Measuring Deformation in the Mouse Optic Nerve Head and Peripapillary Sclera

Abstract

Purpose: To develop an ex vivo explant system using multiphoton microscopy and digital volume correlation to measure the full-field deformation response to intraocular pressure (IOP) change in the peripapillary sclera (PPS) and in the optic nerve head (ONH) astrocytic structure.

Methods: Green fluorescent protein (GFP)-glutamate transporter-GLT1 (GLT1/GFP) mouse eyes were explanted and imaged with a laser-scanning microscope under controlled inflation. Images were analyzed for regional strains and changes in astrocytic lamina and PPS shape. Astrocyte volume fraction in seven control GLT1/GFP mice was measured. The level of fluorescence of GFP fluorescent astrocytes was compared with glial fibrillary acidic protein (GFAP) labeled astrocytes using immunohistochemistry.

Results: The ONH astrocytic structure remained stable during 3 hours in explants. Control strain-globally, in the central one-half or two-thirds of the astrocytic lamina-was significantly greater in the nasal-temporal direction than in the inferior-superior or anterior-posterior directions (each P≤ 0.03, mixed models). The PPS opening (perimeter) in normal eye explants also became wider nasal-temporally than superior-inferiorly during inflation from 10 to 30 mm Hg (P = 0.0005). After 1 to 3 days of chronic IOP elevation, PPS area was larger than in control eyes (P = 0.035), perimeter elongation was 37% less than controls, and global nasal-temporal strain was significantly less than controls (P = 0.007). Astrocyte orientation was altered by chronic IOP elevation, with processes redirected toward the longitudinal axis of the optic nerve.

Conclusions: The explant inflation test measures the strain response of the mouse ONH to applied IOP. Initial studies indicate regional differences in response to both acute and chronic IOP elevation within the ONH region.

Figure 1

(A) The inflation test chamber for mouse eyes with two tilt-correcting gears mounted on the imaging stage. (B) Optic nerve head area (arrow) facing the objective lens with the globe cannulated. (C) The maximum-intensity projection of a Z-stack from imaging the astrocytes in the ONH and surrounding sclera (see Supplementary Movie S3 for a video in color through the Z-stack) at 10 mm Hg. (D) Second harmonic generation image of PPS surrounding the ONH at 10 mm Hg. This is a summation of the slices through Z projection image of the PPS. Superior (S) and temporal (T) margins are indicated. The inner scleral contact zone is indicated by the arrow and one of three vascular channels passing through the inferior sclera is marked by an asterisk.

Figure 2

(A) Control GLT1/GFP optic nerve area of a 2D Z-averaged intensity projection of the astrocytic lamina with its perimeter delineated in yellow (superior [S] and nasal [N]) at baseline pressure. (B) An ellipse was fitted to the marked perimeter (yellow line) and an inner ellipse (red line) was calculated to include one-half of the optic nerve area.

Figure 3

(A) A 2D Z-averaged intensity projection of a control GLT1/GFP astrocytic lamina at baseline pressure with the optic nerve area outlined in red and oriented with the superior (S) on top and nasal (N) on the right. (B) For a Z-stack imaged at 10 and 30 mm Hg, the area in which DVC correlation was accurate is shown in the colored area, and the degree/direction of displacement indicated by DVC is shown by the color shading in the contour plot. The ONH area is outlined in red. Note that correlation was least likely in the more peripheral ONH.

Figure 4

Two-photon fluorescence images illustrating the detailed structure of mouse ONH astrocytes remaining stable for 3 hours in control GLT1/GFP explants (A, C = lower magnification; B, D = higher magnification) at 10 mm Hg. When IOP was increased from 10 mm Hg (E, F) to 30 mm Hg (G, H) for 2 hours, visible changes were detected in astrocyte somal shape (F, H: astrocyte soma elongates) and process orientation (E, G: astrocyte process straightens; arrows; magnification factor bar indicates 100 μm in A and C and 20 μm in B, D–H).

Figure 5

Digital volume correlation calculated displacements and strains of one control GLT1/GFP ONH astrocytic lamina (mouse 2; superior at top of images) between 10 and 30 mm Hg illustrating the (A) nasal-temporal (N-T) displacement field, (B) inferior-superior (I-S) displacement field, (C) anterior-posterior (A-P) displacement field, (D) normal strains along N-T, (E) normal strains along I-S, (F) normal strains along A-P. The color bar for the displacement plots (A, B, C) is in microns.

Figure 6

Mean percent change in the maximum ONH diameter (N-T) of control GLT1/GFP at each of 10 locations, equally spaced in the Z-stack from nearer to the retina (right) to the posterior astrocytic lamina (left), for IOP change between 10 and 30 mm Hg. The parameter here was the maximum diameter of the perimeter of the ONH analyzed as an ellipse.

Figure 7

Example from one control GLT1/GFP ONH of displacements (A, B, C) and strains (D, E, F) in the X, Y, and Z directions (A, D: X = nasal-temporal direction; B, E: Y = inferior-superior direction; and C, F: Z = anterior-posterior direction) between 10 and 20 mm Hg. The superior ONH (at the top of the images) successfully correlated, along with the inferior and nasal portions of the PPS. The temporal portion of the PPS did not correlate (at left of each image). The color bar for the displacement plots (A, B, C) is in microns.

Figure 8

Confocal images of control (A, B) and chronic IOP elevation (C, D) in cryosections of ONH (GFP, green; GFAP, red; 20×). Although astrocyte structure appears generally in the plane of section in control, the change in orientation in chronic IOP elevation eye leads to oblique sections through many astrocytes.