This web audiovisual is based on a presentation given by C.D. Fermin, Ph.D. to audience of the Symposium below. Dr. Fermin is a professor of Pathology and Otolaryngology at Tulane Medical School. Phone 504 584-2521 FAX 587-7389. email:fermin@tmc.tulane.edu. In depth description of figures & scientific interpretation is available upon request.

Presented at the Phase-1 NASA-MIR Symposium, JSC Houston Tx 8/5/97-8/7/97.The Symposium was organized and supervised by J. Uri, Ph.D. and J. Valverde at JSC

The work presented in this report was made possible by a NASA grant NAGW2-999 to C.D. Fermin, Ph.D. Additional NASA grants supported the other investigators. Data in this report is © and requires permission before use! Additional references about this type of work can be extracted from selected references cited in (Dr. Fermin's Publications).

Introduction-Pair 1

In this presentation the research of 10 investigators participating in the SLM project and whom work in different fields is summarized. Special thanks to NASA AMES (ARC) personnel for their continued and devoted efforts to ensure that all investigators fulfilled the requirements needed to satisfy regulatory and governmental policies: Dr. K. Souza, Dr. G. Jahns, S. Piert, K. Lagel, & T. Schnepp; in NASA headquarters to Dr. Schneider and J. Vernikos for supporting this important avian project; and to the Russian team Dr. V. Sychev, Dr. T. Gurieva, Dr. O. Dadasheva and Dr. D. Lychakov for their invaluable help during numerous debriefing and dissection of every specimen returned.

This presentation was accessed via URL http://www.som.tulane.edu/ferminlab. The views presented here are those of the author and not of Tulane Medical school.

The Project & Investigators-Pair 2

A team of investigators with diverse experience in anatomy, molecular biology and animal husbandry earned peer acceptance and approval of 10 distinct projects to investigate tissues of specimens flown to the MIR space station and returned via the US space Shuttle. Upon return of specimens from MIR, investigators met at NASA ARC to dissect and divide the specimens according to funded protocols. Specimens were shared with Russian investigators involved in the program. Studies proposed were justified by previous work on the STS-29 Shuttle that showed specific effect of microgravity upon the development of sensory structures in the inner ear responsible for the correct posture and balance of the body (Discussion of STS-29 Findings). Balance of erect animals such as bipedal birds and primates is a complex process that depends greatly on visual and vestibular cues shaped by gravity on earth. Analysis of the absence of gravity is only possible in space where it can be removed for prolonged periods of time. Brief removal of gravity possible in KC flight is not sufficient to study embryological processes that require weeks to complete.

Objectives-Pair 3

 The original objectives of the projects were changed slightly after unexpected problems with the performance of the Russian incubator were detected in flight. In addition, specimens were shared with the Russian colleagues reducing the available number of specimens to satisfy statistical testing of the data. The overall objectives of the SLM project were met and investigators were able to evaluate egg fertility and viability, organ formation, eye and ear structures development, bone formation, vascular development and calcium utilization. Development of US controlled hardware will infuse much needed flexibility in the development of payloads that maximize science return.

Avian Model-Pair 4

Besides being precocious, quail and chicken hatchlings are ideal models for space experimentation. Being precocious afford birds fully functional sensory structures upon hatching. Thus, hatchlings can walk and balance, and have fully functional vision. Other attractive features of the avian developmental model are that it has been studied for centuries with a wealth of information accumulated on its development, behavior and survival. Furthermore, fertile eggs can develop in space in self-contained system (shell) that do not require crew time, a valuable commodity in space. Birds can also potentially provide a rich source of protein for long duration flights.

Acceleration & Vibration-Pair 5

Ascent vibration & acceleration of the space vehicles at launch demand special protection of its cargo. Eggs with fragile and thin shells would seem unsuited to withstand the forces created by the ascent acceleration of the Space Shuttle. However, controls on both variables showed that eggs withstand both physical parameters and develop normally. Synchronous controls subjected to similar vibration and acceleration as those encountered in space but developing under 1.0G load developed normally, whereas those that developed in space in the absence of gravity showed significant modifications of their sensory structures. In particular, structures responsible for balance and equilibrium.

Afferent Neurons-Pair 6

Immunohistochemical staining (More on Inner Ear Antibody Labeling via this link) of inner ear afferent neurons with anti-neurofilament protein antibody suggested that chick embryos exposed to microgravity for only 5 days during development had more afferent nerve terminals in their sensory epithelia. Similar increase in afferent terminals was reported by Dr. M. Ross in rats flown in space and indicates that sensory structures responsible for keeping the body erect at 1.0G on earth are affected by reduced gravity.

Behavioral Measures-Pair 7

One of the Russian investigator involved with this research showed previously that quails hatched in space required time to readapt to the earth environment. In addition, the quails showed ataxia and desorientation similar to that exhibited by humans upon return from space flights. It is believed that behavioral deficits of posture and balance are related to modifications induced by microgravity to the sensory neurons that process sensory perception of the environment and tell the brain the body position in space. Modifications of the sensory pathways requires time to correct. Fertile eggs flown into space and returned to earth were allow to hatch. Vestibular thresholds were measured on the hatchlings non-invasively weeks after returned to 1.0G and showed thresholds shift in space and not in ground synchronous controls. Such delayed effect suggests that readaption of certain behavior requires weeks to complete. Anecdotal accounts by Russian cosmonausts indicate that even 6 months after return to 1.0G following long term stay in the MIR station there were occasional miss judgments in sensory perception. It is clear that future studies of the avian model should include behavioral testing for comparing responses of flight and ground synchronous controls and for validating the morphological changes already documented.

Hardware-Pair 8

There is a considerable difference between the technologies used by the US and the Russian space agencies. The incubator used for the STS-29 experiment was not available for the MIR project. Instead a Slobak incubator used by the Russian in previous flights and certify to flight on MIR was used. The Slovak incubator consisted of a cylinder with cells for positioning the fertile eggs. The US incubator approved to flight in the Shuttle was a sophisticated instrument with temperature and humidity control. Future avian experiments with fertile eggs should insist on proven technology that delivers humidity and temperature control within a reasonable and acceptable margin of error. With today technology, the incubator should be able to store data on such important parameters of egg development to evaluate upon return to earth.

Embryonic Membranes-Pair 9

Preliminary results from Dr. Lelkes & Unsworth suggests that the vasculature in the choriollontoic embryonic membrane (CAM) of synchronous and control embryos may be affected by variables other than microgravity alone. The CAM is responsible for gas exchange/oxygenation of the embryo. This group uses morphometric analyses aided by imaging technology to study changes in the embryonic membranes that nourish the embryo development. This and other work suggest that certain critical developmental changes occur during narrow windows of time (critical periods). Accurate knowledge of critical periods of development of cells, tissue formation and organogesis will permit investigators to design experiments that maximize embryo survival and availability of viable materials.

Bone-Mineral-Pair 10

Dr. Doty from the Special Surgery Hospital in NY uses Transmission electron microscopy and elemental analysis of thin sections to detect changes in mineral composition in bone of flight and synchronous controls. There seem to be critical windows of development when primarily the utilization of calcium and phosphorous differs between the two groups. This is an important part of the SLM-1 research because bone retains its structural architecture even after the sub optimal fixation that is possible in space where the hazardous chemical of ground laboratory can not be used. All fixative used on earth to produce acceptable ultrastructural preservation of cells and tissues are off-limits in the Shuttle and MIR due to their toxicity and hazardous properties. In addition, volume and weight remain important variables for space flight payloads prohibiting the volume:solid ratios of fixative:tissue required for optimal fixation. The compromise reached for the MIR project proved acceptable but not optimal and future analyses will require modifications of present protocols.

Bone-Mineral-Pair 11

Dr. Hester & Dr. Orban evaluated the mineral content of the shell to determine calcium and phosphorous utilization during development. Dr. Hester's results on calcium utilization from shell and those of Dr. Doty agree on the existence of critical periods of development for the formation and refinement of many developmental processes. Both, the study of bone and shell take advantage of the fact that bone and shell retain their structural integrity even following sub optimal fixation. Both Dr. Doty (Left Figure) and Dr. Hester/Orban analyses (Right Figure) are essential in space exploration because bone and shell retain structural integrity better than soft tissues.

Eyes-Pair 12

Dr. Conrad & Dr. Barrett takes advantage of the resilience of the cornea to withstand pressure and resist damage to determine variability in its morphology. Besides evaluating the innervation pattern of synchronous and flight embryos, this group takes advantage of an ossicle ring that surrounds the cornea of quails and provides a landmark for detecting variability in the formation of the eye. Other aspects of quail development will be evaluated and presented in future gathering. In the next slide sets I will describe properties and changes of the avian inner ear (Dr. Fermin's expertise) that justify the chick as a model for space experimentation.

Tissue Density-Pair 13

Fixation of inner ear structures is difficult even on the earth laboratories. The temporal bone that houses the inner ear is the hardest bone of the body. The inner ear tissues are heterogeneous and surrounded by fluids. Not only are the tissues hard to reach (Left Image), but once the fixing solution reaches the inner ear it can be diluted by the endolymph and perilymph before reaching the cells. This difficulty of fixative penetration into the inner ear can be appreciated from the composite diagram illustrating several layers of hard tissue from the skin toward the brain fixing solutions must diffuse through before reaching the inner ear structures. Fixatives must also penetrate the CAMs and are diluted by unused egg white. The membranous labyrinth of birds is similar to the mammals except for the presence of an otoconia containing organ at the end of a straight auditory organ. In mammals, the auditory organ is coiled (cochlea), but the sensitivity needed to detect vestibular and auditory stimuli to keep the body in tune with the environment is comparable in birds and mammals.

Ideal-Real-Pair 14

Space experiments meant to analyze cell integrity provide a reality check to the researcher that is accustomed to ground laboratories. Even after aldehyde fixation, the structural integrity of cells fixed in space may not match that of tissues fixed on the ground because the ideal ratio of 1:20 (tissue:fluid) can not always be met due to payload restrictions. The ideal preservation of inner ear gravity detectors (linear accelerometers) is shown on the left after fixation of inner ear tissues by substituting the blood of the chicks with saline and then with fixative through a heart perfusion. The procedure delivers fixative to the space between cells where it can cross link the proteins of the cytosol and preserve the cell integrity. The distance between the epithelia and the otoliths (membrane with crystals that at 1.0G provide load to deflect the stereocilia of the receptor hair cells) was maintained due in part to the embedding of structures in methacrylate plastic instead of wax embedding (More on Inner Ear Attributes) . The slide on the right shows extraction of cytosol from the epithelia due to weak cross linking of proteins in the cytosol and extraction of wax. Wax embedding of tissues yields more reliable antibody-antigen recognition than plastic embedding and thus the compromise. Nonetheless, analysis of organs, tissues and cells is possible in space fixed tissues (Right Figure).

Angular Acceleration Detectors-Pair 15

The arrangement of afferent nerve fibers in the crista of the ampulla of birds is identical to that previously demonstrated for mammals shown on the right. Different types of afferent neurons contact different types of detectors hair cells to provide adequate information to the brain about the position of the body in space. Anti-neurofilament staining of the fibers on chicks returned from the STS-29 experiments (Fixed on the ground immediately after return from space) demonstrated that labeling of the fibers in well fixed samples returned from space is possible and yield important information about morphological properties of afferent neurons developed at 1.0G and in microgravity.

Linear Acceleration Detectors-Pair 16

The arrangement of afferent nerve fibers in the macula of the utricle and saccule of birds is identical to that previously demonstrated for mammals shown on the left. Different types of afferent neurons contact different types of detectors hair cells to provide adequate information to the brain about the position of the body in space. Anti-neurofilament staining of the fibers on chicks returned from the STS-29 experiments demonstrated that labeling of the fibers in well fixed samples returned from space is possible and yield important information. Count of fibers inside the epithelia suggests that microgravity induces sprouting of afferent terminals around the receptor hair cells. This observation requires corroboration because it may explain the delay recovery of certain vestibular functions at 1.0G after prolong space flights. In other words, rewiring of the circuits may be necessary both during flight in space and after return to 1.0G (see pair 7).

Neuronal body (Perikarya)-Pair 17

Besides the well documented sprouting of afferent fibers inside the sensory epithelia (see Pair 6), there seems to be an increase in the surface area (that may reflect an increase in volume) of the perikarya (soma) or bodies (left Figure, and left histogram of right Figure). There was however no difference in the afferent fibers (Right Figure) of the in flight (top) when compared to ground (bottom) embryos outside the basement membrane of the sensory epithelia. In the ear these bipolar neurons (two axons) connect the hair cells to the brain processing center for detection of linear and angular acceleration. An increase in volume of the perikarya where the metabolic machinery of the cell resides may be related to hastened modifications of the neuronal functioning brought about by a changed environment. These type of observations must be corroborated in future experiments that are only possible in the planned space station, where prolonged periods of reduced gravity will be possible and where an on board 1.0G centrifuge may be available.

Subtle Modifications-Pair 18

The original planned morphological analyses of space and ground synchronous controls were based on the assumption that functional alteration of human vestibular system leading to motion sickness would have morphological correlates of similar magnitude. However, many of the functional modifications observed in space and after return to earth at 1.0G may be subtle and have little or no visible morphological modifications, specially those that are reversible such as latent synapse activation . It is known that inactive synapses may be activated during hastened functional demands and deactivated thereafter. Such process of activation-deactivation is economical to systems, as opposed to formation of new neurons. Subtle differences between these STS-29 macular epithelia structures are seen at the level of the support cells nuclei and the distance between the basement membrane and the lumen of the epithelia. Such changes may not yield statistically significant differences but are nevertheless representative of actual metabolic modifications of the system and justify further analyses. Left top flight and bottom control. Right top control and bottom flight.

Rocks in the Ear-Pair 19

All vertebrate have crystalline structures inside the inner ear that have been retained throughout evolution as necessary loading masses to deflect the stereocilia of hair cells (More on Inner Ear). This is a requirement for proper transduction of mechanical into neural energy. If you recall the discussion introduced with the Tissue Density(Pair 13), the inner ear tissues are difficult to fix in space where perfusion of chemical hazardous chemical through the heart is not possible. Assuming a reasonable fixation by passive diffusion as permitted in space, one has to accept other inherent difficult to control variables of the system. On the left the crystalline nature of the otoconia (hexagonal crystal (Otoconial Membrane as loading Masses) obtained on earth fixed controls embedded in plastic un calcified. Unless the tissues are decalcified with chelators such as EDTA, sectioning at 1-10 micrometer thickness is not possible. Incomplete penetration of the plastic embedding medium (methacrylate) into the dense otoconial membrane is common. While the preservation of cells on the sensory organs of flight materials shown at right is not comparable to the grown shown at left, the architecture of components is evident, and the cupula and otoconia retain their organiztion.

Ultrastructural details-Pair 20

The main problem with mild fixation such as is possible in flight, is that the cytosolic components are only weakly cross-linked and are therefore easily extracted during dehydration. To successfully cut soft tissues as thin as 60 nm, proteins must be fixed in place, lipids oxidized and water must be removed. All these procedures are harsh and usually extract those components of the cells that are not strongly immobilized. These transmission electron micrographs (Compare with Pairs 10-11) of space fixed tissues demonstrate that there is sufficient ultrastructural details to evaluate cellular changes. However, the axoplasm (See Pairs 15-16) of the afferent fibers was extracted. In other words, the antigens present in the sections of Pairs 15-16 were extracted by the harsh procedure needed to prepare electron micrographs. Since knowing the molecules present in cells and understanding their topography are both important, a multidisciplinary approach is needed to maximize science return from space experiments and compromises such as this are inevitable.

Gross morphological changes-Pair 21

On the left figure (bottom right) a flight embryo inner ear shows bending of the horizontal canal whereas the synchronous control has no such bending. Such gross morphological changes are rare and the impact of the observation must be tested statistically. Sharing of specimens among numerous investigators is a great mean to ensure a multidisciplinary approach for evaluating the effects of microgravity upon developing vertebrates, but sharing should be assessed carefully to ensure that sufficient samples remain in each group to permit acceptable and meaningful statistical tests. Gross examination of brain showed little differences between flight (Right) and ground controls. Microdissection of individual inner ear organs yield no difference either. However, the wiring of cells inside individual organs may be altered and requires further evaluation.

Discussion & Lessons Learned-Pair 22

The results obtained from the MIR avian project are encourashing because they demonstrated that fertile eggs can be incubated and fixed in an orbiting space station and then returned to earth via the space Shuttle for scientific analyses. The ability to incubate eggs in space and eventually produce eggs in space is necessary to remove gravity from the developmental process. Fertile eggs produce at 1.0G on earth and brought to the space station via the Shuttle are already 22 hours post fertilization and thus have experience the gravity vector of the earth environment. True lack of gravity as contributing variable for vertebrate development of avian embryos will be possible with eggs produced in space. Results obtained thus far permitted scientists to focus aims to maximize the science-return from future space experimentation.

Future Considerations-Pair 23

The complexity of the space program and the need to mix politics with scientific design for successful completion of the SLM project suggests that cooperation between the partners of the planned international space station is a reality. Life science projects that evaluate alteration that lack of gravity may cause to vertebrates tissues and cell should be a priority on the list of things to do on the upcoming international space station. If changes that lack of gravity induce to tissues and cells were only chronic, traditional histological observations would suffice to demonstrate their severity. Unfortunately, many changes are transitory, reversible or simple sporadic and their scientific evaluation requires multidisciplinary approaches that incorporate cellular, molecular & behavioral analyses.