateral ventricle (ICV) for test article or vehicle administration and to the lumbar spine (IT-L) for serial CSF sampling as previously described [26] . Briefly, prior to catheter implantation, animals had MRI scans to determine coordinates for proper ICV catheter placement. Under sedation with ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, Iowa) and isoflurane (MWI, Meridian, Idaho) anesthesia, a polyurethane catheter (SAI Infusion Technologies) was introduced through a craniotomy into the left ventricle with the aid of stereotaxic techniques. Catheters were anchored with dental acrylic (Reliance Dental Manufacturing, Company Worth, Illinois) and a subcutaneous titanium access port (MINLOVOL; Access Technologies) was attached to the catheter. For lumbar catheter placement, an incision was made over the dorsal process at L4, L5, or L6 and the polyurethane catheter (SAI Infusion Technologies) was inserted and advanced to the area of the thoraco-lumbar junction via a hemilaminectomy. A subcutaneous titanium access port (MINLOVOL; Instech Solomon) was attached to the catheter and proper placement was confirmed by the ability to sample CSF. Animals were provided a post-surgical recovery period of at least two weeks prior to dose administration.

Both male and female wild-type (WT) C57BL/6J mice between 6 and 10 weeks of age purchased from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study. For all procedures, anesthesia was achieved by intraperitoneal injection of 100 mg/kg ketamine hydrochloride (Fort Dodge Animal Health, Overland Park, KS, USA) and 10 mg/kg xylazine (Anased, Akorn, Decatur, IL, USA), and pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon, Fort Worth, TX, USA). All animal experiments were approved by the University of Virginia Institutional Animal Care and Use Committee and was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

Unilateral optic nerve crush surgery was performed using a custom-made forceps (Sautter and Sabel, 1993). Briefly, the rats were anesthetized by intraperitoneal injection of Ketavet (0.75 mL/kg) (ketamine hydrochloride; Zoetis Deutschland GmbH, Berlin, Germany) and Dormitor (0.5 mL/kg) (medetomidine hydrochloride; Orion Corporation, Espoo, Finland). The optic nerve crush was made with calibrated cross-action forceps (KLS Martin Group, Tuttlingen, Germany) applied for 30 seconds at 2–3 mm from the eye with the forceps jaws spaced 0.2 mm apart to produce a moderate crush. Initially, connective tissue was detached from the sclera and a sharp needle (0.8 mm diameter) was used to puncture it, followed by insertion of a Hamilton syringe with blunt cannula for injection into the vitreous. For the ex vivo study, 3 μL of CaspNPs were injected into the vitreous body of both sides (n = 3) or, for control purposes, blank NPs (loaded with PBS) (n = 3), non-silencing NPs (n = 3), caspase-3 siRNA-Ca²⁺ particles (n = 3), or PBS (n = 3). For the in vivo study, bilateral ONC was performed, and immediately thereafter, the left eye was treated with 3 μL of CaspNPs and the right eye was treated with 3 μL blank NPs suspended in phosphate buffered saline (PBS, pH 7.4, 0.1 M) as control.