Retinal glia are classified into two groups: macroglia (Müller cells and astrocytes) and microglia. Each glial subtype differs markedly in distribution, morphology, and pathophysiology. Some studies reported that glial fibrillary acidic protein (GFAP), a glial cell marker, is a sensitive indicator of CNS injury. GFAP is increased in glial cells in patients after 1 to 3 months of uncontrolled diabetes, with pathological potential reduction after longer durations of the disease [
65,
66]. Pathological changes in retina glial cells in DR are shown in Table
3.
Table 3
Changes of retina glial cells and neurons in DR
Müller cell | Nuclear chromatin dispersion, nuclear granulation electrondense | ↑ | GFAP↑ | Proinflammatory | iNOS/COX2 | Rat/rMC-cells | |
Angiogenic | PEDF | Rat |
Caspase-1/IL-1β | Rat |
Wnt/β-catenin | Mice |
p38 MAPK/NF-κB/IL-6 | Human Müller cells |
Astrocyte | Axonal bundles are scanty, starlike cell bodies are irregularly distributed | ↓ | GFAP↓ | Anti-angiogenic | COX-2/EP3/PGE2 | Human | |
Microglia | Cell bodies appear larger and bore long blunt ruffles with thin thread-like projections | ↑ | CD45, CD68, HLA-DR | Proinflammatory | MAPK | Rat | |
Angiogenesis | P2 receptors/Ca2+ | Rat |
NF-κB/TNF-α, IL1-β | Mice/rat |
Ganglion cell | Axonal swellings and associated constriction enlarged cell bodies, increased dendritic branches and terminals | ↓ | Thy1 | Neurodegeneration | ERK1/2/COX-2/PGE2 | Rat | |
MAPK | Mice |
NF-κB | Mice |
Müller cells
Müller cells are the major glial cell type in mammalian retina, which span the entire depth of the neural retina. Müller cell somata are located in the inner nuclear layer (INL), from which two major trunks extend in opposite directions. The outer trunk forms a network of adherent junctions known as the outer limiting membrane between Müller cells and photoreceptors [
67]. In vascularized retina, the end feet contact and surround blood vessels within the retina. The secondary processes branching from the main trunk of Müller cells form extensive sheaths that surround neuronal cell bodies, dendrites, and the axons of ganglion cells [
68]. In the normal retina, Müller cells limit the spread of excitatory neurotransmitters such as glutamate, provide metabolic support for a subset of inner retinal cells, and maintain the stability of the extracellular environment [
68,
69]. In diseases, Müller cells possess a marked capacity to respond to a wide variety of environmental insults with pathophysiologic and biosynthetic changes [
69].
The density of Müller cells is significantly increased at 4 weeks of diabetic rats. The expression of GFAP in Müller cells is not detectable at 4 weeks (early stage) but the expression becomes prominent at 12 weeks. It is noteworthy that hyperplasia of Müller cells precedes GFAP overexpression in the diabetic retina [
70]. On electron microscopy, Müller cells in diabetic rats exhibit dispersion of nuclear chromatin and electrondense nuclear granulations, with the presence of increased glycogen, dense bodies, and lysosomes in the cytoplasm [
71]. Diabetic retina shows edematous Müller cell end feet in the nerve fiber layer, ganglion cell loss, intercellular space increase in the inner and outer nuclear layers, and outer retina degeneration due to apoptotic cell death as a result of overexpression of caspase-3 [
72]. Müller cells are major sources of inflammatory mediators [
73] and become “activated” or “reactive” in response to virtually all pathological changes in the retina [
74]. By using high-throughput techniques, diabetes-induced alteration of gene expression profile in Müller cells reveals that among 78 altered genes, one third are associated with inflammation [
75], suggesting that Müller cells contribute to inflammatory responses during the development of DR. VEGF is rapidly released from Müller cells in early DR, enhancing perfusion by locally increased permeability of blood vessels with concomitant decrease in anti-angiogenic pigment epithelium-derived factor [
76,
77]. In VEGF knockout mice, diabetes-induced retinal inflammation, vascular leakage, and vascular degeneration exhibit a significant reduction [
76]. In Müller cells cultured in high glucose, the levels of histone acetylation at histone H3 (AcH3K9), AcH3K18, AcH2BK5, and AcH4K8 are increased, with upregulated mRNA of inflammatory genes, such as VEGFR1, IL1-β, ICAM-1, TNF-α, and MCP-1 (CCL2) [
78]. These findings suggest that elevation of histone acetylations in Müller cells plays an important regulating role in the inflammatory response under diabetic conditions. The expression of VEGF and pigment epithelium-derived factor (PEDF) in Müller cells is disregulated in high glucose concentration, which contributes to retinal neovascularization in DR [
79]. The anti-angiogenic P60, a PEDF derivative, reduces vascular leakage by increasing tight junction proteins in retina vessels through Müller glia signaling and by reducing the levels of inflammatory cytokines that promote vessel abnormalities. The neuroprotective P78, another PEDF derivative, is more effective in the prevention of cell dropout and inner plexiform layer (IPL) thinning with reduction of vitreous levels of TNF-α and IL-2 and activation of the PI3K/AKT pathway in Müller glia [
80].
In an STZ-induced diabetic mouse model, disruption of β-catenin in Müller cells attenuates the overexpression of inflammatory cytokines and ameliorates pericyte dropout in the retina. Thus, Müller cell-derived β-catenin is an important contributor to retinal inflammation in DR, and the Wnt/β-catenin pathway is activated in DR model mice [
81].
Müller cells produce IL-1 and exert an inhibitory activity on Ag- and IL-2-driven proliferation of T helper cell lines. Under conditions where the inhibitory capacity of Müller cells is suppressed, the cells display APC function to show a dual effect on autoimmune T helper lymphocytes [
82]. Müller cells have been reported to produce increased amount of IL-1β when exposed to high glucose in vitro [
83], in which caspase-1/IL-1β signaling plays an important role in diabetes-induced retinal pathology [
19]. IL-1β has also been reported to induce IL-6 production by Müller cells predominantly through the activation of p38 MAPK/NF-κB signaling pathway [
74].
Studies of our laboratory have shown that hyperglycemia induced the overexpression and activation of HMGB1 in Müller cells. HMGB1 mediates toll-like receptor 4 (TLR4)-dependent angiogenesis [
84]. The expression of TLR4 was markedly increased in fibrovascular membranes from DR patients and in retinal vascular endothelial cells of diabetic mice [
85]. We therefore speculate that Müller cells are involved in inflammation-driven angiogenesis.
Retinal Müller cells (rMC-1) cultured in high glucose increase their production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. In vitro results suggest that hyperglycemia-induced increase in NO in retinal Müller cells promotes the production of cytotoxic prostaglandins via COX-2. iNOS appears to account for the increased production of NO by Müller cells [
86].
Exposure of Müller cells to high glucose also induces their expression of RAGE and S100B. RAGE signaling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevents cytokine production induced by high glucose and S100B in Müller cells [
87].
Müller cells regulate the level of substances in the neuronal microenvironment. One of the most characterized functions of Müller cells is the regulation of K
+ in the retina [
88]. The accumulation of K
+ in extracellular space leads to changes in neuronal excitability. Müller cells may also control neuronal activity more directly. When sufficiently depolarized, glutamate uptake by salamander Müller cells is reversed and glutamate is released into extracellular space [
89]. Additionally, glycogen stores in the retina are restricted to Müller cells. Furthermore, Müller cells also regulate blood flow in retinal vessels in response to the changes in neuronal activity.
Astrocytes
Astrocytes are the primary glia in the brain, constituting approximately one third of the brain mass [
90]. Astrocytes in the retina show a stellate morphology, with somata located in the ganglion cell layer and nerve fiber layer (NFL). In the monkey retina, GFAP-positive astrocytes are found ubiquitously in the NFL. Astrocytes are absent in avascular foveal region. The concurrence of retinal astrocytes and intraretinal vascularization may be a common feature for many mammalian species [
91]. Despite the fact that astrocytes are far less pervasive in the retina than in the brain, these cells play an important role in the development and maintenance of retinal neurons and blood vessels. They provide energy substrates to neurons and regulate the production of trophic factors and antioxidants in retinal microenvironment [
92].
Astrocytes show opposite reactions as compared with Müller cells in response to hyperglycemia. The density of Müller cells is increased, whereas the number of astrocytes is decreased in diabetic retinas. In 4-week diabetic rat retina, astrocyte density is significantly reduced in the peripapillary region and in the far periphery [
70]. Astrocytic profiles, notably the processes investing axonal bundles, are scanty in rat diabetic tissue, and the starlike cell bodies are irregularly distributed [
70]. In addition, recent study demonstrates that exosomes from retinal astrocytes contain multiple anti-angiogenic components that inhibit laser-induced choroidal neovascularization in model mice [
93].
Astrocytes are the major cell population in the optic nerve head and are responsible for the remodeling of the lamina cribrosa structure [
94]. Astrocytes are important in stress over-activation of inflammatory responses in glaucoma that leads to local axonal damage within the optic nerve head [
95]. Astrocytes have the potential to secrete a wide array of mediators [
96]. COX-2 can be constitutively produced by astrocytes and is generally considered as an “immediate early response gene” following damage to the CNS [
97]. As an acute phase gene, COX-2 is readily induced in a variety of cells by inflammatory and mitogenic stimuli, including cytokines and growth factors [
98]. Overexpression of transforming growth factor-alpha (TGF-α) and epidermal growth factor receptor (EGFR) occurs in active astrocytes [
99]. EGFR-dependent induction of COX-2 occurs early in astrocytes following optic nerve injury [
100]. COX-2 and COX-2-induced PGE2 participate in DR and regulate the expression of VEGF [
101]. In human diabetic retina, COX-2 is induced in astrocytes and contributes markedly to preretinal neovascularization in ischemic retinopathies. This effect appears to be PGE2-mediated mostly via prostaglandin E receptor 3 (EP3) implicating a new interaction through thrombospondin-1 (TSP-1) and CD36 [
102].
IL-1β induces its own synthesis in the retinal vascular endothelial cells, Müller cells, and astrocytes. The combination of high glucose stimulation and the upregulation of IL-1β in the diabetic retina is responsible for sustained IL-1β overexpression in astrocytes [
103].