Fig. 2
Intermittent intraocular hypertension and analysis of the optic nerve axon density. a An exemplary profile of 12 iOHT eyes conducted at the ninth day of the experiment shows the effective mean IOP (ordinate) during simulation of a 1-h wavelike IOP profile (abscissa). IOP reduces to 10 mmHg after anesthesia, and IOP is raised to 35 mmHg for 20 min (interval I), followed by 45 mmHg for another 20 min (interval II). The loop was released to simulate a pressure drop. IOP was reset to 35 mmHg for 20 min (interval III), and IOP decreased to 12.5 mmHg during recovery phase. Arrows indicate important pressure changes before and after iOHT. b The survival of optic nerve axons was evaluated as the number of axons in an area of 0.05 mm2. Injured eyes of iOHT group showed a significant loss of optic nerve axons compared with fellow eyes in the same group (gray bar, p < 0.01) and control eyes (black bar, p < 0.01). Additionally, axon densities of injured eyes of iOHT group were significantly reduced compared with fellow eyes of iOHT + Belimumab group (grey bar, p < 0.01) but not to injured eyes of the iOHT + Belimumab group (horizontal lines, p = 0.05). Note that no significant loss of axonal density was observed for injured eyes of the Belimumab-treated group compared with fellow eyes (p = 0.5) and with control eyes (p = 0.07). Significances are indicated as follows: **p < 0.01. c A representative p-phenylenediamine-stained ultrathin transverse cross-sections (×10 magnification) shows the area where 15 high-resolution pictures were taken for the optic nerve axon density analysis. In total, 32 % of the optic nerve was analyzed for axon density survival. Scale bar represents 200 μm. d–h Representative high-resolution images (×100 magnification) show different stages of degeneration in optic nerve cross-sections. Healthy axons were present in control optic nerves (ON, d), while fellow eyes of iOHT ON (e) and iOHT + Belimumab (g) showed a slight damage, including swollen and collapsing axons indicated by arrows. Major axon degeneration with bulged myelin sheaths around axons (arrows) and gliotic areas (triangle) were observed in injured ONs of iOHT group (f) and iOHT + Belimumab group (h). Scale bar represents 20 μm