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The Impact of Hindlimb Suspension on the Rat Eye: A Molecular and Histological Analysis of the Retina

Corey A. Theriot
Corey A. Theriot
, 
Patricia Chevez-Barrios
Patricia Chevez-Barrios
, 
Thomas Loughlin
Thomas Loughlin
, 
Afshin Beheshti
Afshin Beheshti
, 
Nathaniel D. Mercaldo
Nathaniel D. Mercaldo
 and 
Susana B. Zanello
Susana B. Zanello
   | Sep 18, 2021
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Article Category: Research Note
Published Online: Sep 18, 2021
Page range: 86 - 103
DOI: https://doi.org/10.2478/gsr-2021-0007
Keywords
© 2021 Corey A. Theriot et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Histological examination of young female rat HS + 90 days recovery eye sections. (A) Low power magnification (2X) H&E stain shows residual peripheral normal retina (arrows) with a posterior thin retina forming the chorioretinal scar. The lens (L), vitreous (V) and sclera (S) are unremarkable. (B) High magnification (20X) H&E stain of a chorioretinal scar. (C) 40X magnification view of the retina in the scar immunostained with anti-GFAP antibody shows the irregular proliferation of glial cells in the inner layers (facing vitreous (V)). Immunohistochemistry, DAB chromogen, Glial fibrillary acidic protein antibody. (D) 40X magnification of the edge of the retinal scar immunostained with vimentin highlights the proliferating and activated Müller cells. Vitreous (V). Immunohistochemistry, DAB chromogen, vimentin antibody.
Histological examination of young female rat HS + 90 days recovery eye sections. (A) Low power magnification (2X) H&E stain shows residual peripheral normal retina (arrows) with a posterior thin retina forming the chorioretinal scar. The lens (L), vitreous (V) and sclera (S) are unremarkable. (B) High magnification (20X) H&E stain of a chorioretinal scar. (C) 40X magnification view of the retina in the scar immunostained with anti-GFAP antibody shows the irregular proliferation of glial cells in the inner layers (facing vitreous (V)). Immunohistochemistry, DAB chromogen, Glial fibrillary acidic protein antibody. (D) 40X magnification of the edge of the retinal scar immunostained with vimentin highlights the proliferating and activated Müller cells. Vitreous (V). Immunohistochemistry, DAB chromogen, vimentin antibody.

Figure 2

Histologic effects of HS in the rat retina of young male and female rats. (A) H&E staining at 20X magnification of peripheral retina showing few inner layer ganglion cells (arrow) and some condensation of vitreous (V). (B) Images of retina from YM at 180 days at equator is thicker but also shows some drop in ganglion cells (arrow) and condensed band of vitreous (V). H&E, original magnification 20X. (C) Immunohistochemistry example of GFAP staining of the glial and Muller cells in equatorial retina a male hind limb suspended (HS + 14 days recovery). Immunohistochemistry, DAB chromogen, GFAP antibody, 40X magnification. (D) Immunohistochemistry example of vimentin staining of Muller cells in the peripheral retina of a male hind limb suspended (HS+90 days recovery). Immunohistochemistry, DAB chromogen, vimentin antibody, 40X magnification.
Histologic effects of HS in the rat retina of young male and female rats. (A) H&E staining at 20X magnification of peripheral retina showing few inner layer ganglion cells (arrow) and some condensation of vitreous (V). (B) Images of retina from YM at 180 days at equator is thicker but also shows some drop in ganglion cells (arrow) and condensed band of vitreous (V). H&E, original magnification 20X. (C) Immunohistochemistry example of GFAP staining of the glial and Muller cells in equatorial retina a male hind limb suspended (HS + 14 days recovery). Immunohistochemistry, DAB chromogen, GFAP antibody, 40X magnification. (D) Immunohistochemistry example of vimentin staining of Muller cells in the peripheral retina of a male hind limb suspended (HS+90 days recovery). Immunohistochemistry, DAB chromogen, vimentin antibody, 40X magnification.

Figure 3

Histologic effects of HS in the rat optic nerve. (A) Image of a young male rat at 14 days HS at 40X magnification showing the posterior retina surrounding the optic nerve with intense immunoreactivity to GFAP antibody in the head of the nerve. Immunohistochemistry, Permanent Red chromogen, GFAP antibody.
Histologic effects of HS in the rat optic nerve. (A) Image of a young male rat at 14 days HS at 40X magnification showing the posterior retina surrounding the optic nerve with intense immunoreactivity to GFAP antibody in the head of the nerve. Immunohistochemistry, Permanent Red chromogen, GFAP antibody.

Figure 4

Microarray analysis of rat experimental cohorts. (A) Experimental groups represented in the microarray analysis. Groups for which no retinas were available are marked with a red X. (B) Principal component analysis (PCA) of gene expression in all experimental groups shown in A. Left, color coded for treatment; middle, color coded for gender; right, color coded by cohort (YM, YF, OM). (C) PCA showing clustering by treatment of YM and YF at 14 days of treatment.
Microarray analysis of rat experimental cohorts. (A) Experimental groups represented in the microarray analysis. Groups for which no retinas were available are marked with a red X. (B) Principal component analysis (PCA) of gene expression in all experimental groups shown in A. Left, color coded for treatment; middle, color coded for gender; right, color coded by cohort (YM, YF, OM). (C) PCA showing clustering by treatment of YM and YF at 14 days of treatment.

Figure 5

Heatmap of the significantly regulated genes (FDR < 0.05). Euclidean clustering was used for clustering the genes.
Heatmap of the significantly regulated genes (FDR < 0.05). Euclidean clustering was used for clustering the genes.

Figure 6

Upstream regulator analysis utilizing Ingenuity Pathway Analysis (IPA) on the significantly regulated genes (FDR < 0.05). The results are shown only for the common upstream regulators across 14 and 90 days as a heatmap of the activated z-scores.
Upstream regulator analysis utilizing Ingenuity Pathway Analysis (IPA) on the significantly regulated genes (FDR < 0.05). The results are shown only for the common upstream regulators across 14 and 90 days as a heatmap of the activated z-scores.

Figure 7

Network diagram of “Key Genes” comparing HS vs controls for females for (A) 14 days, (B) 90 days, (C) 104 days, and (D) 180 days. For young males for (E) 14 days, (F) 90 days, and (G) 104 days. For old males for (H) 14 days, (I) 104 days, and (J) 180 days. Transcriptionally upregulated genes in red, downregulated genes in green and connectivity with predicted relationships between these key genes are displayed using Ingenuity Pathway Analysis software. The figure legend indicates the line relationship with solid lines representing known direct relationships between the genes and dash lines representing the known indirect relationships. The arrows in the networks represent which direction the genes are acting on each other.
Network diagram of “Key Genes” comparing HS vs controls for females for (A) 14 days, (B) 90 days, (C) 104 days, and (D) 180 days. For young males for (E) 14 days, (F) 90 days, and (G) 104 days. For old males for (H) 14 days, (I) 104 days, and (J) 180 days. Transcriptionally upregulated genes in red, downregulated genes in green and connectivity with predicted relationships between these key genes are displayed using Ingenuity Pathway Analysis software. The figure legend indicates the line relationship with solid lines representing known direct relationships between the genes and dash lines representing the known indirect relationships. The arrows in the networks represent which direction the genes are acting on each other.

Figure 8

The network view of the key related genes CSNK1A1, TP53, and EGR1 interaction predicted using STRING. The figure legend displays the meaning of the line colors and symbols.
The network view of the key related genes CSNK1A1, TP53, and EGR1 interaction predicted using STRING. The figure legend displays the meaning of the line colors and symbols.

Average IOP values by age, side, and time.

Time (days) YM Cohort OM Cohort Cross-sectional Age Comparisons (Old–Young)
Left Eye (95% CI) Right Eye (95% CI) Left Eye (95% CI) Right Eye (95% CI) Difference (95% CI) p-value (p-FDR)
0 18.0 (16.9, 19.0) 18.1 (17.0, 19.1) 19.1 (17.7, 20.6) 19.3 (17.8, 20.7) 1.19 (−0.51, 2.89) 0.168 (0.213)
45 24.6 (23.2, 26.0) 24.7 (23.3, 26.1) 22.0 (20.6, 23.3) 22.1 (20.7, 23.4) −2.62 (−4.51, −0.72) 0.007 (0.011)
90 21.1 (20.0, 22.3) 21.2 (20.1, 22.4) 24.2 (22.9, 25.5) 24.3 (23.0, 25.6) 3.03 (1.40, 4.65) <0.001 (0.001)
135 22.7 (21.4, 24.0) 22.8 (21.5, 24.1) 22.0 (20.5, 23.5) 22.1 (20.6, 23.6) −0.69 (−2.61, 1.24) 0.482 (0.557)
180 26.2 (23.8, 28.7) 26.3 (23.9, 28.8) 18.7 (15.9, 21.5) 18.8 (16.0, 21.6) −7.53 (−11.17, −3.88) <0.001 (<0.001)

HS-induced IOP temporal changes by age cohort.

Temporal Comparison (days) YM Cohort OM Cohort Change Comparisons by Age Group (Old–Young)
Estimate (95% CI) p-value (p-FDR) Estimate (95% CI) p-value (p-FDR) Difference (95% CI) p-value (p-FDR)
45 vs. 0 6.62 (5.05, 8.20) <0.001 (<0.001) 2.82 (1.17, 4.46) 0.001 (0.002) −3.81 (−6.08, −1.53) 0.001 (0.002)
90 vs. 0 3.19 (1.96, 4.41) <0.001 (<0.001) 5.02 (3.43, 6.61) <0.001 (<0.001) 1.83 (−0.17, 3.84) 0.073 (0.098)
135 vs. 90 1.54 (0.43, 2.65) 0.007 (0.011) −2.17 (−3.44, −0.91) 0.001 (0.002) −3.71 (−5.40, −2.03) <0.001 (<0.001)
180 vs. 90 5.08 (2.47, 7.69) <0.001 (<0.001) −5.48 (−8.46, −2.49) <0.001 (0.001) −10.55 (−14.52, −6.59) <0.001 (<0.001)

Average IOP values by sex, side, and time.

Time (days) YM Cohort YF Cohort Cross-sectional Sex Comparisons (Female–Male)
Left Eye (95% CI) Right Eye (95% CI) Left Eye (95% CI) Right Eye (95% CI) Difference (95% CI) p-value (p-FDR)
0 17.9 (16.9, 18.9) 18.1 (17.1, 19.1) 19.1 (18.2, 19.9) 19.3 (18.4, 20.1) 1.15 (−0.08, 2.39) 0.067 (0.091)
7 19.4 (18.5, 20.3) 19.6 (18.7, 20.5) 19.7 (18.9, 20.5) 19.9 (19.1, 20.7) 0.31 (−0.80, 1.43) 0.582 (0.644)
14 20.8 (19.9, 21.7) 21.0 (20.1, 21.9) 20.3 (19.5, 21.2) 20.5 (19.7, 21.4) −0.48 (−1.65, 0.68) 0.411 (0.486)
28 23.2 (22.0, 24.3) 23.4 (22.2, 24.5) 21.4 (20.4, 22.5) 21.7 (20.6, 22.7) −1.73 (−3.24, −0.22) 0.025 (0.036)
45 24.5 (23.2, 25.9) 24.7 (23.4, 26.1) 22.4 (21.2, 23.6) 22.6 (21.4, 23.8) −2.16 (−3.90, −0.42) 0.015 (0.023)
90 21.1 (20.1, 22.1) 21.3 (20.3, 22.3) 22.6 (21.7, 23.5) 22.8 (21.9, 23.7) 1.51 (0.18, 2.83) 0.026 (0.037)
97 20.8 (19.8, 21.9) 21.1 (20.0, 22.1) 22.6 (21.7, 23.6) 22.8 (21.9, 23.8) 1.77 (0.42, 3.13) 0.011 (0.016)
104 20.8 (19.8, 21.9) 21.1 (20.0, 22.1) 22.7 (21.7, 23.6) 22.9 (21.9, 23.9) 1.84 (0.46, 3.21) 0.009 (0.014)
180 26.2 (23.9, 28.4) 26.4 (24.1, 28.7) 24.2 (21.8, 26.7) 24.4 (22.0, 26.9) −1.94 (−5.27, 1.40) 0.254 (0.307)

HS-induced IOP temporal changes by sex cohort.

Temporal Comparison (days) YM Cohort YF Cohort Change Comparisons by Sex Group (Female–Male)
Estimate (95% CI) p-value (p-FDR) Estimate (95% CI) p-value (p-FDR) Difference (95% CI) p-value (p-FDR)
7 vs. 0 1.49 (1.15, 1.83) <0.001 (<0.001) 0.64 (0.34, 0.95) <0.001 (<0.001) −0.84 (−1.30, −0.39) <0.001 (0.001)
14 vs. 0 2.91 (2.25, 3.57) <0.001 (<0.001) 1.27 (0.67, 1.87) <0.001 (<0.001) −1.64 (−2.53, −0.75) <0.001 (0.001)
28 vs. 0 5.27 (4.08, 6.46) <0.001 (<0.001) 2.39 (1.31, 3.47) <0.001 (<0.001) −2.89 (−4.49, −1.28) <0.001 (0.001)
45 vs. 0 6.62 (5.15, 8.10) <0.001 (<0.001) 3.31 (1.97, 4.65) <0.001 (<0.001) −3.31 (−5.31, −1.32) 0.001 (0.002)
90 vs. 0 3.19 (2.03, 4.34) <0.001 (<0.001) 3.54 (2.51, 4.57) <0.001 (<0.001) 0.35 (−1.20, 1.90) 0.656 (0.700)
97 vs. 90 −3.68 (−5.15, −2.22) <0.001 (<0.001) 0.25 (−1.09, 1.59) 0.714 (0.742) 3.93 (1.95, 5.92) <0.001 (<0.001)
104 vs. 90 −3.68 (−5.21, −2.16) <0.001 (<0.001) 0.31 (−1.08, 1.71) 0.659 (0.700) 4.00 (1.93, 6.07) <0.001 (<0.001)
180 vs. 90 1.64 (−0.76, 4.04) 0.180 (0.223) 1.86 (−0.70, 4.42) 0.155 (0.201) 0.22 (−3.29, 3.73) 0.902 (0.902)
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