Iridectomy

The following zebrafish strains were used in this study: wild-type AB (Oregon) strain, Ekkwil (EK) strain, transgenic strain cmlc2::DsRed2-nuc [26 (link)] to visualize cardiomyocytes nuclei, and transgenic strain cmlc2::EGFP [27 (link)] to analyze the injured area on the whole hearts. Fish aged 6-18 months were anesthetized in 0.1% tricaine (Sigma Aldrich) and placed ventral side up in a damp sponge. A small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. The tip of the cryoprobe was 6 mm long with a diameter of 0.8 mm, the handle of the cryoprobe was 4 cm long with a diameter of 8 mm and was covered with a plastic surface. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. For heart resection surgeries, ventricular muscle was removed at the apex with iridectomy scissors as previously described [15 (link)]. Animals were allowed to regenerate for various times at 26.5°C. Experimental research on animals has been approved by the cantonal veterinary office of Fribourg.

The tunica albuginea of dissected murine testis was opened with a ∼1 mm linear incision with iridectomy scissors and fixed in a 4% solution of PFA or Bouin’s fixative at 4°C for 24 h. After the first pass of fixation, the testes were hemisected and fixed for an additional 48 h under the same conditions before washing and paraffin embedding according to the standard procedures. Embedded tissues were sectioned to a thickness of 5 µm, mounted on statically charged slides, dewaxed, and rehydrated. Sequentially, the prepared slides were stained with hematoxylin (#S211-16OZ, Poly Scientific, Bay Shore, NY, USA) and periodic acid Schiff’s reagents (#1090341000, Millipore, Burlington, MA, USA) according to the standard protocols, and then imaged at 63× using a Leica DMi8 S Platform inverted microscope system. Lastly, for the visualization of acrosomal granule displacements in Actl7a^−/− spermatids, we utilized the innate fluorescence of the PAS stain in Bouin’s fixed testis using a Y5/CY5 red filter cube with an excitation filter of 620/60 nm, a dichromatic mirror for 660 nm, and a suppression filter of 700/75 nm.

A fornix-based flap was dissected followed by mobilization of Tenon’s capsule. A sponge soaked with MMC (0.2 mg/mL) was inserted under the conjunctiva for 3 min, followed by intensive rinsing with 30 mL saline solution. A 4 × 4 mm scleral flap of one-third scleral thickness was created, and a temporal paracentesis was made. An anteriorly placed trabeculectomy was conducted and a peripheral iridectomy was created. The scleral flap was closed with four 10–0 nylon sutures, two at the edges and two at the sides. The conjunctiva was closed with meander-shaped sutures described by Pfeiffer and Grehn [14 (link)]. To re-check the bleb and tightness of the sutures the anterior chamber was inflated with a balanced salt solution.
Following both procedures, 4 mg dexamethasone was injected under the conjunctiva of the inferior fornix.

Patients were prepped and draped in a usual sterile fashion. A fornix based conjunctival peritomy was performed in the superonasal quadrant in the majority of the cases with wide blunt dissection extending posteriorly. Wet-field cautery was utilized to achieve hemostasis. An orthogonal 3 × 4 mm (TF) or 4 × 4 mm (FT) partial thickness scleral flap was dissected extending through limbus into cornea. A sponge soaked in MMC was applied under the conjunctiva for 0.5–3 min. The duration and concentration of MMC application was at the discretion of the surgeon with 0.2 mg/mL for 2 min (TF) or 0.3 mg/mL for 3 min (FT), representing the most prevalent choices. The surgical area was then copiously irrigated with balanced salt solution (BSS). A paracentesis was created to establish access to the anterior chamber, the anterior chamber was entered under the scleral flap and trabeculectomy was performed utilizing a Kelly-Descemet’s punch or a surgical blade. An iridectomy was then performed with Vannas scissors. Two interrupted 10.0 Nylon adjustable sutures (TF) or three interrupted 10.0 Nylon sutures (FT) were used to secure the scleral flap. The anterior chamber was re-inflated with BSS and the suture tension was adjusted to allow slow egress of aqueous without collapse of the anterior chamber. The sutures were locked with two additional throws and subsequently rotated to bury the knots. Finally, the conjunctiva was closed with two interrupted 8.0 Vicryl sutures (Ethicon Inc., Johnson & Johnson, Somerville, NJ, USA). A 9.0 Vicryl running suture on a BV needle (TF) (Ethicon Inc., Johnson & Johnson, Somerville, NJ, USA) or an 8.0 Vicryl running suture (FT) was used to close the wings of the conjunctival incision in a watertight fashion. An additional 9.0 or 8.0 Vicryl mattress suture was passed parallel to the limbus to decrease the incidence of early aqueous leaks. The anterior chamber was formed with BSS and the incisions were examined for leaks. Patients received a subconjunctival injection of 0.4 mL dexamethasone disodium phosphate (4 mg/mL) and of 0.4 mL gentamicin sulfate (40 mg/mL) at the end of the case. Post-operative management with regards to medication selection and additional interventions was at the discretion of the surgeon and did not follow a specific protocol. In combined cases phacoemulsification was initially performed through a separate temporal clear cornea incision that was sutured with a single 10.0 Nylon suture followed by a trabeculectomy as described above. Intraocular lens selection was at the discretion of the surgeon.