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3R and welfare perspective of a rat non-invasive hypoxia model for neuroscience and other research application

Perinatal hypoxia and premature birth are common causes of neurological disorders and other impairments of physiological function in newborns and children, often with consequences present later in the life of adults. Regarding this, we wanted to create a non-invasive model of moderate perinatal hypoxia in rats and then use different research methods to research specifically the disturbances in brain development and its consequences. We designed a multimodal research approach with 3R principles applied that allowed us to gain insight into mild and moderate brain deficits using a minimal number of laboratory animals and the same animals in multiple experimental procedures. Since an alternative model is not available, reducing the number of animals, reusing animals, and refining the procedure in conducting the experiments were imperative. The same animals were used for behavior and then in a non-return experiment for MRI and histological investigations. The refinement of this research was the least invasive methods of hypoxia and animal marking, the usage of the nest and mothers’ cage bedding in all experiments, and the shortest possible separation of pups from dams. The application of modern knowledge about the welfare of laboratory animals – health supervision, better housing conditions, education of the personnel and the experimenter, and application of modern methods aimed at reducing pain, suffering, and/or anxiety of laboratory animals are our standard practices when animals are used in scientific research. In study 48 of RccHan:WIST rats at postnatal day 1 (P1) were sex-determined, weighed, and randomly divided into hypoxic or control groups, keeping both sexes represented equally per experimental group (4 females and 4 males). Rat pups were placed in a hypoxic chamber and subjected to hypoxic (8%O2 in 92%N2) or normal conditions (21%O2 and 78%N2) for two hours. Subsequently, all rat pups were marked by a permanent toe tattoo. Twelve pups per group were decapitated immediately, and blood was collected to analyze the acid-base status using an i-STAT Alinity gas analyzer. Twelve pups per group were used for behavioral studies from P3 to P15 to investigate possible deficits in various reflexes, motor, sensory, memory, and learning disorders. These animals afterward were used for brain MRI at P15. Eight animals per experimental group were scanned using 7TMRI with several imaging modalities. All MRI-scanned animals were then sacrificed, and the brains were isolated for immunohistochemical labeling. Examination and analysis of brain sections were performed in search of histological indicators of brain injury at the molecular, cellular, and tissue level. The acid-base status measurements have confirmed a moderate shift from aerobic to anaerobic metabolic state in animals after hypoxia. The early postnatal behavior shows a significant increase in body weight and mild motor and sensory deficits in animals subjected perinatally to hypoxia. Histological and immunobiological labeling show no significant changes in cortical cyto- or histo- architectonics. The MRI-volumetric measurements indicate some increase in the volume of neocortical regions in hypoxic animals. The results obtained with this optimized research design were sufficient for statistical analysis and firm scientific conclusions. Funding: project CSF-IP-2019-04-3182 and project GA KK01.1.1.01.0007.

reducing, reusing, refining, postnatal behavior, 7TMRI

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