AEM hardware

Three ground government support equipment AEM units (Figure 5) were used for the feeding studies of rats and mice. AEM units were built according to flight configurations which, for mouse studies, delineates that each AEM is loaded with two water bags in the water box container with approximately 2 liters of heat-sterilized distilled water per bag. Water consumption was derived as a difference in the water bag mass before and after the test. Each AEM cage was fitted with a cage divider that provided two separate and equal compartments for mice. In each half of the AEM cage, two food plates were provided with one attached to the water box side, and one attached to the cage side. For the rat study, water was provided with two external graduated cylinders connected with plastic tubes through the water box to lixits. Control animals were handled and provided the same environment and provisions as flight configuration. Water consumption was recorded daily and fresh heat-sterilized water was added as needed. No divider was used in the cage for the rat study due to the size of the animals, therefore only five rats were housed in each AEM. Each AEM was instrumented with humidity (Vernier Software and Technology Type RH-BTA) and temperature probes (Vernier Software and Technology Type TMP-BHA) and a gas port for periodic sampling for ammonia (NH 3 ) and carbon dioxide (CO 2 ). NH 3 and CO 2 were measured using appropriate Draeger tubes and calibrated SKC Personal Sampling Pumps (model 222). Pressure drops across the exhaust filter were also measured at the gas port using an inclined Dwyer manometer accurate to 0.1 inches of water. The temperature and room humidity data were logged using LabView (National Instruments, Austin, TX, USA) software running on two laptop computers. The sensor to laptop connections used Vernier Software and Technology ‘SensorDAQ’ (Vernier Software & Technology, Beaverton, OR, USA) interfaces. During the test period, the AEMs were placed on vertical stands and covered with shrouds at all times except for daily health checks and any lighting was provided by the internal AEM light fixtures.

Figure 5 Schematic outlining the Animal Enclosure Module key features. Full size image

Diets

The NASA Type 12 NuRFB, the official approved rodent flight diet, were used for the reported studies and a detailed description of the NASA NuRFB has previously been reported.17–19 Diet requirements for the 35-day study were estimated based on the AEM flight payload preparation formula, which states that the average daily NuRFB and water consumption requirement are 4.0 g and 4.5 ml, respectively, per mouse per day, and 40 g and 50 ml per rat per day. In the shuttle flight payload operations, an additional 20% mass of NuRFB and water were loaded as waste allowance for both the mice and rats.

In the mouse study, each AEM cage was fitted with a cage divider, providing two separate areas for the mice. In each half of the AEM cage, two food plates were provided: one attached to the water box and the other to the cage side. Each water box diet plate accommodated four pieces of NuRFB, and each cage diet plate accommodated five pieces of NuRFB, for a total of 9×Type 12 M NuRFB (20.32 cm×2.54 cm×3.05 cm) per half cage, or ~2.24 kg per half cage (4.48 kg per AEM). In the rat study no cage divider was utilized in the AEM and the remaining NuRFB on the food plates were replaced with new food plates on day 21.

Animal study

All procedures used in this study conformed to the NRC Guide for the Care and Use of Laboratory Animals20 and Title 14 of the Code of Federal Regulations.21 The animal protocols for mouse and rat studies were reviewed and approved by the Institutional Animal Care and Use Committee at the NASA Ames Research Center prior to the experiments.

For the mouse study, female C57BL/6 mice (~35 days old) were acquired from Charles River (Wilmington, MA, USA) 4 weeks prior to the start of the test. Female C57BL/6 mice (body mass 15.2±0.8 g) were used in this study because they are a strain commonly used in the NASA flight experiments.2–10 For the rat portion of the study, male Sprague–Dawley rats aged 12 weeks (body weight 159±8.6 g) were obtained from Charles River (Wilmington, MA, USA). Young male Sprague–Dawley rats were used in the study because many spaceflight and ground-based spaceflight simulation studies have been conducted using young male rats and they are the strain likely to be used in future NASA rat flight experiments.

At the NASA Ames Research Center Animal Care Facility, animals were housed in standard polypropylene ‘shoebox’-style cages in a room maintained at a temperature of 24±1 °C, relative humidity of 40–70% and lighting which allows for a 12 h light/dark cycle. The control animal lighting cycle in the vivarium cages was synchronized with the AEM hardware operations.

Upon receipt, the animals were randomly assigned to groupings of six mice or five rats, housed in vivarium cages with microisolator tops, and provided distilled water via standard water bottles and sipper tubes. The animals were continued on the same food diet as at the vendor’s facility. After 3 days of acclimation, the animals were implanted subcutaneously with microchips (Biomedic Data Systems IMITM 1000, Biomedic Data Systems, Inc., Seaford, DE, USA) under anesthesia for identification purposes, a standard practice in AEM flight payload operations. Then, animals were provided distilled water via water bottles with stoppers fitted with AEM-like lixits that were refurbished prior to the test. The diet was transitioned slowly from the standard rodent diet over the course of 5 days until the animal diet consisted entirely of the NuRFB, at which point all animals were fed with only NuRFB until the end of the study. Water was provided ad libitum throughout the entire study duration. Diets were provided ad libitum throughout the entire study duration but removed overnight before the end of day 35 due to blood draw taken at time of tissue collection.

In the mouse study, following a 10-day acclimation, female C57BL/6 mice at 9 weeks old were randomly assigned (n=36 per group) to AEM units or vivarium cages. There were no significant differences in the initial body masses of mice in vivarium (19.0±0.5 g) or AEM cages (19.0±0.5 g).

In the rat study, following a 10-day acclimation, male Sprague–Dawley rats age 13–14 weeks old were randomly assigned (n=15 per group) to AEM units or vivarium cages with 5 rats per cage. There were no significant differences in the initial body weights of rats in vivarium (275.8±11.4 g) or AEM cages (274.2±11.3 g). The cages remained untouched during the course of the study, except for on day 21 when the AEM were opened, the NuRFB plates were removed and weighed, new NuRFB plates were added, and the animals were given a general health check-up by an on-site veterinarian while body mass measurements were recorded.

Animal water consumption was measured daily for the rats, while animal body mass and food intake were measured at day 21 and at the end of the study (day 35). CO 2 and NH 3 measurements were recorded on test days 1, 16, 28, and 35. NASA Animal Care Facility staff and Science team personnel performed a daily visual animal health evaluation. The attending veterinarian performed a detailed individual animal check and health evaluation on all of the animals at animal receiving, animal load at test start (day 0), during NuRFB replenishment (day 21), and animal unload at test end (day 35).

Organ mass measurements and serum clinical chemistry

One day before the end of the study (day 34), the animals were fasted overnight and then on day 35 the animals were anesthetized with isoflurane, blood was collected via cardiac puncture, and then the subjects were euthanized via isoflurane overdose paired with decapitation (rats) or cervical dislocation (mice). The kidneys, lungs, spleen, thymus, adrenals, and heart were dissected and mass measurements were recorded for each organ.

For clinical chemistry measurements, blood was collected in vacutainer tubes and serum was obtained by centrifugation at 1,500 g for 10 min at 4 °C. Serum samples were stored on ice and shipped to IDEXX Veterinary Services, Inc (West Sacramento, CA, USA). Serum triglycerides, cholesterol, fasting glucose, globulin, creatine kinase, and bicarbonates were measured by colorimetric enzyme assays using an automated Clinical Chemistry Analyzer (Olympus AU 400, Olympus America Inc, Center Valley, PA, USA). Colorimetric enzyme assays were also used to measure serum biomarkers of liver function of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and direct and total bilirubin. Biomarkers of renal function included the measurement of albumin, creatinine, total protein, blood urea nitrogen, calcium, phosphorus, and electrolytes (potassium, sodium, and chloride) also by colorimetric assays using the Clinical Chemistry Analyzer.

A Hematology Analyzer (Bayer Advia 120, Global Medical Instruments, Ramsey, MN, USA) was also used to determine red blood cell count, mean red blood cell volume, hemoglobin, mean corpuscular hemoglobin, mean cell hemoglobin concentration, white blood cell count, percent lymphocytes, neutrophils, monocytes, eosinophils, basophils, hematocrit, prothrombin time, partial thromboplastin, and fibrinogen.

Statistical analysis

Animal body mass measurements, food consumption, water consumption, and organ mass data normalized to total body mass following euthanasia were analyzed using one-way analysis of variance with JMP 7.0 software (JMP, Cary, NC, USA). For all tests, P<0.05 was considered significant. Values in all figures are expressed as mean±s.d. unless specified.