Ocular hypertension was induced in Group 2 by ic injection of microbeads as previously described.34 (link) Anesthesia was induced with 5% Isoflurane (Baxter Healthcare Corp, Deerfield, IL, USA)/1.5 L per minute O2 and maintained at 3.5% throughout the procedure. Using a 15° blade (Fine Science Tools, Reading, PA, USA) a small 2-mm incision was made at the peripheral cornea and aqueous humour was allowed to exude. Using the same incision site, a 10-μL solution of microbeads was administered with a glass micropipette, produced in-house from a glass capillary rod (Harvard Apparatus, Kent, UK) using a Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA, USA). The microbead solution was loaded into the microneedle immediately before injection and consisted of 5 μL of 6-μm beads (polybead polystyrene, Cat#07312; Polysciences, Inc., Warrington, PA, USA) followed by 5 μL of 10-μm beads (polybead polystyrene, Cat#17136; Polysciences, Inc.), both at concentrations of 2 × 108/mL. Administration was made slowly and the needle was retracted with a 2-minute delay to minimize leakage. Due to the variable translucency of the eye after microbead injection, reliable ERG and optical coherence tomography (OCT) measurements were not possible.
Glass capillary rod
Manufactured by Harvard Apparatus
Sourced in United Kingdom
Glass capillary rod is a cylindrical glass tube with a small internal diameter. It is used for various laboratory applications that require precise fluid handling, sample manipulation, or specialized equipment components.
Automatically generated - may contain errors
Lab products found in correlation
Flaming brown micropipette puller, by Sutter Instruments (4 mentions)
Glass micropipette, by Harvard Apparatus (2 mentions)
Polybead polystyrene, by Polysciences (1 mentions)
Isoflurane, by Baxter (1 mentions)
Pulled glass micropipette, by Harvard Apparatus (1 mentions)
Fg solution, by Biotium (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
5 protocols using glass capillary rod
1
Ocular Hypertension Induction by Microbeads
2
Intravitreal Virus Injection Protocol
3
Optic Nerve Crush and Fluorescent Tracing
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After anaesthetic induction as described above and a subcutaneous injection of buprenorphine (0.1 ml/100 g; National Veterinary Supplies) animals were secured in a head-holding frame. Intraorbital left ONC was performed in Group 1b and 2b 8–10 week old rats as described previously [31] (link). Briefly, the optic nerve was exposed and crushed using forceps 1 mm posterior to the lamina cribrosa, completely closed around the optic nerve for 5 seconds, without damaging the central retinal artery (confirmed by lack of ischemia in eyes 7–21 days after ONC). After surgery, animals were placed in warmed (30°C) recovery cages and closely monitored until the return of normal behaviour, when they were transferred to home cages. Two days before tissue harvest, all Group 1 animals were re-anaesthetised and the optic nerves re-exposed as above and 2 µl of 4% FG solution (Biotium, Hayward, CA) in sterile phosphate-buffered saline (PBS) was injected directly into the right and left nerves distal to the lamina cribrosa (proximal to the crush site in the left optic nerves in Group 1b), using a glass micropipette, produced in-house from a glass capillary rod (Harvard Apparatus, Kent, UK) using a Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA). The injected FG is incorporated into axons and retrogradely transported axonally to RGC somata.
4
Corneal Incision for Intracameral Injections
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At day 0, after the first IOP reading, the heads of anaesthetized rats were fixed in a stereotactic frame. The cornea was incised with a disposable 15 degree blade to create a self-sealing two-step incision as previously described [34 (link)]. The first and all subsequent IC injections were administered through the incision using a sterile glass micropipette, produced in-house from a glass capillary rod (Harvard Apparatus, Kent, UK).
5
Intraocular Pressure Modulation by TGF-β1 and MSC
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Following anaesthetic induction, IOP were recorded for all rats using an icare tonometer (Tonolab, Helsinki, Finland). Rats were then secured in a head-holding frame for IC injections of TGF-β1 (Peprotech, London, UK) through a single corneal incision, 2mm anterior to the limbus using a 15° blade (BD Ophthalmic System, Warwickshire, UK). Using the same incision site a glass micropipette, produced in-house from a glass capillary rod (Harvard Apparatus, Kent, UK) using a Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA) was used to inject 3.5µl of 5µg/ml activated TGF-β1 IC into all 12 rats. Contemporaneously, while the animals were still anaesthetised, a glass micropipette preloaded with 150,000 MSC suspended in 5µl of PBS, was used to inject living or dead cells (killed by heating for 30min at 80°C), into the vitreous of the eye (Fig. 1). A cell dosage of 150,000 was the maximum number of cells that could be suspended in 5µl while still allowing easy passage through the fine tip of the glass micropipette. After surgery, animals were placed in warm recovery cages and monitored for recovery of normal behaviour before being returned to their home cages. IOP recordings and IC injections of TGF-β were repeated bi-weekly 0-35d throughout the study. A separate 6 rats (Intact Group) received biweekly 0-35d IC injections of PBS alone.
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