reports the results of the analysis performed on the blood of mice that returned alive from space (MDS-ISS) and of the MDS-ground and vivarium controls. All 3 MDS-ISS mice have a higher erythrocyte concentration (RBC) and RDW% with a hematocrit near or above 50% compared to reference values and controls. Moreover PTN-2 and WT-2 MDS-ISS mice showed also a very high platelet concentration; on the contrary the Hb content did not show any alteration related to space flight.
Hematological parameters of flown and control mice.
These data are in agreement with observations made in humans where increased blood concentration are probably due to body fluid shift,renal function, and to a dysfunction of erythropoiesis. A highly random distribution of red cell width is also observed, probably due to a higher hemolysis 
Previous experiments, conducted during a 12 days space flight, showed an increase of RBC concentration and a parallel decrease of red blood cell distribution width (RDW) and mean corpuscle value (MCV) as result of water loss during space flight 
. Our data from blood of animals, that remained in space for 3 months (MDS-ISS, PTN and WT), are different for what RDW is concerned indicating that the long duration microgravity exposure influenced the erythropoiesis and/or increased cell aging.
Oxidative stress has been correlated with several diseases and morbidities. Space radiation, microgravity and their effects on living organisms might generate oxidative stress responsible of space anemia 
. The MDS mission offered to us the possibility, for the first time to measure in vivo oxidative stress after a long permanence of the animals on board the ISS.
For these reasons, we determined the Thiobarbituric Acid Reactive Substances (substances formed as byproducts of lipid peroxidation – TBARS) content as an indicator of oxidative stress and the activities of antioxidant enzymes in the mice erythrocytes.
We observed in the PTN MDS-ISS mice a statistically significant increase of TBARS in erythrocytes mice compared to both Earth controls (PTN MDS-ground and vivarium), . The same trend was observed for the wild type mice. These data indicated that during space flight mice underwent oxidative stress, which generated lipid peroxidation products such as malonildialdehyde.
Thiobarbituric acid reactive substances (TBARS) content (pmol/mg Hb, mean± SD) in PTN and wild type mice erythrocytes after space flight. MDS-ISS (black bars), Ground MDS controls (line bars) and vivarium (white bars).
By analyzing the content of erythrocyte glutathione, the major antioxidant present in these cells, we observed that the total content of this thiol was significantly increased after space flight compared to control in PTN MDS-ISS mice (). In addition, the enzyme involved in glutathione utilization, the glutathione peroxidase, which eliminates hydroperoxides from lipids, was significantly more active in PTN MDS-ISS mice. On the contrary, the activity of glutathione reductase, a very important enzyme that regenerates glutathione after its oxidation was not modified during the space flight.
Antioxidant enzymes and glutathione content in PTN and wild type mice erythrocytes after space flight.
Several antioxidant enzymes can limit the availability of ROS, including superoxide dismutases (SOD), which transforms superoxide anion to hydrogen peroxide, and Catalase, that removes hydrogen peroxide.
Both enzymes had a significantly higher activity in flown PTN mice erythrocytes (). The same trend was observed for WT mice, even if no statistics may be performed on these data.
Catalase (Panel A, U/mg Hb+SD) and Superoxide dismutase (Panel B U/g HB+SD) activities in PTN and wild type mice erythrocytes after space flight.
Moreover it seems that flown PTN mice reached an higher level of TBARS, GSH and GSH peroxidase after space flight; these may be related to an higher sensitivity of PTN mice to oxidative stress. In fact in primary culture of human osteoblasts from osteoporotic tissue it was recently demonstrated that a down regulation of PTN is paralleled by an up regulation of genes involved in response to reactive oxygen species probably due to increased exposure of osteoblasts to oxidative stress in osteoporotic tissue 
The increase of these enzymatic activities and of glutathione intracellular content may indicate an induction of these proteins and may be also correlated to the age of newly synthesized erythrocytes; in fact it is demonstrated that during RBC aging the level and the activity of these enzymes significantly decrease 
An induction of antioxidant enzymes has been reported in the liver of mice flown for 13 days in previous space missions 
. Moreover, simulated microgravity and chronic stresses induced antioxidant responses in lymphocytes, brain and muscles 
Finally, we investigated the lipid composition of RBC membrane phospholipids. The membrane phospholipid content and composition of mice erythrocytes is reported in .
Content of phospholipids and percentage distribution of each phopholipid class in cell membrane of mice erythrocytes.
As the lipid membrane composition may be greatly influenced by food quality and consumption 
we first compared the fat composition of food bars used in the MDS facility during flight and ground control (produced by Mucedola, Italy) with the one of the standard food utilized for vivarium mice (Global Diet 2018, Harlan, Italy). As the fat content was different between the two diets, we considered for statistical comparison only the membrane lipid composition of MDS-ISS mice and MDS-ground control mice that had the same diet.
In the RBC membrane of MDS-ISS mice there was an increase of phospholipids, statistically significant for the PTN mice, with changes in their relative percentage composition. In particular, the content of phosphatidylcholine (PC) was significantly reduced, whereas the phosphatidyl serine content was increased in MDS-ISS PTN mice. Moreover, we purified the single phospholipid classes and analyzed the fatty acid composition of each phospholipid. We noticed a significant decrease of the total polyunsaturated fatty acids of the omega-3 series in PTN mice (from 15.39% in MDS-ground mice to 12.89%in MDS-ISS mice). In particular, in the purified PC the amount of docosaexahenoic acid (DHA) was significantly reduced from 2.45 to 0.62% ().
Total omega-3 fatty acid content (black bars) and phosphatidylcholine DHA content (white bars) in PTN mice erythrocytes after space flight (MDS-ISS, Ground MDS controls).
These data are in agreement with the increase of TBARS, which could have been generated from oxidative damage of membrane polyunsaturated lipids and may be related to changes in membrane fluidity of erythrocyte or may be the results of microgravity adaptation 
It is noteworthy that, in addition to structural roles, omega 3 fatty acids, especially DHA, play major roles in signal transduction and are increasingly being recognized as reservoirs of lipid messengers. Specific precursors are cleaved from membrane phospholipids, in particular from PC, upon stimulation by neurotransmitters, neurotrophic factors, cytokines, membrane depolarization, ion channel activation, etc.
Impaired polyunsaturated fatty acid (PUFA) status is observed, and measured in erythrocyte, in numerous non physiological states and chronic diseases, like heart disease, metabolic syndrome, rheumatoid arthritis, and other inflammatory conditions, pulmonary disorders, and some psychiatric disorders 
There are several potential mechanisms that mediate the effects of PUFA on human health including antithrombotic and anti-arrhythmic effects, decreased heart rate variability and resting blood pressure, decreased serum low density lipoproteins (LDL) cholesterol and triglyceride content and increased insulin sensitivity. Other possible mechanisms of omega-3 PUFA are favorable effects on endothelial function, anti-inflammatory effects as well as neuroendocrine influences including modulation of the hypothalamic-pituitary-adrenocortical axis activity 
The operation of the space station and the human exploration will require long duration missions. However, mission duration will be limited by the degree of knowledge on the level of the physiological adaptation as well as on the acceptable limits of exposure to specific conditions during the permanence in space environment; our observations may be of particular importance for future manned long term space flight to prepare and implement a nutritional and nutraceutical program to maintain crew health.