Molecular Effects of Spaceflight in the Mouse Eye after Space Shuttle Mission STS-135

Space exploration involves challenges for an extended human presence. The space environment exposes the human body to a unique combination of physical and psychological stressors. Most noteworthy are the effects of microgravity and high charge/high energy (HZE) particle radiation with a potential for hypoxia (in off-nominal situations), occasional hyperoxia, dietary restrictions, and possible nutritional deficiencies as significant hazardous confounding factors (Sonnenfeld et al., 2003; Taibbi et al., 2013; Taylor et al., 2013; Sides et al., 2005; Mader et al., 1999; Planel et al., 1989; Reitz et al., 2009; Cucinotta et al., 2001; Cucinotta et al., 2008). Microgravity induces cephalad fluid shifts and physical unloading, leading to deleterious effects such as muscle atrophy, loss of bone mass, cardiovascular alterations, postflight orthostatic intolerance, and immunosuppression (Pietsch et al., 2011; Sundaresan and Pellis, 2009; Blaber et al., 2013). More recently, ocular changes associated with spaceflight have been reported in astronauts exhibiting alterations in near visual acuity and other ocular-based signs and symptoms (Alexander et al., 2012). These medical data describe evidence of optic disc edema, choroidal folds and posterior globe flattening, hyperoptic shift in vision, optic nerve sheath distension, and in some cases, optic nerve kinking and empty sella (Kramer et al., 2012; Mader et al., 2011), possibly associated with intracranial pressure increase. Identifying causative factors and investigating microgravity’s influence as a source of stress for the eye have acquired importance.

Previous basic research investigating the effects of spaceflight on eye structures and general ocular health is scarce and limited to basic histopathology on space-flown animal models (Marshall-Bowman et al., 2013). Neonatal rats flown for nine days onboard the Space Shuttle mission STS-72 displayed clear evidence of retinal degeneration (Tombran-Tink and Barnstable, 2006). In another study, adult rats flown onboard Russian Cosmos satellites for approximately 14 days exhibited cellular swelling in the retina and retinal disruption (Philpott et al., 1980).

Recently, our group investigated the effects of spaceflight on the eyes of mice flown onboard Space Shuttle mission STS-133 and, for the first time, reported molecular and cellular changes correlating to the histopathologic outcomes (Zanello et al., 2013). Flight samples from that study stained positive for β-amyloid and glial fibrillary acidic protein (GAFP) in the optic nerve. Retina from mice flown on STS-133 also showed increased levels of 8-hydroxy-2’-deoxyguanosine (8OHdG), an oxidized form of guanosine that serves as a standard biomarker of oxidative DNA damage, and of caspase-3 immunoreactivity, suggesting an early step both in the extrinsic (death ligand) and intrinsic (mitochondria) apoptosis pathway. In addition, upregulated levels of oxidative and cellular stress response genes were detected. Herein, we report the findings of a subsequent animal experiment onboard STS-135 in which we expanded the molecular aspects of the impact of spaceflight on retinal biology by performing differential gene expression profiling between mice flown onboard STS-135 and their ground control counterparts.

Our work explores the possible effects of the spaceflight environment on retinal biology, in an effort to shed light on the confounding factors and processes that may contribute to vision risks in space. Corollary to a previous report on the effects of spaceflight on the retina of BALB/cJ mice (Zanello et al., 2013), we now report results of a similar experiment carried out on C57BL mice onboard STS-135. The main findings in this work demonstrate evidence of increased oxidative stress-DNA damage, histological changes, as well as potential effects of spaceflight on retinal biology based on microarray gene expression profiling. Similar results have been reported by an independent group (Mao et al., 2013); however, the gene expression profiling performed in the cited work originated from non-dissected eyes that included all eye tissues and structures. The eye is a complex organ with a heterogeneous tissue composition due to its various structures: sclera, cornea, retina, lens, choroid, retina microvasculature, aqueous humor production, and drainage system. Therefore, gene expression derived from the entire eye globe does not precisely reflect the tissue of origin of the genetic responses. Our work presents, for the first time, comparative whole genome expression profiling of retinas dissected from mice exposed to spaceflight and their ground controls. Even in this case, we must bear in mind that the retina is composed of various cell types from both neural and vascular cellular populations.

A number of activated caspase-3 positive cells were observed, in particular, in the RGC layer of FLT samples, but due to the limitation in the number of sections, our examination was limited to a qualitative comparison. Overall, caspase-3 immunoreactivity in the FLT samples was observed to be localized at moderate levels in the RGC, with light levels seen throughout the photoreceptor inner segments. Of note, cytological alterations in the photoreceptor layer from FLT mice in this study were in line with previous reports in the literature of spaceflight induced damage to the ganglion cell layer and disruption of the plexiform layers and photoreceptors of rat neonates (Tombran-Tink and Barnstable 2006). Quantification of 8OHdG immunoreactivity showed statistically significant (P<0.05) increase in DNA damage in the INL of FLT mice compared to AEM controls. Similar findings were observed in the previous experiment in BALB/cJ mice; however, BALB/cJ mice showed larger damage in the photoreceptor layer upon exposure to spaceflight. The propensity of this strain to develop retinal degeneration due to light (Song et al., 2012; Hadziahmetovic et al., 2012; Whelan and McGinnis 1988; Chang et al., 2002) (and probably other kinds of electromagnetic radiation) may provide an explanation for this difference.

Pathway analysis derived from differential gene expression of microarray data revealed processes related to neuronal and glial cell loss, mechanisms of apoptosis, and ER stress. Evidence supporting these processes included, among others, the downregulation (-1.65 fold FLT vs. AEM) of XBP1, a potent transcription factor involved in the unfolded protein response (UPR). Processing of XBP1 mRNA is an obligate step in the UPR pathway and the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER (Calfon et al., 2002; Lee et al., 2002; Sriburi et al., 2004). In addition, Hspa5, a member of the Hsp70 family that also resides in the ER and participates in protein folding, was also downregulated -1.69 fold, in agreement with a possible impairment of the ER stress response, which other studies have linked to increased susceptibility to Alzheimer’s disease (Hebert and Molinari 2007; Guzel et al., 2011; Hsu et al., 2008). A result of cellular insults that cause prolonged ER stress include apoptosis through caspase-7-mediated caspase-12 activation and lead to neuronal and glial cell loss (Nakagawa et al., 2000; Szegezdi et al., 2003).

Interestingly, XBP1 is also implicated in processes related to the reactivation of herpes virus (Lawson et al., 2008; Dalton-Griffin et al., 2009; Wilson et al., 2007). It is well known that virus reactivation is a common phenomenon associated with spaceflight, but the mechanisms leading to it are not understood (Brinley et al., 2013; Mehta et al., 2000; Mehta et al., 2004; Stowe et al., 2001). We must bear in mind that the retina is mostly composed of neural cells and that the herpes virus is neurotropic. A putative implication of XBP1 as a transcription factor crucial in the protection against viral reactivation in space is not implied, but could be further investigated to shed some light into possible targets for mitigation against the risk of viral reactivation in space.

Other differentially expressed genes between FLT and AEM retinas suggested pathways of neuronal cell loss and degeneration. SLC1A1, a member of the high-affinity glutamate transporters that play an essential role in transporting glutamate across plasma membranes, was upregulated +1.64 in FLT compared to AEM retinas. In the brain, these transporters are crucial in terminating the postsynaptic action of the neurotransmitter glutamate and in maintaining extracellular glutamate concentrations below neurotoxic levels (Sheldon and Robinson 2007; Deng et al., 2007; Arnold et al., 2006). It has also been shown to be involved in RGC death (Martins et al., 2006; Sheldon and Robinson, 2007) and its upregulation in FLT samples may suggest that glutamate level increase may be associated with space environmental factors. Concurrently, the transporter lipocalin 2 (LCN2) showed a marked upregulation of +2.75 fold in the FLT group. LCN2 has been shown in the literature to be related to inflammation in the retina following ischemia-reperfusion injury (Abcouwer et al., 2013) and may serve a similar role in modulating an inflammatory response to spaceflight.

Beta-amyloid deposits have previously been suggested as related to RGC and optic nerve degeneration (Zanello et al., 2013). Alpha-2-macroglobulin (A2M) inhibits many proteases, including trypsin, thrombin, and collagenase and is implicated in Alzheimer disease due to its ability to mediate the clearance and degradation of A-beta, the major component of beta-amyloid deposits (Liao et al., 1998; Blacker et al., 1998). Our results show that A2M was induced +1.72 fold in FLT compared to ground AEM, possibly suggesting the triggering of clearance mechanisms to reduce an increase in any deposits.

The RNA used in this study originated from dissected retina containing a mixed cell type population. While the major representation is of neural cells of various types, it is expected that some relevant genes were represented due to their expression in other retinal cell types. Endothelial cells from the retinal microvasculature are one example. S1PR3 encodes a member of the EDG family of receptors, which are G-protein coupled receptors (Crousillac et al., 2009). This protein has been identified as a functional receptor for sphingosine 1-phosphate and likely contributes to the regulation of angiogenesis and vascular endothelial cell function (Sanchez et al., 2007; Daum et al., 2009). We observed a significant downregulation of -2.26 fold in FLT retinas versus AEM, a change that may suggest vascular remodeling could be taking place in conjunction with some degree of retinal degeneration and neural remodeling and should be investigated in future studies. Changes in the expression of genes related to axonal guidance were also observed in FLT compared to AEM such as GNA13 and KALRN (Lee and Luo, 1999; Mandela et al., 2012), in agreement with the concept of vascular and neural guidance as coordinated processes (Suchting et al., 2006; Eichmann et al., 2005; Park et al., 2003). A significant upregulation of two key genes implicated in reorganization of the cytoskeleton, cell shape, and cell-cell adhesion by desmosomes, guanine nucleotide binding protein (G protein) alpha 13, and desmoplakin (+1.89 and +4.01 fold, respectively) also suggests that remodeling processes were taking placed in the stressed retinas of FLT mice.

In summary, these results bring light to some of the molecular processes taking place in the retina when mice are exposed to spaceflight environmental conditions. While we have highlighted the pathways that may be biologically relevant and correlated with the changes observed in the histomorphology of retinal tissues of animals exposed to spaceflight, this work provides a catalog of differentially expressed genes that can be used to explore various hypotheses related to the pathophysiology of visual alterations in spaceflight and translated to possible ocular implications for human crews in long-duration space exploration missions.