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Neuroscience xx (2008) xxx

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ON-INVASIVE VISUALIZATION OF CNS INFLAMMATION WITH

UCLEAR AND OPTICAL IMAGING

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. WUNDER,* J. KLOHS AND U. DIRNAGL

enter for Stroke Research Berlin (CSB), Department of Experimentaleurology, Charité-University Medicine Berlin, Charitéplatz 1, 10117 Ber-

in, Germany

bstract—Inflammation is crucially involved in many diseasesf the CNS. Immune cells may attack the CNS, as in multipleclerosis, and therefore be responsible for primary damage.

mmune cells may also be activated by injury to the CNS, as forxample in stroke or brain trauma, secondarily enhancing le-ion growth. In general, CNS inflammation involves a complex

nterplay of pro- and anti-inflammatory cells and molecules. Thelood–brain barrier loses its integrity, plasma proteins leak intohe CNS parenchyma, followed by invasion of blood-borne im-une cells, and activation of resident microglial cells and as-

rocytes. However, inflammation not only exacerbates CNS dis-ase, it is also indispensable in containment and resolution ofissue damage, as well as repair and regeneration. The timeourse and the contribution of inflammatory processes to theathophysiology of the disease depend on several factors in-luding the type of injury and the time point after injury, and canxhibit a high individual variability. Imaging technologies thatnable specific visualization of these inflammatory processeson-invasively are therefore highly desirable. They provideowerful tools to further evaluate the contribution of specificrocesses to the pathophysiology of CNS disease. Moreover,hese technologies may be valuable in detecting and assessingisease progression, in stratifying patients for therapy, and inonitoring therapy. Among the existing non-invasive imagingethods to visualize neuroinflammation in the CNS, we here

eview the current status of nuclear and optical imaging tech-iques, with particular emphasis on the sensitivity, specificity,s well as the limitations of these approaches. © 2008 IBRO.ublished by Elsevier Ltd. All rights reserved.

ey words: neuroinflammation, PET, SPECT, optical imaging,ioluminescence, fluorescence.

Corresponding author. Tel: �49-30-450-560-206; fax: �49-30-450-60-915.-mail address: andreas.wunder@charite.de (A. Wunder).bbreviations: AD, Alzheimer’s disease; BBB, blood–brain barrier;LI, bioluminescence imaging; CCD, charge-coupled device; CNS,entral nervous system; EAE, experimental autoimmune encephalo-yelitis; FMT, fluorescence-mediated tomography; FRI, fluorescence

eflectance imaging; MCAO, middle cerebral artery occlusion; MRI,agnetic resonance imaging; MS, multiple sclerosis; NIRF, near-in-

rared fluorescence; PD, Parkinson’s disease; PET, positron emissionomputed tomography; SBE, Smad-binding element; SPECT, singlehoton emission computed tomography; TFI, transillumination fluores-ence imaging; TSPO, translocator protein 18-kDa; 18F-FDG,-[18F]fluoro-2-deoxy-D-glucose; 68Ga-EDTA, 68Ga-ethylenediami-etetraacetic acid; 99mTc-DTPA, 99mTc-diethylenetriaminepentaaceticcid; 99mTc-HMPAO, 99mTc-hexa-methyl-propylene-amine-oxime;9m 99m 111 111

STcO4, Tc-pertechnetate; In-oxine, indium-111-oxine; In-tro-

onolate, indium-111-troponolate; 123I-IMP, 123I-iodoamphetamine.

306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.oi:10.1016/j.neuroscience.2008.10.005

1

Contentsuclear imaging approaches to visualize neuroinflammation

non-invasively 2on-invasive imaging of increased vascular permeability 2on-invasive imaging of increased glucose metabolism 3on-invasive imaging of leukocyte infiltration 3on-invasive imaging with targeted compounds 5ptical imaging approaches to visualize neuroinflammation

non-invasively 6LI of neuroinflammation 7IRF imaging of neuroinflammation 8onclusions 11cknowledgments 11eferences 11

cute and chronic pathological processes of the brainnduce brain inflammation, which contributes to damage asell as repair and regeneration (e.g. Ekdahl et al., 2008;cGeer and McGeer, 2001; Minagar et al., 2002; Ker-

chensteiner et al., 2008; Vezzani and Granata, 2005;cColl et al., 2007; Wang et al., 2007). Thus, inflammation

s a highly relevant therapeutic target in practically allisorders of the CNS. Only recently it has become clearhat inflammation of the brain is Janus-faced (Dirnagl,004): destruction of brain tissue and the containment ofamage, or even regeneration, may be governed by theame inflammatory molecules and mechanisms. In theserocesses, time, cellular context, and stimulus intensity are

mportant switches between destruction and repair.The important role of inflammation in brain disorders

hould not come as a surprise: inflammation is the basicechanism by which tissues of multi-cellular organisms

espond to injury. Inflammation first eliminates pathogensr noxious agents and clears debris (Napoli and Neumann,008), then it controls the restoration of tissue integrity andunction (Oberyszyn, 2007; Cui et al., 2008). The role ofnflammation in brain disorders and after brain lesions is a

ultifaceted process, which is also strongly affected by theeripheral immune status of the patient (e.g. atherosclero-is, infection, Perry et al., 2007; McColl et al., 2008; Teel-

ng and Perry, 2008). Reciprocally, CNS disorders andesions themselves affect peripheral immunity (e.g. CNS-nduced immunodepression, Meisel et al., 2005; Klehmett al., 2008; Riegger et al., 2008; Urra et al., 2008).

Numerous pro- and anti-inflammatory cells and mole-ules are involved in CNS inflammation (Ridder and

chwaninger, 2008), which is characterized by blood–

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rain barrier (BBB) perturbations (Candelario-Jalil et al.,008), leakage of plasma proteins into the CNS paren-hyma, infiltration of blood-borne immune cells, and thectivation of resident microglial cells and astrocytes (deloppo, 2008). Imaging technologies that non-invasivelyisualize specific biological events using target specificompounds might be powerful tools to evaluate the role ofnflammation in brain disorders. Furthermore, these tech-ologies would also prove valuable in diagnosing CNS

njury-induced inflammation, in assessing disease progres-ion, in stratifying patients for therapy, and in monitoringesponses to therapeutic interventions. In recent years, aapidly increasing number of target specific compoundsave been developed, enabling non-invasive imaging ofathophysiological processes, including inflammation. Inhese approaches different imaging technologies such asositron emission computed tomography (PET), singlehoton emission computed tomography (SPECT), mag-etic resonance imaging (MRI), ultrasound, and optical

maging methods have been successfully applied to visu-lize inflammation of the brain in vivo in preclinical diseaseodels as well as in patients.

The review by Stoll and Bendszus (2008) in this issuef Neuroscience explores the technical basics of iron-ontrast-enhanced MRI and its application to the in vivomaging of inflammation in the peripheral and CNS. Weere review the current status of two other imaging modal-

ties which have great potential in the visualization of brainnflammation, and are highly complementary to MRI: nu-lear imaging using PET and SPECT, as well as opticalmaging such as near-infrared fluorescence (NIRF) imag-ng and bioluminescence imaging (BLI). Issues of quanti-ation, specificity, sensitivity, and practicability will ulti-ately decide whether these approaches will benefit pa-

ients with CNS disorders, and will therefore be particularlyonsidered.

NUCLEAR IMAGING APPROACHES TOVISUALIZE NEUROINFLAMMATION

NON-INVASIVELY

uring the past decade, the performance as well as thevailability of clinical and small animal PET- and SPECT-canners has improved substantially. PET and SPECTffer high sensitivity, which is orders of magnitude greaterompared with MRI or CT. Exceedingly low amounts of theignal molecule (radioisotope) can be detected by SPECT,nd especially by PET. This is particularly important whennly low amounts of the imaging agent can be delivered to

he target tissue, for instance due to low expression of themaging target, or biological barriers limiting the number ofignal molecules reaching the target tissue. Clinical PET-nd SPECT-scanners usually have a spatial resolutionf 5 and 10 mm, respectively. The newest generations ofET- and SPECT-scanners resolve below 5 mm. Cur-

ently, small animal imaging PET-systems can resolvemm, a resolution which is even surpassed by small

nimal SPECT systems. MRI and CT compensate by

uch higher spatial resolution, but PET- and SPECT- i

Please cite this article in press as: Wunder A, et al., Non-invasive visuaing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

canners can profit from the combination with CT or MRI,llowing precise anatomical localization of tracer uptake,hich can be quantified (Beekman and Van der Have,007; Rowland and Sherry, 2008).

A wide variety of approaches have been taken to visu-lize neuroinflammatory processes using nuclear imagingechniques. These include imaging of increased vascularermeability, imaging of increased glucose metabolism,

maging of leukocytes infiltrating the CNS lesion, and im-ging of radiolabeled compounds designed for specificargeting of molecules involved in the inflammatory pro-esses.

NON-INVASIVE IMAGING OF INCREASEDVASCULAR PERMEABILITY

n acute and chronic inflammation, vascular permeability isarkedly increased, leading to extravasation of plasma

omponents and leukocyte infiltration into areas of tissueamage. In the CNS, injury and subsequent inflammation

eads to impairment and leakiness of the BBB, which is aallmark of several neurological disorders (Ballabh et al.,004; Zlokovic, 2008). Alterations of BBB integrity areften mediated by pro-inflammatory molecules and are aart of the inflammatory response (Persidsky et al., 2006).herefore, non-invasive, highly sensitive localization anduantification of vascular leakage can be a valuable tool tovaluate neuroinflammation in the CNS. Several PET- andPECT-radiopharmaceuticals are used to image BBB dis-

uption. Typically, the tracers for imaging BBB disruptionave a comparatively low molecular weight, are non-lipidoluble molecules, and are unable to cross the intact BBB.xamples include 99mTc-pertechnetate (99mTcO4) or 99mTc-iethylenetriaminepentaacetic acid (99mTc-DTPA) for SPECT-

maging and 68Ga-ethylenediaminetetraacetic acid(68Ga-DTA) for PET imaging. In contrast, tracers such as 123I-iodo-mphetamine (123I-IMP), 99mTc-hexa-methyl-propylene-amine-xime (99mTc-HMPAO) or 99mTc-ethyl-cisteynate dimer (99mTc-CD) are lipophilic, can freely cross the BBB, and can besed to image perfusion (Saha et al., 1994).

Numerous brain perfusion imaging studies have beenerformed with nuclear imaging (Wintermark et al., 2005),hereas PET or SPECT is rarely used for the imaging ofBB disruption in neurological disorders. In ischemictroke, neurovascular perturbations lead to BBB disrup-ion, edema, hemorrhage, influx of leukocytes into therain parenchyma and progressive inflammatory pro-esses, starting a few hours after symptom onset, andersisting for several days (Rosell and Lo, 2008). Lorber-oym et al. (2003) studied BBB impairment in 30 patientsith acute middle cerebral artery infarct (between 48 and2 h after the onset) by injecting 99mTc-DTPA followed byPECT imaging (Fig. 1A). They found that enhanced

racer uptake is significantly related to clinical outcome andhat areas with high signal intensity are associated with aore severely damaged tissue. They defined a so-calledisruption index as the ratio of the signal intensity in the

nfarcted region compared with the corresponding con-

lization of CNS inflammation with nuclear and optical imag-

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ralateral side. Patients with a disruption index thresholdmaller than 2.5 had a significantly better prognosis thanatients with a disruption index higher than 2.5. All sevenatients who did not show enhanced tracer uptake in the

esions (disruption index equals 1) had a very good prog-osis. Interestingly, the disruption index was not correlated

o infarct volume. Since hemorrhagic stroke leads to annflammatory response including BBB disruption and for-

ation of a peri-hematomal edema (Wang and Doré,007; Thiex and Tsirka, 2007), the same group examinedBB impairment in 27 patients with acute hemorrhagictroke of the middle cerebral artery (24–48 h after thenset), again using 99mTc-DTPA and SPECT imaging (Fig.B). In this study they also found a significant correlationetween the SPECT imaging data and the late functionalutcome (Lampl et al., 2005).

BBB disruption is one of the hallmarks of multipleclerosis (MS), which occurs early in disease progression,nd correlates well with disease activity. However, it is notompletely understood whether the loss of BBB integrity ishe initial event leading to lesion formation, or a conse-uence of cellular CNS infiltration (Waubant, 2006). Theumber of reports investigating BBB disruption with nu-lear imaging techniques in MS is limited. However, weound two studies: Pozzilli et al. (1988) examined 15 pa-ients with MS using 68Ga-EDTA and PET imaging. In fourut of eight patients with disease exacerbation they no-iced areas with signal enhancement, which also appearedn CT. In a more recent study, Terada and Kamata (2003)ound a strong lesional uptake of 201TI and 99mTcO4 in twoatients with MS (Fig. 1C). Alterations of the BBB havelso been reported in neurodegenerative diseases such aslzheimer’s disease (AD) and Parkinson’s disease (PD),hich may be involved in disease progression (Desai etl., 2007). Studies that support these findings with non-

nvasive imaging are limited (Wang et al., 2006; Kortekaas

ig. 1. Nuclear imaging of BBB disruption. SPECT image of a patien9mTc-DTPA. Patients with a disruption index smaller than 2.5, defineontralateral side (see marked regions of interest), had a significantly brom Lorberboym et al. (2003) (A). SPECT (top row) and CT (bottom rowrtery 2 h after injection of 99mTc-DTPA. Note the rim of high signal intPECT imaging correlated significantly with functional outcome of theultiple sclerosis after injection of 99mTcO4 (top) and Thalium-201 (2

racer uptake was detected after injection of both tracers, which was fo2003) (C).

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NON-INVASIVE IMAGING OF INCREASEDGLUCOSE METABOLISM

levated glycolysis has been reported for cancer cells butlso for several immune cells including T cells, macro-hages, and neutrophils, in particular if they are activated.herefore, PET imaging of increased glucose metabolismsing 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) as a

racer can be used as a marker for imaging inflammation.DG is transported into the cell by specific glucose trans-orters, gets trapped after phosphorylation, and thereforeccumulates inside the cell (Jalilian et al., 2006; Bleeker-overs et al., 2008). In a recent paper, Radu et al. (2007)sed FDG-PET to visualize neuroinflammation in an ani-al model of MS. Using a high-resolution PET/CT scan-er, they found increased tracer uptake in areas of inflam-atory infiltrates within the spinal cord. Moreover, the

ignals detected were sensitive to systemic immunosup-ression with dexamethasone (Fig. 2). It should be notedhat the application of FDG-PET for imaging of neuroin-ammation is limited by several factors (Herholz and He-

ss, 2004): due to the exceedingly high energy consump-ion of the organ, 18F-FDG uptake in the brain is generallyigh. Second, glucose consumption of particular brain re-ions is activation dependent. Third, neurodegenerationecreases glucose consumption.

NON-INVASIVE IMAGING OF LEUKOCYTEINFILTRATION

fter injury, blood-borne immune cells such as lympho-ytes, granulocytes, neutrophils or monocytes/macro-hages infiltrate the CNS parenchyma. To monitor thisrocess by non-invasive imaging, autologous leukocytesan be labeled for detection with nuclear imaging, usingifferent techniques and radioisotopes including indium-11-oxine (111In-oxine), indium-111-troponolate (111In-tro-

te ischemic stroke of the middle cerebral artery 2 h after injection ofal intensity in the infarcted region compared with the corresponding

gnosis than patients with a disruption index greater than 2.5. Adaptedof three patients with acute hemorrhagic stroke of the middle cerebral

rrounding the core of the hemorrhage, especially in the middle panel.Adapted from Lampl et al. (2005) (B). SPECT images of a patient withacer with potassium like characteristics (bottom). Increased lesional

higher after injection of 99mTcO4. Adapted from Terada and Kamata

t with acud as signetter pro) images

ensity supatients.01TI), a trund to be

onolate), or 99mTc-HMPAO for scintigraphic or SPECT

lization of CNS inflammation with nuclear and optical imag-

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maging. Isolated leukocytes can also be labeled with 18F-DG for PET imaging. The underlying principles of theifferent labeling techniques, are nicely described by Ball-

nger and Gnanasegaran (2005).Several studies addressed the recruitment of leuko-

ytes at sites of inflammation using nuclear imaging tech-iques in different disease conditions, including neuroin-ammation. Pozzilli et al. (1985) radiolabeled circulatinghite blood cells of patients with brain infarction in vitroith 111In-troponolate, and re-injected them i.v. during therst 2 weeks after the onset of neurological symptoms. Inrder to evaluate BBB permeability, the patients were also

njected with 99mTcO4. In seven out of eight patients theyound a well-defined area of increased radioactivity in thenfarcted hemispheres with scintigraphy 2 and 20 h afternjection. In all eight patients they found increased tracerptake in the infarcted area after injection of 99mTcO4 atoth time points. Kao et al. (1991) studied the distribution

ig. 2. Nuclear imaging of neuroinflammation with FDG-PET. Increasn EAE-affected mice. [18F]FDG PET/CT scans (coronal and transvmmunized mouse at various time points before and after EAE onset.lycolysis in the cervical, thoracic, and lumbar spinal column segments

maged between days 13 and 21 after immunization in four independeverity measured by using the clinical score (C). Figure from Radu e

f injected autologous white blood cells labeled with 99mTc- c

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MPAO within the brain of 26 patients with acute cerebralnfarction. Poor outcome was associated with higher signalntensities in the ischemic compared with the contralateralemispheres as determined by SPECT imaging. The sameroup examined 19 patients with acute hemispheric isch-mic stroke within the first week after onset and three patientsith chronic hemispheric ischemic stroke of durations longer

han 7 months after injection of 99mTc-HMPAO-labeled leu-ocytes with brain SPECT. In contrast to the patients withhronic stroke, the patients with acute stroke showedigher signal intensities in the ischemic compared with theontralateral hemispheres after SPECT imaging. This ef-ect persisted for no fewer than 5 weeks after the onset oftroke and then declined (Wang et al., 1993). Akopov et al.1996) studied the distribution of radioactivity in the brainfter injection of polymorphonuclear leukocytes labeledith 99mTc-HMPAO in 88 patients from 3 h to 90 days aftertroke onset. The accumulation of activity in the regions of

lysis at anatomical locations corresponding to the spinal cord/columntions) of a non-immunized mouse and of a MOG35-55/CFA/PTX-H, heart; SC, spinal column; BL, bladder; BF, brown fat (A). Elevatedffected mice, as well as in the entire (C1–L6) spinal column. Mice wereriments (B). Glycolysis in the spinal column does not reflect disease7).

ed glycoerse secB, brain;of EAE-a

erebral infarction correlated with the severity of brain

lization of CNS inflammation with nuclear and optical imag-

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issue damage and poor neurological outcome. Stevenst al. (1998) injected 99mTc-HMPAO-labeled leukocytes

nto patients with middle cerebral artery infarction 5 to 30ays after onset of the first symptoms. SPECT imagingas performed 4 h after injection. One to three days later,

hey injected 57Cobalt followed by SPECT imaging after4 h. Cobalt isotopes have been proposed as marker toisualize calcium-mediated damage, which may be ex-lained by neuronal influx of the tracer, using routes anal-gous to that of calcium ions. In addition, 57Cobalt uptake

n damaged brain tissue may be due to infiltrating leuko-ytes or protein-bound cobalt. In 5 out of 10 patients,nhanced uptake in the infarcted brain area has beenetected with SPECT for both tracers, whose uptake cor-elated significantly. Additionally, enhanced uptake corre-ated significantly with clinical prognosis. More recently,rice et al. (2004) showed that significant amounts of

adioactivity in the ischemic hemisphere can be detectedfter i.v. injection of 111In-troponolate-labeled neutrophilsith SPECT imaging in 9 out of 15 patients with acuteerebral ischemia within 24 h of stroke onset. In this study,nter-individual accumulation of radioactivity in the isch-mic hemispheres did not correlate with infarct volume andutcome, but appeared to be correlated with infarct expan-ion (Fig. 3).

A key problem within all of these approaches is theelease of the radiolabel from the cells in vivo, which canead to signals not related to specific distribution of the

ig. 3. Nuclear imaging of neuroinflammation with radiolabeled leukocschemic stroke of the cortical middle cerebral artery at 16 h after clininset injected with 111In-troponolate-labeled neutrophils (B). Manually

D) and posterior (E) planar SPECT images at 46 h after clinical onset. Co-regiransaxial SPECT image (42 h after clinical onset) viewed caudo-rostrally from

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adiolabeled cells. Especially in approaches using 99mTc-MPAO labeled cells, hydrophilic 99mTc-HMPAO decom-osition products occur in vivo, which are unable to crossn intact BBB. In case of BBB impairment, these productsan enter the brain parenchyma, confounding the interpre-ation of the results. Compared with 99mTc-HMPAO label-ng, 111In-oxine and indium-111In-troponolate labeling is

ore stable, but these techniques fall short because of theomparatively long half-life of 111In (67 h versus 6 h of9mTc), which allows injection of only low amounts of ra-ioactivity in patients leading to a worse image quality. Theore recent leukocyte labeling techniques with 18F-FDG

how similar problems. Besides difficulties with leakage ofhe label, the short half-life of 18F (110 min) may not allowufficient time for cell tracking. A further general con-ounder in cell labeling approaches is that one cannotiscriminate between intraparenchymal activity and intra-ascular signal from circulating label (Ballinger andnanasegaran, 2005).

NON-INVASIVE IMAGING WITH TARGETEDCOMPOUNDS

or imaging of inflammation, compounds binding with highpecificity to molecules crucially involved in inflammationan be used. For imaging neuroinflammation, radioligandshat bind to the peripheral benzodiazepine receptor (PBR),ecently renamed as translocator protein 18-kDa (TSPO),

ired radiological format non-contrast CT image of a patient with acute(A) and corresponding transaxial SPECT image at 42 h after clinical

emispheric region of interest (ROI), planar SPECT image (C). Anterior

ytes. Pacal onsetdefined h

stered image of non-contrast CT image (16 h after clinical onset) androstral (top) to caudal (bottom) (F). Figure from Price et al. (2004).

lization of CNS inflammation with nuclear and optical imag-

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re most extensively studied. TSPO is largely, but notxclusively, located in the outer mitochondrial membrane.nder physiological conditions in the CNS, only low-levelSPO expression can be found in the ependymal cells

ining the ventricles, in the olfactory bulb, and in the choroidlexus. A low expression of TSPO can also be detected inlial cells including astrocytes and microglia. High levels ofSPO expression have been demonstrated in several pe-ipheral tissues including glandular and secretory tissues,he kidneys, the heart, the lungs, and in various blood cellsuch as monocytes. After brain injury and inflammation,SPO expression is strongly upregulated in activated mi-roglia cells, and in blood-borne monocytes infiltrating therain parenchyma, making TSPO a suitable target for im-ging of neuroinflammation. Several radiolabeled TSPO

igands have been developed and studied as a biomarkeror non-invasive imaging of reactive gliosis and inflamma-ion in experimental and clinical studies in a variety ofeuropathological conditions, including stroke, MS, AD,nd PD (for review see Venneti et al., 2006; Cagnin et al.,007). Furthermore, TSPO-specific ligands may also haveherapeutic implications in brain injury and repair (Chennd Guilarte, 2008).

Of these ligands designed for imaging, the lipid solublesoquinoline carboxamide derivative PK11195, whichinds with high affinity to TSPO, is the most extensivelytudied for visualization of neuroinflammation. For bindingssays, autoradiography, and non-invasive PET or SPECT

maging, the ligand can be labeled with 3H, 11C, 123I, and25I. However, there are several limitations confoundinghe interpretation of the in vivo imaging results. Addition-lly, there are sensitivity issues. Because of the high abun-ance of TSPO in peripheral organs, the injection of radio-

abeled PK11195, results in comparatively low tracer con-entrations in the brain and in signal-to-noise ratios whichan be rather low, limiting the sensitivity of the approachor detecting regions of TSPO expression. Furthermore,he tracer shows low brain permeability and needs to over-ome the cell membrane in order to bind to the target. As

consequence of the comparatively low expression ofSPO in the CNS, distribution effects not related to specificinding, such as vascular distribution and tissue inhomo-eneity may become prominent. For these reasons, sev-ral other TSPO-ligands with higher affinity and lower li-ophilicity have been developed and evaluated, promising

mproved properties for imaging (Venneti et al., 2006; Cag-in et al., 2007; Chen and Guilarte, 2008).

In patients with ischemic stroke, binding of radiola-eled PK11195 becomes significant after a few days oftroke onset, subsequently increases within about a week,nd declines after 3 to 4 weeks as shown by PET imagingFig. 4). Signals were detected from regions identified asnfarction core, peri-infarct zone, and contralateral hemi-phere (Pappata et al., 2000; Gerhard et al., 2000, 2005;rice et al., 2006). In MS, increased binding of radiola-eled PK11195 compared with normal white matter can beetected with PET imaging in areas of active focal inflam-atory lesions, i.e. at sites of BBB impairment as as-

essed by gadolinium-enhanced MRI. Old structural le- (

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ions with severe tissue destruction, appearing as “blackoles” in T1-weighted MRI, show little binding. During re-

apse, PK11195 binding in these lesions increases, sug-esting renewed inflammatory activity. In contrast, other1 hypointense regions did not show relapse-associatedhanges. PK11195 binding in T2 hyperintense areas wasound to be low, but increases during relapse. Duringisease progression, increased PK11195 binding can beetected in normal-appearing white matter (Vowinckelt al., 1997; Banati et al., 2000; Debruyne et al., 2003).

Radiolabeled PK11195 was also evaluated in demen-ias. In patients with PD, Ouchi et al. (2005) found that theK11195 binding potential in the midbrain contralateral to

he clinically affected side was significantly higher in PDhan in age-matched healthy subjects as shown by PETmaging. They demonstrated an inverse correlation of mid-rain PK11195 binding potential and the binding potentialf a dopamine transporter marker (radiolabeled CFT) inhe putamen. PK11195 binding in the midbrain was posi-ively correlated with clinical motor scores. Gerhard et al.2006) detected increased PK11195 binding in the pons,asal ganglia, as well as in frontal and temporal corticalegions. Unlike the study performed by Ouchi et al., levelsf PK11195 binding did not correlate with the clinical se-erity of the disease or uptake of [18F]-dopa in the puta-en. In AD, significantly higher binding levels of PK11195

ompared with normal individuals can be detected withET and SPECT in the entorhinal, temporoparietal, andingulated cortices, brain regions that are involved in theathology of the disease (Cagnin et al., 2001; Versijpt etl., 2003).

Taken together, PET and SPECT allow imaging ofnflammation in patients with CNS disorders at high sensi-ivity, and using targeted probes, with high specificity. Nu-lear medicine approaches are orders of magnitude moreensitive than current MRI, and they provide true signaluantitation. Major drawbacks that remain are the rela-ively low spatial resolution compared with MRI and CT,he use of ionizing radiation, the complexities of radio-hemistry, regulatory issues, and the high cost of thequipment. Nevertheless, PET and SPECT are the onlyechniques available that allow non-invasive imaging ofNS pathophysiology in patients at the molecular level.e predict that the increasing availability of PET-CT (and

oon PET-MRI) combinations, together with 68Ga-labelingechniques, superseeding the need for complex radiophar-acology (Maecke et al., 2005), will rekindle the interest in

hese techniques. This will continue to tremendously con-ribute to CNS pathophysiology in general, and CNS in-ammation in particular.

OPTICAL IMAGING APPROACHES TOVISUALIZE NEUROINFLAMMATION

NON-INVASIVELY

n recent years, optical technologies such as NIRF imagingnd BLI have emerged that enable non-invasive imaging ofhysiology and metabolism down to the molecular level

Ntziachristos, 2006; Contag, 2007). In contrast, intravital

lization of CNS inflammation with nuclear and optical imag-

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icroscopy approaches are invasive optical techniquesnd overviews of their application in neuroscience can be

ound elsewhere (Kubes and Ward, 2000; Misgeld anderschensteiner, 2006). Non-invasive optical imaging of-

ers several advantages including high sensitivity, the usef non-ionizing radiation, the requirement for relatively in-xpensive, simple instrumentation, and high-throughputapabilities. In addition, they have the potential to deliveruantitative data sets. Like all non-invasive imaging meth-ds, optical techniques have some specific limitations.ainly due to scattering of light in biological tissues, the

patial resolution of optical imaging systems is limited tobout 1 mm, decreasing with the depth of the fluorescentource. The penetration depth is limited by scattering andbsorption of photons, which can be several millimeters or

ig. 4. Nuclear imaging of neuroinflammation targeting TSPO. Radiolerebral artery infarction 2, 13, and 30 days after ictus (shown top to bRI showing core infarct ROI (middle column). The right-hand columnOI (dotted red), and peripheral ROI (pale green). Figure from Price

entimeters depending on the localization of the fluores- l

Please cite this article in press as: Wunder A, et al., Non-invasive visuaing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

ent source. Penetration depth is enough to reach mostissues in rodents and even subsurface structures in hu-ans (Ntziachristos, 2006; Contag, 2007). A growing num-er of researchers utilize optical approaches to study phys-

ology and pathophysiology. As described in the following,ome of these studies demonstrate that specific imaging ofeuroinflammation using optical imaging techniques in an-

mal models of neurological disease, such as experimentalutoimmune encephalomyelitis (EAE, a model of MS) orouse models of stroke is feasible.

BLI OF NEUROINFLAMMATION

LI is based on the ability of several organisms to produceight by an enzyme-catalyzed reaction of the substrate

mat binding potential maps (left column) of a patient with a left middlespectively) presented alongside co-registered structural T2-weightedtrates significant binding potential within core ROI (blue), dilated core06).

ogical forottom, redemons

uciferin by luciferase in the presence of oxygen and ATP.

lization of CNS inflammation with nuclear and optical imag-

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A. Wunder et al. / Neuroscience xx (2008) xxx8

ARTICLE IN PRESS

or imaging purposes, a “luciferase” gene (bacterial lux,refly luc or Renilla Rluc) is inserted into the genome ofells under the control of a promoter of a gene of interest.henever the promoter is active, luciferase is expressed,

esulting in the emission of light in the presence of itsubstrate luciferin that needs to be administered. The emit-ed light can then be detected with a highly sensitiveharge-coupled device (CCD) camera. BLI can be used toisualize gene expression, to study protein–protein inter-ctions, and for tracking cells non-invasively (Welsh anday, 2005; Contag, 2007).

Costa et al. (2001) demonstrated that BLI can be usedo study the fate of CD4� T-cells in EAE. CD4� T-cellsere transduced to express luciferase and were adoptively

ransferred into naïve mice and mice immunized with my-lin-basic protein. In both groups, luminescent cells wereetected in lymphoid tissues such as lung, spleen, and

ymph nodes within 24 h of transfer. In immunized mice 3ays after transfer, a significant increase in biolumines-ence in the brain was found which was still detectable 50ays after transfer. To study neuroinflammation, severalroups have used transgenic mouse lines that carry the

uciferase gene under the transcriptional control of theFAP promoter (GFAP-luc) (Zhu et al., 2004; Luo et al.,007, 2008; Cordeau et al., 2008). In a study by Zhu et al.2004), the detection of reactive astrocytes as a result ofntracranial kainic acid injection, which causes neuronalell death and gliosis, was demonstrated with BLI inFAP-luc mice at 24 and 48 h after injection. GFAP-lucice were also used to visualize gliosis in EAE. In thispproach it was shown that gliosis and neuroinflammationrecede clinical signs of EAE (Fig. 5). An increase of theLI signal was detected in the brains and spinal cords of

mmunized mice at day 3 and 7 after immunization, whichreceded the onset of clinical symptoms by 4 days. TheLI signal correlated significantly with clinical score, weight

oss, and immunohistological markers of activated astro-ytes (GFAP) and microglia (Luo et al., 2007, 2008). Tolucidate the role of TGF-1 signaling in the EAE model,mad-binding element (SBE)-luc mice were developed by

he same group. Bioluminescence was detected in SBE-uc mice at day 7 after immunization. Treatment with IN-130 blocked TGF-mediated ALK5-dependent signalingnd resulted in reduced BLI signals in SBE-luc and GFAP-

uc mice, reduced presence of CD3� T-cells, and amelio-ated disease severity (Luo et al., 2007).

In a study by Cordeau et al. (2008), gliosis as result oftroke-induced inflammation was detected with BLI in aouse model of cerebral ischemia. GFAP-luc mice under-ent 1 h of middle cerebral artery occlusion (MCAO). NoLI signal in the brain was observed after injection of

uciferin at 6–8 h after reperfusion, but bioluminescenceignificantly increased at 24 and 72 h. At 24 and 72 h,ignals were higher in female mice out of estrus comparedith male, age-matched littermates, demonstrating an ef-

ect of estrogen on gliosis. The BLI signal declined 5 and 7ays after reperfusion.

BLI is a powerful tool to study physiology and patho-

hysiology. However, there are several limitations. The o

Please cite this article in press as: Wunder A, et al., Non-invasive visuaing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

echnique requires the insertion of reporter luciferaseenes into cells or whole organism, which clearly restrictshe use of this technology to experimental models. Further-ore, the insertion of foreign genes into the genome of

ells might lead to alterations in transcriptional profiles (Wut al., 2006) and physiology (Cao et al., 2007). The need to

nject the substrate luciferin for the enzymatic reaction inhe presence of oxygen and ATP bears some of the sameonstraints that are known for the use of conventional

maging probes. Identical levels of luciferase expressionould result in different bioluminescence if the local avail-bility of the substrate is altered by inflammation. Afterystemic injection, luciferin normally reaches only low con-entrations in the brain. The detection of bioluminescencemission from the brain requires exposure times in theange of several minutes, thereby limiting the temporalesolution of the method (Lee et al., 2003). BLI has thedvantage that it does not require a light source for illumi-ation, which allows several animals to be imaged at theame time and thereby increases through-put. While biolu-inescence signals are comparatively weak, the back-round due to auto-luminescence is low and hence goodignal-to-background ratios can be achieved (Troy et al.,004). Most of the published BLI approaches were doneith two-dimensional imaging systems, but currently tech-iques that deliver three-dimensional bioluminescence im-ges and quantitative data sets are being developed, aseviewed by Wang et al. (2008).

NIRF IMAGING OF NEUROINFLAMMATION

n non-invasive NIRF imaging, the distribution of a fluores-ent probe in vivo is visualized by illuminating the animalith a light source (usually a laser) and detecting the

esulting fluorescence emission with a highly sensitiveCD camera. Various NIRF imaging devices with differentetups, modes of data collection, and features have be-ome commercially available. The vast majority of NIRF

maging systems are planar NIRF systems: in fluorescenceeflectance imaging (FRI) the light source and the detectorre on the same side of the object. In transilluminationuorescence imaging (TFI) the source and detector are onpposite sides. In contrast to the planar modes FRI andFI, fluorescence-mediated tomography (FMT) makes usef multiple illumination and/or detection sources, whichequentially illuminate the object and whose resultingmission patterns are collected by the detector(s). Dedi-ated algorithms then allow three-dimensional reconstruc-ions of fluorochrome distribution. This opens the potentialor quantitation of the signal, another important advantagever planar NIRF imaging (Ntziachristos et al., 2005).ovel fluorescent probes are being developed, enabling

he detection and imaging of molecular events in various ofxperimental disease models (Licha and Olbrich, 2005;lohs et al., 2008a).

Only a few studies report the application of non-inva-ive NIRF imaging to study neuroinflammation (Berger etl., 2007; Klohs et al., 2008b). Berger et al. (2007) dem-

nstrated that the infiltration of T-lymphocytes in the acute

lization of CNS inflammation with nuclear and optical imag-

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A. Wunder et al. / Neuroscience xx (2008) xxx 9

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nflammatory phase of EAE can be visualized with NIRFmaging. The indocyanine dye Cy5.5, conjugated to theI-virus derived cell permeable peptide, TAT, was extra-orporally loaded into T-lymphocytes. The labeled cellsere then adoptively transferred into rats immunized toevelop EAE. FRI revealed high fluorescence intensities

ig. 5. Optical imaging of neuroinflammation using bioluminescencen GFAP-luc mice with MOG35-55 emulsified in CFA plus PT, and biolay before (�1) and at 3, 7, 10, or 14 dpi. Time course of bioluminescens fold induction plotted with clinical score (B). Mice were killed at indt different time points were examined for neuroinflammation, and

mmunohistochemistry as a function of astrogliosis (GFAP), microgliosrom Luo et al. (2007).

ver the head with a good correlation between signal t

Please cite this article in press as: Wunder A, et al., Non-invasive visuaing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

trength and neurological scoring. Confocal imaging re-ealed a co-localization of Cy5.5 with markers of lympho-ytes (CD3) and macrophages (ED1). This indicates thathagocytosing cells might have taken up the label from

ymphocytes that have died during the course of the study,hich precludes longitudinal studies over a long period of

ating that gliosis can be visualized non-invasively. EAE was inducednce was recorded in living mice injected with luciferin (150 mg/kg) 1ed in a representative mouse (A) and the bioluminescence expressede points. Sagittal brain sections from control (�1) or EAE mice killedwere taken from cerebellum. Neuroinflammation was assessed by, and T lymphocyte infiltration (CD4) (C). Scale bars�100 �m. Figure

demonstruminescece record

icated timimages

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ime. In a recent study, we demonstrated that a CD40-

lization of CNS inflammation with nuclear and optical imag-

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A. Wunder et al. / Neuroscience xx (2008) xxx10

ARTICLE IN PRESS

ig. 6. Optical imaging of neuroinflammation using NIRF imaging demonstrating that the inflammatory CD40 receptor can be specifically visualizedon-invasively. Wild type or CD40 knock-out mice were subjected to MCAO or sham-operated and were i.v. injected with Cy5.5-labeled CD40-antibodyr non-specific IgG 80 h after reperfusion. Target-to-background ratios (TBR) from non-invasive NIRF images were calculated by dividing theuorescence intensities in rectangular regions of interest (ROIs) selected over the left and the right hemisphere. Non-invasive NIRF imaging of theead of mice (top row) and ex vivo NIRF imaging of the extracted brains (bottom row) 16 h after injection of the compounds (A) showed significantlyigher fluorescence intensities over the ischemic hemisphere compared with the contralateral side (A, B). Selective plane illumination microscopyemonstrated vascular and parenchymal cellular distribution of the injected CD40-antibody in the ischemic region (C). Confocal microscopy showedartial co-localization of the signal from the injected compound with activated microglia/macrophages (Iba1 positive cells) (D). The study demonstrates

hat after injection of the fluorescent-labeled CD40-antibody most of the detected signal is due to specific binding and/or uptake of the compound rather

han due to leakage. Figure adapted from Klohs et al. (2008b).

Please cite this article in press as: Wunder A, et al., Non-invasive visualization of CNS inflammation with nuclear and optical imag-ing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

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A. Wunder et al. / Neuroscience xx (2008) xxx 11

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argeted fluorescent antibody enables specific detection ofhe inflammatory receptor CD40 after cerebral ischemia inice with non-invasive NIRF imaging (Klohs et al., 2008b)

Fig. 6).The examples reported above all used planar NIRF

maging. Planar NIRF imaging is highly sensitive in detect-ng trace amounts of fluorescent dyes in the brains of live

ice (Klohs et al., 2006). However, planar NIRF imagingas several limitations such as single projection viewing,he non-linear relationship between signal intensity andepth, and the inability of absolute quantification of theuorochrome concentration. Three-dimensional recon-truction of probe distribution with quantification has beeneported for FMT-systems (Ntziachristos et al., 2005). Weave recently described the development of a fluores-ence tomography system dedicated to visualize the three-imensional distribution of fluorochromes within the brainf live mice (Bourayou et al., 2008). A clear advantage ofuorescence imaging is that after non-invasive imaging theistribution of injected fluorescent probes in tissues can beerified with high resolution using fluorescence micros-opy. Additional immunohistochemistry can identify cellypes that bind or take up the imaging agent, as demon-trated in Fig. 6.

NIRF imaging of small animals offers several advan-ages, making it a well-suited tool to evaluate targets andigands in model systems. Its application in clinical settingss more challenging. However, several applications haveeen described (Frangioni, 2003; Ntziachristos, 2006).ven though depth-resolved in vivo NIRF measurementsere performed in the brain of human adults after i.v.

njection of indocyanine green, the ultimate depth limit ofhe excitation and emission light restricts the application toortical structures (Liebert et al., 2006; Steinbrink et al.,008).

Taken together, non-invasive optical imaging hasroven useful in monitoring of pathophysiology, includingrain inflammation, in various preclinical models of CNSisorders. Optical imaging is relatively inexpensive, is sim-le, can be highly sensitive, and enables exact localizationf the probe ex vivo with microscopy, which is highlyelevant for validating specificity and cellular localization.he major drawbacks of non-invasive optical imaging, are

ts low spatial resolution and the current limitation to neo-ortical processes in humans.

CONCLUSIONS

pecific imaging of physiological and pathophysiologicalrocesses is challenging. A major confounder can be com-artmental distribution of the injected compounds. Evenighly target-specific compounds with high affinity for the

arget might generate signals in vivo, which are not attrib-ted to specific binding. This is particularly true in diseaseshere blood–tissue barriers are altered. Especially under

nflammatory conditions in the CNS, the BBB shows anncreased permeability, allowing cells and soluble factorso reach the sites of inflammation. In imaging approaches,

his leads to an increased extravasation and distribution of

Please cite this article in press as: Wunder A, et al., Non-invasive visuaing, Neuroscience (2008), doi: 10.1016/j.neuroscience.2008.10.005

he injected compound in the inflammatory tissue not re-ated to specific binding, an effect that can be formidablend must be considered. On the other hand, this effect ishe basis of contrast generation using “unspecific” com-ounds. Furthermore, it needs to be proven that the imag-

ng signal truly reflects or correlates with the biologicalrocess intended to be imaged. Eventually, the signalhould correlate with a clinical parameter, making the im-ging technique valuable to translational studies. Other

ssues are the ability to visualize dynamic processes overime, the temporal resolution, the sensitivity, and the abilityf the method to quantify. Several compounds have beeneveloped demonstrating successfully that specific imag-

ng of neuroinflammation with various non-invasive imag-ng modalities is feasible. These approaches show prom-se that the time course and the contribution of specificnflammatory processes to the pathophysiology of CNSiseases can be imaged in the individual patient, improvingur possibilities to selectively modulate these processes,nd therefore lead to novel therapeutic strategies based ondeeper knowledge of pathophysiology.

cknowledgments—The authors are supported by grants from theechnology Foundation Berlin (A.W., U.D., J.K.) the Federal Min-

stry for Education and Research-BMBF (U.D.), the German Re-earch Foundation-DFG (U.D.), the Herman and Lilly Schillingtiftung (U.D.), and the European Union’s Seventh Frameworkrogramme (FP7/2008–2013) under grant agreements No.01024 and No. 202213 (European Stroke Network).

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(Accepted 1 October 2008)

lization of CNS inflammation with nuclear and optical imag-