microglia account for approximately 10% of the adult brain cell population and represent the first and main form of immune defense in the central nervous system ( cns ; lawson et al .
microglia become activated and they can be identified and distinguished from their resting phenotype based on a combination of morphological and immunophenotypic changes ( dheen et al . , 2007 ; ransohoff and perry , 2009 ) .
microglia initiate immune responses by enhancing the expression of toll - like receptors ( tlr ) and a wide range of pro - inflammatory mediators such as tumor necrosis factor - alpha ( tnf ) , interleukin ( il)-1 and il-6 for the removal of the cns threat ( suzumura et al . , 1996 ;
microglia may also fulfill a neuroprotective role via the release of neurotrophic factors and promotion of neurogenesis for the restoration of normal physiology ( stadelmann et al . , 2002 ) .
hence , the acute inflammatory response is generally beneficial , as it tends to minimize injury and promotes tissue repair .
however , chronic neuroinflammation is closely related to various neurodegenerative disorders such as parkinson s disease ( pd ) , huntington s disease ( hd ) , dementias , and multiple sclerosis ( ms ) , although the consequences of sustained microglial activation in these diseases is unclear .
activated microglia upregulate expression of the 18-kda translocator protein ( tspo ; chen and guilarte , 2008 ; cosenza - nashat et al .
tspo are found in abundance throughout the body in peripheral organs ( i.e. , liver and adrenals ) , and hematogenous cells , but are present at very low levels in the normal healthy cns ( banati , 2002 ) .
functionally , tspo has several biological functions including the control of cholesterol transport and neurosteroid synthesis ( papadopoulos et al . , 2006 ) , and
may also be involved in the release of pro - inflammatory cytokines during inflammation ( choi et al . , 2002 ; wilms et al . ,
enhanced tspo expression can be detected in vivo by using positron emission tomography ( pet ) imaging with the selective tspo radioligand c - pk11195 ( benavides et al .
, 1993 ; banati et al . , 1999 ) , with evidence that increases in c - pk11195 binding potential ( bpnd ) correspond to activation of microglia ( stephenson et al . , 1995 ; conway et al .
although tspo is also expressed by reactive astrocytes , a c - pk11195 pet study of patients with hippocampal sclerosis , a condition histopathologically characterized by marked astrogliosis , did not yield results that were significantly different to healthy normal controls ( banati et al .
this is consistent with the view that reactive astrocytes , in vivo , do not significantly contribute to the c - pk11195 signal .
therefore , in the absence of invading blood borne cells or severe focal leakage of blood - brain barrier , the increased pk11195 binding is likely to indicate the transition of microglia from a resting to an activated state , and is due to an increase in the number , rather than the affinity , of tspo ( banati et al . , 2000 ) .
hence , the measurement of tspo uptake using pet provides an in vivo tool to monitor progression and severity of neuroinflammation and is a useful indicator of active cns disease .
this article aims to review the use of pet imaging to promote the understanding of activated microglia in neurodegenerative disease .
parkinson s disease is the second most common neurodegenerative disorder of the elderly and is associated with the motor symptoms of tremor , bradykinesia , and rigidity .
it is characterized by the extended loss of dopaminergic neurons in the substantia nigra pars compacta , resulting in a deficiency of dopamine in the striatum ( braak et al . , 2006 ) , and the presence of alpha - synuclein ( -synuclein)-containing lewy bodies .
pd is the most common of a group of parkinsonian movement disorders that also includes multiple system atrophy ( msa ) , corticobasal degeneration ( cbd ) , and progressive supranuclear palsy ( psp ) .
the presence of activated microglia close to dopaminergic neurons in post - mortem pd patient brains ( mcgeer et al . , 1988a ; mogi et al . , 1994 ; langston et al . , 1999 ; imamura et al . ,
2003 ) , and pd animal models ( czlonkowska et al . , 1996 ; kim et al . , 2009 ) suggests a close relationship between neurodegeneration and neuroinflammation in pd .
numerous investigations have proposed a deleterious role of microglial activation in pd based on the vulnerability of dopaminergic neurons to various microglia - derived pro - inflammatory cytokines ( ferrari et al . , 2006 ; stone et al . , 2009 ; de lella ezcurra et al . , 2010 ) , while -synuclein can directly induce activation of microglia ( zhang et al . , 2005 ) .
however , it seems that the plasticity of microglia must be considered with regards to their contribution in pd , and their role ; whether beneficial or detrimental , it may depend on the stimuli present and the stage of disease ( li et al .
, 2007 ; michelucci et al . , 2009 ; sanchez - guajardo et al . ,
further clues regarding the role of activated microglia has also come from in vivo pet imaging studies ( table 1 ) .
significant microglial activation , as reflected by an increase in c - pk11195 bpnd was reported in the midbrain and putamen of pd patients when compared to controls , and was found to correlate positively with the motor severity of parkinsonism ( ouchi et al . , 2005 ; bartels et al . , 2010
these findings suggest that activated microglia has a pathogenic importance in the disease and indicate that the early introduction of a neuroprotective drug to suppress microglial activation could be favorable in pd . additionally , pd patients exhibited significantly increased c - pk11195 bpnd in the basal ganglia , pons , and frontal and temporal cortical regions ( gerhard et al . , 2006a ) .
in this study , the increased microglial activation remained unchanged for 2  years , while the patients deteriorated clinically during this period .
hence , it is likely that microglia are activated early in pd , where they remain activated for longer periods and possibly drive progression of the disease ( gerhard et al . , 2006a ) .
bpnd , binding potential ; cbd , corticobasal degeneration ; msa , multiple system atrophy ; nc , normal control ; pd , parkinson s disease ; psp , progressive supranuclear palsy .
multiple system atrophy is a sporadic neurodegenerative disorder involving a progressive akinetic - rigid syndrome , autonomic failure , and cerebellar dysfunction .
it is associated by the appearance of abnormal glial cytoplasmic inclusions ( gci ) containing ( -synuclein aggregates and neuronal loss within the nigrostriatal and olivopontocerebellar regions ( lantos and papp , 1994 ) .
the presence of activated microglia is also a prominent feature of msa ( schwarz et al . , 1998 ) .
in an in vivo pet study of msa patients , significant c - pk11195 bpnd was observed in the putamen , pallidum , pons , substantia nigra pars compacta , and dorsolateral prefrontal cortex , reflecting the known distribution of neuropathological changes in msa ( gerhard et al . , 2003 ) . although the role of microglia in msa is inconclusive , microglial activation localization correlated significantly with the locations of gcis in specific neuroanatomical systems affected in msa ( ishizawa et al . , 2004 ) .
a correlation between extent of microglial activation and dopaminergic neurodegeneration has also been reported ( stefanova et al . , 2007 ) .
progressive supranuclear palsy is an adult - onset progressive neurodegenerative disease of unknown cause , characterized by pd - like symptoms such as postural instability and bradykinesia .
the pathological hallmark of the disease is neurofibrillary tangles consisting of hyperphosphorylated tau , accompanied by neuronal loss in the thalamus , basal ganglia , and specific brainstem regions ( hauw et al .
several early studies including immunohistochemical investigations have confirmed the possible involvement of activated microglia in psp ( kida et al .
, 1992 ; komori et al . , 1998 ; ishizawa et al . , 2000 ; ishizawa and dickson , 2001 ) .
c - pk11195 pet have also reported significant levels of activated microglia in brain regions known to be affected by the disease process such as the midbrain , cerebellum , pons , frontal lobe , and basal ganglia ( gerhard et al . , 2006b ) . although these results were unable to support a direct causal contribution to neurodegeneration in psp , they are at least suggestive of a role of microglia in the disease .
corticobasal degeneration is a neurodegenerative disorder that affects both cortical and basal ganglial regions , with considerable clinical heterogeneity between patients .
typically , cbd features an asymmetric hypokinetic - rigid syndrome , coupled with alien limb phenomenon and cortical sensory impairment that is unresponsive to dopaminergic therapy ( rebeiz et al . , 1968 ; gibb et al . , 1989
information on the association of activated microglia in cbd is limited , and mainly coming from immunohistochemical - based assessments ( armstrong et al . , 2000 ; ishizawa and dickson , 2001 ) .
however , more recent in vivo pet investigations have attempted to quantify microglial activation in cbd patients .
increased c - pk11195 bpnd was observed in regions such as the caudate nucleus , putamen , substantia nigra pars compacta , pons , and pre- and post central gyrus ( gerhard et al .
2004 ) that correspond to the expected neuropathological changes seen in cbd ( ishizawa and dickson , 2001 ; dickson et al . , 2002 ) .
huntington s disease is an autosomal , dominant inherited progressive neurodegenerative disorder associated with motor , cognitive , and psychiatric symptoms .
it is caused by an abnormal polyglutamine - repeat expansion on the it15 gene that codes huntingtin , and involves the progressive loss of medium spiny dopaminergic receptor - bearing striatal gaba - ergic neurons ( vonsattel and difiglia , 1998 ) .
although the role of chronic neuroinflammation in the hd pathogenesis is not fully understood , post - mortem assessments have reported high levels of activated microglia close to degenerating neurons ( mcgeer et al .
upregulated inflammatory cytokines have also been detected in the striatum and plasma , indicative of an inflammatory component in hd ( dalrymple et al . , 2007 ;
imaging studies using c - pk11195 pet have found increased microglial activation in both premanifest hd gene carriers and manifest hd patients when compared to healthy controls ( table 2 ; pavese et al .
, 2006 ; tai et al . , 2007 ; politis et al . , 2008 , 2011 ) . in premanifest
hd patients , significant increases in c - pk11195 bpnd in the striatum and hypothalamus was reported , which correlated inversely with neuronal dysfunction as measured by c - raclopride ; a marker of dopaminergic d2/d3 receptor availability ( tai et al . , 2007 ; politis et al . , 2008 ) .
interestingly , microglial activation in the striatum , and regions related to cognitive function has been shown to predict the 5-year disease clinical onset in premanifest hd patients ( tai et al .
these results imply that microglial activation is an early event in the hd disease course , with a possible pathogenic involvement that is associated with a subclinical progression of the disease .
bpnd , binding potential ; hd , huntington s disease ; nc , normal control . in manifest hd patients ,
significant c - pk11195 bpnd in the striatum , hypothalamus , and various cortical regions was found , that correlated with greater disease burden and higher motor disability ( pavese et al .
the cortical microglial activation is likely to indicate the involvement of cortical neurons in hd , a well - recognized phenomenon as the disease progresses .
collectively , these findings are consistent with the post - mortem studies ( messmer and reynolds , 1998 ; sapp et al . , 2001 ) and suggest a detrimental microglial contribution to the ongoing neuronal degeneration in hd .
dementias are a group of disorders that are expected to affect more than 100 million people by 2050 raising remarkable financial costs for healthcare ( wimo et al . , 2003 ) .
ad is the most common cause of dementia and is the most common neurological disorder of the elderly .
ad is characterized by the presence of amyloid plaques , neurofibrillary tangles , and activated microglia ( for review , see hardy and selkoe , 2002 ) .
there is a plethora of evidence from post - mortem human ad studies ( mcgeer et al . , 1988a ; venneti et al . , 2009 ) and animal models ( frautschy et al . , 1998 ; stalder et al
, 1999 ; leung et al . , 2011 ) reporting a high accumulation of activated microglia in close proximity with the amyloid plaques , and upregulated levels of pro - inflammatory cytokines ( akiyama et al . , 2000 ; eikelenboom et al . ,
positron emission tomography enables a broad range of functional processes to assess the ad brain in vivo ( table 3 ) .
c - pk11195 has been used to demonstrate increased levels of activated microglia in both ad animal models ( venneti et al . , 2009 ) and ad patients ( cagnin et al . , 2001 ; edison et al . , 2008 ; yokokura et al . , 2011 ) . in ad patients ,
significant c - pk11195 bpnd was consistently observed in the temporal , parietal , and occipital cortices , regions known to be affected by ad pathology ( cagnin et al . , 2001 ;
the increased activated microglia also inversely correlated with the patient mini - mental state examination ( mmse ) scores , which is compatible with a role of microglia in neuronal damage ( edison et al . , 2008 ) .
interestingly , elevated levels of activated microglia were also detected in patients with amnestic mild cognitive impairment ( mci ; okello et al . , 2009a ) , although this was not observed in another study assessing mci patients ( wiley et al .
mci could represent an early precursor stage of ad , since it was found that mci patients with increased amyloid load were significantly more likely to clinically convert to ad within 3  years ( okello et al . , 2009b ) .
therefore , microglial activation could be an early event in the ad pathogenesis that begins at the mci stage .
ad , alzheimer s disease ; bpnd , binding potential ; ftld , frontotemporal lobar degeneration ; mci , mild cognitive impairment ; mmse , mini - mental state examination ; nc , normal controls . despite the evidence suggestive of a pathogenic role of activated microglia in ad
, it is hypothesized that the accumulation of amyloid plaques is actually due to a failure in microglial clearance mechanisms that would normally remove the protein ( bornemann et al . , 2001 ;
this indicates a beneficial , rather than detrimental role of microglia in ad . notwithstanding the abundance of activated microglia close to senile plaques , they maybe inefficient in the clearance of amyloid , hence , resulting in aggregate formation ( bolmont et al . , 2008 ) .
it has been shown that in the presence of pro - inflammatory cytokines , phagocytic functions of microglia are compromised ( koenigsknecht - talboo and landreth , 2005 ) .
therefore , microglia may confer a dichotomous role in ad , where early microglial activation is possibly neuroprotective involving the removal of amyloid . however , chronic neuroinflammation may downregulate amyloid clearance mechanisms , thus , promoting aggregation and progression of disease .
frontotemporal lobar degeneration ( ftld ) which includes frontotemporal dementia is the name given to a group of pathologically , clinically , and genetically heterogeneous disorders involving focal atrophy of the frontal and temporal lobes , while unlike ad , with sparing of the parietal and occipital regions ( neary et al . , 1998 ) .
another important dissimilarity between ad and ftld pathology is the absence of amyloid plaque formation ( paulus et al . , 1993 ;
rather , the key histopathological features of ftld , depending on subtype , includes tau deposition ( including pick bodies ) and ubiquitin - positive , tau - negative inclusions ( munoz et al . , 2003 ; uchihara et al . , 2003 ) .
in vivo pet imaging of ftld patients detected enhanced microglial activation in the expected frontotemporal regions ( cagnin et al . ,
2004 ) . in the same study , significant c - pk11195 bpnd in the bilateral putamen is also consistent with previous neuropathological data showing the involvement of the basal ganglia in ftld ( mirra and hyman , 2002 ) .
these observations indicate the presence of an active microglial response that reflects progressive neuronal degeneration .
importantly , the detection of increased microglial activation in affected regions in ftld suggests that microglial responses occur independently of amyloid deposition , and that neuronal loss alone is enough to induce activation ( cagnin et al . , 2004 ) .
multiple sclerosis is a disease characterized pathologically by inflammatory demyelination and axonal transection , and is the most common cause of non - traumatic disability in young adults ( compston and coles , 2008 ) .
the involvement of activated microglia has long been proposed in ms ( benveniste , 1997 ) .
post - mortem investigations have detected activated microglia in the cortical gm of ms patients ( de groot et al .
2002 ) , while histopathological studies have implicated microglia in lesion pathogenesis ( for review , see lassmann , 2008 ) .
an observed correlation between neuronal loss and microglial activation was reported in animal experimental ms ( rasmussen et al . , 2007 ) .
significant levels of activated microglia was also found in ms patients , especially in the progressive forms of disease that are associated with neurodegeneration ( kutzelnigg et al . , 2005 ; magliozzi et al . ,
2010 ) , and selective ablation of parenchymal microglia was able to prevent demyelination and axonal damage ( heppner et al . , 2005 ) .
pathological aspects of ms such as neuroinflammation , demyelination , and neurodegeneration may be explored in vivo with pet ( for review , see kiferle et al . , 2011 ) .
pet with c - pk11195 and other tracers has demonstrated inflammatory processes with microglial involvement in ms ( figure 1 ; table 4 ) . in animal
experimental ms and human post - mortem brains it has been shown that c - pk11195 uptake corresponds to the distribution pattern of activated microglia ( banati et al . , 2000 ) .
it has also been demonstrated that there is increased c - pk11195 bpnd in areas of focal pathology identified by t1- and t2-weighted mri ( vowinckel et al . , 1997 ; banati et al . , 2000 ) and in gadolinium - enhanced t1-weighted mri ( debruyne et al . , 2003 ) .
increased c - pk11195 bpnd was observed in normal - appearing gray and white anatomical structures ( banati et al .
this is in line with the hypothesis that inflammatory processes initiated by microglia early in ms may constitute the real burden of disease , associated with invisible microglia - mediated damage that occur independently of relapses ( kesselring , 1990 ; confavreux et al . , 2000 ) .
a positive correlation has been suggested between ligand uptake and disease duration , disability , and brain atrophy ( banati et al . , 2000 ;
however , recent data from our group found a significant association between high c - pk11195 bpnd in the cortical gray matter and disability in patients with secondary progressive ms , and with higher c - pk11195 bpnd in the secondary progressive group than the relapse - remitting ms group ( politis et al .
enhanced microglial activation in ms has also been detected using the more recently developed tspo tracers c - vinpocetine and c - pbr28 ( vas et al . , 2008 ; oh et al . , 2011 ) .
positron emission tomography images showing increased c - pk11195 bpnd in a multiple sclerosis patient ( a ) when compared to a healthy normal control ( b ) . color bar represents intensity of c - pk11195 tracer binding ( bpnd ) .
bpnd , binding potential ; gm , gray matter ; mri , magnetic resonance image ; ms , multiple sclerosis ; nawm , normal - appearing white matter ; nc , normal control ; pp , primary progressive multiple sclerosis ; rr , relapse - remitting multiple sclerosis ; sp , secondary progressive multiple sclerosis . microglial activation may contribute to the mechanism of axonal injury via the release of soluble factors that may either directly or indirectly cause neuronal dysfunction ( peterson et al .
, 2001 ; barnett and prineas , 2004 ; dutta and trapp , 2006 ; zipp et al . , 2006 ; dal bianco et al . , 2008 ; lassmann , 2008 ; magliozzi et al . , 2010 ) ,
however , activated microglia may also exert protective functions with ms through the release of neurotrophic factors ( stadelmann et al .
, 2002 ; napoli and neumann , 2009 ) , and triggering of remyelination mechanisms ( li et al . , 2005 ; setzu et al . ,
c - pk11195 was the first tracer to be consistently used for the study of activated microglia and neuroinflammation in vivo .
however , limitations associated with the application of c - pk11195 include a high level of non - specific binding ( petit - tabou et al . , 1991 ) , and a poor signal to noise ratio , which complicates its quantification ( boutin et al . , 2007 ) .
this has prompted the search for novel pet tracers ( termed , second generation radioligands ) with improved capacities to quantify tspo expression .
radioligands such as c - pbr28 , c - daa1106 , f - fedaa1106 , and f - pbr111 have recently been developed to image tspo in vivo ( gulys et al . , 2002 ; ikoma et al . , 2007 ;
, 2011 ; for a review , see chauveau et al . , 2008 ) .
published data using the second generation ligands c - daa1106 ( ikoma et al . , 2007 ) and f - fedaa1106 ( fujimura et al . , 2006 ) in humans were promising , with both tracers showing significantly higher cerebral uptake than c - pk11195 .
furthermore , increased c - daa1106 binding was reported in ad patients ( yasuno et al .
, 2008 ) that were similar to the previous studies that used c - pk11195 ( cagnin et al . , 2001 ) . the only published study using the second generation radioligand c - pbr28 found areas of focal increases in radiotracer binding in the brain of ms patients ( oh et al . , 2011 )
interestingly , the increased focal c - pbr28 binding preceded the development of some gadolinium - enhancing lesions . brain parenchymal c - pbr28 binding in ms patients was positively correlated with the duration of the disease , however it was not significantly higher than that of healthy volunteers . interpretation of these results is limited by the lack of characterization of the binding affinity pattern , which might have significantly affected the comparison between subjects .
it has been recently demonstrated that there are three different affinity patterns for second generation tspo ligands in healthy volunteers as well as patients with ms , which was evident with all the ligands tested ( c - pbr28 ; c - pbr06 ; f - pbr111 ; owen et al . , 2010 ) .
this presents a methodological problem , as differences in pet signal across subjects can not be safely interpreted as differences in target density , but may reflect differences in the affinity pattern . a possible approach to solve this problem
is based on the use of peripheral binding affinity , which can be characterized to classify subjects into one of the groups , as differences in affinity status between individuals have been shown to be present on peripheral cells as well ( owen et al .
interestingly , the difference in binding patterns observed with second generation radioligands was not observed with c - pk11195 .
also , in vitro autoradiography data using c - pk11195 suggest a receptor density ( bmax ) significantly higher than that found using second generation ligands .
it could be speculated that c - pk11195 and newer ligands bind to distinct sites within the tspo molecule .
although , data obtained from first generation studies have been promising and suggested that c - pk11195 could be useful to image acute inflammatory lesions and microglial activation in ms , a conclusive demonstration of the potential of tspo imaging for the application as disease biomarker , indicative of microglial activation in ms , is still lacking .
furthermore , despite second generation ligands constituting a potential improvement relative to c - pk11195 at least from a methodological point of view , a clear advantage in their clinical application as disease biomarkers has not been demonstrated yet .
for these reasons , we aim to characterize a second generation tspo pet radioligand in vivo in humans , and to evaluate its application as a disease biomarker in ms . among second generation tspo tracers ,
f - pbr111 presents different advantages , as there is low difference in its affinity for tspo between high , medium , low affinity binders .
also , it could be potentially used in clinical applications as it is labeled with fluorine-18 .
promising preclinical data , and ongoing studies in neurological patients , suggest it could be a good choice amongst second generation tspo ligands to progress into studies in ms patients .
inflammation coupled with the presence of activated microglia seems to be a common feature of a wide range of cns diseases .
however , despite a large number of research studies , the exact role of microglia in chronic neurodegenerative diseases remains uncertain . in line with the high plasticity of microglia that allows them to perform numerous cns functions , microglia are likely to play a dichromatic role in disease , depending on signals present in their microenvironment and the duration of activation . while early microglial activation could represents a beneficial response ( i.e. , removal of cns threat , promoting tissue repair and removal of misfolded protein ) , chronic exposure could induces detrimental effects by promoting neuronal death ( i.e. , through the sustained release of neurotoxic factors ) , thus , contributing to progression of disease . pet imaging with the use of tspo radioligands
provides a valuable tool that allows us to track the progression and severity of neuroinflammation in the living brain , and is a useful indicator of active cns disease .
therefore , the early detection of microglia using pet could offer opportunities for pharmacological interventions to limit the potential disruptive effects of chronic microglial activation .
furthermore , with the development of newer tspo tracers , the potential for pet imaging research to promote our understanding of activated microglia in cns disease can only increase .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 microglia constitute the main immune defense in the central nervous system . in response to neuronal injury , 
 microglia become activated , acquire phagocytic properties , and release a wide range of pro - inflammatory mediators that are essential for the annihilation of the neuronal insult . 
 although the role of microglial activation in acute neuronal damage is well defined , the pathophysiological processes underlying destructive or protective role to neurons following chronic exposure to microglial activation is still a subject of debate . 
 it is likely that chronic exposure induces detrimental effects by promoting neuronal death through the release of neurotoxic factors . 
 positron emission tomography ( pet ) imaging with the use of translocator protein ( tspo ) radioligands provides an in vivo tool for tracking the progression and severity of neuroinflammation in neurodegenerative disease . 
 tspo expression is correlated to the extent of microglial activation and the measurement of tspo uptake in vivo with pet is a useful indicator of active disease . 
 although understanding of the interaction between radioligands and tspo is not completely clear , there is a wide interest in application of tspo imaging in neurodegenerative disease . in this article , we aim to review the applications of in vivo microglia imaging in neurodegenerative disorders such as parkinson s disease , huntington s disease , dementias , and multiple sclerosis . 
Introduction
Parkinsons Disease and Related Disorders
Huntingtons Disease
Dementia
Multiple Sclerosis
New TSPO Ligands
Conclusion
Conflict of Interest Statement