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2. MATERIALS AND METHODS

2.1. STS-72 In-Cabin Payloads

STS-72 was launched at the Kennedy Space Center on a 9 day Mission on January 11, 1996. The space shuttle carried the NIH.R3 Life Science Payload, a proof-of-concept study designed to test whether the Animal Enclosure Module (AEM-NF) nursing facility was capable to support nursing rats and neonates, to evaluate maternal behavior of rat dams in the cages, and to verify the retrieval of clinically healthy animals post flight. Six litters of Sprague-Dawley rats were used in the experiment. Each litter consisted of one nursing dam and 10 neonates. Two identically age and weight-matched litters were launched for each neonate ages at post-natal days 5 (PN5), 8 (PN8), and 15 (PN15). Similar groups were house on earth as ground controls.

2.2. In-Flight Rodent Conditions

All animals were fed normal chow or nursed and exposed to normal on/off light cycles. In flight activities consisted of daily health checks, water refill, and videotaping the animals. The rats were in flight for 9 days. STS-72 orbital data were: Altitude: 250 nautical miles; (288 statute miles) Inclination: 28.45 degrees. Number of Orbits: 142; Duration: 8 days, 22 hours, 01 minutes, 47 seconds; Distance travelled: 3.7 million miles.

2.3. Post Flight Evaluation of the Rodents

Post-flight, animals were removed from the AEM-NF and assessed for survival rate and health. It was reported that 6 out of 20 PN5 neonates, 19 out of 20 PN8 neonates, and all of the PN15 neonates survived the 9 day Space Mission. All flight groups weighed less than the corresponding ground controls, possible due to cage effect and independent of gravity or microgravity.

All surviving flight animals were in good condition as described by the inspecting veterinarian. The AEM-NF supported rat neonates aged PN8 or older at launch, but was unsuitable for the support of PN5 or younger rodents at launch. Because of the sample size of only 2 dams, it was difficult to determine whether the poor survival of the PN5 group was due to poor dam behavior.

2.4. Dissection and Preservation of the Rodent Eyes Post Flight

Healthy rodents were sacrificed immediately after the shuttle returned to earth and the eyes from both experimental and ground controls (caged and uncaged) were dissected and placed in 4% paraformaldehyde. The samples were archived at the Ames Research Laboratories until histological and morphometric studies were performed.

2.5. Preparation of Retinal Tissue for Histological Examination

The NIH.R3 neonatal rodent eyes were obtained from the Ames Research Laboratories, after inspection by Drs. Paul Callaghan and Alison French. Age and weight matched

controls were also obtained for comparison in our study to determine the effects of space flight conditions on retinal cytoarchitecture.

The anterior segment of each eye was removed with a sharp razor blade and the posterior segment cryoprotected overnight in 30% sucrose. The eyecups were place in freezing molds containing 100% OCT and frozen overnight at -80°. Samples were cryosectioned at 15 mm and thawed onto subbed slides. Sections were stained with hematoxylin/eosin for five minutes at room temperature. After rinsing, sections were dehydrated, cleared, and mounted. Sections were viewed with brightfield and DIC optics using a Zeiss microscope.

2.6. Immunocytochemistry, Lectin, and Morphometric Studies

Sections were rehydrated in PBS, preincubated in 5% normal goat serum, 0.1% Triton X-100 in PBS (G-PBS) to block non-specific binding and then incubated in primary antibody overnight at 4°C. After washing in PBS, sections were incubated for 45 min at room temperature in Cy3-labeled secondary antibody diluted in G-PBS. After washing with PBS, sections were mounted in VectaShield with DAPI, coverslipped and viewed with a Zeiss widefield microscope equipped with DIC and epifluorescence optics. Control sections were incubated in normal mouse or rabbit serum diluted to an equivalent immunoglobulin concentration as test antibodies.

Calibrated images of sections were collected from several regions of the central retina of each section. The thickness of various retinal layers was measured in microns. At least three non-adjacent sections of each eye were measured.

3. RESULTS

The focus of this study was to examine the histological appearance of the retinas of rodents exposed to space environment. The results presented below represent the first findings of photoreceptor outer segment loss and other disruption of normal retinal development in the orbiting eyes. The results were obtained after examination of a large number of retinal sections obtained from at least 3 neonate rats at 3 different stages of development flown on orbital flight (STS-72) for 9 days.

3.1. Loss of Photoreceptor Outer Segments and Disruption of the RPE Monolayer in the Retina of Neonatal Rodents During Orbital Space Flights

The most striking difference among the space eyes and controls was the decreased length of rod photoreceptor outer segments. In normal rat retinal development, the outer segments of photoreceptor cells start to develop at about PN5 and reach their full length by about PN28 (Obata and Usukura, 1992; Bumsted et al., 2001). In figure 1, we show the length of the photoreceptor outer segments in rodents at PN5 launch (Fig 59.1A), PN8 launch (Fig 59.1B) and PN15 launch (Fig 59.1C). At all three postnatal days, the photoreceptor outer segments (OS) were either absent or shortened in animals exposed to 9 days of space travel environment. In addition, we observe that immature rod cell bodies, labeled with the RET P1 opsin antibody, present in the inner nuclear layer (INL) of both ground controls, are absent in the experimental animals. The histological preparations (Fig 59.2) show disruption of the retinal pigment epithelium (RPE). In most cases the RPE layer is

A

B

C

Figure 59.1. RET-P1 labeling showing differences in length of photoreceptor outer segments in female Sprague Dawley rats. A. PN5 launch, dissected PN14 GAS1:ground control (no cage), wt-10.9 g. AFS1: flight animals, wt10.1 g; VS4: control (ground-cage), wt-10.7 g. B. PN8 launch, dissected PN17. GAM1:ground control (no cage), wt-15.6 g. AFM1: flight animals, wt-16.5 g; VM1: control (ground-cage), wt-16.2 g. C. PN15 launch, dissected PN24. GAL1:ground control (no cage), wt-29.7 g. AFL1: flight animals, wt-29.2 g; VL1: control (ground-cage), wt-30 g.

discontinuous or is not seen attached to the retina in the flight animals. There is little difference in the length of the inner segments of rod photoreceptors in the ground control and in flight rodents. Measurements of the average length of photoreceptor outer segments in control and experimental groups for each age are presented in Figure 59.3.

3.2. Disruption of Normal IPL Development

The IPL contains the synapses that link the bipolar cells to the amacrine and ganglion cells of the inner retina. In normal development conventional synapses in the IPL occurs

Figure 59.2. Hematoxylin and Eosin staining of the PN5-PN14 animal series. Note disrupted choroid (Ch) and choroidal vessels, lack of distinct RPE layer (RPE), and thinner IPL between ground control and flight tissue.

between PN3-PN10 and ribbon synapses between PN11-15 (Weidman and Kuwabara, 1968; Robinson, 1991). PN15 marks the time of eye opening in the animals and at this time there is a sharp reduction in both types of synapses. The widths of the IPL in ground controls and in flight rat neonates as well as those for the IS and OS are given in Figure 59.3. At PN5, PN8, and PN 15, the average width of the IPL in ground controls are approximately 48 mm, 58 mm, and 61 mm, respectively. In neonates exposed to the space flight environment, however, there is a significant decrease in the IPL at all three postnatal time points with the most dramatic reduction seen at PN15. The thinner IPL is obvious in the H&E stained micrographs of Fig 59.2. The micrographs also show large spaces in the IPL in the retina of the inflight animals suggesting that there is shrinkage and degeneration of the neuropil comprising the IPL.

3.3. Loss of Ganglion Cells

As many as 25 different ganglion cell types are found in the mammalian retina. These are the first cells to differentiate in the retina and vary in cell body size, dendritic arborization, and laminar branching patterns. The appearance of the cells in the ganglion cell layer of the retina of ground-control rodents are morphologically distinct from those seen in the retina of the matched in flight neonates (Figure 59.2). The ganglion cell layer of the control animals had more cells that were round with abundant cytoplasm. In the retinas of the space flight animals, the ganglion cells are elongated and form a discontinuous layer. The ganglion cells have scanty cytoplasm, appear detached from the neuropil of the IPL, and generally appear unhealthy. This suggests that the space flight animals have extensive damage to the ganglion cell layer.

Figure 59.3. Thickness of retinal layers in the three sets of animals examined. There is a general shortening of outer segments and thinning of the inner plexiform layer in space flight animals. IPL, inner plexiform layer; IS, rod inner segments; OS, rod outer segments. Top panel, PN5 launch; AFS-3, AFS-4 and AFS-5 are the flight animals. Middle panel, PN8 launch; AFM-1, AFM-3 and AFM-5 are the flight animals. Bottom panel, PN15 launch; AFL-1, AFL-3 and AFL-4 are the flight animals. All other animals are ground controls.

4. DISCUSSION

Experimental studies have linked the perception of “light flashes” and hallucinations by astronauts in flight to the penetration of ionizing nuclei through the nervous tissue. There is some evidence that cosmic rays induced cell death in the outer nuclear layer of rats flown in space (Philpott et al., 1978), that microgravity induces intraocular pressure and vascular changes in the eye (Mader et al., 1993), and promotes apoptosis in astrocytes (Uva et al., 2002).

This study shows abnormal development of the retina and loss of photoreceptor outer segments but has not allowed us to determine whether the effects we observed in the

neonatal rodent retina are transient, reversible, or dependent on flight duration. Nor does it answer the question as to whether these changes are triggered by a warp in gravitational force, solar radiation, impact of launching or reentry into the earth’s atmosphere, or fluctuations in oxygen level.

It is possible that neonatal retinas do not adapt as well to the hazards encountered in the space travel environment as adult retinas. The impact on the neural retina in neonates, however, highlights the importance of developing more rigorous research efforts to study how the eyes adapt or respond to various space related assaults since the duration of manned space journeys will be significantly increased in the near future and could have irreversible adverse effects on human health and performance.

Knowledge of how this unusual environment affects molecular mechanisms and pathways of the CNS are key to accelerating development of appropriate physiological risk mitigation measures to remove biological barriers that could impede the astronauts’ ability to survive and function in future long-term human space exploration. Such studies could lead to information that is important to understanding and treating similar earth-based retinal disorders as well.

5. ACKNOWLEDEMENTS

We thank Drs. Paul Callahan and Alison French (Ames Res Center, CA) and Dr. Louis Ostrach (NASA, Washington, DC) for making this study possible. Supported by NIH, the Connecticut Lions and RPB Inc.

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