Rees Lab ~ Major Projects

There are several major projects in the Rees Lab:

• Project 1 - Intrauterine infection and hypoxemia: damage in the developing brain and retina.
• Project 2 - Investigation of cerebral development and injury in the prematurely born primate by magnetic resonance imaging and histopathology.
• Project 3 - Late gestation fetal alcohol exposure: Physiological, histological and biochemical studies.
• Project 4 - Neurodevelopmental hypothesis of schizophrenia: myelin-related changes and neuroprotection.
• Project 5 - The role of erythropoietin in experimental diabetic retinopathy.

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PROJECT 1 - Intrauterine infection and hypoxemia: damage in the developing brain and retina and neuroprotective therapies.

Investigators: Professor Sandra Rees, Dr Jhodie Duncan, Dr Michelle Loeliger, Mr Todd Briscoe, in collaboration with Prof Richard Harding ( Monash University )

Prenatal insults such as placental insufficiency or intrauterine infection have detrimental effects on the developing fetal brain with the potential to impair neurological development and result in disorders such as cerebral palsy. In an ovine model we have seen a striking link between intrauterine infection (mimicked by exposure to the endotoxin lipopolysaccharide, LPS) and the presence of cerebral white matter injury, similar to that reported in some cases of cerebral palsy. Using techniques including immunohistochemistry, biochemistry, microarray analysis, cytokine assays, stereology and ultrastructural analysis in this model we are currently investigating the contribution of different mechanisms including hypoxemia and proinflammatory cytokines in the manifestation of this injury. The ultimate aim of these studies is to develop intervention strategies to prevent or limit prenatal brain injury.

Photomicrographs of transverse sections from the cerebral hemispheres in control (A, C, E) and LPS-exposed (B, D, F) fetuses at 105 days of gestation. Following LPS-exposure injury was present in both the subcortical (B) and periventricular (F) white matter. (Duncan et al., 2002. Ped. Res. 52: 941-949)

Very low birth weight (VLBW) and small for gestational age (SGA) infants have a increased risk of visual impairment, however little information is available on the aetiology of these impairments. We have shown that a compromised prenatal environment, including hypoxia and infection affects development of the retina and optic nerve. Dopaminergic amacrine neurons have been of particular interest as they are reduced in several models of prenatal compromise and these neurons may be involved in the mechanisms underlying contrast sensitivity, a parameter that is affected in VLBW infants.

Supported by the National Health and Medical Research Council of Australia

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PROJECT 2 - Investigation of cerebral development and injury in the prematurely born primate by magnetic resonance imaging and histopathology

Investigators: Professor Sandra Rees, Dr Michelle Loeliger, in collaboration with Assoc Prof Terrie Inder (Royal Children's Hospital), Assoc Prof Gary Egan and Dr Hong Wang (Howard Florey Institute), Drs Bradley Yoder and Don McCurnin (Southwest Foundation, San Antonio, Texas, USA)

The neurodevelopmental outcome of very preterm infants (< 30 weeks gestational age) is of major concern. Whilst advances in perinatal care have significantly improved the survival of the prematurely born infant, it is recognized that up to 50% of these infants face adverse motor, cognitive and behavioral deficits as they approach school age. Periventricular leukomalacia (PVL), the most common cerebral neuropathology observed in premature infants, is thought to underlie these neurobehavioural deficits. Cerebral injury to the hippocampus, cortex and deep grey matter, may also contribute to subsequent neurologic impairments.

The survival rates of very premature infants have improved dramatically as a result of advances in perinatal care, in particular, respiratory support. It is critical that we understand how particular ventilatory therapies may alter the nature and severity of cerebral injury in preterm infants. To provide insight into the relationship between neonatal respiratory care and cerebral injury, we have established a model of premature birth in a non-human primate, the baboon ( Papio sp.).

In a histological and immunohistochemical study of the brain, we have defined the ontogeny of the cerebral hemispheres and cerebellum and the pattern of cerebral injury in the premature baboon nursed with identical neonatal intensive care to that of the human premature infant. The animals sustained a spectrum of neuropathologies including hemorrhage, white matter injury and ventriculomegaly; which are frequently observed in the human premature infant.

Ontogeny of cortical development (Dieni et al., 2004. JNEN. 63(12):1297-1309) Pattern of brain damage (Dieni et al., 2004. JNEN. 63(12):1297-1309)

Ontogeny of cortical development (Dieni et al., 2004. JNEN. 63(12):1297-1309) Pattern of brain damage (Dieni et al., 2004. JNEN. 63(12):1297-1309)

Project A: We are now assessing how the pattern of cerebral injury varies in relation to specific ventilatory regimes including: inhaled nitric oxide, positive pressure ventilation, continuous positive airway pressure and high frequency ventilatory oscillation. We hypothesise that the nature and severity of brain development will vary between animals in relation to their neonatal ventilatory therapy.

Project B: In collaboration with investigators at the University of Washington, St Louis (Assoc Professor Jeff Neil and Dr Chris Kroenke) we are correlating neuropathological alterations with conventional and advanced MRI imaging in the prematurely delivered baboon brain. In addition we are using diffusion anisotropy to monitor the ontogenetic development of the normal primate cerebral cortex. This projects is supported by the National Institutes of Health, USA ; grant RO1 HL074942-01A1

Anisotrophy of the primate brain at 125 days of gestation.

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PROJECT 3 - Late gestation fetal alcohol exposure: Physiological, histological and biochemical studies

Investigators: Ms Penny Dalitz (PhD Student), Professor Sandra Rees, in collaboration with Prof Richard Harding and Dr Megan Cock ( Monash University )

Alcohol is a well established teratogenic drug. For many decades it has been known that fetal alcohol exposure can result in cognitive impairment, behavioural disturbances, physical abnormalities and neurological damage. Though the detrimental effect of alcohol consumption in pregnancy has been established, the combined rate of Fetal Alcohol Syndrome (FAS) and Alcohol Related Neurodevelopmental Disorder (ARND) is still estimated to be at least 9/1000 live births. Substantial research into fetal alcohol exposure in the past has focused on neuronal loss, interference of neuronal migration and alcohol direct toxic effect on cells in small animal models or in cell culture. This project aims to characterise central nervous system (CNS) damage following a repeated "binge" pattern of alcohol exposure in a large animal model, at a biologically relevant dose. Experiments to date have particularly focused on increased apoptosis and white matter damage in the cerebral hemispheres and cerebellum following alcohol exposure in late gestation. With our observations of CNS damage from this model, we hope to further identify potential mechanisms of alcohol-induced fetal brain damage.

Project supported by the Pratt Foundation, Victoria, Australia.

Section of fetal brain indicating sites of damage in EtOH exposed fetuses (Dalitz, 2004).

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PROJECT 4 - Neurodevelopmental hypothesis of schizophrenia: myelin-related changes and neuroprotection.

Investigators: Professor Sandra Rees, Dr Mary Tolcos, Mr Todd Briscoe, Ms Elizabeth Bateman

Schizophrenia is one of the most debilitating of human mental disorders and affects about 1% of the population. The age of onset is most commonly 15-25 years of age and it is thought that the illness proceeds with apparently minimal neurodegenerative changes. The early neurodevelopmental hypothesis of schizophrenia posits that, changes in utero disrupt normal brain development, creating an underlying vulnerability, and predisposing an individual with risk factors, such as a genetic inheritance, to develop the typical symptoms of schizophrenia in later life.

Our laboratory has developed a guinea pig model of chronic placental insufficiency (CPI) to investigate the early neurodevelopmental hypothesis of schizophrenia. The strength of the model is that it mimics a situation that can occur in pregnant women. namely chronic placental insufficiency. We have already shown that aspects of the neurostructural (ventriculomegaly) and functional abnormalities in the model resemble alterations seen in disorders including schizophrenia.

Micrographs of control (left) and prenatally compromised (right) guinea pig brains. In thionin stained sections note the enlargement of lateral ventricle and reduction in basal ganglia in the PC brain (right) compared to control (left).

Tyrosine hydroxylase immunoreactivity in the hippocampus of the guinea pig at 8 weeks of age.

New directions for 2005: Recent studies by others have identified reduced levels of myelin and oligodendrocyte proteins and genes in post-mortem brains of schizophrenic patients. As we have not studied the effects of CPI on oligodendrocyte lineage and myelination in the adolescent prenatally compromised guinea pig, we now plan to undertake such a study.

The next important step is to attempt to ameliorate abnormal brain development as currently there is no effective treatment; one avenue might be by the administration of therapeutic agents after birth. As axons and dendrites continue to grow in the postnatal period, this is an ideal process to target when investigating intervention therapies. In 2005 we will trial several therapeutic agents which have been shown to penetrate the blood-brain barrier when delivered peripherally and target a number of cell types including neurons, astrocytes and oligodendrocytes.

Supported by the Melbourne Early Career Researcher Grant Scheme ( University of Melbourne ) and the ANZ trustees Medical Research and Technology in Victoria Program - The William Buckland Foundation awarded to Dr Mary Tolcos.

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PROJECT 5 - The role of erythropoietin in experimental diabetic retinopathy.

Dr Mary Tolcos , Professor Sandra Rees , Ms Kathryn Munro in collaboration with Prof Mark Cooper and Dr Merlin Thomas (Baker Medical Research Institute) and Assoc Prof Algis Vingrys and Dr Bang Bui (Department of Optometry and Vision Sciences, University of Melbourne)

Diabetic retinopathy is the leading cause of blindness in the working-age population. Research into diabetic retinopathy has focussed primarily on alterations to the vasculature, with the assumption that these changes lead to alterations in neuronal function and thus a loss of vision. However, an increasing body of evidence supports the view that altered neuronal function and viability is an important component in the pathogenesis of diabetic retinopathy beginning shortly after the onset of diabetes. Considering these neural alterations manifest early in non-proliferative diabetic retinopathy, these changes should be targeted for the generation of new therapies to complement existing treatments. We are now exploring the therapeutic potential of a novel neuroprotective factor and naturally-occurring hormone, erythropoietin (Epo), to protect retinal structure and function. Much interest has recently been generated in the potential use of recombinant human Epo to treat an increasing number of neurological disorders following its successful use in the treatment of hypoxic-ischaemic brain damage, retinal ischaemia, light-induced retinal degeneration, diabetic neuropathy and stroke in both humans and animals. This project is supported by the Diabetes Australia Research Trust awarded to Dr Mary Tolcos.

Structural arrangement of the retina. (A) Methylene blue stained semi-thin section of guinea pig retina showing the structural arrangement of neuronal and plexiform layers. (B) Schematic diagram of retinal connections.

This project is supported by the Diabetes Australia Research Trust.

Purkinje cell dendritic trees.