Retinal And Brain Microglia in Multiple Sclerosis And Neurodegeneration Part 1

Abstract:

Microglia are the resident immune cells of the central nervous system (CNS), including the retina. Like brain microglia, retinal microglia are responsible for retinal surveillance, rapidly responding to environmental changes by altering morphotypes and functions. Microglia become activated in inflammatory responses in neurodegenerative diseases, including multiple sclerosis (MS). 

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When activated by stress stimuli, retinal microglia change their morphology and activity, with beneficial or harmful consequences. In this review, we describe characteristics of CNS microglia, including those in the retina, with a focus on their morphology, activation states, and function in health, aging, MS, and other neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, glaucoma, and retinitis pigmentosa, to highlight their activity in condition. We also discuss contradictory findings in the literature and the potential ways of reducing inconsistencies in the future by using standardized methodology, e.g., automated algorithms, to enable a more comprehensive understanding of this exciting area of research.

Keywords:

Retina; microglia; neurodegeneration; multiple sclerosis; retinal microglia; microglia morphotype.

1. Introduction

Microglia are resident immune glial cells of the central nervous system (CNS). They dynamically shift into various morphologies, which have also been associated with specific activation states that may be related to neuroprotective and/or neurotoxic functions in response to stimuli, injury, or insult [1–6]. These morphological and functional changes are essential to support a healthy CNS by contributing to homeostasis [2,7,8]. However, emerging evidence has started to show the involvement of microglia in disease whereby microglial dysfunction may be caused by disease and/or cause augmented disease-associated pathologies [2]. 

Nevertheless, the exact degree of involvement and mechanisms of how microglia may influence health and disease is unknown and currently being investigated. The purpose of this review was to compile information on the characteristics of CNS microglia, including those in the retina, with a focus on their activation states, morphology, and function, about health and disease. In the first section, we have compiled an extensive description of the main features of microglia in the general CNS and the retina. 

We also explain some of the microglial changes that occur throughout normal development and the aging process. In the next section, we discuss the current understanding of multiple sclerosis (MS) as an autoimmune disease, the surprisingly common ocular manifestations of MS, microglia in MS, and finally, microglia in other neurodegenerative diseases. Finally, we provide insight as to why there may be contradicting findings about the characteristics of CNS and retinal microglia. Here, we suggest different methods of experimentation such as using automated algorithms in light of producing more conclusive and consistent results.

2. Microglia

The CNS is made of several different types of cells, 5–10% of which are microglia, the resident immune cells [9]. Microglia were originally thought to exist as quiescent or “resting” cells that continuously survey their microenvironment for any stimuli or injury that may be harmful [5]. Conversely, more recent findings have suggested explanations for their dynamic properties. Microglia can shift into different morphological states, referred to as morphotypes. Each morphotype has been correlated to different activation states, which have also been associated with unique functions required to preserve a physiologically “normal” environment [5].

Once microglia are activated from their resting state, they can further differentiate into two main phenotypes: M1 and M2 [6]. Although there is a lack of understanding of the specific mechanisms that induce this differentiation, M1 and M2 microglia have also been associated with distinct cytokines, chemokines, and trophic factors [6]. Pro-inflammatory responses are associated with the “classically” activated M1 microglia, which encourage neuroinflammation as a response to insult or injury, creating a neuro-toxic environment and removing dysfunctional fragments of cellular debris [6]. This may occur as a result of inflammatory factors such as interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNFalpha), and inducible nitric oxide synthase (iNOS) [6,10]. 

Conversely, the “alternatively” activated M2 microglia are notoriously responsible for anti-inflammatory responses that encourage neuroprotective and restorative processes [6,10]. More recently, at least three more sub-phenotypes of the M2 type (M2a-c) have been found [11] whereby, more specifically, the M2a type secrete anti-inflammatory factors such as IL-10 and insulin-such as growth factor-1 (IGF-1), promoting cell debris removal and neuroprotection [6,11,12]. 

The M2b is said to be stimulated by inflammatory factors such as IL-1ß and lipopolysaccharides (LPS), which may also increase the expression of IL-10 [11]. These M2b microglia have been found to have phagocytic properties in brains modeled for Alzheimer’s disease (AD) and express high levels of CD64 [11]. The M2c “acquire deactivation” by IL-10 or glucocorticoids, in turn increasing expression of growth factors such as TGFß [11]. Despite these differences, M1 and M2 activation are functionally required to guarantee the removal of dysfunctional cells or noxious aggregates of cellular debris [6]. M1 microglia are usually involved in the clearance of cell debris, and this inflammatory response must be controlled by M2 microglia to avoid needlessly prolonged inflammation [6].

Often in pathological processes, the typical balance of M1 and M2 polarisation seen in normal conditions may be affected [6]. This can result in the clearance of healthy cells due to excessive M1 inflammation and the M20 s dampening effect of M1s may be overwhelmed causing further damage [6]. This often occurs in neurodegenerative illnesses and therefore, some therapeutic candidates that target M1 and M2 polarisation have been proposed [13]. Despite this, there is also emerging evidence that suggests that the M1/M2 polarisation may be outdated. It was first introduced and used to accommodate easier methods of data interpretation [14]. 

However, recent technological advances have revealed overlapping morphological and genetic characteristics between M1/M2 types, suggesting a need to reevaluate microglia types [14]. More recently, disease-associated microglia (DAM) have also been recognized as a unique microglia type seen in disease [14]. DAMs are characterized by microglia that express low levels of surveillant and homeostatic genes and high levels of markers associated with degeneration such as triggering receptors expressed on myeloid cells 2 (TREM2) [14,15].

With regards to morphology, there are about five main microglial morphotypes that have been recognized, including the ramified, hyper-ramified, activated, amoeboid, and rod types. Under non-primed or “inactive” conditions, microglia appear “ramified”. They are distributed evenly like a “mosaic”, with each consisting of a small and round cell body to which are attached several thin and long processes that constantly extend and retract to facilitate their surveillant functions (Figure 1) [2,5,16]. 

Experimental in vivo imaging of the brain has shown that these dynamic processes come into proximity with neurons, glia, and blood vessels, suggesting that microglia actively cooperate with other parts of the cortex to sustain a physiologically normal CNS environment [17]. Sometimes, ramified microglia may recognize microenvironmental changes and respond by changing into “hyper-ramified” microglia typically defined by more abundant processes that are longer and thicker, attached to larger, lobular, and irregularly shaped cell bodies (Figure 1) [16,18]. Most types of primed “non-ramified” microglia, including hyper-ramified cells, are scattered in the CNS in an irregular and “clustered” distribution [2]. 

Hyper-ramified microglia may also shift into the activated form upon exposure to significantly noxious stimuli, which also have similar cell bodies to hyper-ramified cells whilst having much fewer processes that are thicker and shorter (Figure 1) [2,16,19]. When noxious stimuli are extensively prolonged, activated microglia may morph into the “amoeboid” state with a rounder, larger, and more regularly shaped cell and very few or no processes (Figure 1) [16,18]. A recently re-discovered morphotype is “rod” microglia characterized by a long, sausage-shaped cell body with a few processes that may not always extend beyond the length of ramified microglia (Figure 1) [20,21]. 

Although emerging evidence has shown rod microglia localizing near neurons and aligning themselves along the nerve fibers, their exact function is still yet to be discovered [20–23]. The final type is the amoeboid microglia, often referred to as the phagocytic type that moves to the site of damage and phagocytoses dead or dying neurons and cell debris (Figure 1) [5,24]. Recently emerging evidence has led to a theory that the hyper-ramified, activated, and rod morphotypes may be “transitioning” forms that exist between the ramified and amoeboid states [5,25].

2.1. Retinal Microglia

The retina is an essential part of the CNS. Due to its transparent nature to light, it is possible to use less invasive and high-resolution imaging modalities to visualize the retina [26,27]. It is predominantly known for its involvement in converting light energy into electrical signals [28]. In humans, the retina develops from the first month embryologically to the end of the first year, originating from the neuroectoderm [28,29]. The retina consists of several distinct layers with the innermost layer being the retinal nerve fiber layer (RNFL), then the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL) and the final layer the retinal pigment epithelium (RPE) (Figure 2) (29]. Throughout these layers exist several types of cells, including amacrine cells, Muller cells, astrocytes, horizontal cells, rod and cone photoreceptors, and bipolar cells, all of which may also be found in the rest of the CNS [29].

Approximately 0.2% of the total retinal cells are made of microglia, of which 50% usually reside in the IPL whilst the rest reside in the OPL (Figure 2) [2]. Through development and homeostasis, microglia dynamically move through the different layers of the retina, although avoiding the ONL [2]. Furthermore, many retinal diseases and retinal injury models have shown that microglia may migrate towards the region of degeneration, become activated and proliferate [30,31], as described in more detail later. Although both retinal and brain microglia are developed from the primitive yolk sac, the appearance of each morphotype may vary depending on the region of the CNS [10]. 

For example, microglia in the striatum, hippocampus, and frontal cortex have larger cell bodies with more processes compared to those in the cerebellum [32]. Additionally, there is evidence that distinct layers of the cerebral cortex contain microglia of different sizes [32]. Morphotype appearances may also vary depending on the methods used or the axes of dissection. For instance, ramified microglia may appear horizontally ramified, which, in a cross-sectional observation, appear as one horizontally long cell whilst in a whole-mount observation, appear as and like the previously mentioned standard morphological description of ramified microglia [2,33]. 

When microglia are primed and become hyper-ramified, their processes extend radially, reaching across different layers, which is more visualizable in cross-sectional dissection compared to whole-mount observations [2,33]. Despite these differences, retinal and brain microglia have been found to share the expression of several transcription factors [2]. Numerous studies have also been able to observe each morphotype in both the brain and the retina [2,4,5,22,33,34]. However, it is still not clear whether or not each morphotype in both CNS regions shares the same features [2].

2.2. Molecular Markers and Stimuli Affecting Microglial Morphology

Similar to the brain, individual microglia morphotypes in the retina may result from microglial responses to various cytokines, chemokines, or damage-associated molecular patterns present in its microenvironment, suggesting heterogeneity in microglial genetic expression [9]. For instance, ramified microglia are said to have high expressions of P2RY12, which is often associated with surveillant functions, whilst amoeboid microglia have been found to express high levels of CD68—a notorious marker of phagocytosis [35,36]. 

Differential expression levels of microglial ion channels and surface receptors can also interact with such molecules (e.g., cytokines) in the microglial microenvironment, resulting in microglial changes including density, spatial distribution, activation state and morphotype, and disease pathogenesis [9,37]. For instance, transforming growth factor beta (TGFß) is an important microglial cytokine, pleiotropically involved in the physiological development of retinal neurons and vessels [37]. Ma et al., observed iba-1 positive microglia, from whole-mounted retinas of tamoxifen-induced ablation of TGFßR2 (TGFß receptor) in 2-month-old Cx3cr1CreER/+, Tgfbr2flox/flox mice [37]. 

The microglial morphology appeared ramified at 1 day post TGFßR2 ablation (PTA), which then became less ramified with “stubby” processes by 2–5 days PTA and finally appeared with lengthy processes that were aligned along the retinal blood vessels by 3–10 weeks PTA [37]. Real-time polymerase chain reaction (RT-PCR) analyses of TGFßR2 ablated retinal microglia revealed decreased expression of growth factors (e.g., BDNF, PDGFA) and increased expression of inflammatory activation markers (e.g., MHCII, CD68). This was not observable in healthy microglia. 

Additionally, whilst the TGFßR2 ablated animals showed no effects on the retinal vasculature, they experienced retinal thinning and amplified rates of pathological choroidal neovascularisation in response to injury [37].

2.3. Extracellular Vesicles: Effects on Microglia

Retinal (and brain) microglia also secrete extracellular vesicles (EVs), which are membrane-bound particles composed of mRNA, miRNA, DNA, cytokines, lipids, and proteins, regarded as a biopsy of its origin cell [6,38,39]. EVs are involved in cell-to-cell communication by transporting their neuro-protective/toxic components in response to intracellular and extracellular cues as they move through the bloodstream and cerebrospinal fluid (CSF) to reach other cells within close and distant proximity [6,38,39]. 

EVs include exosomes and exosomes, which consist of apoptotic bodies and microvesicles [39]. Firstly, many endosomal vesicular bodies fuse to form exosomes (40–160 nm in size) which are then released for intercellular communication [39]. Secondly, microvesicles instead originate from the plasma membrane undergoing outward budding (100–1000 nm) [38]. Finally, apoptotic bodies are >1000 nm in size and form through membrane blebbing of disintegrating, e.g., retinal microglia [38]. Components of apoptotic bodies such as phosphatidyl serine modulate microglial phagocytosis [38,39]. As a result of these diverse features, microglial EVs have recently started to be investigated for neurodegenerative diseases.

2.4. Microglia and Ageing

Microglial cells in the retina of newborn and postnatal rats have a round or amoeboid form and show pseudopodal processes involved in cell debris phagocytosis and developmental synapse remodeling [2]. Steadily, as the second and third week of the postnatal period approaches, microglial cell bodies become smaller with fine ramifications [2], assuming a highly ramified phenotype with the progress in brain development [40]. This progression is also reflected in brain regions, including the cerebellum, which suggests that microglia are actively involved through the maturation of the CNS.

A study investigated cortical microglia characteristics in old adult (24 months) and young adult mice (6 months) to find decreased microglial density and irregularly distributed clusters [41]. However, retinal microglia studies have revealed some contradictory results. 

Firstly, similar age groups of mice as the previous study were examined (18–24 months vs. 3–4 months) to find that the older adults had significantly elevated retinal microglia densities compared to that of younger adult mice [42]. This suggests that the microglial responses to aging may be region-specific. Further inspection of real-time retinal imaging showed that most of the old adult microglia appeared to have fewer and shorter processes, suggestive of activated or amoeboid microglia, than those in the young mice [42]. 

These were found to be arranged in a mosaic distribution. A more recent study looked at the morphological and marker expression differences of whole-mounted retinal microglia in old (15 months) and young (age not specified) mice [43]. Unlike the results from Damani et al., this study found no significant differences in iba-1 positive retinal microglia cell density and area covered by the processes between the two age groups, whilst the cell soma area in the OPL, IPL, NFL, and GCL and the number of vertical processes had significantly increased [42,43]. 

Additionally, the young microglia cells were mostly expressing P2RY12 and no CD68, whilst the aged microglia were CD68+ and appeared amoeboid [43]. These results, instead, imply that with aging, microglia may shift their morphologies and genetic profile to accommodate the aging process [43]. FernándezAlbarral et al., however, do not disclose the exact ages, species of mice, or the quantitation methods used. Such differences between studies may impact the results.

Aged microglia become less dynamic, showing significantly slower process motilities compared to those in their younger counterparts, which likely compromises their ability to continuously survey and interact with their environment [42]. Post-mortem hippocampal and cortical investigations in “young”, “middle”, or “old” (20–69 vs. 70+ vs. 90+ years old) adults revealed more region-specific microglial responses to aging [4]. Rod-shaped microglia were found to be significantly more abundant in the “middle” compared to “young” adults, although this was only seen in the hippocampus whilst the “old” adults only had significant rod increases in the hippocampus [4]. Other cortical and hippocampal studies found similar microglial trends with age, which suggests that aged microglia may have diminished surveillant capabilities required for maintaining a healthy CNS, which in turn may increase the risk of developing neurodegenerative diseases [44–46].

The myelinated axons of neurons are an essential feature of the CNS that enables efficient action potential conductance [47]. Whilst myelin is produced by many oligodendrocyte cells, it undergoes constant renewal, with myelin debris being directly removed and indirectly replaced by microglia [47,48]. However, it has been proposed that changes in myelin debris formation with aging may also result in age-related dysfunction of CNS immune cells such as microglia [49]. Hence with age, there are more impaired myelin-associated molecules, and a higher myelin protein turnover rate is required [49]. 

This leads to an increased rate of myelin breakdown, causing burdensome myelin accumulation, which then forms insoluble lysosomal aggregates within microglia cells [49]. TREM2 is expressed on microglial cell surface membranes; however, its deficiency or mutation can result in disease caused by excessive demyelination [50]. Poliani et al. found “aged” TREM2 deficient brains had demyelination with dystrophic and amoeboid-looking microglia [50]. It is also said that TREM2-positive amoeboid microglia may then morph into different shapes as it starts to produce factors such as TNF and IL-1, which are considered to be “pro-regenerative” factors [48]. 

Thus, the extracellular matrix is modified to trigger modifying the extracellular matrix to attract and activate oligodendrocyte precursor cells (OPCs) which then remyelinate the axon [48]. Furthermore, healthy microglial responses to demyelination were found to increase expression of genes associated with activation, phagocytosis, and lipid metabolism, whilst that of TREM2 deficient microglia were found not to [50]. Other studies have shown that although debris clearance by microglial phagocytosis is more significant with the increase in age, “younger” myelin phagocytosis was more proficient than with “older” myelin [51]. These age-related changes were also correlated with the more frequent appearance of “dystrophic” non-ramified microglia [51]. There are not yet many investigations of myelin debris-related retinal microglial phagocytosis, possibly due to the lack of myelin in the retina itself.

With aging, the microglial populations become dystrophic and undergo structural and morphological changes. The cytoplasm starts to fragment, and their cell processes gradually lose the fine ramifications and show spheroidal swellings [52]. In addition, their constitutive microglial function starts to decline and show abnormal microglial injury responses. These alterations, combined with molecular and gene expression aging changes within microglia, result in their reduced capability of maintaining homeostasis in the immune environment, and this may contribute to neuronal impairments, cognitive decline, and age-related diseases [5,27,53–55].

2.5. Genetic Factors: Effects on Ageing Microglia

Age is a well-known risk factor for neurodegenerative diseases [56–58]. Therefore, the change of gene expression in the aging retina has become of significant interest. For instance, Chen et al. investigated the total retinal RNA of 3-month- and 20-month-old C57BL/6 mice [57]. With the increase in age, 298 genes, including those related to stress response and glycoprotein synthesis, were upregulated more than two-fold whilst 137 genes, including those related to immune and defense responses, had also been downregulated more than two-fold [57]. 

Additionally, RT-PCR analyses showed increases in inflammatory cytokine-, chemokine-, or complement activation-associated genes, e.g., chemokine (C-C motif) ligand 2 (CCL2), CCL12 or complement component 3 (C3) [57]. The authors then hypothesized that this might reflect microglial activation, which was supported by the immunohistological observation of isolectin B4+ amoeboid microglia in the IPL of aged mice only [57]. Another study was able to specifically investigate transcriptional changes in the aging retinal microglia by comparing the RNA extracted from isolated retinal microglia of 3-, 12-, 18-, and 24-month-old C57BL/6 mice [58]. 

A total of 719 differentially expressed genes were identified and were functionally associated with microglial immune regulation, e.g., IL3 and IL7, angiogenesis, e.g., vascular endothelial growth factors, and trophic growth factors, e.g., neurotrophin [58]. Interestingly, like Chen et al., expression of C3, a gene associated with age-related macular degeneration (AMD), increased with age which implied that senescence-associated retinal microglia transcriptional changes might contribute to AMD pathogenesis [58].

3. Multiple Sclerosis

3.1. Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic inflammatory disease of the CNS, affecting approximately 100,000 patients in the U.K. and 2,500,000 patients globally [59]. Interestingly, it can affect up to three times as many women as men, with an average age of onset in early adulthood [60]. Whilst the initial records of pathophysiological features related to MS date back to 1838, their pathological and clinical presentations were first identified as “MS” in 1863 by Jean-Martin Charcot [61]. 

Since then, the complex pathologies of MS have been revealed. Frequently occurring symptoms of MS involve the ocular pathway such as RNFL thinning, optic neuritis (ON) characterized by inflammatory damage to the optic nerve, and uveitis characterized by intraocular inflammation of the vitreous body, retina, and uveal tract [62–66]. It has also been reported that such retinal changes may occur before changes in the rest of the CNS in many neurodegenerative diseases, including MS [26]. 

These recent findings have led to much interest in retinal research. Despite the complex processes involved in MS development, it is a disease defined by autoimmune responses through activation of immune cells such as T-lymphocytes, B-lymphocytes, microglia cells and macrophages, demyelination, remyelination and neurodegeneration [12,67–69].

A well-recognized hypothesis of MS is that it develops through two main phases. Firstly, T-cells and B-cells mediate inflammatory responses by releasing cytokines that induce activation of inflammatory cells such as microglia behind a “closed” blood-brain barrier (BBB) [68,70–72]. Chronic inflammation can then result in mitochondrial dysfunction, causing energy deficiency. Secondly, the neuroprotective signals are overridden, which impairs the ability to repair demyelination, damaged axons, and neurodegeneration [68,70]. As a result, there are significant blockages of axonal conductance whereby eventually, the patient is left with irreversible lesions of the CNS such as the BBB, causing it to become “leaky” [68]. This, however, remains a theory due to recent emerging evidence showing that some patients respond better to therapeutics agents that target B-cells as opposed to those that target T-cells [70]. Although there have been multiple investigations using ever-evolving methods, there is a lack of consensus due to contradictory scientific results [73].

3.2. Sub-Types of Multiple Sclerosis

There is a spectrum of severity for symptoms and rates of progression, reflecting the heterogeneity of MS [70]. The earliest presentation of MS patients can be recognized as a clinically isolated syndrome (CIS) [67]. CIS patients have monophasic and mono-focal symptomatic episodes, experiencing, e.g., ataxia, photophobia, or areflexia, which lasts between 24 h at 3 weeks [67]. Primary progressive MS (PPMS) affects around 15% of MS patients who usually experience a steady and progressive decline in health [74]. Progressive relapsing MS (PRMS) is the least common form of MS, which only affects around 5% of patients that experience a steady decline in health with unexpected spikes of deterioration and recovery [74]. Relapse remitting MS (RRMS) is a more common form, affecting 80–90% of patients that experience unforeseen surges of disability [70]. 

Usually, these surges are succeeded by complete recoveries, but as patients age and progress to later stages, these recoveries become more partial [70,74]. This may be justified by many RRMS patients proceeding to develop secondary progressive MS (SPMS), where patients may experience fewer spikes of impairment and start to mimic the disease trajectory of PPMS [70,74]. Recently, wider interests in genetic studies have enabled MS-specific alleles and gene variants to be recognized, especially through the genome-wide association study [70,75]. Large amounts of data have accommodated more sophisticated analyses that revealed that specific gene variants were correlated to the various types of MS [76]. These gene variants were predominantly found physically and functionally near to immunomodulatory genes associated with MS pathogenesis [70,75].

Despite this, the profiles of these subtypes are only descriptive as there is a lack of sufficient evidence to make accurate pathophysiological distinctions [77]. For instance, a large proportion of MS patients experience asymptomatic phases whereby MS-associated lesions and other pathophysiological changes may occur silently [78]. Some post-mortem brain studies have even shown that there were distinctive MS-associated pathologies seen in subjects who had been considered “healthy” during their lifetime [78]. More recently, references to these sub-types have therefore evolved, distinguishing between disease activity or no disease activity with details of “surges” or steady progression and observations of new CNS lesions [77].

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