Inflammatory mechanisms after ischaemic stroke: characterisation of central nervous system (CNS) border-associated macrophages (BAMs) and dimethylarginine dimethylaminohydrolase (DDAH2)

Gerganova, Gabriela (2025) Inflammatory mechanisms after ischaemic stroke: characterisation of central nervous system (CNS) border-associated macrophages (BAMs) and dimethylarginine dimethylaminohydrolase (DDAH2). PhD thesis, University of Glasgow.

Abstract

Central nervous system (CNS) border-associated macrophages (BAMs) are a rare and specialised population of immune cells in the brain with emerging functions in ischaemic stroke. Preliminary work from our group has identified BAMs as CD45+highCD11b+CD206+Lyve1+ cells by flow cytometry and indicated increased numbers of BAMs 24 hours following stroke in a mouse model of focal ischaemic stroke (transient middle cerebral artery occlusion, tMCAo). Work presented in this thesis establishes the accumulation of BAMs beyond the period of infarct development. The numbers of BAMs in the ipsilateral hemisphere after tMCAo increased in the acute phase after stroke, peaked one week later and subsequently declined in the chronic stages. BAMs were identified in perivascular and in meningeal spaces as CD206+aSMA+ and CD206+Lyve1+ cells and their localisation with respect to the infarct and peri-infarct zones was investigated.

The signalling molecule nitric oxide (NO) has deleterious roles in ischaemic stroke and macrophages are an immune cell source of inducible nitric oxide synthase (iNOS)-produced NO. Work from Prof Leiper’s lab has shown that dimethylarginine dimethylaminohydrolase (DDAH2) enzymatically regulates NO signalling in hypoxic and septic conditions and thereby controls macrophage function. Work presented here demonstrates that Ddah2 mRNA expression increased following oxygen and glucose deprivation in RAW264 macrophage cell line, while DDAH2 protein levels remained unchanged after tMCAo in C57BL6/J mice. The expression of the pro-inflammatory genes Il-1b, Tnfa, Il-6 and Ccl2 was reduced in peritoneal macrophages from naïve monocytes/macrophages specific Ddah2 knockout mice, indicating that DDAH2 may regulate the inflammatory phenotype of these cells.

To explore the potential implications of DDAH2-mediated altered macrophage phenotype on ischaemic stroke outcomes, a study was conducted in monocytes/macrophages specific Ddah2 knockout mice. Preliminary evidence suggests that infarct and oedema volume did not differ between knockout and control mice. There was a slight reduction in neurological deficit and reduced levels of apoptosis in knockout mice after tMCAo. The rate of immune cell infiltration into the brain after tMCAo was not altered, as the numbers of total leukocytes, myeloid cells and lymphocytes were comparable between genotypes.

This thesis reinforces the emerging significance of BAMs in the pathophysiology of ischaemic stroke. Through detailed characterisation and temporal mapping, it establishes BAMs as dynamic responders that accumulate during the acute and subacute phases of stroke and recede in the chronic stages. Although modulation of nitric oxide signalling via DDAH2 showed limited impact on infarct size and immune infiltration, subtle improvements in neurological deficits and reduced apoptosis suggest that DDAH2 may fine-tune the inflammatory profile of macrophages in ways that are neuroprotective. These findings open a window into targeted immune modulation as a therapeutic avenue in stroke recovery.

This work points to a paradigm shift in how we understand neuroimmune regulation in stroke. Far from being passive residents at CNS borders, BAMs may play active, time-sensitive roles in brain injury and repair. By linking BAM behaviour with nitric oxide signalling and the regulatory role of DDAH2, the thesis lays groundwork for future exploration into cell-specific interventions—aimed not at altering infarct size, but at refining the inflammatory environment to promote neurological resilience. The immune landscape of the brain is more intricate than previously assumed, and the key to healing might lie not in suppressing inflammation broadly, but in sculpting it precisely.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the British Heart Foundation.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Funder's Name: British Heart Foundation (BHF)
• Supervisor's Name: Miller, Dr. Alyson and Leiper, Professor James
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85427
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 03 Sep 2025 10:22
• Last Modified: 03 Sep 2025 10:25
• Thesis DOI: 10.5525/gla.thesis.85427
• URI: https://theses.gla.ac.uk/id/eprint/85427

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