The generation and role of complement factor C5a during cerebral brain ischemia/reperfusion

Pavlovski, Dale (2013). The generation and role of complement factor C5a during cerebral brain ischemia/reperfusion PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Pavlovski, Dale
Thesis Title The generation and role of complement factor C5a during cerebral brain ischemia/reperfusion
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Trent Woodruff
Thiruma Arumugam
Steve Taylor
Total pages 199
Total colour pages 36
Total black and white pages 163
Language eng
Subjects 110903 Central Nervous System
110707 Innate Immunity
1115 Pharmacology and Pharmaceutical Sciences
1109 Neurosciences
1107 Immunology
111502 Clinical Pharmacology and Therapeutics
Formatted abstract
Following Stroke inflammation contributes to progressive neurological injury. The complement cascade is influential in the modulation of inflammation through, neutrophil chemotaxis, generation of reactive oxygen species, platelet activation and adhesion molecule upregulation. Following ischemic stroke, a focal upregulation of the complement C5a anaphylatoxin receptors, CD88 and C5L2, is seen in the infracted brain. This enhanced complement has been demonstrated to play a role in cerebral ischemia/reperfusion injury.

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a which activates these receptors is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucosedeprivation, or oxygen-glucose deprivation (OGD), neurons demonstrated a) increased apoptosis, b) up-regulation of CD88 and c) increased levels of C5a in the media. Exogenous murine C5a added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53, significantly blocked ischemia-induced apoptosis, and neurons from CD88-/- mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion, C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88-/- mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences.

As previously demonstrated and consistent with the literature the complement anaphylatoxin C5a has been implicated with stroke pathology; however the role of the C5a receptor, C5L2, remains to be investigated. The current study demonstrates that animals deficient in C5L2 show functional improvement when subjected to cerebral ischemia/reperfusion injury. Pathophysiological improvements in C5L2-deficient animals were also demonstrated by preserved blood brain barrier integrity, decreased immune cell infiltration and reduced apoptosis during cerebral ischemia. These findings for the first time identify a key role for C5L2 in acute ischemic stroke pathology.

The present findings that inhibition of C5a receptors during murine cerebral ischemia/reperfusion improves pathophysiological and functional outcome indicates a posisble therapeutic for stroke. Entertaining the well-founded theory that multiple target therapy is the best intervention for the plethora of deleterious mechanisms occurring during stroke specific CD88 inhibition was combined with clinical hypothermia. By using in-vitro ischemic-like conditions it was demonstrated that pharmacological blockade of CD88 enhances hypothermia-induced neuroprotection of primary cortical neurons under glucose deprived conditions. The findings were confirmed by culturing the neurons of CD88 deficient animals, with CD88-/- cultures showing neuroprotection under normothermic conditions and further enhanced hypothermia-induced neuroprotection.

Taken together these results demonstrate that neurons have the capability to generate C5a following ischemic stress and this has the potential to activate CD88, with deleterious consequences. In addition for the first time the role of C5L2 during cerebral ischemia/reperfusion was addressed and results demonstrated that it plays a role in stroke injury. By inhibiting C5a receptors during cerebral ischemia/reperfusion these results have indicated a potential therapeutic, further, by combining CD88 inhibition with hypothermia therapy it is demonstrated that hypothermic neuroprotection can be improved illustrating that pharmacological blockade of CD88 is also a candidate for the popular multiple target stroke therapy theory.
Keyword Complement factor 5a (C5a)
Ischemic Stroke
Neuronal cell death

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Created: Wed, 19 Jun 2013, 23:06:38 EST by Dale Pavlovski on behalf of Scholarly Communication and Digitisation Service