Discuss this statement in relation to normal homeostasis and the pathophysiological roles of these cells

Microglia is dynamic cells that act as pathological sensors within the adult nervous system.Discuss this statement in relation to normal homeostasis and the pathophysiological roles of these cells?

In my essay I will be focussing on how resting microglial cells function as sensors and scan the tissue in the normal brain and how this microglia change in morphology and transform into activated microglia in response to injury. I will also focus on how these activated microglial cells respond to injury and the immune responses involved in clearing cellular debris, aid tissue repair by producing growth factors and engulf invading microorganisms. In the central nervous system, microglia were found to be the third glial cell type by Del Rio Hortega amongst all of the possible glial cell types (Tanaka et al, 2003). In 1932, Rio-Hortega described these microglia in silver carbonate stained brain preparation as consisting of long and branched processes. They are mesodermal, monocytic or ectodermal in origin (Lee et al, 2002). The brain, the spinal cord parenchyma and the CNS proper consists of microglias which are a group of cells related to monocytes and dendritic cells and are intrinsic immune cells. These groups of cells provide a first line of defence against neural infection. The microglia makes up 5-20% of all glial cells in the normal adult brain and is found uniformly in a scattered array.Microglial cells play an important role as immunocompetent and phagocytic cells in the CNS (Perry and Gordon, 1988; McGeer and McGeer, 1995).In the early stages of brain development, microglia demonstrate a macrophage-like morphology with a big cell body and short processes. These microglia are referred to “amoeboid microglia” and its essential function is to phagocytose dying cells, including neurons that have already undergone apoptosis during late embryonic to early postnatal stages (Nakajima et al, 2001).These cells also clear debris and dead cells for example degenerating axons and myelin and assist in the early recruitment of T cells following a minor injury(Raivich,2005).As the brain develops, the numbers of amoeboid microglia decline and become replaced by large numbers of ramified microglia.These ramified microglia consist of tiny cell bodies with long branched processes consisting of a tertiary and quaternary branch structure. Each branch can cover an area of 30-50µm wide in the brain and this never touches the other branches of surrounding sister cells. This complicated branching structure impairs the ability of microglia to survey the surrounding tissue. Ramified microglia are described as inactive and is associated with the resting phenotype and are usually called the ‘resting’ microglia.Although ‘resting’ microglia are not dormant, studies involving in vivo 2-photon microscopy in transgenic mice expressing the green fluorescent protein in the Cx3cr1 locus(which encodes CX3CR1,the chemokine receptor for CX3CL1) demonstrated enhanced motile microglial processes. Microglial processes are being continuously removed as well as mobile filopodium like protrusions and also microglial processes are being continuously replaced with de novo formation as shown by time lapse-imaging (Hanish et al, 2007).

This dynamic and careful reorganisation permits the ‘resting’ microglia to survey the brain parenchyma every few hours without causing disruptions in the fine wired neuronal structures. Since approximately 20% of the brain is extracellular space, the neural parenchyma is completely surveyed every few hours. When retraction of thick processes occurs in one cell, border zones between adjacent microglial cells were liable to change and cause alterations in favour of neighbouring microglial cells (Nimmerjahn et al, 2005).

When the processes of neighbouring cells meet each other, the endings of these projections repel each other.

The resting behaviour of the microglia has a ‘housekeeping’ function and these microglial cells regulate the microenvironment by removing the parenchyma of low diffusible metabolic substances and waste tissue products (Nimmerjahn et al, 2005).

Hence the highly ramified resting microglia provides the brain with a very dynamic and proficient surveillance system. These microglial cells take turns to survey the brain parenchyma randomly without any contact and when microlesions are introduced in a site of injury, microglia are targeted towards this injury site. The targeted movement of the microglia relies on purinoreceptor stimulation and is assisted mainly by astrocytes (Hanish et al, 2007).

When the brain is in a healthy condition, the density of the microglial cells and its ramified morphology is maintained. Experiments have shown that ‘resting’ microglia which is mesodermal in origin is highly motile in the mature and uninjured adult brain (Nimmerjahn et al, 2005).This experiment used the CX3CR1::EGFP mice and this study used the brain microglia which expresses an enhanced green fluorescent protein (EGFP) which is under control of the fractalkine receptor promoter (CX3CR1). In depth, time lapse series of fluorescence images were obtained using trans-cranial 2-photon laser scanning microscopy which reduces photodamage to living tissue. This is vital in the sense that microglia are the main sensors of pathology in the brain and damage to the brain will induce loss of the resting phenotype and cause secondary activation. The results demonstrated that the cell body of the resting microglia is stationary but regardless of this there seems to be continuous and enhanced motile extension and retraction of processes (Raivich, 2005). These processes derived from each branch order, ranging from primary to quaternary branches and above, with speeds of 1.2µm/min to 1.5µm/min and slightly rapid movement for tiny and transient processes of the branches. These microglial projections survey the extracellular environment randomly and at a high turnover rate of approximately 15% per hour (Raivich, 2005). Microglial cells consist of small rod shaped somata and from the somata, thin and ramified processes extend symmetrically. Time-lapse imaging experiments demonstrate that somata of microglial cells remain stationary with little evidence of migration (Nimmerjahn et al, 2005).

Microglial processes consist of enhanced mobile filopodia like protrusions of different shapes, which form bulbous endings. There is continuous appearance of protrusions at various places along the main processes and at their terminal endings .The activity of these protrusions stops for a few minutes when there is further extension or retraction occurring.

There is evidence in the healthy brain microglia can interact with other cortical elements because the processes and protrusions of these microglial cells can directly contact astrocytes, neuronal cell bodies and blood vessels.

The severity of the injury determines the number of responding microglial cells Experiments show that ...

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The severity of the injury determines the number of responding microglial cells Experiments show that microglial cells close to the microlesion are activated and microglial cells further from the microlesion did not activate.

The continual structural alterations of resting microglial cells serve an immune surveillance function.

In the case of brain injury, ramified microglia change into “activated microglia” or “reactive microglia” in a few hours which demonstrates motile branches, migration of somata, retracted processes and magnified cell bodies. At the affected sites, these microglia proliferate to increase in number (Nakajima et al, 2001).

In most pathological states, the microglia is functionally stimulated and during infection, inflammation, trauma, ischemia, the microglia play a role as scavenger cells. There are two distinct forms of activated microglia. The first one is the hypertrophic, non-phagocytic microglia with a raised expression of various marker molecules for example MHC antigens and the latter is the phagocytic microglia (Gehmann et al, 1996).

During the prenatal and early postnatal stages of brain development microglial cells phagocytise cellular debris. As the CNS develops the overproduced neurons and glial cells are removed by axonal degeneration and neuronal cell death, which occurs as a genetically programmed event (Kim et al, 2005).

The monocyte-macrophage lineage cells invaded into the CNS due to the stimulus provided by the dead and dying neurons and their degenerating processes.

The stimulation of microglia leads to transformation of cells to phagocytes which in turn releases oxygen radicals, nitric oxide, proteases and proinflammatory cytokines which are potential cytotoxic substances (Banati et al, 1993).Examples of proinflammatory cytokines are IL-1, IFN-γ, TGF-β, IL-1β, IL-6, IL-12 and TNF-α which can induce cytotoxic effects in the CNS (Lee et al, 2002).These cytokines signal to T lymphocytes (Kettenmann, 2006).

Figure 1: This figure shows the interactions between microglia, T cells, astrocytes and oligodendrocytes (Benveniste, 1997).

Microglial activation which occurs in a graded fashion is commonly followed by neuronal and glial cell destruction in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and stroke and in several of these neurological disorders there is presence of activated microglia in pathological lesions. The bone marrow derived monocytes are the origin of resident microglia and at early embryonic stages, monocytes pass the blood brain barrier and accumulate in the brain parenchyma, where they either go through or not to the stage of amoeboid microglia and mature into ramified microglia. When the monocytes differentiate into resident microglia during embryonic development they express many of the cell surface antigens found commonly in macrophages. These shared antigens include β2-integrins (CD11a, CD11b, CD11c, CD18), LCA (CD45), CD64, CD68, CR3, B7-2 and MHC class I and II antigens. So these microglia display surface expression of immunoglobulin and receptors for complement and apoptotic cell surface markers. ICAM-1 is the ligand for CD11a and complement proteins are the ligands for CD11b and CD11c.The receptors for Fc chain of immunoglobulin are increasingly expressed in microglia. In reactive microglia, MHC class II antigens are particularly expressed (Woodroofe et al, 1991). Some microglia display both MHC class I and class II antigens and interact with T helper and T cytotoxic classes of lymphocytes.

Figure 2: Figure 3b shows that parenchymal microglia or macrophages in their resting state do not express CD14 and following traumatic brain injury, the numbers of CD14 increased in brain parenchyma. In figure 3c following injury, the early expression of CD14 forms an important part of the acute inflammatory CNS response (Beschorner et al, 2002).

In human microglia, B7-1 and B7-2 are expressed in a cytokine-regulated manner. B7-1 and B7-2 are part of the members of the B7 family of cell surface glycoproteins. Activated microglia-derived macrophages express the antigens and at sites of inflammation cell mediated immune responses occur. Many cytokines such as IL-2, IL-4, IFN-γ and GMCSF increase the expression of B7 on antigen presenting cells and is downregulated by IL-10 (Azuma et al, 1993).

Figure 3: This figure shows the expression of various cytokines and chemokine receptors in microglia (Hanisch, 2002).

Microglial cells are characterized by monocyte/macrophage antigens. As well as the monocyte-origin theory, there is also a neuroectodermal-origin theory which suggests that microglia are born in neuroectodermal derived glioblasts.Microglia is the main defence line of the brain because when the central nervous system is infected, ramified microglia change into phagocytes and engulf the infectious microbes. Dying cells present in the central nervous system are phagocytosed by ramified microglia-derived macrophages. When the blood brain barrier is disturbed, microglia work as antigen presenting cells in the immune system.

Furthermore, microglia releases many biologically active substances which stimulate inflammation and cell death and therefore maintain regenerative processes. Hence microglia acts as a “sensor” for pathological events in the brain.

During brain injury, resident ramified microglia convert into “stimulated microglia”.These activated microglia enhance numerous cellular antigens for example complement receptor3,MHC class I and II molecules and activity of the enzyme 5’-nucleotidase.These are all associated with the functional state of the microglia. Microglia work as antigen presenting cells by way of interacting with invading immune cells via the MHC type II protein and therefore starts an immune response. (Kettenmann et al, 2006). There is little knowledge about the factors that cause microglial activation or the factors that guide microglial cells to the injury site.

In acute injury such as after transection of facial nerve and administration of toxic ricin into facial nerve,microglia present in the facial nucleus transform morphologically and after 2-3 days proliferate(Nakajima et al,2001).Over a period of 1-2 weeks, the population of stimulated microglia increase and eventually congregate around the injured motoneuron cell bodies. After the sustained activation, microglia slowly regenerate and return back to the original ramified microglia.

Figure 4: This diagram shows that in the facial nucleus there is presence of ramified microglial cell (Bouscein et al, 2000).

In the axotomized facial nucleus, activated microglia is present in two states. These two activated states are split into being phagocytic or non-phagocytic.These activated microglia originate from resident ramified microglia because transection of the facial nerve at the stylamastoid foramen does not induce blood brain barrier injury of the axotomized facial nucleus and there is no entry of peripheral monocyte lineage cells into the brain parenchyma.

In chronic inflammatory diseases such as Alzheimer’s disease and multiple sclerosis, microglia in its active form is seen in the affected site. These resident microglia as well as monocytes and macrophages are stimulated in these chronic diseases and function as immune and/or inflammatory cells.

Resting microglia demonstrate a down regulated immunophenotype which is specifically adapted to the microenvironment of the central nervous system. These microglia can respond to numerous signalling molecules quickly as they are extremely vigilant and can sense alterations in their extracellular environment.

In response to injury, microglia are activated in their early stages. The stimulation of microglia comes before any reactions of the cell type in the brain. Microglia respond to modifications in the brain’s structural integrity and minute changes in the brain’s microenvironment such as an imbalance in ion homeostasis (Kreutzberg et al, 1996).

Microglia consists of receptors for central nervous system signalling molecules for example ATP,cGRP,Ach and noradrenaline and can be able to react both with alterations in their extracellular ionic environment and by stimulation of transcriptional processes.Microglia can respond specifically to molecules involved in neurotransmission and this permits them in their ‘resting’ state to survey the physiological integrity of their environment on a continual basis and to react quickly if there were any pathological events induced.

Microglia contains surface receptors such as purinoceptors, receptors for complement fragments, immunoglobulins, adhesion molecules and inflammatory stimuli, which can sense alterations in their microenvironment. P2Y12 receptors, a group of purinergic receptors are crucial in activating resting microglia to injury and guiding them towards the site of action (Kettenmann et al, 2006).

Figure 5: This diagram shows that after administering 10 µm and 1Mm ATP to purified microglial cells, oxidized ATP (a P2X7 antagonist) decreases the long lasting increase in [Ca2+] after 1 Mm of ATP administration. This shows that microglia display P2X7 receptors which mediate fura-2-efflux once stimulated by mM amounts or continual administrations of µm ATP amounts (Verderio et al, 2001).

Microglial cells are targeted towards sites of injury because there are many potential factors which act as attractants. ATP is a factor, which is released from damaged or injured cells (Kettenmann et al, 2006). ATP mediates rapid microglial response to local brain injury in vivo.

Figure 6: This diagram shows within 30 minutes of laser induced injury, microglial processes of neighbouring cells combine together and form a sphere around it which is shown in figure d. Also during this time, the cells consist of retracted processes which lay in opposite directions previously to the injury site which is shown in figures d and e. Within the first one to three hours, the cellular content of each of the immediate neighbours was guided towards the damaged area which is shown in figures e and f.During mechanical injury, there were similar microglial responses as shown with laser induced injury. This was seen in figures g and h.Immediately upon injury, microglial processes assumed the bulbous terminology at their termini and rapidly migrated into the damaged tissue (Davalos et al, 2005).

Microglia display a variety of ATP-sensitive purinergic receptors of both P2X (cation channel) and P2Y (G-protein coupled receptor) families.

The activation of purinergic receptors can initiate IL-1β and IL-10 release or reduce the release of the proinflammatory cytokines TNF-α or IL-6 by stimulated microglia. In cultured microglial cells, ATP is a chemoattractant (Honda et al, 2001). In culture, P2Y receptors are vital in the rapid morphological transformation of microglia initiated by ATP and also for a ruffling motion of the flattened projections.

In vitro, the P2Y12 receptor is vital for microglial mobility. There is also genetic evidence that the P2Y12 is the predominant site at which ATP acts to initiate microglial activation in response to local CNS injury in vivo.

These experiments showed that after injury microglial cells changed over a 24-hour period from ramified form to the amoeboid form. The expression of P2Y12 receptor decreases with microglial stimulation. Microglia becomes more ramified as the expression of P2Y12 receptor increase (Kettenmann et al, 2006).

In vivo studies showed that after lipopolysaccharide injection (trigger for inflammatory response) into the striatum for four days, P2Y12 expression is absent in the amoeboid, activated microglia. The P2Y12 receptor is only expressed in the resting microglia and the receptor is down regulated during their stimulation.

Tissue damage through release of intracellular high energy purine nucleotides ATP, UDP, UTP induces microglial activation. Tissue damage by way of laser microlesion produced a quick response in microglial branches which were targeted towards the lesion site at 1.0-1.5 µm/min and enclosed the lesion site with dense sphere of arborizations.Local injections of ATP,ADP,UTP mimicks this response and inhibitors of P2Y receptors blocks this response. This response was blocked by apyrase which is an enzyme that destroys ATP,ADP and UTP.The injection of apyrase caused a considerable decrease in the motility of the resting microglia demonstrating ATP involvement in the normal brain(Raivich,2005).The effect of ATP analogues such as AMPPNP/ATPγS can be completely blocked using apyrase or connexin-hemichannel inhibitors(Davalos et al,2005).This points to a requirement for augmentation of ATP,UDP,UTP signals which are released through the connexin-hemichannels as a downstream regulator of motility and chemotaxis.

Figure 7: This diagram shows that local administration of ATP induces a quick chemotactic response of microglial processes. These images show fast extension and retraction of microglial processes over a few seconds. There is no movement of cell bodies (Farber et al, 2006).

Because in astrocytes, connexin-hemichannels are increasingly expressed and not in ‘resting’microglia, these studies show that the significant role of other glial types in surveying the immune system. So in this case, surrounding astrocytes instruct microglial branches which guides them to targeted sites needed for surveying.

Figure 8: This diagram demonstrates that spontaneous calcium increases in the astrocyte (green trace) initiates a long-lasting [Ca2+] I rise in microglia (blue trace).The results demonstrate that the rise in calcium and membrane permeability of microglial cells is regulated by the paracrine stimulation of microglial P2X7 receptors induced by raised or low ATP concentrations continually released from astrocytes (Verderio et al, 2001).

Experiments show that macrophages, which enter the CNS after injury, did not display P2Y12 receptors when they were in the resting phenotype.

P2Y12 protein was restricted to the microglial cells surface and also the ramified processes. Studies were conducted to observe the change in microglial morphology in response to injury and these type of studies involved using imaged microglia in brain slices.

Highly motile protrusions continuously inspect the environment and pick up the tissue debris. The movement and growth of microglia is driven by filamentous actin, which are held in large amounts in the microglia.

Microglia has a neuroprotective function because microlesion experiments show that injured sites are shielded. The early appearance of inclusions shaped as spheres demonstrate that microglia have an immediate phagocytic function which engulf components and clear out damaged tissue or leaked blood components. Therefore this proves that microglia provide the first line of defence.

Studies show that in response to ATP the microglia in rat brain and mouse demonstrates activation of a cation conductance mediated by P2X and delayed stimulation of an outward directed K+ current, which is mediated by P2X receptors (Farber et al, 2006). The stimulation of both P2X and P2Y receptors leads to a rise in intracellular calcium concentration. So in conclusion, microglia in their ‘resting state’ characterised by their ramified morphology are increasingly active and they continuously survey their microenvironment with their mobile processes and protrusions. In a response to any pathological events, such as stroke or trauma, the resting microglial cells alter into an activated amoeboid form, where they can proliferate, migrate chemotactically to sites of injury and secrete many cytokines and chemokines and so this demonstrates that microglial cells are able to sense factors involved in the pathological point of view. This microglia changes their behaviour from patrolling to shielding of the injured site. So therefore microglia is busy and cautious housekeepers in the brain.

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Discuss this statement in relation to normal homeostasis and the pathophysiological roles of these cells

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