First Proof CRISPR Can Be Safe in Cancer Therapy – Published: Feb 07, 2020 – By Mark Terry @ The future of humanity is genetic engineering and neural implants @ New technique allows scientists to ‘listen in’ on cancer cells @ Is the Law of Conservation of Energy Cancelled? Maybe energy can be created and destroyed, or maybe the notion doesn’t quite make sense @ Spaceflight Effects and Molecular Responses in the Mouse Eye: Preliminary Observations After Shuttle Mission STS-133 @ ´´Before humans risked their lives launching into space aboard the Vostok Program and Mercury Project, various organisms were flown to determine if it was possible for life to survive spaceflight. For decades, NASA, along with international partners, have continued sending biological experiments to space. Potential biological hazards from spaceflight include decreased gravity, increased exposure to radiation, altered light-dark cycles, and loads experienced during launch and landing.´´ @ VERY IMPORTANT SOCIAL NETWORKS, WEBSITES, LINKS AND IMAGES OF THE WORLD

Do the downloads!! Share!! The diffusion of very important information and knowledge is essential for the world progress always!! Thanks!!

  • – > Mestrado – Dissertation – Tabelas, Figuras e Gráficos – Tables, Figures and Graphics – ´´My´´ Dissertation @ #Innovation #energy #life #health #Countries #Time #Researches #Reference #Graphics #Ages #Age #Mice #People #Person #Mouse #Genetics #PersonalizedMedicine #Diagnosis #Prognosis #Treatment #Disease #UnknownDiseases #Future #VeryEfficientDrugs #VeryEfficientVaccines #VeryEfficientTherapeuticalSubstances #Tests #Laboratories #Investments #Details #HumanLongevity #DNA #Cell #Memory #Physiology #Nanomedicine #Nanotechnology #Biochemistry #NewMedicalDevices #GeneticEngineering #Internet #History #Science #World

Pathol Res Pract. 2012 Jul 15;208(7):377-81. doi: 10.1016/j.prp.2012.04.006. Epub 2012 Jun 8.

The influence of physical activity in the progression of experimental lung cancer in mice

Renato Batista Paceli 1Rodrigo Nunes CalCarlos Henrique Ferreira dos SantosJosé Antonio CordeiroCassiano Merussi NeivaKazuo Kawano NagaminePatrícia Maluf Cury


GRUPO_AF1 – GROUP AFA1 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO AFAN 1 – GROUP AFAN1 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO_AF2 – GROUP AFA2 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO AFAN 2 – GROUP AFAN 2 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

Slides – mestrado – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto



Avaliação da influência da atividade física aeróbia e anaeróbia na progressão do câncer de pulmão experimental – Summary – Resumo – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto


Lung cancer is one of the most incident neoplasms in the world, representing the main cause of mortality for cancer. Many epidemiologic studies have suggested that physical activity may reduce the risk of lung cancer, other works evaluate the effectiveness of the use of the physical activity in the suppression, remission and reduction of the recurrence of tumors. The aim of this study was to evaluate the effects of aerobic and anaerobic physical activity in the development and the progression of lung cancer. Lung tumors were induced with a dose of 3mg of urethane/kg, in 67 male Balb – C type mice, divided in three groups: group 1_24 mice treated with urethane and without physical activity; group 2_25 mice with urethane and subjected to aerobic swimming free exercise; group 3_18 mice with urethane, subjected to anaerobic swimming exercise with gradual loading 5-20% of body weight. All the animals were sacrificed after 20 weeks, and lung lesions were analyzed. The median number of lesions (nodules and hyperplasia) was 3.0 for group 1, 2.0 for group 2 and 1.5-3 (p=0.052). When comparing only the presence or absence of lesion, there was a decrease in the number of lesions in group 3 as compared with group 1 (p=0.03) but not in relation to group 2. There were no metastases or other changes in other organs. The anaerobic physical activity, but not aerobic, diminishes the incidence of experimental lung tumors.

@ ´´Spaceflight exploration presents environmental stressors including microgravity-induced cephalad fluid shift and radiation exposure. Ocular changes leading to visual impairment in astronauts are of occupational health relevance. The effect of this complex environment on ocular morphology and function is poorly understood. Female 10-12 week-old BALB/cJ mice were assigned to a flight (FLT) group flown on shuttle mission STS-133, Animal Enclosure Module ground control group (AEM), or vivarium-housed (VIV) ground controls. Eyes were collected at 1, 5, and 7 days after landing and were fixed for histological sectioning. ´´

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First Proof CRISPR Can Be Safe in Cancer Therapy

Published: Feb 07, 2020 By Mark Terry


Although CRISPR gene editing is touted as likely to revolutionize medicine, the actual proof of its effectiveness and safety in treating diseases has been slow in coming. At least until now. Sort of.

Researchers with the Abramson Cancer Center of the University of Pennsylvania, led by Carl Junepublished results from the first U.S. Phase I trial of CRISPR-Cas9-edited T-cells in humans with advanced cancer. The data was published in the journal Science.

The trial involved three patients with refractory cancer, two women and one man, all in their 60s. One of the patients had sarcoma and two had multiple myeloma. The approach was similar to that seen in CAR-T therapy, where the patient’s own T-cells are recovered, engineered to express a specific receptor that can detect and kill cancer cells, then reinfused into the patient.

In the case of this trial, instead of engineering the T-cells with a receptor to a protein like CD19, they used CRISPR to remove three genes from the T-cells. Two edits removed the T-cell’s natural receptors, which could then be reprogrammed to express a synthetic T-cell receptor called NY-ESO-1. The third edit eliminated PD-1, a checkpoint receptor that allows cancer cells to hide from T-cells.

The researchers are presenting the data as a positive because it appears to be safe. June told Genetic Engineering & Biotechnology News, “CRISPR technology has proven safe in patients with advanced refractory and metastatic cancer. Our results demonstrate the ability to precisely edit the DNA code at three different genes.”Now HiringCurrently hiring sales representatives, clinical researchers,
engineers, science r&d professionals, and other disciplines.Browse Jobs

In an accompanying article, Jennifer Hamilton and CRISPR pioneer Jennifer Doudna wrote, “These findings provide a guide for the safe production and non-immunogenic administration of gene-edited somatic cells. The clinically validated long-term safety of CRISPR-Cas9 gene-edited cells reported [here] paves the way for next-generation cell-based therapies.”

Before getting overly excited about this, it was also reported that one of the patients has since died and the disease became worse in the other two. June indicated the goal of the study wasn’t to cure cancer, but to show that the CRISPR technique was feasible and safe.

With that goal in mind, it’s safe to say the trial was a success.

“This is a Rubicon that has been decisively crossed,” said Fyodor Urnov, a genome editor at the University of California (UC), Berkeley, in a Science article. He noted the trial was the first of its kind in the U.S. and answered “questions that have frankly haunted the field.”

The research also suggests what the limitations of the approach are, at least currently.

One of the big concerns in using CRISPR is off-target edits. CRISPR is generally pretty precise, but the human genome is quite larger and even a target of 20 or so specific nucleotides in a gene might be duplicated elsewhere, which could have unintended effects. And, studies of the three patients in the study confirmed that CRISPR had resulted in some off-target edits. There weren’t many and the number of cells affected decreased over time.

There have also been questions on how long gene edits last. In theory, they should last indefinitely, but some research has suggested the body tries to fix the edits and return them to their original state. However, this study showed the CRISPR-edited cells continued at least nine months, which is significant compared to about two months in similar CAR-T therapeutic studies.

So this study, which is significant, is more of a starting point for CRISPR-based therapies, particularly given the modest clinical response.

“It wasn’t like you turned off those genes and those T-cells started doing things that were amazing,” Antoni Ribas, a UC Los Angeles oncologist told Science. But it was “a needed start” and going forward, “It’s going to be easier—because they did it first.”




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The future of humanity is genetic engineering and neural implants

Hear why at SXSW in March 2020

Alyse Sue

Alyse SueFollowFeb 9 · 6 min read

SXSW the leading tech, music, and film festival is on March 13–22nd 2020. It will feature talks on cutting edge technology such as genetic engineering and neural implants.

“If software ate the world last decade, biology will dominate the next”

The last decade saw the rise in popularity of software engineering jobs, marked by the increasing number of coding bootcamps, and rush by schools to add coding to the curriculum. This coming decade may see the rise of the genetic engineer. Instead of programming computers, the next decade will be about programming cells. While computers are programmed using ones and zeroes, cells can be programmed with the four letters that make up DNA: A, G, C and T.

Gingko Bioworks is one of the most prominent biotechnology companies in the hot area of synthetic biology. Synthetic biology is programming cells just as we program a computer. According to the National Human Genome Research Institute, it allows us to redesign organisms so that they have new abilities.

Examples include harnessing micro-organisms to clean pollutants in our soil, water and air, modifying rice to produce beta-carotene to prevent vitamin A deficiency, which causes blindness in up to half a million children every year, and engineering yeast to produce rose oil as an eco-friendly substitute for real roses that perfumers use.

Gingko Bioworks’ CEO and Co-founder Jason Kelly argues that we need to think bigger and beyond software to solve our most complex problems: “Websites can’t reverse climate change, apps won’t cure malaria and you can’t eat software.”

What Kelly says is not far from the truth. Some of our global grand challenges include food security, access to water, and global warming which biotechnology may be able to solve.

Gingko Bioworks is a platform that allows genetic engineers to program cells. They have developed automated genetic engineering foundries to speed up the process by using robots, lots of robots, to do the work that a PhD would. This means cell programming can be done much cheaper compared using a lab staffed purely by humans. It has also made it more accessible. Any large company or startup can use the platform for their projects.

Bayer, a multinational pharmaceutical and life sciences company, is currently working with Gingko Bioworks in an $100 million joint venture to develop microbes that allow crops to self-fertilise. Current fertilisation methods for crops mean that fertiliser seeps into waterways and lead to environmental pollution problems. Crops that fertilise themselves are a potential solution.

Synthetic biology not only improves food production and food security, the most exciting areas it could be used in include the production of advanced materials and electronics.

Kelly will be speaking at SXSW in this featured session — You Can’t Eat Software: Biology’s Big Future.

Healing unhealable wounds and 3D printing organs

Two scientists are working at the cutting edge of tissue engineering. Adam Feinberg from Carnagie Mellon University and Ronke Olabisi from UC Irvine are undertaking groundbreaking work in healing ‘unhealable’ wounds with adult stem cells and creating 3D bio-printed materials that can be used in biomedical research to test new drugs and print new organs such as hearts and lungs.

Stem cell therapy is a type of medical treatment where adult stem cells are used to restore damaged tissue to its pre-injured state. Mesenchymal stem cells which make our bone, fat, tendon and cartilage are used for research on the role of stem cells in wound treatment.

3D bio-printing is similar to traditional 3D printing except that cells and growth factors are used, instead of plastics and metals, to manufacture parts that mimic natural human tissue.

This technology can improve the survival rates of critically ill people and improve quality of life for those who have lost function in an organ such as a liver or kidney.

3D organ printing could save the 20 people that die everyday from lack of available organs for transplant. Across America alone, 115,000 people are waiting for an organ transplant so they can make it to their next birthday. The waiting list can be 3–5 years to get a suitable donor organ.

Eventually 3D bio-printing could allow people to upgrade themselves and rebuild their bodies from scratch. As seen in the Netflix series Altered Carbon, the ability to spin up a new body when your old body is destroyed could become a reality… for those that can afford it.

An Australian company, Inventia Life Sciences, has created an award winning 3D bio printer which is able to create realistic 3D models of cells that represent a tumour. It can print samples based on a patient’s actual tumour so that effectiveness of different cancer treatments can be tested. The aim is to use these 3D models to speed up cancer research.

At SXSW, Feinberg and Olabisi will discuss the processes, pitfalls and ethics of their research and how far we are from making this a reality. Hear it all at this featured session — The Future of Healing: Engineering Longer Life.

Over the next ten years, neural implants could become mainstream

Practising neurosurgeon and brain-tech innovator, Dr. Jordan Amadio, from the University of Texas Dell Seton Medical Centre, sees lots of brains. He believes over the next 10 years most of us will likely augment ourselves with neurotechnology.

Elon Musk’s Neuralink is one of the most high-profile neurotechnology companies. It is developing ultra high bandwidth brain computer interfaces which will allow paralysed humans to perform tasks able bodied people take for granted like picking up a glass of water or even operating our mobile phones.

Brain computer interfaces are a system that connects a human to a computer. The device that connects with your brain can be in the form of an implant or a device that you wear on your head. The device measures activity of the brain in the form of electric signals. Devices like these allow you to communicate or control technology, such as driving a car just by thinking about it.

Musk revealed some exciting developments in June 2019. Neuralink had developed an array containing up to 3072 electrodes that can be implanted into the brain’s cortex using nanobots. “Threads” that connect to the brain are thinner than human hair which make it difficult for humans to implant. This is where the surgical nanobots come in. These thin threads are designed to be minimally invasive so that there is less bleeding and bruising to the brain when inserting them.

Neuralink’s technology has been successfully tested in rats and a monkey. It was revealed that the monkey was able to control a computer with its brain. Musk is hopeful the technology will be in human brains via clinical trials planned for 2020.

Brain computer interface technology has been a hot topic for a while. At last year’s SXSW, a panel discussion featured Kernel, a company developing non-invasive neurotechnology. Kernal calls its technology neuroprosthetics, essentially brain augmentations that improve mental function and treat disorders. The talk touched on how its technology could help paraplegics regain movement and treat people with Alzheimer’s.

Amadio might be the first to spot the next generation of neurotechnology companies. As co-founder of NeuroLaunch, an accelerator program for neuroscience startups, Amadio has helped launch and grow 11 startups worth a combined $15 million.

Amadio will show us how our ability to heal and enhance the brain will empower us in this SXSW session — The Future of Your Brain: Neural Implants and More.

Human enhancement technology on the horizon

From overcoming disabilities, enhancing our memory or ultimately, as Elon Musk believes, to keep humans relevant in the age of AI, human enhancement technology has never been more relevant.

Performance enhancement drugs such as Modafinil and Adderall are already being taken by people in the hope it increases their focus at work and school. Exoskeletons are already helping paraplegics walk again. Prescription drugs such as Metformin and Rapamycin are currently being researched for its ability to extend human healthspan.

However it is genetic engineering and neural implants that will truly enable people to be smarter and live longer and healthier.

Genetic engineering could be applied to humans to give them natural immunity to certain diseases, cure disabilities and disease, and to enhance cognitive and physical capabilities.

In November 2018, a scientist named He Jiankui used a gene editing technology called CRISPR-Cas9 to edit the genes of twin baby girls in the hope of providing them immunity to HIV. This type of genetic engineering is prohibited by law in more than 40 countries. However there have been legal CRISPR-Cas9 trials, in the University of Pennsylvania and China, to treat adults with recurring cancers.

Hear Erin Hahn from John Hopkins University Applied Physics Laboratory talk about making yourself smarter, stronger, and even slower to age in the SXSW session — Human Enhancement Tech: How, Why & What Next?

If you’re also making sense of a world impacted by emerging technologies, follow me on Medium where I write about transhumanism and AI.Data Driven Investor

from confusion to clarity, not insanity





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Alyse Sue


Alyse Sue


Freelance Tech Writer, Co-founder Transhumanism AU. Helping people keep up with Transhumanism & AI.

Data Driven Investor

Data Driven Investor


from confusion to clarity, not insanity

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  2.  Oncology & Cancer


FEBRUARY 17, 2020

New technique allows scientists to ‘listen in’ on cancer cells

by Cancer Research UK

Cancer cells
Electron microscopic image of a single human lymphocyte. Credit: Dr. Triche National Cancer Institute

Scientists have a developed a new technique to decipher how millions of individual cells are communicating with each other in miniature tumours grown in the lab, known as organoids, according to new research published in Nature Methods today.

This is the first time that scientists have been able to analyse many different signalling molecules at once in individual cells within replicas of patients’ tumours. Understanding how cells communicate could reveal how tumours are able to evade the immune system and become resistant to treatments.

This could allow scientists to develop more effective new drugs, by revealing why tumours respond the way they do to treatments. It could also help doctors to select the best course of treatment for each individual patient, by testing treatments on a bespoke replica of a patient’s tumour before prescribing them.

The technique rapidly analyses each individual cell in an organoid, looking for the presence of specific signalling molecules—messages that cells send to neighbouring cells, telling them how to behave.

Dr. Chris Tape, lead researcher of the study at UCL, said: “Organoids are already revolutionising cancer research by allowing us to test whether experimental new drugs are effective on lifelike models of tumours. But crucially, this new technique helps scientists to understand why a treatment works or not, by revealing in unprecedented detail how cells are talking to each other”.

In order to listen in on cancer cells, the team grew organoids in the lab. These are self-organising 3-D structures made up of cancer cells alongside other types of cells, such as immune cells and connective tissue. They mimic the behaviour of cancer in the human body much more accurately than cells grown in a dish.

They then modified a complex technique called mass cytometry, which is used to detect and analyse protein molecules. The organoids were broken up into individual cells, then antibodies combined with heavy metal atoms were added. Antibodies are proteins that selectively bind to certain cancer signalling molecules. The scientists nebulised the cells, to convert them into a fine mist, and electrically charged the heavy meal atoms, so that a magnetic field could be used to separate out the different signalling molecules.

The researchers tested this technique in bowel cancer cells and were able to simultaneously detect 28 key signalling molecules, across 6 different cell types, in over 1 million cells. They found indications that the cancer cells themselves, as well as immune cells and connective tissue, had ‘rewired’ the normal signalling networks of bowel tissue, allowing tumours to grow unchecked.

The next steps will be to use this technique to look for ways to block the communications between cells that allow them to withstand treatment. The team also hopes to test this new technique in different types of cancer.

Dr. Emily Armstrong, research information manager at Cancer Research UK, said: “Having a better understanding of this complex communication between cancer cells and other types of cell that make up a tumour could reveal secrets of how cancer comes back after treatment and spreads around the body.

“While this technique is in the early stages of development right now, in the future we may be able to grow replicas of individual patients’ tumours, to identify early signs that a drug won’t work for them so we can personalise their treatment plan. We hope this could one day help more people to survive cancer“.

Explore furtherZooming in on breast cancer reveals how mutations shape the tumour landscape

More information: Xiao Qin, Jahangir Sufi, Petra Vlckova, Pelagia Kyriakidou, Sophie E. Acton, Vivian S. W. Li, Mark Nitz, Christopher J. Tape, Cell-type-specific signaling networks in heterocellular organoids, Nature Methods (2020). DOI: 10.1038/s41592-020-0737-8 , information:Nature MethodsProvided by Cancer Research UK392 shares

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Research Article
Spaceflight Effects and Molecular Responses in the Mouse Eye: Preliminary
Observations After Shuttle Mission STS-133
Susana B. Zanello1
, Corey A. Theriot2
, Claudia Maria Prospero Ponce3
, and Patricia Chevez-Barrios3,4
Division of Space Life Sciences, Universities Space Research Association, Houston, TX;
Wyle Science,
Technology and Engineering, Houston, TX, Department of Preventive Medicine and Community Health, University
of Texas Medical Branch, Galveston, TX;
3 Pathology and Laboratory Medicine and Ophthalmology, Weill Medical
College of Cornell University, The Methodist Hospital, Houston, TX; 4 Department of Pathology and Genomic
Medicine, The Methodist Hospital, Houston, TX
Spaceflight exploration presents
environmental stressors including microgravityinduced cephalad fluid shift and radiation
exposure. Ocular changes leading to visual
impairment in astronauts are of occupational
health relevance. The effect of this complex
environment on ocular morphology and function
is poorly understood. Female 10-12 week-old
BALB/cJ mice were assigned to a flight (FLT)
group flown on shuttle mission STS-133, Animal
Enclosure Module ground control group (AEM),
or vivarium-housed (VIV) ground controls. Eyes
were collected at 1, 5, and 7 days after landing
and were fixed for histological sectioning. The
contralateral eye was used for gene expression
profiling by RT-qPCR. Sections were visualized
by hematoxylin/eosin stain and processed for 8-
hydroxy-2′-deoxyguanosine (8-OHdG), caspase-3,
and glial fibrillary acidic protein (GFAP) and βamyloid double-staining. 8-OHdG and caspase-3
immunoreactivity was increased in the retina in
FLT samples at return from flight (R+1)
compared to ground controls, and decreased at
day 7 (R+7). β-amyloid was seen in the nerve
fibers at the post-laminar region of the optic nerve
in the flight samples (R+7). Expression of
oxidative and cellular stress response genes was
upregulated in the retina of FLT samples upon
landing, followed by lower levels by R+7. These
results suggest that reversible molecular damage
occurs in the retina of mice exposed to spaceflight
and that protective cellular pathways are induced
in the retina and optic nerve in response to these
The space environment creates challenges for
extended human spaceflight and presents a unique
combination of stressors: microgravity, highenergy-particle radiation, nutritional deficiencies,
hypobaric hypoxia, intermittent hyperoxia, and
psychological stress. Lack of gravity implies
reduced physical loading, fluid shift, and
incompletely understood cellular responses that
are reflected by a number of detrimental changes,
such as muscle atrophy and loss of bone mass,
immunosuppression, and overall gene expression
changes (Pietsch et al., 2011; Sundaresan and
Pellis, 2009). Ground models of simulated
Key words: Spaceflight; Retina; Cornea;
Oxidative Stress; Visual Impairment;
Intraocular/Intracranial Pressure; BetaAmyloid; Mouse
Correspondence to: Susana Zanello
Universities Space Research Association
Lyndon B. Johnson Space Center
2101 NASA Parkway, Mail CodeSK
Houston, TX 77058
Phone: 281.244.6779
Gravitational and Space Research Volume 1 (1) Oct 2013 — 29
Zanello … al. — Spaceflight-Induced Ocular Changes in Mice
microgravity, namely hindlimb suspension (HS)
and bed rest, induce a fluid shift and concomitant
vascular pressure and flow alterations (Hargens
and Watenpaugh, 1996; Wilkerson et al., 2002),
affecting not only cardiovascular physiology but
also inducing genome-wide gene expression
changes in the central nervous system (Frigeri et
al., 2008).
Ocular changes have been reported related to
exposure to the space environment. In humans,
the direct effect of radiation in the lens results in
cataract formation (Cucinotta et al., 2001), which
manifests with a higher incidence and earlier
onset in the astronaut population. Light flashes in
the eye are an occurrence that has been observed
by astronauts since the Apollo program (Sannita
et al., 2006) — a phenomenon not completely
Most importantly, recent medical data from
astronaut cohorts have reported the development
of optic disc edema, choroidal folds, posterior
globe flattening, and a resulting hyperopic shift
(Kramer et al., 2012; Mader et al., 2011) in a
fraction of the astronaut population upon return
from missions longer than 30 days (NASA, 2010).
No clear etiology has been established for these
cases, but it is hypothesized that microgravity, the
ensuing cephalad fluid shift, and venous
congestion may play a role. The perturbations
observed in some individuals of the astronaut
cohort resemble those found in papilledema
associated with idiopathic intracranial
hypertension (IIH) also known as pseudotumor
cerebri (Friedman, 2007; Kramer et al., 2012;
Mader et al., 2011). Because the etiology is still a
matter of speculation, investigating whether
exposure to microgravity represents a source of
stress for the eye is an issue of critical
occupational health importance. To this aim, this
project examines the effects of spaceflight on the
rodent eye and the responses that occur when
challenged with exposure to microgravity in
combination with other stressors during
Previous spaceflight studies performed on
rodents found evidence of retinal degeneration in
neonatal rats aboard shuttle mission STS-72
(Tombran-Tink and Barnstable, 2006), and of cell
swelling and disruption in rats aboard two
experiments on Russian Cosmos satellites
(Philpott et al., 1980; Philpott et al., 1978).
However, these studies were limited to structural
histopathologic observations of the eye. In the
present work, we expand the
immunohistopathologic analysis to investigate the
effects of spaceflight and the elicited responses
observed in the eyes of mice aboard shuttle
mission STS-133, focusing, for the first time, on
molecular and cellular processes subjacent to the
histopathologic changes.
This work consisted of a tissue sharingderived project that used specimens collected
from a parent animal experiment aboard shuttle
mission STS-133. The original experiment
included animals infected with respiratory
syncytial virus immediately after return to Earth
(study led by independent investigator Dr.
Roberto Garofalo, from the University of Texas
Medical Branch in Galveston). However, the work
discussed in this article only included the noninfected control animals. Animal procedures were
approved by the NASA Ames Research Center
and Kennedy Space Center institutional animal
care and use committees. The STS-133 mission
occurred from February 24 to March 9, 2011, for
a total duration of 12 days and 19 hours. Female
10 to 12 week-old BALB/cJ mice were assigned
to one of three experimental groups: Flight (FLT),
Animal Enclosure Module (AEM) ground
controls, and vivarium-housed (VIV) ground
controls. The flight animals (FLT) were housed in
AEMs identical to the ground controls. The AEM
is a self-contained habitat that provides
ventilation, waste management, food, water, and
controlled lighting (Naidu et al., 1995). It has
previously been used in experiments studying
rodent biology during spaceflight. The AEM
flight unit is located in the middeck locker of the
shuttle and its temperature is set at 3° to 8°C
above the environmental middeck temperature.
Lighting of 14 lux is set to a 12 hour day/12 hour
night cycle. AEM ground controls were
maintained in identical conditions at the Space
Life Sciences Laboratory, Kennedy Space Center.
Vivarium ground controls were housed in
standard vivarium cages and conditions, on a 12-
hour day/12-hour night light cycle at 200 to 215
lux. In view of the housing and lighting conditions
30 – Gravitational and Space Research Volume 1 (1) Oct 2013
of the vivarium, the proper ground controls that
allow measuring the effects attributed to
spaceflight are the AEM-housed ground controls.
After sacrifice, one eye of each mouse from
the three groups (FLT, AEM, and VIV) was
collected at 1, 5, and 7 days after landing, and was
fixed for histological examination. The
contralateral eye was stored in RNALater and
used for gene expression profiling by RT-qPCR.
The histological 4% paraformaldehyde-based
fixative was obtained from Excalibur Pathology,
Inc., Oklahoma City, OK. Goat polyclonal
antibody to 8-hydroxy-2′-deoxyguanosine
(8OHdG) (ab10802) and rabbit polyclonal
antibody to activated caspase-3 (ab52181) were
purchased from Abcam Inc., Cambridge, MA.
Mouse monoclonal antibody to β-amyloid 1-16
was obtained from Millipore (Temecula, CA) and
rabbit polyclonal antibody against glial fibrillary
acidic protein (GFAP) was purchased from Dako,
Carpinteria, CA. Paraffin embedding and
histologic sectioning were contracted from
Excalibur Pathology. qRT-PCR reagents were
purchased from Qiagen Inc., Valencia, CA and
BioRad, Hercules, CA. Tissue samples were
assigned a different number for
immunohistochemistry evaluation and gene
profiling to perform a masked analysis.
Histology and Immunohistochemistry
Fixed eyes were paraffin embedded, sectioned
at 5 µm thickness, and stained with standard
hematoxylin-eosin (H&E) for histologic
examination. Four immunohistologic stains were
performed: 8OHdG to detect oxidative-related
DNA damage, activated caspase-3 to study
apoptosis, and double stain using β-amyloid as a
marker of neuronal and axonal injury and GFAP
as an indicator of glial activation. All
immunostains had negative (omitting primary
antibody) and positive (using known tissue that
reacts with the antibody of interest) controls. For
8OHdG and caspase-3 staining, sections were
equilibrated in water after deparaffinization and
treated sequentially in 3% hydrogen peroxide, 1%
acetic acid, and 2.5% serum (Vector Labs,
Burlingame, CA) before incubating with the
diluted primary antibody for either 2 hours at
room temperature or overnight at 4ºC. After
washing in phosphate buffer saline (PBS), the
specimens were incubated with Vector ImmPress
detection kit corresponding to the primary
antibody’s host and counterstained with
hematoxylin. For the double stain with β-amyloid
and GFAP, antigen retrieval was performed with
Dako target retrieval solution (a modified citrate
buffer from Dako, Carpinteria, CA), steaming for
25 minutes, and then treated with peroxidase
blocking buffer as above, and endogenous biotin
blocked with Vector Avidin/Biotin blocking kit
(Vector, Burlingame, CA). Staining for β-amyloid
was done with the mouse-on-mouse peroxidase kit
according to the manufacturer’s instructions
(Vector Labs). Diaminobenzidine (DAB) was
used for color labeling for β-amyloid (brown). For
GFAP immunostaining, Dako’s streptavidin
phosphatase kit was used with permanent red
(red) as the chromophore.
Qualitative Detection
Morphology and histology were interpreted
by an ophthalmic pathologist (masked for specific
study groups) on H&E slides. Immunostained
slides were evaluated for positivity of stain in a
graded scale from 0 to 3+, where 0 indicated
absence of staining and 3+ indicated marked
positivity and more than 3 positive cells per layer.
Immunoreactivity was evaluated in the corneal
epithelium and endothelium, iris, lens, choroid,
retinal ganglion cell (RGC) layer, inner nuclear
layer (INL), outer nuclear layer (ONL), and optic
Quantitative Detection
To quantify oxidative-related DNA damage in
the retina, densitometric quantification of 8OHdG
immunohistochemistry was performed. Briefly,
digital color images of the retina were processed
using NIH ImageJ ver.1.68 (Abramoff et al.,
2004) and converted to an 8-bit inverted grayscale image for analysis. Regions of interest were
selected from each retina section, corresponding
to the RGC, INL, and ONL as well as nearby
areas without immunoreactivity for background
measurements. Five sections were analyzed for
each sample, for which the mean density per unit
area (minus mean background density) was
To quantify apoptosis in the retina, activated
caspase-3 positive cells were identified for each
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
retinal sample and expressed over the total
number of cells in each of the following retinal
layers: RGC, INL, and ONL. Cellular number was
determined with the cell counting plug-in for
ImageJ ITCN (Byun et al., 2006).
Gene Expression Analysis
Mouse retina was microdissected and placed
in RNAlater (Life Technologies, Grand Island,
NY). Total RNA was then isolated using the
AllPrep DNA/RNA Micro kit (Qiagen, Valencia,
CA) and analyzed for quality using an Agilent
2100 Bioanalyzer. All samples used reported a
RNA Integrity Number (RIN) >7.0. The
Quantitect Reverse Transcriptase kit (Qiagen) was
then used to generate cDNA templates for
subsequent real-time qPCR analysis. Fifty
nanograms of RNA were used in each reverse
transcriptase reaction in a total reaction volume
scaled to 30 μL according to manufacturer’s
instructions, and the synthesis reaction was
allowed to proceed for 2.5 hours. qPCR
amplifications were done in a total volume of 20
μL using 1 μL of a 1:10 dilution of the cDNA
pool obtained in the previous step and SYBR
Green qPCR mastermix (BioRad, Hercules, CA)
on a Bio-Rad CFX96 real-time PCR detection
system. Samples were run in three technical
replicates each. Primers (Qiagen) were selected to
hybridize with genes specific for various cellular
response pathways according to relevant findings
in the literature that reported known roles in
retinal stress, degeneration, oxidative stress,
inflammation, and death/survival (Table 1). Three
housekeeping genes (Hprt1, Rplp0, and Rpl13)
were selected according to previously reported
expression stability (van Wijngaarden et al.,
2007). Normalization to the housekeeping genes
was performed using the geNorm algorithm
(Vandesompele et al., 2002) built into the CFX96
software, which computes a normalization factor
for each sample from the contribution of each
housekeeping gene.
Histological Analysis of Eye Specimens
Results are summarized in Table 2. All groups
showed corneal acanthosis, defined as thickening
of the epithelium of more than 5 layers of cells,
and edema defined as clearing of cytoplasm with
enlargement of the cell. However, irregular
acanthosis, irregular increment of cell layers, with
pronounced edema was present in the VIV group
at R+7 (mice #41, 42). All mice had inflammatory
cells either in the anterior chamber or vitreous,
regardless of the group. Focal cortical cataracts,
disrupted fibers, and formation of globules in the
cortex of the lens, which is located between the
nucleus and the epithelium, were present in
several mice. As shown in Figure 1, full cortical
cataracts were seen only in the two mice of the
FLT group at R+7 group and this was associated
with caspase-3 2+ staining. The VIV group at R+7
had no morphologic changes of cataract but had
caspase-3 2+ staining as well (see below).
Apoptosis of neurons defined as shrinkage of the
cytoplasm with hyperchromatic nuclei and
degenerated chromatin was observed in some
mice. These findings were quantified using
immunohistochemistry and they are discussed
below. Some slides showed artifacts in the
histology (possibly due to traumatic enucleation)
that precluded complete interpretation. These
findings are not included in the interpretation.
Only those findings that are clear and not affected
by processing are reported.
Oxidative Stress: 8OHdG
8OHdG immunoreactivity was positive in all
mice in the acanthotic areas of the cornea. In the
FLT group, positivity was evidenced in the
corneal epithelium and endothelium, but we were
not able to document significant differences
compared to AEM and VIV controls with the
present data.
Retina and Optic Nerve
Figure 2 summarizes 8OHdG data. The two
mice in the FLT group at R+1 showed frank
positivity for 8OHdG in the neuronal layer. One
of these also evidenced 8OHdG in some vessels
over the ON head. Digital quantitative analysis of
immunoreactivity in the retinal layers was more
prominent in the RGC of FLT samples at R+1
(Figure 2B). Comparing FLT samples at the
different tissue collection time points, 8OHdG
immunoreactivity decreased from R+1 to R+7
(Figure 2B, C, D, and E). All mice were negative
at the level of the optic nerve.
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Table 1. 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
(Lohr et al., 2006)
Bcl2-associated X protein
B-cell lymphoma 21
Bcl2-associated athanogene 12
Autophagy related 123
Cellular Stress response Hsf1
Nfe2l2 (Nrf2)
Heat shock transcription factor 1
Heat shock 70kDa protein 1A4
Sirtuin 15
Nuclear factor (erythroid-derived 2)-like 26
Oxidative stress response Hmox1
Heme-oxygenase 17
Superoxide dismutase 2, mitocondrial8
Glutathione peroxidase 49
Peroxiredoxin 1
Inflammation Nfkb1
Nuclear factor of kappa light polypeptide gene
enhancer in B-cells 110
Transforming growth factor beta 111
Normalizing genes Rpl13
Ribosomal protein L13
Ribosomal protein, large, P0
hypoxanthine phosphoribosyltransferase 1
1 (Godley et al., 2002)
2 (Liman et al., 2008)
3 (Wang et al., 2009)
4 (Awasthi and Wagner, 2005)
5 (Chen et al., 2009)
6 (Wei et al., 2011)
7 (Zhu et al., 2007)
8 (Justilien et al., 2007)
9 (Ueta et al., 2012)
10 (Wise et al., 2005)
11 (Gerhardinger et al., 2009)
Apoptosis: Caspase-3
Activated caspase-3 appeared positive in the
cornea of all mice with the same intensity.
Two mice of the FLT group at R+7 had
cataract formation associated with caspase-3 2+
staining (Figure 1). The VIV group at R+7 had no
morphologic changes of cataract but had caspase3 2+ staining as well.
Retina and Optic Nerve
Detection of apoptosis by activated caspase-3
immunoreactivity was performed on retinal
sections and compared in the different specimens
(Figures 1 and 3). All mice showed positivity in
the neuronal layer regardless of day of sacrifice.
Digital image quantification of caspase-3
immunoreactivity revealed that VIV samples had
the highest percentage of apoptotic cells in the
INL and RGC layer, followed by FLT samples, at
day R+1 and R+7. Comparatively, VIV and FLT
retina samples showed more caspase-3 positive
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
Table 2. 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.
cells than AEM samples at R+1, except for the
INL in the AEM group at R+7. VIV samples also
tended to increase their percentage of apoptotic
cells at day R+7, as seen in qualitative analysis.
Retinal pigment epithelium (RPE) of the FLT
group at R+1 and one mouse at R+5 showed
positivity with caspase-3, and one mouse AEM
R+7 showed only rare and focal RPE staining
(Figure 1). Qualitative and quantitative evaluation
of ON immunoreactivity was inconclusive.
β-amyloid and GFAP
β-amyloid and GFAP stains were studied in
the retina and optic nerve only and
immunostained retina sections are shown in
Figure 4. With regard to the retina, all mice were
positive in the neuronal layer for β-amyloid.
Overall, the vivarium mice showed a slightly
higher positivity in both RGC and INL compared
to the rest of the mice (VIV animals showed 2-3+
positivity at R+1 and R+5, more than any other
group; one FLT animal at R+7 showed similar 2+
reactivity). GFAP was present in astrocytes of the
retinal neuronal layer in at least one mouse of
each group, except in the FLT group at R+5,
where it was absent. No activation (positivity) of
Muller cells was noted in any of the eyes.
While results were not conclusive from these
retinal findings, it is important to note that only
the FLT group at R+1 were positive for all stains
at the retinal neuronal layer: 8OHdG, caspase-3,
β-amyloid, and GFAP.
At the level of the optic nerve, only the FLT
group at R+7 showed positivity for both βamyloid in the axons and GAFP in the astrocytes
either at the level of the lamina cribrosa or distal
to it (Figure 4). No co-expression was seen of
GFAP and β-amyloid in same cell type.
Cellular Responses Identified by Gene
Expression Analysis
Gene expression profiling on STS-133 flight
samples and their AEM and vivarium ground
controls was performed targeting a set of genes
focused on cellular death and survival, oxidative
stress and cellular stress response, and
inflammation. Results are shown in Figure 5 and
Figure 6 and expressed as comparative normalized
expression across the individual specimens at R+1
and R+7 for all groups. Due to the limited sample
size, statistical analysis was not possible and these
results are mainly descriptive.
Activation of Oxidative Stress Response and
Pro-Inflammatory Genes
Figures 5 and 6 (see section below) plot gene
expression data measured by real time qPCR.
Several genes coding for key antioxidant enzymes
(Hmox1, Sod2, Cat, Gpx4, Cygb, Prdx1) were
elevated in retina samples obtained immediately
after flight (Figure 5B), but this elevation returned
Cornea Lens Retina ON
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
Day 5
FA and basal E FA, E 1+ Central E Nml Focal cortical C Nml Nml Nml Nml Nml Nml Nml FA Intranuclear inclusions, A 1+, E 2+ FA Nml Focal cortical C Anterior subcapsular C Nml Nml Nml Nml Nml Nml Day 7 FA FA Irregular A 1+ E 3+ Cortical C Nml Nml Nml Nml Nml Nml Nml Nml A 1+, E 2+ FA Irregular A 1+
E 2+ Cortical C Nml Nm Nml Nml Nml Nml Nml Nml
(A)= acanthosis, (C)= cataract, (E)= edema, (FA)= focal acantosis, (Nml)=normal, anterior subcapsular C (anterior subcapsular
cataract is disruption of the fibers with proliferation of the epithelium in the anterior subcapsular áreas of the lens)
Comments: Anterior chamber 1+ cell 34 – Gravitational and Space Research Volume 1 (1) Oct 2013 Zanello et al. — Spaceflight-Induced Ocular Changes in Mice to levels closer to AEM ground control values at 7 days post-landing. A similar trend was observed for inflammatory mediators Nfkb1 and Tgfb1 (Figure 5A). Hmox1 showed the highest levels in those samples for which a higher evidence of stress was observed (FLT samples at R+1 and VIV ground controls). 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).
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
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).
36 – Gravitational and Space Research Volume 1 (1) Oct 2013
Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
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
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
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 βamyloid (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.
Cell Death and Survival Genes
The proapoptotic gene Bax was elevated in
one flight sample (#13) at day R+1 and
moderately elevated in one flight sample (#52) at
R+7. Vivarium mice showed a higher expression
of Bax at all collection time points compared to
AEM ground controls. FLT samples at R+1 and
VIV samples exhibited higher levels of the
autophagy marker Atg12 and the survival genes
Bcl2 and Bag1, suggesting that cellular protection
mechanisms may be triggered as a response to
cellular stress (Figure 6A).
Activation of Cellular Stress Genes
The cellular stress response genes Hsf1and
Nrf2 (Nfe2l2) were expressed slightly higher in
VIV samples compared to AEM controls. Among
the FLT mice, there was a tendency to higher
expression at R+1 than R+7 (Figure 6B). The
Hsf1 activator sirtuin 1 (Sirt1) did not show major
differences across the various samples.
Interestingly, the heat shock protein 70KDa
Hsp1a1 was expressed at a lower level in mouse

13 that exhibited, overall, the highest signs of

38 – Gravitational and Space Research Volume 1 (1) Oct 2013
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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.
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
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.
40 – Gravitational and Space Research Volume 1 (1) Oct 2013
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While the spaceflight results reported herein
represent pilot data due to the small sample size,
these data offer, for the first time, direct evidence
suggesting that oxidative stress, neuronal damage,
and mechanical injury take place in the retina,
lens, and optic nerve of rodents flown in lowEarth orbit for a period under two weeks. Several
previous studies have shown the occurrence of
oxidative stress during spaceflight (Stein, 2002),
however, our work gives a first insight into the
impact of space-associated factors on biological
processes like cell death, oxidative stress, and
probable mechanical injury in the rodent eye.
Because the BALB mouse strain used in the
STS-133 experiment is susceptible to lightinduced retinal degeneration (LaVail et al., 1987),
we speculate that this particular strain exhibits an
enhanced sensitivity to oxidative stress and/or a
reduced stress response, making it a suitable strain
in which to identify alerting evidence of risks
previously unrecognized in the retinal tissue,
while impacting its value as a model for the study
of the human changes seen in-flight.
8OHdG, a product of deoxyguanosine
oxidation, is a marker of oxidative stress-induced
DNA damage. This damage has been observed in
mouse cornea exposed to dryness (Nakamura et
al., 2007), ultraviolet radiation (Tanito et al.,
2003), and in mouse retina exposed to intense
light (Tanito et al., 2002; Wiegand et al., 1983). In
our study, 8OHdG was present in all acanthotic
areas of the cornea. Irregular acanthosis with
visible edema was only seen in the VIV samples
at R+7, and it was only in this group where
positivity at the corneal endothelium was
observed since day 1, suggesting an impaired ion
and water transport in the cornea.
The retinal response to intense light in
susceptible mice has been studied before and has
been found to be related to lipid peroxidation at
the ONL (Tanito et al., 2002; Wiegand et al.,
1983). Likewise, radiation-induced retinopathy is
an ocular complication in cancer patients that
receive radiation therapy (Parsons et al., 1996).
The processes involved in the damage by highenergy-particle radiation in these cases may share
commonalities (direct DNA damage and oxidative
stress) with exposure to radiation present during
spaceflight. The present work shows evidence of
both oxidative stress-induced DNA damage in the
neuronal layers of flight mice retinas and of an
oxidative stress response induced at the gene
expression level in these mice. Short-term
responsiveness to DNA oxidation followed by
DNA repair has been studied longitudinally in
blood of trauma patients (Oldham et al., 2002),
suggesting that the attenuated DNA damage
observed after one week of return from flight may
be the result of DNA repair.
Of note, the ground controls kept in the
vivarium exhibited a comparable level of retinal
oxidative stress to the samples from flight,
especially at longer exposures (day R+7). This is
likely due to the fact that the illumination
conditions in a standard vivarium room are
approximately 15-fold in light flux compared to
the illumination of an AEM, even if both maintain
a 12 hour light-12 hour dark cycle.
Caspase-3 is a pro-enzyme that is activated in
the intrinsic apoptotic pathway in all mammals
(D’Amelio et al., 2010). In this study, all mice
showed positivity for caspase-3 at the level of the
cornea. This may be explained by the fact that
caspase-3 immunoreactivity in the stratified
epithelium of the cornea serves as an internal
positive control due to the natural differentiation
process that the basal cells suffer towards
cornification. Apoptosis can be triggered by
oxidative stress, brain trauma, or ischemia. In a
model of brain ischemia, the area of neuronal
apoptosis has been identified not in the infarct
region but in the surrounding area, where the
oxygen tension is decreased, but not absent
(Pulsinelli et al., 1982). The presence of activated
caspase-3 is thus related to hypoxic environment
and radiation exposure. In our study, the FLT
group at R+1 showed higher positivity compared
to the rest of the groups. This may be related to
radiation and microgravity exposure during
spaceflight. It is important to point out that the
effect of high-energy-particle radiation may be
overall increased in this susceptible mouse strain.
Qualitative examination revealed that VIV
and FLT groups showed more caspase-3-positive
cells at the retinal layers than AEM retinas. This
may suggest that the damage caused by visible
light radiation in the albino strain in the vivarium
conditions may be comparable to the damage
caused by the exposure to spaceflight
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Zanello et al. — Spaceflight-Induced Ocular Changes in Mice
environmental factors. We also observed positive
microglial (astrocytes) but not Muller cell
activation in VIV specimens, which may support
the notion of visible light radiation effects as the
triggering factor in inner layers of the retina only
in these mice (Song et al., 2012).
Both mice in the FLT group at R+1 and one
mouse at R+5 showed evidence of apoptosis in the
RPE. Apotosis in the RPE has been identified in
ocular pathologies like age-related macular
degeneration (AMD) secondary to exposure to
activated monocytes (Yang et al., 2011), or
triggered by oxidative stress with H2O2,
lipofuscin, or light irradiation (Sparrow et al.,
2000). This data also suggest oxidative stress may
be an important component in the retinal damage
in these mice. Of note, in vitro experiments with
human RPE cells cultured in simulated
microgravity generated by a NASA-bioreactor
resulted in DNA damage and inflammatory
response in these cells (Roberts et al., 2006).
Retinal pigment epithelium attenuation has been
related to retinal choroidal folds previously found
in astronauts (Mader et al., 2011). It is yet to be
determined whether or not increased RPE
apoptosis may contribute to the formation of
choroidal folds or if it increases the risk for AMD
in astronauts.
Several advances in immunohistochemistry
have led to the identification of β-amyloid in
traumatic brain injury in humans (Iwata et al.,
2002), rats, and pigs (Smith et al., 1999), by
tracing not only the full-length protein but also
small aminoacid peptides. β-amyloid was present
in areas of the brain as soon as one day after brain
trauma was provoked by pressure injection of
saline into the cranium in a rat model (Pierce et
al., 1996). Moreover, β-amyloid deposits showed
evidence of optic nerve injury in cases of shakenbaby syndrome (Gleckman et al., 2000). Previous
studies in animal models have shown distribution
of β-amyloid in the mouse retina that suggests its
involvement in the pathophysiology of glaucoma
(Kipfer-Kauer et al., 2010). We report that βamyloid deposition was present in the neural
retina of mice in all treatment groups and that the
VIV mice showed a slightly higher positivity in
both RGC and INL compared to the rest of the
mice. Interestingly, β-amyloid was present in the
optic nerve of both mice in the FLT group at R+7
and had the unique characteristic of being at the
level of lamina cribrosa or immediately distal to
it. This compares with the findings in traumatic
injury in children of shaken-baby syndrome where
most of the axonal changes are seen in the
postlaminar region (Gleckman et al., 2000). This
may be associated to the anatomy of this region
where the nerve is anchored by the fibers of the
lamina cribrosa but immediately posterior to this
or beyond this area the nerve can move freely.
Thus, in the event of mechanical trauma the
immediate fibers in the postlaminar region may be
the ones demonstrating more damage. The trauma
may include increased intracranial pressure that is
transmitted into the nerve, positional or whiplash
(similar, although in a less intense manner to what
happens in shaken baby syndrome), or vibration
(as the one occurring during launch or landing).
However, there is the need to further investigate
the nature of the changes through additional
experimental work.
GFAP is an intermediate filament protein
known to be present in astrocytes, Muller cells,
and oligodendrocytes in the post-laminar optic
nerve. GFAP is elevated when there is stress in
the central nervous system and has been shown in
the injured retina mostly present in the activated
Muller cells (Lewis and Fisher, 2003). In this
paper, we show that the optic nerves of several
mice were positive for GFAP and β-amyloid;
however, it was only the FLT group at R+7 that
showed increased expression of GFAP at the
postlaminar optic nerve. These findings suggest
that the astrocytes and oligodendrocytes were
activated in this region probable secondary to
mechanical trauma. The causes of this, either
vibration or fluid shift-related, need to be further
In addition, only FLT mice sacrificed at day 1
(FLT R+1) were immunoreactive in the neuronal
layer for all β-amyloid, GFAP, caspase-3, and
8OHdG, suggesting increased oxidative and
possibly mechanical damage. This may be
explained by the possible correlation of β-amyloid
deposition and activation of astrocytic cells, both
triggering reactive oxygen species production
(Lamoke et al., 2012).
The gene expression profiling results with
BALB mice in flight STS-133 support the
immunohistopathologic findings and suggest that:
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a) Oxidative stress-induced DNA damage was
higher in the FLT samples compared to controls
on R+1, and decreased on R+7. A trend toward
higher oxidative and cellular stress response gene
expression was also observed on R+1 compared to
AEM controls, and these levels decreased on R+7.
Several genes coding for key antioxidant
enzymes, namely, heme-oxygenase-1,
peroxiredoxin, and catalase, were among those
elevated after flight. Likewise, the inflammatory
response genes Nfkb1and Tgfb1 were elevated
after flight. The fact that only two mice flown on
STS-133 were genetically analyzed per day of
sacrifice creates a major limitation in any
statistical analysis. However, this does not
preclude the comparisons of samples. b) There is
an apparent correlation trend in the stress
parameters measured in the different animals and
there is certain variability in the stress response
among the individual animals. For example,
mouse # 13 in the FLT group at R+1 suffered
from overall elevated stress, demonstrated by the
highest 8OHdG levels, induction of antioxidant
enzymes, induction of Nfkb1, and concomitant
lower levels of the cytoprotective heat shock
protein Hsp1a1. Sirtuin 1 gene expression results
were non-conclusive, but further analysis is
required to determine if translocation of sirtuin 1
may occur and how this may affect the expression
of downstream cellular stress response genes
(Jaliffa et al., 2009; Ozawa et al., 2010).
c) Spaceflight represents a source of
environmental stress that translates into oxidative
and cellular stress in the retina, which is partially
reversible upon return to Earth. Also, retinas
from VIV control mice evidenced higher
oxidative stress markers, Nfkb1 and Tgfb1, likely
due to the more intense illumination in vivarium
cages versus the AEM.
In addition, mice in FLT group at R+7 were
positive for both β-amyloid and GFAP, and it was
only in these mice that there was increase in
GFAP staining adjacent to lamina cribrosa in the
optic nerve. We suspect some long term damage
in the optic nerve may be seen after spaceflight
because this did not resolve after seven days on
Earth. Additional quantitative experiments are
needed to give a better understanding on this
These preliminary data suggest that
spaceflight represents a source of environmental
stress that directly translates into oxidative and
cellular stress in the retina, which is partially
reversible upon return to Earth. Moreover, the
optic nerve findings suggest that the lesion may be
mechanical in nature and that does not resolve
after return to Earth, at least in the animals
studied. Further work is needed to dissect the
contribution of the various spaceflight factors
(microgravity, radiation) and to evaluate the
impact of the stress response on retinal and optic
nerve health. These preliminary results should
inform investigators on the design of future
studies utilizing a more suitable mouse strain
devoid of photic degeneration predisposition,
male animals that better reflect the astronaut
population, and statistically powered larger
sample sizes.
We would like to recognize Richard Boyle for
tissue sharing and collection, Audrey Nguyen for
help with digital image analysis, and James
Fiedler for graphic work. This work was funded
by the NASA Human Research Program.
Abramoff, M.D., Magalhaes, P.J., and Ram, S.J.

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    Zanello et al. — Spaceflight-Induced Ocular Changes in Mice

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Physiological discoveries abound within NASA samples

News and Views

Elizabeth Keller
NASA Ames Research Center, CA, USA

Ryan Scott
NASA Ames Research Center, CA, USA

Before humans risked their lives launching into space aboard the Vostok Program and Mercury Project, various organisms were flown to determine if it was possible for life to survive spaceflight. For decades, NASA, along with international partners, have continued sending biological experiments to space. Potential biological hazards from spaceflight include decreased gravity, increased exposure to radiation, altered light-dark cycles, and loads experienced during launch and landing. It is imperative to understand the basic science and health risks associated with spaceflight, along with developing countermeasures, as humanity ventures back to the Moon, and then to Mars and beyond.

NASA Ames Research Center houses an Institutional Scientific Collection (ISC) of spaceflight biological specimens and tissues. Through NASA’s Biospecimen Sharing Program (BSP), the samples in this biological repository are available for request by all researchers, including those based outside the United States.

A number of newly identified biological knowledge gaps and astronaut health risks have emerged from extended exposure to microgravity from long-duration missions. Of particular concern to NASA is the loss of visual acuity, which a significant number of astronauts have unexpectedly experienced. NASA is keen on getting researchers to work on elucidating the underlying cause(s) of this issue. In an effort to further the research progress on this topic, the NASA BSP coordinated the sharing of mouse eyes from the STS-133 Shuttle mission. The study led by Susana Zanello1 conducted histological examinations of the mouse eyes from post-flight days 1, 5 and 7. Gene expression analysis suggested that reversible molecular damage occurs in the retina of mice exposed to the spaceflight environment, and that protective cellular pathways are induced in the retina and optic nerve in response to these changes. While correlation of research findings in the mouse tissues to human astronauts presents a set of challenges, the mouse model is an excellent way to characterise underlying changes at the cellular and molecular levels that are simply not available with the human crew.

The costly effort of sending organisms into space makes space-flown biological specimens a rare and valuable resource. Quite often, when life science samples are retrieved from completed missions, there are surplus tissues which remain unused by the Principal Investigators and collaborators. In this scenario the tissues are harvested, preserved, and archived in the NASA ISC at the Ames Research Center to ensure the maximum scientific return from space missions in the belief that new discoveries can be made from the samples.

An important recent finding was made possible by the study of what would have previously been considered waste: rodent fecal pellets. Through NASA’s BSP, Martha Vitaterna, from Northwestern University obtained mouse fecal pellets from a 37-day mission aboard the International Space Station and examined them for the effects of spaceflight on gastrointestinal microbiota2. Findings from previous studies have demonstrated a change in the gut microbial diversity and community structure during spaceflight, but it was unclear what the functional relevance of those microbiome changes were. Using 16S rRNA gene amplicon sequencing, Vitaterna and her team profiled the microbiome of the fecal samples. They then compared the microbiome changes to other relevant datasets and integrated the gut microbiome data with publicly available transcriptomic data in the liver of the same animals for a systems-level analysis. Observations from her analyses shed light on the specific environmental factors that contributed to a robust effect on the gut microbiome during spaceflight, with important implications for mammalian metabolism.

Available tissues for physiology research
The NASA ISC at Ames Research Center currently stores over 32,000 specimens. Most come from Shuttle and International Space Station flight investigations, but also included in the collection are ground-based specimens from spaceflight-model experiments. Tissues are predominantly from mice and rats, though samples are also available from bacteria and quail. The specimens include tissues from many systems including musculoskeletal, neurosensory, reproductive, respiratory, circulatory, and digestive. The samples are stored at -80°C, -20°C, or +4°C, depending on the fixative used. Detailed metadata are available for all samples. Historically, these tissues have been used for a wide range of analyses, including histology, genomics, and transcriptomics. The NASA Ames Life Sciences Data Archive (ALSDA) has been shipping samples to investigators since 1995.

How to request tissues from the NASA ISC
Tissue requests are initiated by submitting an online Biospecimen or Data Request. If the requested tissues are available, the requestor will be sent instructions for submission of a short proposal. Visit the NASA ISC website for more information: Contact the ALSDA team:


  1. Zanello SB et al. (2013). Spaceflight effects and molecular responses in the mouse eye: observations after shuttle mission STS-133. Gravitational and Space Research 1(1), 29 – 46.
  2. Jiang P et al. (2019). Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight. Microbiome 7(1), 113. DOI: 10.1186/s40168-019-0724-4



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