Anterior Optic Nerve Head Perfusion is Dependent on Adjacent Parapapillary Choroidal perfusion

Recent studies reported that parapapillary microvascular dropout (MvD) was significantly associated with glaucoma and glaucoma progression. To understand the clinical relevance/importance of MvD, it is essential to know the exact vascular anatomy of optic nerve head (ONH). Although it is known that parapapillary choroid and the deep ONH structure including prelaminar tissue are both supplied by branches of short posterior ciliary artery, it remains controversial whether parapapillary choroid provides a major contribution to the prelaminar tissue perfusion. This study investigated perfusion within and around the ONH using indocyanine green angiography. Thirty-three eyes from 33 patients with primary open-angle glaucoma and 10 eyes from 10 normal subjects were included. The temporal sequence of dye appearance in various tissues was analyzed. We also sought the microvessels directly responsible for blood supply to the prelaminar tissue. The perfusion of the prelaminar tissue, which occurred in a sectoral fashion, was dependent on the dye appearance in the adjacent parapapillary choroid. In addition, microvessels crossing over the optic disc margin from the parapapillary choroid to the ONH were found. The findings suggest that the centripetal flow from the parapapillary choroid is an important source of prelaminar tissue perfusion.

ONH filling. The ONH filling occurred in a sectoral fashion and propagated uniformly from the disc margin to the central retinal vein (i.e., in a centripetal direction) both in normal subjects (Fig. 1, Supplemental video 1) and glaucoma patients (Figs 2 and 3). The time interval between when the dye first appeared within the ONH and peak intrapapillary dye filling was 10.46 ± 2.85 seconds in POAG patients and 9.83 ± 3.43 seconds in normal subjects.
Temporal relationship between the filling of the optic nerve head and other vascular structures. The order in which dye appeared in various vessels varied markedly among subjects (Fig. 4). CRA, cilioretinal artery, and parapapillary choroidal arteriole were filled without a regular order. It contrasted to parapapillary choroidal arteriole filling being universally seen at the same angiographic frame (Fig. 4A) or earlier ( Fig. 4B) than the appearance of microvessels crossing over the optic disc margin. Regions of the ONH were not perfused until the visualization of those microvessels (Figs 1-3, Table 2). The microvessels appeared at the early stage of ONH filling, followed by diffuse filling of the adjacent parapapillary choroid, which probably represented the filling of the choriocapillaris. Occasionally, sectoral delayed filling was found (i.e., the microvessels were not evident even after the diffuse filling of the adjacent parapapillary choroid; Fig. 5). In those cases, the adjacent ONH filling was also delayed until the visualization of microvessels crossing over the disc margin (Fig. 5).
Regional absence of microvessels between the parapapillary choroid and the onH. Microvessels crossing over the optic disc margin were not visualized regionally throughout the entire ICGA session in 15 POAG eyes (45.5%). Such finding was not observed in any of the healthy eyes. In those regions the corresponding ONH sector was not perfused until the end phase of ICGA. Sectoral nonperfusion was also observed in the corresponding parapapillary area (Fig. 6, Supplemental video 2).

Discussion
The temporal sequence of first dye appearance in the retinal, choroidal and ciliary artery was diverse among subjects. The CRA, SPCA and cilioretinal arteries are all branches of ophthalmic artery. The interindividual variation of the first dye appearance in those branch vessels reflects variation in the length of the path of blood vessels and in the velocity of the blood flow in those vessels.
An important finding of this study is that the parapapillary choroidal arteriole was universally filled before the adjacent ONH filling. The parapapillary choroid and the prelaminar tissue share the common source of perfusion (i.e. SPCA). If the parapapillary choroid and the prelaminar tissue are supplied by separate branches of SPCA, the time sequence of perfusion between the former and latter may be varying among individuals as is seen in other branches of ophthalmic artery in the present study. However, the dye appeared in the ONH in a sectoral fashion only after the substantial filling of the adjacent choroid in all eyes. These data suggest that the prelaminar tissue is likely to be distal to the parapapillary choroid in perfusion. If not, the branches supplying the parapapillary choroid and the prelaminar tissue should have a comparable length and similar velocity along their paths from their common origin (i.e., more proximal branch of SPCA) in all cases, which is not likely. The sectoral ONH perfusion was initiated by the appearance of microvessels crossing over the optic disc margin. Moreover, when these vessels were not visible at any time point in the ICGA session, the corresponding  www.nature.com/scientificreports www.nature.com/scientificreports/ sector of the ONH was also not perfused. These finding suggests that these microvessels are directly responsible for the ONH perfusion. The origin of these microvessels is of particular interest. It is obvious that they are not the branches of retinal vessels since their appearance was not related with the dye appearance in the CRA. Then, they can be either choroidal or scleral vessels. It is known that vessels below the choroid are not easily visible on ICGA 20,21 . Scleral vessels such as Zinn Haller circle were reported to be only visible in ICGA in the area of parapapillary atrophy 22 . If the microvessels crossing over the optic disc margin were underneath the choroidal layer, they should be observed more easily through the window of choroidal perfusion defect. This was not the case: the microvessels were not visible in any case of choroidal perfusion defect. Therefore, it may be reasonably proposed that they are in the choroidal layer and dropped out in eyes with choroidal perfusion defect (i.e., MvD). Taken together, our data suggest that ONH perfusion, which occurs in a sectoral fashion, is largely dependent on the centripetal flow from the parapapillary choroid.
Cioffi et al. demonstrated small vessels between the parapapillary choroid and ONH using corrosion casting 23 . We consider that the microvessels crossing over the optic disc margin are the correspondent of the small vessels from the parapillary choroid and ONH identified by corrosion casting. Although Cioffi et al. asserted that the contribution of these centripetal small vessels to the prelaminar region is minor compared to the direct branches from the SPCA 23 , our data suggest that those microvessels may give a critical contribution to prelaminar tissue perfusion. This notion is consistent with other studies which demonstrated the dependency of ONH perfusion to the adjacent parapapillary choroid in experimental occlusion model of posterior ciliary artery 24 or in eyes with nonarteritic anterior ischemic optic neuropathy 25,26 .
This study has a limitation. The sample size was small. A larger study is needed to investigate whether there is an interindividual variation in the dependency of ONH blood supply to parapapillary choroid. However, universal dependency of sectoral ONH perfusion on the dye appearance in the adjacent parapapillary choroid and microvessels crossing over the disc margin from the parapapillary choroid suggests that this is likely the predominant variation if variations exist.
In conclusion, we have demonstrated that the perfusion of the ONH sector was dependent on the dye appearance in the microvessels crossing over the optic disc margin from the parapapillary choroid to the ONH. The findings suggest that the centripetal flow from the parapapillary choroid is an important source of anterior ONH perfusion.

participants. This prospective study enrolled POAG patients who visited Seoul National University Bundang
Hospital between November 2014 and May 2015. Healthy control subjects were recruited by advertisement and also among patients who visited the clinic due to nonglaucomatous problem (e.g., incipient cataract, dry eye). Written informed consent to participate was obtained from all subjects. This study was approved by the Seoul Figure 4. Relative time to perfusion in the branches of ophthalmic artery. Although the order in which dye appeared in various vessels varied markedly among subjects, choroidal arteriole filling was seen at the same angiographic frame (A) or earlier (B) than the appearance of microvessels crossing over the optic disc margin in all cases. (A) Cases where first dye appearances in the parapapillary choroidal arteriole and the microvessels crossing over the optic disc were simultaneous (17 cases in total). In 11 eyes, the central retinal artery (CRA) filling appeared at the same frame with the filling in the parapapillary choroidal arteriole and the microvessels. In 4 eyes, the CRA filled earlier than the latter and vice versa in 2 eyes. (B) Cases where first dye appearance in the parapapillary choroidal arteriole was earlier than that in the microvessels (26 cases in total). The first dye appearances in the CRA and cilioretinal artery (cilioretinal a.), in relation with those in the parapapillary choroidal arteriole and the microvessels were highly variable. www.nature.com/scientificreports www.nature.com/scientificreports/ National University Bundang Hospital Institutional Review Board and followed the tenets of the Declaration of Helsinki.
POAG was defined as the presence of an open iridocorneal angle, glaucomatous optic neuropathy such as notching, rim thinning, and RNFL defect, and corresponding defects in the visual field. A glaucomatous visual field defect was defined as (1) outside the normal limits on the glaucoma hemifield test; (2) three abnormal points, with a probability of being normal of P < 0.05, and one abnormal point with a probability of being normal of P < 0.01 by pattern deviation; or (3) a pattern standard deviation of <5% confirmed on two consecutive reliable tests (fixation loss rate of ≤20%, and false-positive and false-negative error rates of ≤25%). The normal controls had an IOP of ≤21 mmHg, no history of increased IOP, a normal appearing optic disc, and a normal visual field.
To be included, eyes had to have a BCVA of 20/40 or better, a spherical equivalent range from −8.0 diopters to o 3.0 diopters, cylinder correction within ±3.0 diopters, and no history of intraocular or corneal refractive surgery. The exclusion criteria were combined retinal or neurologic diseases that could affect visual function, unreliable visual field tests (a fixation loss rate of >20%, or false-positive or false-negative error rates of >25%). When both eyes were eligible, one eye was randomly chosen for ICGA. indocyanine green angiography. The parapapillary and intrapapillary choroidal circulation was examined using ICGA 27,28 . Unlike fluorescein, ICG does not leak from the choriocapillaris due to its binding affinity for plasma proteins 29,30 , allowing better evaluation of the fine microvasculature. ICGA was performed using a scanning laser ophthalmoscope (Heidelberg Retinal Angiograph-2, Heidelberg Engineering) equipped with eye-tracker function. A 20° image was obtained around the optic disc. Images were obtained at the fastest possible rate (approximately every 1 to 2 seconds) from the time when the dye first appeared until the peak angiographic phase, and then every 5 to 10 seconds thereafter until 2 minutes after ICG injection. After 2 minutes, 6 to 8 more images were obtained approximately every 30 seconds 11 . The pattern of ONH perfusion was investigated by examining the serial ICGA images. In addition, the microvessels directly responsible for ONH perfusion were sought.
Temporal relationship between the filling of the optic nerve head and other vascular structures. The times when the dye first appeared within the ONH, CRA, parapapillary larger choroidal arterioles, and cilioretinal artery (when present) were recorded by two masked observers (KML and JMK) who were blinded