Ellis-Behnke, Rutledge G.*,†; Jonas, Rahul A.*; Jonas, Jost B.*
*Department of Ophthalmology, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany
†Department of Brain & Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA
Disclosure: The authors declare no conflict of interest.
Microglial cells function as first responders to signal inflammation, react to injuries by creating a wall to block invaders, and clear debris from the site. To better understand the modulation of microglia in inflammation and injury of eye and brain, we developed a morphological and orienting classification system of each stage of microglia, calling it the ‘Spider Effect’. We transected the olfactory bulb of rats and examined the activation of the microglial system histologically. Six stages of bidirectional microglial activation (A) and deactivation (R) were observed. Our findings support a morphologically defined stepwise activation and deactivation of microglia cells. This relates to inflammation in the eye due to noxious stimuli, injury, or increase in pressure. Future studies may address the reported modulation of the microglial system in retina and optic nerve head in acute and chronic glaucoma.
There are many types of glia in the body. In the peripheral nervous system there are Schwann cells, satellite glia and enteric glia. The glia in the central nervous system (CNS) are differentiated into astroglia, ependymal cells, tanyglia, radial glia, oligodendrocytes, Müller cells and microglia.1,2 Microglia originate from the mesoderm. The cells were originally discovered by Hortega while working with Ramon y Cajal and were referred to as mesoglia. These are the only cells in the CNS that do not arise from the ectoderm or the vasculature.
There is much controversy as to how microglia maintain the CNS tissue and how they are activated and transform into macrophages during times of injury. The microglia has been considered to be the immune system of the CNS, since the cells are located equidistant from each other. With their processes extended, they continually sense the surrounding environment. Microglia represent 20% of all glia in the brain, are involved in synaptogenesis, induce neuronal apoptosis, and clear debris. In addition to inflammation caused by noxious stimuli, microglia react to signals from damaged neurons. Once they receive the signal, there is also an autocrine signal function which activates itself to fully activate macrophages. The microglia also receive signals from astrocytes; e.g., pro-inflammatory cytokines such as TNF alpha, IL-6 and nitric oxide. Microglia also have a phagocytic function and have been implicated in wound healing, angiogenesis, and extracellular matrix formation.
The microglia in the retina reside in the layers between the nuclear layers. In addition, they are found in the choroidal capillaries, but are not in direct contact with blood vessels. In the brain as well as in the eye, the blood-brain barrier consists of endothelial tissue with pericytes surrounded by astrocytic end feet. There also appear to be perivascular macrophages between the endothelial cells and the astrocytic end feet, but the microglia do not come in direct contact with the blood vessels. Microglia, like monocytes, adult dendritic cells, perivascular macrophages, choroidal plexus macrophages and meningeal macrophages, arise early in development from the extra-embryonic yolk sac myeloid cells.3 In contrast to macrophages, dendritic cells and monocytes, which enter the circulatory system, microglia become resident in the CNS and the eye. Origin from a relatively primitive progenitor cell may explain how microglia are able to transform back to a more spheroidal form — the macrophage. Microglia have also been implicated in stabilizing and removing synapses. Microglia have been referred to as the ‘gardeners of the brain’ implicating them in being able to engulf synaptic material and thus remove splines, as well as be one of the primary mediators of plasticity in early development.4 This may have implications for glaucoma, since the number of synapses and connections of the retinal ganglion cells to other retinal cells begin to decrease before the cells die in glaucoma.
There are many markers to identify microglia from the first silver stain that Hortega used, to Coronin-1a, IBA-1, ED-1, HLA-DR (MHC II receptor), CD-68, CD-45, F4/80 (mouse/rat), CD11b (Mac-1), tomato lectin, Ox-42 (mouse/rat), MHC II (lower levels than infiltrative macrophages) and Isolectin B4, to name a few. Each of these has its benefits and drawbacks, and they do not necessarily label only microglia. For example, lectin also labels the vasculature, so it is not a marker to identify microglia only. We have found that Iba-1 will label most of the activated microglia, while ED-1 is a label of all activated macrophages. Iba-1 and ED-1, when used in combination, can differentiate infiltrating macrophages from microglia which are fully activated.
The field of glia and microglia is vast, but the goal of this paper is to focus on several examples of what microglia look like when they are resting or activated in the brain and in the retina and to provide a basic understanding of the activation and transformation of microglia as they transform to clean up damage and react to noxious stimuli.
THE SPIDER EFFECT
We have found that during activation, the microglia transform from highly ramified cells to spherical cells that resemble macrophages. We have further delineated and morphologically characterized these changes into 12 steps. The first six steps include the full spectrum from the resting cell stage 1A to fully activated stage 6A (‘A’ for ‘Advancing’), which resembles a fully activated macrophage that has migrated to the site of injury. After the cell has cleared some of the debris and has gathered multiple nuclei, stage 6R (‘R’ for ‘Returning’) has developed. We have followed the microglia morphologically back through each of the six returning stages. It appears that the microglia not only return to their original shape 1R (or 1A) but that they also return to their original location to take up their sentinel duty. We have termed this the ‘spider effect’, comparing the microglial cell in stage 1A with a spider sitting in the middle of the web. When the spider feels vibration, it proceeds to the area of disturbance, detects and consumes the intruder at that location, and returns to the center of the web, resuming its sentinel duty.5 It appears that the microglia are able to transform to a more differentiated phenotype than macrophages, and the microglial cells are also able to revert back to their original location and formation (Fig. 1). It appears that the microglial cells in their advancing and returning stages of activation do not cause secondary inflammation.
INJURY VERSUS INFLAMMATION
The microglial response to physical injury is a transformation from a highly ramified cell shape in stage 1A to the cell form of a macrophage in stages 6A and 6R and then back to the original location and shape in stage 1R. Some studies have suggested that the nuclei previously consumed by the microglia in stages 6A and 6R are handed over to invading macrophages from the bloodstream during the clearing of the debris.6 As in injury, the modulation of the microglial cells in inflammatory conditions such as in Alzheimer’s disease or inflammation induced by bacterial lipopolysaccharides can cause a similar transformation and activation of the microglia (Fig. 2). A specific finding in Alzheimer’s disease is that when the microglial cells encounter the Tau protein, they attempt to consume it but fail and appear to become fragmented. When, however, the microglia cells encounter the A-beta protein, the microglial cells do not appear to react and remain in the 1A stage, although the A-beta protein has been considered to be a toxin.7 When lipopolysacharides were experimentally injected into the eye, the microgial cells became fully activated (stage 6A) and remained so until they cleared the inflammation. Adding lyceum barbarum, lipopolysaccharides inactivated the microglia cells and the loss in retinal ganglion cells was significantly decreased. That effect could be removed by adding MIF (macrophage migration inhibitory factor).8 In our experiments, we were able to reduce the amount of damage and facilitate healing in the CNS by reducing microglial activation. The modulation of the microglia is generally a delicate balancing act depending on the type of stimuli; the microglial cells become pro-inflammatory M1 macrophages. By reducing the activation level, one may be able to modulate not only tissue damage, but also preserve the structure of the retina in the brain. The reaction of the microglial cells to a physical trauma in the olfactory bulb5 was similar to the response of the microglia to an increase in intraocular pressure.
In conclusion, although Hortega discovered the microglia nearly 100 years ago, the microglial system was almost neglected and forgotten until in the last two decades. An intensive research has started to focus on microglia. The studies have shown that the microglial system is far more robust than first thought; that it is essential during the early development of the brain, eye and spinal cord; that it plays an essential role in neurodegenerative disorders; and that its role in retinal and optic nerve diseases such as diabetic retinopathy and glaucomatous optic neuropathy still has to be explored.
1. Hanisch UK, van Rossum D .Larry RS .Microglia Properties.Encyclopedia of Neuroscience. 2009; .Oxford:Academic Press; 853–859.
2. Kettenmann H, Hanisch UK, Noda M, et al .Physiology of microglia.Physiol Rev. 2011; 91:461–553.
3. Ransohoff RM, Cardona AE .The myeloid cells of the central nervous system parenchyma.Nature. 2010; 468:253–262.
4. Paolicelli RC, Bolasco G, Pagani F, et al .Synaptic pruning by microglia is necessary for normal brain development.Science. 2011; 333:1456–1458.
5. Jonas RA, Yuan T-F, Liang Y-X, et al .The spider effect: morphological and orienting classification of microglia in response to stimuli in vivo.PLoS ONE. 2012; 7:e30763
6. Petersen MA, Dailey ME .Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices.Glia. 2004; 46:195–206.
7. Streit WJ, Braak H, Xue QS, et al .Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer’s disease.Acta Neuropathol. 2009; 118:475–485.
8. Chan HC, Chang RC, Koon-Ching Ip A, et al .Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.Exp Neurol. 2007; 203:269–273.