Effects of age on retinal macrophage responses to acute elevation of intraocular pressure
Introduction
Aging is the biggest risk factor for neurodegenerative diseases including Alzheimer's disease, age-related macular degeneration (AMD) and glaucoma. There is increasing evidence that aging shifts the central nervous system (CNS) milieu towards a proinflammatory or ‘para-inflammatory’ state, which is characterized by increased reactivity of microglia, enhanced inflammatory cytokine production and a reduced phagocytic function of CNS macrophages (Ardeljan and Chan, 2013; Perry et al., 1993; Wong, 2013; Xu et al., 2009). Since neuronal-microglial interactions are essential for tissue homeostasis in the CNS, increased microglial reactivity in the normal aged brain or retina may alter retinal ganglion cell (RGC) survival and tissue damage in response to injury. Thus, alterations in the functional capacity of senescent microglia may have serious implications for age-related diseases affecting CNS tissues.
Numerous alterations to microglial morphology and function with aging have been reported. These include increased steady-state expression of activation markers such as MHC class II and CD11b (Frank et al., 2006; Ogura et al., 1994; Perry et al., 1993; Wong, 2013), reduced process complexity, reduced phagocytic ability, altered granularity and the accumulation of lipofuscin granules (Sierra et al., 2007), as well as reduced branching of processes accompanied by slower motility of processes (Damani et al., 2011). Microglia in aged animals also change their inflammatory profile and functional phenotype. Namely, microglia from the aged mouse brain constitutively produce greater amounts of TNFα, IL1β, IL6, IL10 and TGFβ1, relative to microglia from the younger mouse (Njie et al., 2012; Sierra et al., 2007). Taken together these data suggest an over-responsive or ‘primed’ microglial phenotype with increasing age, which may have major implications on the CNS microenvironment and thus, the microglial response to injury, inflammation or disease. Indeed, studies of microglia in the aging CNS have already described the dysregulation of microglial responses to injury such as facial nerve axotomy (Miller and Streit, 2007), cortical impact injury (Kumar et al., 2013), cerebral ischemia (Lee et al., 2010) and laser-induced retinal injury (Damani et al., 2011), as well as a reduced phagocytic capacity of microglia to clear cellular and other debris in the aging brain (Reviewed in Lourbopoulos et al., 2015; Mosher and Wyss-Coray, 2014; Theriault and Rivest, 2016).
To assess the potential role of aging on the retinal microglial response to acute intraocular pressure (IOP), we used an established model of retinal injury whereby the anterior chamber is cannulated and IOP elevated to 50 mmHg for 30 min (Kong et al., 2009, 2012). Previous studies in our laboratory using electroretinography have found that the duration and level of pressure used in this experimental model produces maximum functional impairment of the inner retina (retinal ganglion cells) whilst maintaining outer retinal function (Kong et al., 2009). Additionally, 50 mmHg produces consistent oxidative stress without the induction of ischemia, as confirmed by measurement of inner retinal capillary blood flow using a Heidelberg retinal flow meter (Kong et al., 2012), and does not induce significant levels of retinal ganglion cell death in young or middle-aged mice (Chrysostomou and Crowston, 2013). Given that the susceptibility of retinal ganglion cell damage appears to increase with age (Katano et al., 2001; Wang et al., 2007), we considered this model as a valuable tool to assess the way in which ocular responses to injury or stress change during normal aging. Indeed, middle-aged (12 month old) and older (18 month old) C57BL/6J mice suffer greater loss of inner retinal function and impaired functional recovery in response to acute IOP elevation when compared to younger (3 month old) mice (Kong et al., 2012).
We have previously described the retinal macrophage response to cannulation of the anterior chamber and the acute elevation of IOP using the above model and demonstrated microglial activation, increased density of retinal hyalocytes and the accumulation of retinal macrophages in the subretinal space (Kezic et al., 2013b). Additionally, we showed that cannulation alone induces inflammatory changes in the retina, however macrophage reactivity is higher following IOP elevation, as demonstrated by a greater number of rounded macrophages and a higher level of expression of CD68 and MHC Class II (Kezic et al., 2013b). Based on the hypothesis that macrophage/microglial reactivity increases with age, or alternatively, their functional capacity decreases, in the present study we set out to determine whether the retinal macrophage response to an acute retinal insult differs in the middle-aged (12 month old) mouse when compared to macrophages in the younger (3 month old) mouse. Further, as part of a longer-term exploration of strategies to attenuate detrimental macrophage responses to injury in the aging eye, whole-body irradiated adult mice received bone marrow replenishment from either young or middle-aged donor mice. These experiments were carried out to address the question of whether the age of bone marrow alters the macrophage response to injury. Various studies in mice have already demonstrated the benefits of “young” bone marrow therapy in rejuvenating the aged heart (Li et al., 2018), preserving learning and memory in old mice (Das et al., 2019) and reducing behavioral deficits and diminishing the immunological response after stroke in aged mice (Ritzel et al., 2018).
Section snippets
Animals
C57BL/6J mice of 3 and 12 months of age were bred and housed at the Royal Victorian Eye and Ear Hospital (RVEEH) and were maintained in a 22 °C, 12-h light (~40 lux)/12-h dark environment with standard murine chow (WEHI breeder mix; Barastoc, VIC, Australia). We chose 12 months as our time point to examine aging macrophages as previous studies in our laboratory have demonstrated a greater loss of inner retinal function and impaired recovery in response to acute IOP elevation in mice from 12
Effects of age on the retinal microglial response to acute IOP elevation
Confocal microscopic assessment of Iba-1+ retinal microglia in the outer plexiform layer (OPL) in 3 month old (Fig. 1A–C) and 12 month old (Fig. 1D–F) mice revealed morphological changes to a subpopulation of microglia one week after cannulation (Fig. 1B, E) and experimental elevation of IOP (Fig. 1C, F). Namely, the highly branched appearance of quiescent microglia (Fig. 1G, high magnification representative image from naïve mouse) was replaced with a more “reactive” phenotype whereby
Discussion
Changes to the distribution, morphology and function of CNS macrophages and microglia that occur during the normal aging process likely influence the response of these cells to injury in the brain and retina. In this study, we investigated the impact of aging on the retinal macrophage and microglial response to an acute IOP injury. We additionally attempted to study this further by transplanting young bone marrow into older animals to see whether replenishing the pool of hematogenous
Conclusions
We report decreased accumulation of subretinal macrophages and reduced gliosis 1 week after IOP elevation in chimeric mice that had received bone marrow from either 8 week old or 12 month old mice, indicating that exposure to irradiation itself rather than age of bone marrow was likely responsible for the observed changes. Preliminary studies whereby mice received sublethal doses of irradiation without bone marrow transplantation prior to IOP elevation confirmed these findings.
Grant information
These studies were supported by The Ophthalmic Research Institute of Australia and National Health and Medical Research Council Centre Project Grant #1033506.
CERA receives Operational Infrastructure Support from the Victorian Government.
Disclosure
The authors declare no competing interests.
Declaration of competing interest
None.
Acknowledgments
These studies were supported by The Ophthalmic Research Institute of Australia and National Health and Medical Research Council Centre Project Grant #1033506.
CERA receives Operational Infrastructure Support from the Victorian Government.
The authors thank staff at the Monash Micro Imaging (MMI) facility, Monash University, for their technical assistance with confocal microscopy.
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- 1
Present address: Department of Histology and Embryology, School of Medicine, University of Zagreb, Šalata 3, Zagreb 10000, Croatia.
- 2
Present address: Centre for Vision Research. Duke-NUS Medical School and Singapore Eye Research Institute, Singapore 169857.