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. 2021 Mar 25;16(3):e0243186.
doi: 10.1371/journal.pone.0243186. eCollection 2021.

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION)

Yan Guo  1 , Zara Mehrabian  1 , Mary A Johnson  1 , Neil R Miller  2 , Amanda D Henderson  2 , John Hamlyn  3 , Steven L Bernstein  1   4
Affiliations

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION)

Yan Guo et al. PLoS One. .

Abstract

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SD-OCT detectable changes following rNAION.
A-C: Sequential en face views of the rodent retina and intraocular portion of the ON in a pre-induced naïve (A), a 5hr post-induction (B), and a 2d post-induction animal (C). A. Naïve (uninduced). The ONH has a reddish flush, with the ON border is definable as a slightly lighter color against the retina (Ret). Retinal arteries (ar) and veins (ve) are normal in appearance, and are not engorged. B. 5hr post-induction. The ONH is slightly lighter in appearance but still flat against the retina. Retinal veins are slightly engorged. C. 2d post-induction. The ON is edematous and pale. The border of the intraocular ON is poorly defined. D-F: Sequential cross-section individual slice views of the same nerves shown above, using the Heidelberg instrument and a commercially available rodent fundus contact lens. D. Naïve. The intraocular ONH diameter is determined by the INL-INL cross-sectional distance (arrows). E. 5hr post-induction. The ONH diameter is slightly enlarged (arrows), and there is engorgement of the vascular tip above the ONH. F. 2d post-induction. The ONH diameter is significantly expanded in the intraocular region (arrow), and the nerve fiber bundle has become lighter (edema) and the layers less distinct.
Fig 2
Fig 2. Mean post-induction ONH diameter correlates with RGC loss.
A. Correlation of mean post-induction ONH diameter with RGC loss. Post-induction RGC loss is minimal in animals with mean post-ONH diameters <500μm (10/13 animals had RGC loss <20%), whereas relative RGC loss is much greater in induced eyes with mean ONH diameter>500μm; r2 is 0.56. B. No correlation of pre-induction ONH diameter with subsequent RGC loss; r2 is 0.00. C. No correlation of ONH diameter between naïve eye and 2d post-induced eye. The r2 value is 0.01.
Fig 3
Fig 3. rNAION induction severity in response to laser induction time.
A. Comparison of RGC counts in animals induced for 9, 10 and 11 seconds. The X-axis gives the condition, whereas the Y axis gives the % remaining RGCs. Naïve RGC numbers are given as 100±2.5% (SEM). There is a progressive loss of RGCs with increasing laser induction time from 9–11 seconds (Fig 3A). B. Scatterplots of individual retinal counts for each eye. There is considerable variability in RGC loss with induction times <11 seconds, and a threshold effect (RGC loss) is clearly discernable in animals treated for 9 or 10 seconds, with ~half of all animals exhibiting significant RGC loss (defined as <20%).
Fig 4
Fig 4. Information obtained through fluorescein and ICG angiography of uninduced (A-D) retinae and ONH.
All imaging was done at similar times with the same fundus lens. A and B: IVFA and ICGA imaging, 30 sec post-injection. The IVFA reveals inner retinal vasculature (Ar and Ve). The intraocular optic nerve head (ON) appears as a dark center in the IVFA panel. B. ICG of uninduced eye. The dark ON head (ONH; arrow) is surrounded by a ring of peripapillary choroidal (Peripap Chor) fluorescence surrounding the rat ONH. C and D: IVFA and ICGA photos, respectively, of the same animal, taken 6min post injection. The ON and veins (Ve) are barely visible by IVFA, whereas little if any fluorescence remains in the ICGA channel.
Fig 5
Fig 5. Comparison of characteristics of three similarly induced (9 second) rNAION animals.
All animals evaluated at the same time with the same lens and parameters. A-F: paired IVFA (A,C,E) and ICGA (B,D,F), respectively, of three induced eyes 5hr post-induction (ICG 5mg/ml/kg). Diffuse fluorescent signal is present following fluorescein administration above the disk in all three animals, whereas the ICGA photos reveal localized ICG leakage pattern limited to the ONH, the central retinal vascular bundle, and the peripapillary retinal pigment epithelium (RPE). The inner retinal vasculature is apparent in one animal by fluorescein (E; compare with A and C). G-L: Two day post-induction color fundus photo- (G,I,K) and SD-OCT-based cross-sectional image- (H,J,L) pairs taken at the ON centers from the same eyes seen in A-F. There is apparent ON edema in all three eyes (G,I,K) and demonstrable, but varied amounts of expansion of the ON cross-sectional diameter of all three eyes (H,J,L; individual quantification bars seen above the ON). There also is dilation of some retinal veins (arrowheads, G and I) and blurring of the vasculature (see arrows in G and I). There was 46.3%, 60.1% and 28.3% RGC loss at 30 days in these three animals by stereology. M-P: examples of 2d post-induction edema quantification. Following a 150-segment SD-OCT scan using a fundus contact lens, the INL-INL distance from the three middle cross-sections with the greatest distances are averaged. M and N: imaging from a 9 second eye. O and P: Imaging from the contralateral uninduced eye. Scale bars in M-P: 200μm.
Fig 6
Fig 6. Analysis of two 6-second rNAION-induced animals (no RGC loss) at 2 days post-induction.
A-D: 5hr leakage analysis of fluorescein (A and C) and ICG (B and D). Moderate diffuse fluorescein leakage is seen in one animal (A) but almost none in the other animal (C), whereas ICG signal at the same time post-injection reveals a more complex leakage pattern, with dye localization around the ONH. E and G: SD-OCT cross-sectional photos. F and H: Fundus color photos. ONH edema is apparent by quantification of the INL-INL distance (439μm and 441μm, respectively, for F and H). There is mild blurring of the ONH margin in the fundus photo F, and none in H. I. Higher magnification of panel 5D, with increased contrast and signal showing ICG leakage. The circle indicates roughly 500um. There is mild signal at the central vascular bundle (site I) in the center of the ONH. The ONH (site II) itself is relatively dark against the surrounding area, revealing reduced leakage relative to the peripapillary RPE (peripapillary sub-RPE) (site III). Scale bar in I: 400μm.
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