Date Published: January 25, 2019
Publisher: Public Library of Science
Author(s): Rachel Waller, Lynne Baxter, Daniel J. Fillingham, Santiago Coelho, Jose M. Pozo, Meghdoot Mozumder, Alejandro F. Frangi, Paul G. Ince, Julie E. Simpson, J. Robin Highley, Giuseppe Biagini.
Deep subcortical lesions (DSCL) of the brain, are present in ~60% of the ageing population, and are linked to cognitive decline and depression. DSCL are associated with demyelination, blood brain barrier (BBB) dysfunction, and microgliosis. Microglia are the main immune cell of the brain. Under physiological conditions microglia have a ramified morphology, and react to pathology with a change to a more rounded morphology as well as showing protein expression alterations. This study builds on previous characterisations of DSCL and radiologically ‘normal-appearing’ white matter (NAWM) by performing a detailed characterisation of a range of microglial markers in addition to markers of vascular integrity. The Cognitive Function and Ageing Study (CFAS) provided control white matter (WM), NAWM and DSCL human post mortem tissue for immunohistochemistry using microglial markers (Iba-1, CD68 and MHCII), a vascular basement membrane marker (collagen IV) and markers of BBB integrity (fibrinogen and aquaporin 4). The immunoreactive profile of CD68 increased in a stepwise manner from control WM to NAWM to DSCL. This correlated with a shift from small, ramified cells, to larger, more rounded microglia. While there was greater Iba-1 immunoreactivity in NAWM compared to controls, in DSCL, Iba-1 levels were reduced to control levels. A prominent feature of these DSCL was a population of Iba-1-/CD68+ microglia. There were increases in collagen IV, but no change in BBB integrity. Overall the study shows significant differences in the immunoreactive profile of microglial markers. Whether this is a cause or effect of lesion development remains to be elucidated. Identifying microglia subpopulations based on their morphology and molecular markers may ultimately help decipher their function and role in neurodegeneration. Furthermore, this study demonstrates that Iba-1 is not a pan-microglial marker, and that a combination of several microglial markers is required to fully characterise the microglial phenotype.
T2-weighted magnetic resonance image (MRI) white matter hyperintensities are a common feature of the ageing brain . These white matter lesions (WML) are classified based on their anatomical location, with periventricular lesions (PVL) found in white matter (WM) next to ventricles, while deep subcortical lesions (DSCL) occur within the centrum semiovale. DSCL are found in around 60% of the population over 65 years of age and are linked to progressive cognitive decline and depression . The definitive cause(s) of WML are, as yet, unknown, yet there is much evidence to suggest blood brain barrier (BBB) dysfunction , axonal damage [4, 5] and cerebral hypoperfusion  contribute to their pathogenesis.
Microglia are diverse cells that express a range of different markers associated with their particular function and role within the CNS . This current study uses two separate image analysis platforms to quantitate Iba-1, CD68 and MHCII immunoreactivity across control, NAWM and DSCL of an ageing cohort. While NAWM has greater levels of Iba-1 microglial immunoreactivity than control brains, the formation of a DSCL against a background of NAWM appears to be associated with a loss of Iba-1 immunoreactivity in microglia. This conflicts with the recurring acceptance in the literature of Iba-1 as a pan microglial marker . On the other hand, an expected increase in CD68 immunoreactivity was identified with progression from control, to NAWM to DSCL.
In summary, this study identifies that some, but not all, microglia markers correlate with the progression from control WM in the ageing brain to NAWM to fully-developed DSCL. Clearly when using IHC to investigate the proposed function of particular cell types in pathology it is important to look at a range of cellular markers. Initially, with progression from control to NAWM, there is an increase in both CD68+ and Iba-1+ microglia. Following this, with the step from NAWM to DSCL, there is a further increase in the CD68+ signal. However, uniquely the study also shows a distinct population of microglia within DSCL that lose their Iba-1 expression. This finding disputes the existing concept that Iba-1 is a pan-microglial marker. Identifying sub populations of microglia that differ based on the markers they express may ultimately help us to decipher their overall function and explain the underlying pathology of neurological diseases. Therefore, it is important this discovery is explored beyond that of the DSCL in the ageing brain to investigate whether this finding is apparent in all WML and other neurological diseases.