Longitudinal study: measurement of OBF using LSFG

Instead of using typical wooden restraints with the head out, the rabbits were comfortably wrapped in a towel with the paws inside, and the ears free and slightly loose around the neck to keep the animals warm, feeling safe, and comfortable during handling. LSFG measurements were taken from the right eye throughout the study. The baseline readings were randomly taken within one month before surgery, followed by taking readings after the surgery until day 28. The ONH images were captured and the integrated using the LSFG Analyzer software version 3.2.3.0 (Softcare Co, Fukuoka, Japan), which automatically synchronizes the captured MBR images with the beginning and end of the cardiac cycle recorded within 4 s of acquisition time. The synchronized cardiac cycle images were normalized to one image sequence representing a complete cardiac cycle of blood flow in the ONH. A rectangular rubber band was manually set on the ONH region of interest. The same rubber band, at an identical position, was used for the subsequent images of the same rabbit. By using the vessel segmentation function, PWF parameters in the MA, MV, and MT regions were obtained, as presented in Fig. 2b–j. The PWF parameters assessed using LSFG, namely MBR_max, MBR_min, AC, BOS, BOT, RR, FR, FAI, ATI, and RI in all three regions were computed. Figure 3 describes the waveform subdivision area of an average MBR graph plotted against time. The waveform is divided into 12 sections (A1–A12). The blue shaded area represents the S1-area (A3, A5, A6) and S2-area (A7, A10, A11), and the gray-shaded area represent the half width of a complete blood flow wave⁹.

BOS, an index for vessel resistance, indicates how much blood flow is being maintained in the vessel, with (DC) denotes the mean quantity of blood flow, and is defined as

BOT represents the length of time blood flow is maintained at a high level in each heartbeat, signifying the vessel elasticity. The (W) is the half width of the blood flow wave, and (F) is the full width of the blood flow wave and BOT is computed as

RR quantifies the upward area of the wave, whereas FR quantifies the downward area of the wave, with C set as 25 to match the color balance used in the LSFG analyzing software and computed as

FAI indicates the index of the maximum amount of change in MBR, and RI shows the difference of the maximum and minimum MBR by the maximum MBR, and computed as

Thereafter, S1-area and S2-area were compared among baseline, day 14, and day 28 using one-way repeated measure ANOVA. To calculate the area, a second-order trend line was fitted on the MBR heart beat plotted graph to obtain the line regression equation, and the constants a, b, and c [Eq. (⁹)] were individually computed to obtain the exact curve for the area calculation. Referring to Fig. 3, the S1-area and S2-area were calculated as