Hibernation and Radioprotection: Gene Expression in the Liver and Testicle of Rats Irradiated under Synthetic Torpor

Abstract

Hibernation has been proposed as a tool for human space travel. In recent years, a procedure to induce a metabolic state known as "synthetic torpor" in non-hibernating mammals was successfully developed. Synthetic torpor may not only be an efficient method to spare resources and reduce psychological problems in long-term exploratory-class missions, but may also represent a countermeasure against cosmic rays. Here we show the preliminary results from an experiment in rats exposed to ionizing radiation in normothermic conditions or synthetic torpor. Animals were irradiated with 3 Gy X-rays and organs were collected 4 h after exposure. Histological analysis of liver and testicle showed a reduced toxicity in animals irradiated in torpor compared to controls irradiated at normal temperature and metabolic activity. The expression of ataxia telangiectasia mutated (ATM) in the liver was significantly downregulated in the group of animal in synthetic torpor. In the testicle, more genes involved in the DNA damage signaling were downregulated during synthetic torpor. These data show for the first time that synthetic torpor is a radioprotector in non-hibernators, similarly to natural torpor in hibernating animals. Synthetic torpor can be an effective strategy to protect humans during long term space exploration of the solar system.

Keywords: ATM; hibernation; hypothermia; liver; radiation; raphe pallidus; space exploration; synthetic torpor; testicle; torpor.

Figure 1

Thirty-min mean value ± SEM of the brain temperature (Tbr), recorded during the experimental day. Tbr was recorded by means of a thermistor, surgically implanted in the Lateral Hypothalamus. Empty dots: Control (n = 5), filled dots: Hypothermia (n = 5).

Figure 2

Volcano plot representing the expression of the analysed genes in the liver (normothermic vs hypothermic animals) 4 h after a total body irradiation (X-rays, 3Gy) expressed as normalized fold change. In green: genes downregulated (to the left of the left dashed line). In red: genes upregulated (to the right of the right dashed line). Dots above the solid horizontal line are significantly different between the two groups.

Figure 3

Morphology of the hepatic tissue after hematoxylin-eosin staining in irradiated normothermic liver (A,B) and in irradiated liver during synthetic torpor (C,D). The effects of irradiation at normothermia are pronounced compared to the effects detected after irradiation in synthetic torpor. Alteration and disorganization in the hepatic parenchyma were observed in A,B, vacuolization and shrunken nuclei with irregular shape and chromatin condensation (A,B, arrows) were identified in the hepatocytes. The morphology and the organization of hepatic parenchyma irradiated during synthetic torpor (C,D) seems comparable to the normal organization of the liver tissue. Hepatocytes show a dense cytoplasm, round nuclei with dispersed chromatin. An abundance of red blood cells is also observable (C,D arrows). Scale bars = 20 µm.

Figure 4

Volcano plot of the representing the expression of the analysed genes in the testicle (normothermic rat testis vs hypothermic rat testis) 4 h after a total body irradiation (X-rays, 3Gy) expressed as normalized fold change. In green: genes downregulated (to the left of the dashed line). Dots above the solid horizontal line are significantly different between the two groups.

Figure 5

Morphology of cross sections of seminiferous tubules, after hematoxylin-eosin staining, in irradiated normothermic testis (A,B) and in irradiated testis during synthetic torpor (C,D). Alteration on the differentiation and cells stratification were found in the testis of normothermic rats (A,B), in particular disorganization of the germinal cells and of their junctions was present. These effects were not detected in the testis tissue of the rats induced into synthetic torpor, in which the structures could be comparable to the normal organization (C,D).

Figure 6

Experimental protocol. After at least one week of recovery from surgery, rats were moved to the experimental cage and exposed to a lower ambient temperature (15 °C) 48 h prior to the experiment. At 07:00 of the experimental day, rats were randomly assigned to one of the experimental groups: Hypothermia (n = 5) (Group 1), animals underwent multiple microinjections (1/h, 100 nL) of the GABA-A agonist muscimol (1mM) within the RPa, whereas Control (n = 5) (Group 2) were injected with artificial cerebrospinal fluid (ACSF). Four hours after the onset of synthetic torpor in the Hypothermia group, animals underwent a 3 Gy X-rays radiation exposure and were returned to the experimental cage for additional four hours. At 16:00, all animals were euthanized and organs were collected.

Figure 7

Exit dose measured with a radiochromic film under the irradiated animal. Entrance dose can be evaluated from the percentage depth dose curves and the animal thickness.