Characterisation of brain injury and the role of neuroinflammation in a rodent model of preterm hypoxia-ischemia

Carty, Michelle Linda (2011). Characterisation of brain injury and the role of neuroinflammation in a rodent model of preterm hypoxia-ischemia PhD Thesis, School of Medicine, The University of Queensland.

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Author Carty, Michelle Linda
Thesis Title Characterisation of brain injury and the role of neuroinflammation in a rodent model of preterm hypoxia-ischemia
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2011-08
Thesis type PhD Thesis
Supervisor Kathryn Buller
Total pages 226
Total colour pages 12
Total black and white pages 214
Language eng
Subjects 110903 Central Nervous System
110999 Neurosciences not elsewhere classified
Formatted abstract
Hypoxic-ischemic (HI) brain injury is a leading cause of morbidity and mortality in the preterm neonate. A HI episode in the preterm neonate can lead to white matter damage, neuronal loss and an extensive range of functional impairments. However there is currently no clinical treatment available to alleviate HI brain injury and the ensuing long-term functional impairments. As a consequence, considerable educational, health and social resources are required to care for the affected neonates throughout their lives. In order to reduce these burdens it is essential to characterise the neuropathology and mechanisms that contribute to the persistence and progression of HI-induced brain injury. Therefore, the major aim of this thesis was to characterise brain injury, in particular white matter damage, and to investigate the role that neuroinflammation plays in contributing to this injury in a rodent model of preterm HI. Hypoxia-ischemia was produced in the post-natal day 3 (P3) rat pup by ligating the right common carotid artery and exposing the pup to 30 min of 6% oxygen (O2).

The first experimental chapter presents the effects of P3 HI in the immature rat brain on physiological indices of brain injury and hallmark features of white matter damage. The aim of chapter two was to characterise the effects of P3 HI on cerebral hemisphere size, body weight gain, developing oligodendrocytes and myelin content one week after the insult. It was found that P3 HI significantly reduced cerebral hemisphere size ipsilateral to the carotid ligation and there was a significant reduction in body weight gain in P3 HI animals compared to controls. In addition, in P3 HI animals, numbers of O4- and O1-positive oligodendrocyte progenitors were decreased significantly in the cingulum. One week after P3 HI there was also a significant reduction in myelin content in the corpus callosum compared to control animals.

The purpose of chapter three was to investigate the effects of P3 HI on key neuroinflammatory markers. It was found that there were increased numbers of activated microglia, increased cyclooxygenase-2 (COX-2) levels and elevated levels of interleukin-1α (IL-1β) and tumor necrosis factor- α (TNF-α) pro-inflammatory cytokines ipsilateral to the carotid ligation one week after P3 HI. Thus P3 HI induced significant neuroinflammation in the brain therefore I then went on to investigate whether these markers might contribute to the white matter damage observed in chapter two.

Chapter four addressed whether treatment with a potent inhibitor of activated microglia, minocycline, could attenuate neuroinflammation and white matter damage one week after P3 HI. Two different doses of minocycline were administered post-insult for one week; a high dose (45 mg/kg 2 h after HI then 22.5 mg/kg daily) or a low dose (22.5 mg/kg 2 h after HI then 10 mg/kg). The low dose minocycline regimen was as effective as the high dose in ameliorating neuroinflammation after P3 HI. In contrast, only the high dose regimen significantly attenuated P3 HI-induced reductions in O1- and O4-positive oligodendrocyte progenitor cells and myelin content. The low dose only significantly attenuated the reduction in O1-positive oligodendrocyte cell counts. These results suggest that neuroinflammation, in particular activated microglia, may contribute to P3 HI-induced white matter damage. However, although minocycline is a useful tool to inhibit microglia, minocycline is a tetracycline drug and potentially results in adverse effects in neonates and therefore is not usually prescribed to neonates. Other treatments that also target neuroinflammation could be more conducive to use in the clinic.

Ibuprofen may be a clinically desirable alternative anti-inflammatory treatment to minocycline. Therefore the purpose of chapter five was to examine whether ibuprofen, a non-steroidal anti-inflammatory drug (NSAID) that inhibits cyclooxygenase (COX) enzymes, can attenuate neuroinflammation and white matter damage after P3 HI. Post-insult ibuprofen treatment, administered for one week (100 mg/kg 2 h after HI then 50 mg/kg daily), prevented the P3 HI-induced COX-2 expression and IL-1β and TNF-α levels one week after P3 HI. Ibuprofen treatment also attenuated P3 HI-induced reductions in cerebral hemisphere size, O4- and O1- positive oligodendrocyte progenitor cell numbers and myelin content. Thus it appeared that ibuprofen was effective at inhibiting P3 HI-induced neuroinflammation and revealed a new mechanism that may contribute to the demise of white matter determinants after P3 HI.

In addition to the P3 HI-induced neuropathology, it is also important to determine the long-term functional consequences of P3 HI. In chapter six I profiled the effects of P3 HI on certain short- and long-term behavioural outcomes. Short-term neurodevelopmental tests included the righting reflex and negative geotaxis tests. Long-term behavioural testing included open field analyses, wire hanging, beam walking and gait kinematics. Animals subjected to P3 HI took significantly longer than controls to complete the righting reflex task indicative of short term functional impairment. Long-term functional impairments were also observed on the open field test six weeks after P3 HI. In contrast, no differences between the performance of control and P3 HI animals were observed for the negative geotaxis, wire hanging, beam walking and gait tests.

It is becoming increasingly evident that neuronal losses accompany white matter damage however relatively little is known about the effects of HI on specific neuronal phenotypes. In the final experimental chapter, chapter seven, I began to examine which neuronal phenotypes might be affected by P3 HI and whether neuronal changes can lead to long-term effects on neurological performance. Corticotrophin-releasing factor (CRF) and neuropeptide-Y (NPY) neurons were examined as candidate populations that might contribute to neurological impairments after P3 HI. Significant reductions in the number of CRF-positive neurons in the paraventricular nucleus (PVN), central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) ipsilateral to the carotid ligation were observed 1 and 6 weeks after P3 HI. There were also significant reductions in the number of NPY-positive neurons in the PVN, amygdala and BNST ipsilateral to the carotid ligation 1 week after P3 HI. However after 6 weeks, only the number of PVN NPY-positive neurons decreased significantly. Losses of CeA CRF-positive neurons were associated with impaired locomotor and exploratory activity in the open field arena 6 weeks post-insult. However, no significant correlations between neuronal counts and early neurodevelopment tests performed on one week after P3 HI were observed.

In summary, the findings presented in this thesis extend our knowledge on the neuroinflammatory mechanisms underpinning white matter damage, begin to characterise the neuronal phenotypes lost after HI and shed light on two potential anti-inflammatory treatments that could ameliorate brain injury after neonatal HI. These are significant advancements that will hopefully initiate the development of neuroprotective interventions and eventually help reduce the considerable burdens associated with HI-induced brain injury in the preterm neonate.
Keyword Hypoxia-ischemia
Neuronal loss
White matter damage
Additional Notes Printed in colour - page numbers 27, 32, 84-85, 87, 89, 105-108, 120, 185

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Created: Thu, 21 Jun 2012, 10:56:50 EST by Mrs Michelle Carty on behalf of Scholarly Communication and Digitisation Service