Natalie Suff

Continual conscious bioluminescent imaging in freely moving somatotransgenic mice

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.

The power of bioluminescence imaging in understanding host-pathogen interactions

Infectious diseases are one of the leading causes of death worldwide. Modelling and understanding human infection is imperative to developing treatments to reduce the global burden of infectious disease. Bioluminescence imaging is a highly sensitive, non-invasive technique based on the detection of light, produced by luciferase-catalysed reactions. In the study of infectious disease, bioluminescence imaging is a well-established technique; it can be used to detect, localize and quantify specific immune cells, pathogens or immunological processes. This enables longitudinal studies in which the spectrum of the disease process and its response to therapies can be monitored. Light producing transgenic rodents are emerging as key tools in the study of host response to infection. Here, we review the strategies for identifying biological processes in vivo, including the technology of bioluminescence imaging and illustrate how this technique is shedding light on the host-pathogen relationship.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. To further restrict biosensor activity to the CNS, we performed intracerebroventricular injection of each vector. We observed greater restriction of bioluminescence to the head and spine with both vectors. Immunohistochemistry revealed strongest expression in cells of neuronal morphology. LPS administration stimulated a 4-fold increase over baseline bioluminescence. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner using a model of LPS-induced inflammation. This technology employs the 3R’s of biomedical animal research, complements existing germline-transgenic models and may be applicable to other rodent disease models with the use of different response elements.

Gene therapy in argininosuccinic aciduria

Argininosuccinate lyase (ASL) belongs to the liver-based urea cycle detoxifying ammonia, and the citrulline-nitric oxide cycle synthesising nitric oxide (NO). ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia and a multi-organ disease with neurocognitive impairment. Current therapeutic guidelines aim to control ammonaemia without considering the systemic NO imbalance. Here, we observed a neuronal disease with oxidative/nitrosative stress in ASL-deficient mouse brains. A single systemic injection of gene therapy mediated by an adeno-associated viral vector serotype 8 (AAV8) in adult or neonatal mice demonstrated the long-term correction of the urea cycle and the citrulline-NO cycle in the brain, respectively. The neuronal disease persisted if ammonaemia only was normalised but was dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This was correlated with behavioural improvement and a decrease of the cortical cell death rate. Thus, the cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress not mediated by hyperammonaemia, which is reversed by AAV gene transfer targeting the brain and the liver, acting on two different metabolic pathways via a single vector delivered systemically. This approach provides new hope for hepatocerebral metabolic diseases.Argininosuccinate lyase (ASL) belongs to the liver-based urea cycle detoxifying ammonia, and the citrulline-nitric oxide cycle synthesising nitric oxide (NO). ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia and a multi-organ disease with neurocognitive impairment. Current therapeutic guidelines aim to control ammonaemia without considering the systemic NO imbalance. Here, we observed a neuronal disease with oxidative/nitrosative stress in ASL-deficient mouse brains. A single systemic injection of gene therapy mediated by an adeno-associated viral vector serotype 8 (AAV8) in adult or neonatal mice demonstrated the long-term correction of the urea cycle and the citrulline-NO cycle in the brain, respectively. The neuronal disease persisted if ammonaemia only was normalised but was dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This was correlated with behavioural improvement and a decrease of the cortical cell death rate. Thus, the cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress not mediated by hyperammonaemia, which is reversed by AAV gene transfer targeting the brain and the liver, acting on two different metabolic pathways via a single vector delivered systemically. This approach provides new hope for hepatocerebral metabolic diseases.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.Patients with mutations in the ASL gene present with argininosuccinic aciduria characterised by hyperammonaemia and cognitive impairment. Here, the authors show that cerebral disease involves neuronal nitrosative/oxidative stress that is not induced by hyperammonaemia, and that it can be reversed using AAV-ASL directed to liver and brain in mice.

Ascending Vaginal Infection Using Bioluminescent Bacteria Evokes Intrauterine Inflammation, Preterm Birth, and Neonatal Brain Injury in Pregnant Mice

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates.

437. A Light-Producing Model of Infection-Related Preterm Birth

Background:Preterm birth is increasing in incidence and current therapies are relatively ineffective. It is responsible for >1million neonatal deaths per annum worldwide and long term complications in survivors. Approximately 50% of PTBs are preceded by microbial invasion of the intrauterine space; current clinical management centres on diagnosis of intrauterine bacterial presence by identifying the resultant inflammatory response. To investigate the relationship between intrauterine bacterial presence and inflammation we developed two separate gene technology approaches:1. Intravaginal bioluminescent bacteria to measure bacterial ascent into the uterus which mimics the ascending vaginal infection seen in preterm birth.2. Lentiviral gene transfer of an NFKB activated luciferase reporter construct to allow bioluminescent imaging of the subsequent systemic NFKB response.Methods:An NFkB response element was cloned into a lentivirus vector upstream of the genes encoding a codon-optimised firefly luciferase. High titred virus was injected intravenously at birth to neonatal female C57BL/6 J-Tyrc-2J mice to achieve luciferase expression predominantly in the liver (to monitor systemic inflammatory response). These mice received Escherichia coli (non-pathogenic K-12, MG1655 with integrated luxABCDE operon) intra-vaginally once reaching adulthood and intraperitoneal lipopolysaccharide (LPS) three weeks later. Luciferase expression was monitored by whole body bioluminescence imaging. Local inflammation was determined using H&E, ICAM-1 (Intracellular adhesion molecule 1) and Ly6g immunohistochemistry and enzyme-linked immunosorbent assays for serum and uterine TNF-α and IL1-β cytokines.Results:Bioluminescent imaging revealed that C57BL/6 J-Tyrc-2J mice were the most susceptible mice breeds for modelling of ascending vaginal infection with E. coli luxABCDE operon. Intraperitoneal LPS induced an NF-KB response in the liver by biosensing (p<0.01), however intravaginal E. coli administration induced no response. There was evidence of uterine inflammation with an upregulation of ICAM-1 and neutrophils.Conclusion:Although it is possible to detect LPS-induced NFKB inflammation in the liver by biosensing, ascending vaginal infection induced no response. This highlights the clinical challenge of identifying bacterial presence, confined to the uterus, using systemic markers. This model can be used to test new treatments for the prevention of PTB.View Large Image | Download PowerPoint Slide

OS078. Fetal growth restriction: A marker of severity of early-onset pre-eclampsia?

INTRODUCTION Pre-eclampsia (PE), particularly early-onset PE (PE requiring delivery before 34 weeks), is commonly associated with fetal growth restriction (FGR). The evidence for an association between FGR and a more severe PE phenotype is controversial. OBJECTIVES The main aim of this study was to investigate whether the presence of FGR in women with early-onset PE is associated with more severe maternal disease compared to those with appropriately grown fetuses (AGA). METHODS This was a retrospective cohort study of women with early-onset PE between 2001 and 2010 at University College London Hospital. The diagnosis of PE was made according to the criteria defined by the International Society of the Study of Hypertension in Pregnancy (ISSHP). PE was diagnosed when the systolic blood pressure was 140mmHg or more and/or the diastolic blood pressure 90mmHg or more on at least two occasions four hours apart developing after 20 weeks of gestation in previously normotensive women with proteinuria of 300 mg or more in 24h or two readings of at least ++ on dipstick analysis of midstream or catheter urine specimens if no 24-h collection is available. FGR was defined as birthweight less than the 5th centile, with abnormal umbilical artery Doppler (raised pulsatility index, absent or reversed end-diastolic flow). Maternal, fetal and neonatal data were collected and study groups compared using Chi-square test or Fishers exact test (categorical variables), and Mann Whitney-U test (continuous variables). Data analysis was performed using SPSS 16.0. RESULTS In women with early-onset PE (n=134), FGR (n=66, 49%) was associated with significantly higher perinatal mortality (p=0.02). Gestational age at delivery was significantly lower in the FGR group (median, IQR: 29.0 weeks, 28.0-32.8 vs 32.0, 30.0-33.2, p=0.01). However, maternal indicators of PE severity, including blood pressure (3)160mmHg systolic or (3)110mmHg diastolic, ALT>100U/L, platelet count <100×10(9)/L, use of magnesium sulphate or use of antihypertensive therapy, were similar in the two groups. CONCLUSION In women with early onset PE, FGR was associated with worse perinatal outcome, but was not associated with a more severe maternal PE phenotype. This may be because the presence of FGR in these women leads to earlier iatrogenic delivery, before the maternal condition deteriorates, but further studies are needed to investigate this.