Retinal Function Evaluation by ERG

In order to evaluate the condition of the retina, animals underwent a flash ERG, which records the activity of rods and cones in response to luminous stimuli. This procedure was performed 3 times in each animal (n = 24). The first examination was before glaucoma induction, to confirm normal functioning of the retina. The second exam was 3 days after glaucoma induction, to guarantee that the IOP increase had caused cellular damage to the retinal cells. The third time was at 7 days (Groups T7/C7), 14 days (Groups T14/C14), or 21 days (Groups T21/C21) after glaucoma induction to assess the potential beneficial effects of the CS/HA-EPOβ nanoformulation. After this third ERG, the experiment came to end and euthanasia and enucleation were performed.
The ERG protocol was adapted from previously published procedures [31 (link)], which demanded a prior scotopic adaptation of 12 h. General anesthesia was mandatory, and a combination of ketamine (70 mg/kg) and medetomidine (0.8 mg/kg) was administered through intraperitoneal injection. To avoid hypothermia, the animal was placed over a heating pad, and its body temperature was periodically measured. One drop of oxybuprocaine hydrochloride (Anestocil^®, Edol, Lisbon, Portugal) and one drop of a carbomer-based gel (Lubrithal^®, Dechra, Northwich, UK) was applied onto each cornea. Active silver electrodes were placed in contact with both corneas (Figure 7); reference electrodes were placed between the ear and lateral cantus, subcutaneously (Figure 7); and a ground electrode was placed at the tail base. Retinal responses were recorded simultaneously from both eyes. Light stimulation was achieved by means of a MiniGanzfeld device over the animal’s head, with a base luminescence of 3 cds/m² (0 dB). The reference impedance was <5 kohms, and the light frequency was between 0.1 and 1000 Hz.
The ERG examination was divided into 5 parts, and rod function was tested using dim flashes in scotopic conditions, while cone function was tested using bright flashes and flicker in photopic conditions. In the scotopic luminance response (SLR), light flashes of 9 intensities from −35 dB (–3.02 log cds/m²) to +5 dB (0.98 log cds/m²) were delivered 3 times per each intensity level, at a frequency of 0.1 Hz. In the photopic adaptation (PA) step, flashes were delivered 3 times after 0, 2, 4, 8, and 16 min of light adaptation, at a frequency of 1.3 Hz, and the intensity was calculated using the maximum b-wave amplitude of the SLR. The photopic luminance response (PLR) used light flashes of 9 intensities, varying from −35 dB to +5 dB, delivered 3 times at a frequency of 1.3 Hz. The photopic flicker (PF) delivered flashes of 0, −5, −10, and −15 dB, at a frequency of 6.3 Hz, after 10 min of light adaptation. Lastly, the scotopic adaptation (SA) used white dim flashes after 0, 2, 4, 8, 16, and 32 min of dark adaptation, delivered 3 times, at a frequency of 1.3 Hz. The entire ERG exam lasted for 75 min, and anesthesia was reverted with an intramuscular injection of atipamezole (2.5 mg/kg).