Abstract
Luciferase bioimaging in living animals is increasingly being applied in many fields of biomedical research. Rodent imaging usually involves anaesthetising the animal during data capture, however, the biological consequences of anaesthesia have been largely overlooked. We have evaluated luciferase bioimaging in conscious, unrestrained mice after neonatal intracranial or intravascular administration of lentiviral, luciferase reporter cassettes (biosensors); we present real-time analyses from the first day of life to adulthood. Anaesthetics have been shown to exert both neurotoxic and neuroprotective effects during development and in models of brain injury. Mice subjected to bioimaging after neonatal intracranial or intravascular administration of biosensors, targeting the brain and liver retrospectively showed no significant difference in luciferase expression when conscious or unconscious throughout development. We applied conscious bioimaging to the assessment of NFκB and STAT3 transcription factor activated reporters during the earliest stages of development in living, unrestrained pups. Our data showed unique longitudinal activities for NFκB and STAT3 in the brain of conscious mice. Conscious bioimaging was applied to a neonatal mouse model of cerebral palsy (Hypoxic-Ischaemic Encephalopathy). Imaging of NFκB reporter before and after surgery showed a significant increase in luciferase expression, coinciding with secondary energy failure, in lesioned mice compared to controls.
Highlights
The use of germline transgenic and somatic transgenic luciferase bioimaging in mice is well established in the study of cell reprogramming[1], cell engraftment[2], tumour tracking[3], gene therapy[4] and disease pathogenesis[5]
A lentiviral vector containing the constitutive SFFV viral LTR/promoter driving an codon-optimised luciferase transgene linked to an enhanced GFP by a bicistronic linker was pseudotyped with wide tropism VSV-G envelope protein[19]
We have shown that neonatal intracranial administration of lentivirus vector does not induce an immune response and scar the CNS tissue, compared to adult intracranial injections (Supplementary Figure 1), which agree with previous work[20]
Summary
The use of germline transgenic and somatic transgenic luciferase bioimaging in mice is well established in the study of cell reprogramming[1], cell engraftment[2], tumour tracking[3], gene therapy[4] and disease pathogenesis[5]. Targeted perinatal administration enables real-time biosensing of nuclear factor kappa B (NFκB) activity in the brains, liver and lungs of neonatal rodents[6] This technology exploits the fact that gene delivery to neonatal rodents achieves immune tolerance to the transgenic protein[7], in this case being firefly luciferase. Isoflurane exposure has shown to cause microglia activation, cell death and disrupt expression of genes associated with cognitive function and neurodevelopment in newborn male piglet brains[14]. Imaging from visceral organs such as the liver is made possible by using simple light reflection principles We applied these methods of conscious bioimaging of the brain in a neonatal model of Hypoxic Ischemic Encephalopathy (HIE). It is a substantial refinement of animal welfare and encouraging 3Rs principles in state-of-the-art whole-body imaging modalities
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