Intraocular injections, optic nerve injections, I.V. injections, and I.P. injections

We have previously described the procedures for intraocular (IO) and optic nerve injections^32–34,36. Animals received IO injections of each peptide at 3 and 8 days post axotomy, prior to the onset of RGC apoptosis, which occurs at 4–5 days after axotomy. Animals were placed in a stereotaxic frame and anesthetized with isoflurane, delivered through a gas anesthetic mask. The cornea was anesthetized using Alcaine eye drops (Alcon) prior to IO injections. A pulled glass micropipette, attached to a 10-µl Hamilton syringe via a hydraulic coupling through PEEK tubing, was used to deliver 4 µl of a peptide solution (10 mg/ml) into the vitreous chamber of the eye, posterior to the limbus. Care was taken to prevent damage to the lens or anterior structures of the eye. The pipette was held in place for 5 s after injection and slowly withdrawn from the eye to prevent reflux. Injections were performed using a surgical microscope in order to visualize pipette entry into the vitreous chamber and confirm delivery of the injected solution.

PTEN PDZ interactions were antagonized using cell-permeable peptides based on the PDZ-binding motif of PTEN (Table ​(Table1).1). The protein transduction domains consisted of a fragment of the HIV TAT protein (Y-G-R-K-K-R-R-Q-R-R-R^37–39; or a sequence of nine arginine residues (R9))^40–43. Both PDZ peptides were dissolved in normal saline and delivered via IO injection at a concentration of 10 mg/mL (4 µl intraocular) at 3 days following optic nerve transection or ophthalmic artery ligation.

PDZ peptides

The peptides consisted of two components covalently bound together: an active component composed of an amino acid sequence containing the PDZ-binding motif involved in the target PDZ interaction; and a protein transduction component to mediate the transportation of the peptide into the cell

To directly target RGCs, peptide solutions were injected into the transected optic nerve stump using a 10-μl Hamilton syringe. A total of 5 μl of peptide suspension (10 mg/ml) was injected. The majority of the injected fluid will reflux out of the optic nerve during this procedure, so the remaining pool of peptide suspension was left in place when returning the orbital contents to their original positions.

For intravenous injections, a bolus of 1 ml of saline containing 0.5 mg of dissolved peptide was delivered via a tail-vein injection at both 24 h and 8 days following ophthalmic artery ligation (stroke). Injections were delivered using a 25-gauge needle attached to a 5-ml syringe, while animals were anesthetized with isoflurane.

For intraperitoneal (IP) injections, 1 ml of saline containing 0.5 mg of dissolved peptide was delivered via a 25-gauge needle attached to a 5-ml syringe, following rapid anesthesia with isoflurane to incapacitate the animal.

To test the effect of PTEN on regeneration in the axotomized optic nerve, animals were randomized into four groups: a control group that received IO injections of normal saline (control vehicle, n = 8); a group that received treatment of TAT-PTEN via IO injection (n = 6); a group that received Gelfoam soaked in TAT-PTEN solution (n = 6), applied to the cut end of the optic nerve; a group that received R9-PTEN treatment via IO injection (n = 4). To test the effect of PTEN on RGC survival in the ischemic retina, animals were randomized into three groups (n = 7 each): a control group that received IO injections of normal saline (control vehicle) following surgery; a sham-surgery group that received a sham surgery (all steps were followed with the exception of the ligation step) and did not receive IO injections; a treatment group that received ligation surgery followed by IO injections of PTEN (R9-PTEN; Table ​Table11).