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Spaceflight Effects and Molecular Responses in the Mouse Eye: Preliminary Observations After Shuttle Mission STS-133

Susana B. Zanello
Susana B. Zanello
, 
Corey A. Theriot
Corey A. Theriot
, 
Claudia Maria Prospero Ponce
Claudia Maria Prospero Ponce
 y 
Patricia Chevez-Barrios
Patricia Chevez-Barrios
   | 01 jul 2013
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Article Category: Research Article
Publicado en línea: 01 jul 2013
Páginas: 29 - 46
DOI: https://doi.org/10.2478/gsr-2013-0003
© 2013 Susana B. Zanello et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

Histological analysis of H&E and Caspase-3 stained eye samples. Hematoxylin and Eosin stain, original magnification 20X : Panel A. AEM R+7, Epithelium of cornea showing focal edema of cells seen as clearing and enlargement of the cytoplasm in the basal layers (star marks the level of the basal layers) and acanthosis (thickening of more than 5 layers of cells). Panel C. FLT R+1, anterior lens with cortical cataract seen as disorganization of the fibers of the cortex (arrows at the level of the cortex). Notice the displaced nucleus (nucleus of epithelial cells of the lens should only be present in the subcapsular area and not in the cortex in the anterior portion of the lens). Panel E. FLT R+1, retina with an apoptotic neuron seen as a shrunken cell with hyperchromatic condensed nucleus and eosinophilic cytoplasm (arrow head). Remainder of retina appears morphologically unremarkable. Caspase 3 immunostaining: Panel B. FLT R+1 corneal epithelium staining positively with Caspase 3 in the superficial layers and in the basal layers (star). Positive staining of the basal cells of the corneal epithelium is seen in the focal acanthotic areas, and in the upper differentiated layers (internal positive control). Panel D. FLT R+1 lens epithelium staining with Caspase 3; notice that cortex is negative. Panel F. FLT R+1, retina with caspase-3 staining of cytoplasm of neurons (*) predominantly with faint staining of the inner nuclear layer (inl) and inner segments of photoreceptors (pr). The cytoplasm of RPE cells is also staining (arrow).
Histological analysis of H&E and Caspase-3 stained eye samples. Hematoxylin and Eosin stain, original magnification 20X : Panel A. AEM R+7, Epithelium of cornea showing focal edema of cells seen as clearing and enlargement of the cytoplasm in the basal layers (star marks the level of the basal layers) and acanthosis (thickening of more than 5 layers of cells). Panel C. FLT R+1, anterior lens with cortical cataract seen as disorganization of the fibers of the cortex (arrows at the level of the cortex). Notice the displaced nucleus (nucleus of epithelial cells of the lens should only be present in the subcapsular area and not in the cortex in the anterior portion of the lens). Panel E. FLT R+1, retina with an apoptotic neuron seen as a shrunken cell with hyperchromatic condensed nucleus and eosinophilic cytoplasm (arrow head). Remainder of retina appears morphologically unremarkable. Caspase 3 immunostaining: Panel B. FLT R+1 corneal epithelium staining positively with Caspase 3 in the superficial layers and in the basal layers (star). Positive staining of the basal cells of the corneal epithelium is seen in the focal acanthotic areas, and in the upper differentiated layers (internal positive control). Panel D. FLT R+1 lens epithelium staining with Caspase 3; notice that cortex is negative. Panel F. FLT R+1, retina with caspase-3 staining of cytoplasm of neurons (*) predominantly with faint staining of the inner nuclear layer (inl) and inner segments of photoreceptors (pr). The cytoplasm of RPE cells is also staining (arrow).

Figure 2.

8OHdG immunoreactivity in retinal neuronal layers of AEM and FLT mice. Bars indicate the mean of n=2 biological samples. Each individual neuronal cell layer was compared at R+1, R+5, and R+7 in AEM samples (panel A) and Flight samples (panel B). Representative images of 8OHdG stained histological sections of the retina in FLT samples at R+1 (panel C), R+5 (panel D), and R+7 (panel E).
8OHdG immunoreactivity in retinal neuronal layers of AEM and FLT mice. Bars indicate the mean of n=2 biological samples. Each individual neuronal cell layer was compared at R+1, R+5, and R+7 in AEM samples (panel A) and Flight samples (panel B). Representative images of 8OHdG stained histological sections of the retina in FLT samples at R+1 (panel C), R+5 (panel D), and R+7 (panel E).

Figure 3.

Quantification of Caspase-3 immunoreactivity by neuronal layer. Percentage of caspase-3 positive cells in the Inner Nuclear Layer (panel A) and the Retinal Ganglion Cell Layer (panel B) was calculated as described in Methods for day R+1 and R+7 tissue collection time points. Representative images of histological sections stain (red-brown) for caspase-3 of Flight (panel C), AEM (panel D), and Vivarium (panel E) samples at day R+1. Arrows indicate caspase-3 positive stained cells identified in different layers of the retina.
Quantification of Caspase-3 immunoreactivity by neuronal layer. Percentage of caspase-3 positive cells in the Inner Nuclear Layer (panel A) and the Retinal Ganglion Cell Layer (panel B) was calculated as described in Methods for day R+1 and R+7 tissue collection time points. Representative images of histological sections stain (red-brown) for caspase-3 of Flight (panel C), AEM (panel D), and Vivarium (panel E) samples at day R+1. Arrows indicate caspase-3 positive stained cells identified in different layers of the retina.

Figure 4.

Beta amyloid (brown) and glial fibrillary acid protein (GFAP) (red) double staining immunohistochemistry. A: FLT R+1 (mouse #13). Retina with focal positive cytoplasmic staining in neurons of the ganglion cell layer (*) with β-amyloid (brown). Perivascular (arrow) and other astrocytes in the ganglion cell layer stain with GFAP (red). Notice the negative staining of Muller cells with GFAP. B: FLT R+1 optic nerve. Note the staining of the optic nerve (O.N.) in the region posterior to the lamina cribrosa (l.c.) with GFAP and focally with β-amyloid. Non-specific staining of the orbital muscle is also seen with β-amyloid (brown). C: FLT R+1 retina higher magnification of focal positivity with β-amyloid (brown) in ganglion cell layer (*) and GFAP in astrocytes (red). D: FLT R+1 optic nerve higher magnification of immediate post-laminar region. Notice the staining of oligodendrocytes and astrocytes with GFAP (red) and the β-amyloid stain (brown) of the nerve fibers in between the glial cells.
Beta amyloid (brown) and glial fibrillary acid protein (GFAP) (red) double staining immunohistochemistry. A: FLT R+1 (mouse #13). Retina with focal positive cytoplasmic staining in neurons of the ganglion cell layer (*) with β-amyloid (brown). Perivascular (arrow) and other astrocytes in the ganglion cell layer stain with GFAP (red). Notice the negative staining of Muller cells with GFAP. B: FLT R+1 optic nerve. Note the staining of the optic nerve (O.N.) in the region posterior to the lamina cribrosa (l.c.) with GFAP and focally with β-amyloid. Non-specific staining of the orbital muscle is also seen with β-amyloid (brown). C: FLT R+1 retina higher magnification of focal positivity with β-amyloid (brown) in ganglion cell layer (*) and GFAP in astrocytes (red). D: FLT R+1 optic nerve higher magnification of immediate post-laminar region. Notice the staining of oligodendrocytes and astrocytes with GFAP (red) and the β-amyloid stain (brown) of the nerve fibers in between the glial cells.

Figure 5.

Gene expression analysis of inflammatory and oxidative stress response genes. Inflammatory response (panel A) and oxidative stress (panel B) gene expression levels from RNA isolated from retina samples in Flight (FLT), AEM, and Vivarium (VIV) samples at day R+1 and R+7, measured by real time qPCR. Y axis represents the comparative gene expression levels normalized to housekeeping genes.
Gene expression analysis of inflammatory and oxidative stress response genes. Inflammatory response (panel A) and oxidative stress (panel B) gene expression levels from RNA isolated from retina samples in Flight (FLT), AEM, and Vivarium (VIV) samples at day R+1 and R+7, measured by real time qPCR. Y axis represents the comparative gene expression levels normalized to housekeeping genes.

Figure 6.

Gene expression analysis of cell death and survival and cellular stress response genes. Cell death and survival (panel A) and cellular stress (panel B) gene expression levels from RNA isolated from retina samples in Flight (FLT), AEM, and Vivarium (VIV) samples at day R+1 and R+7, measured by real time qPCR. Y axis represents the comparative gene expression levels normalized to housekeeping genes.
Gene expression analysis of cell death and survival and cellular stress response genes. Cell death and survival (panel A) and cellular stress (panel B) gene expression levels from RNA isolated from retina samples in Flight (FLT), AEM, and Vivarium (VIV) samples at day R+1 and R+7, measured by real time qPCR. Y axis represents the comparative gene expression levels normalized to housekeeping genes.

Histologic interpretation with Hematoxylin-Eosin. Data arranged according to group (FLT, AEM, VIV) and day of sacrifice: 2 mice per group at R+1, +5, or +7, respectively.

Cornea Lens Retina ON
FLT AEM VIV FLT AEM VIV FLT AEM VIV FLT AEM VIV
Day 1 FA and E FA FA Anterior subcapsular C Nml Anterior subcapsular C Nml Nml Nml Nml Nml Nml
Bullae*, A 1+, E 2+ basal layer calcification A* 2+ FA Nml Nml Anterior subcapsular C Nml Nml Nml Nml - Nml
FA and basal E FA*, E 1+ Central E Nml Focal cortical C Nml Nml Nml Nml Nml Nml Nml
Day 5 FA Intranuclear inclusions, A 1+, E 2+ FA Nml Focal cortical C Anterior subcapsular C Nml Nml Nml Nml Nml Nml
FA FA Irregular A 1+ E 3+ Cortical C Nml Nml Nml Nml Nml Nml Nml Nml
Day 7 A* 1+, E 2+ FA Irregular A 1+ E 2+ Cortical C Nml Nm Nml Nml Nml Nml Nml Nml

Genes of interest evaluated for expression changes in the mouse retina. Grouping was done according to relevant cellular processes and complete gene name with gene symbol are provided, as well as references reporting possible relevant roles in retina physiology.

Process Gene Symbol Gene name
Cell death and survival Bax Bcl2-associated X protein
(Lohr et al., 2006) Bcl2 B-cell lymphoma 21
Bag1 Bcl2-associated athanogene 12
Atg12 Autophagy related 123
Cellular Stress response Hsf1 Heat shock transcription factor 1
Hspa1a Heat shock 70kDa protein 1A4
Sirt1 Sirtuin 15
Nfe2l2 (Nrf2) Nuclear factor (erythroid-derived 2)-like 26
Oxidative stress response Hmox1 Heme-oxygenase 17
Cat Catalase
Sod2 Superoxide dismutase 2, mitocondrial8
Gpx4 Glutathione peroxidase 49
Prdx1 Peroxiredoxin 1
Cygb Cytoglobin
Inflammation Nfkb1 Nuclear factor of kappa light polypeptide gene
enhancer in B-cells 110
Tgfb1 Transforming growth factor beta 111
Normalizing genes Rpl13 Ribosomal protein L13
Rplp0 Ribosomal protein, large, P0
Hprt hypoxanthine phosphoribosyltransferase 1
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