Neuroinflammation Extends Brain Tissue at Risk to Vital Peri-Infarct Tissue: A Double Tracer [ 11 C]PK11195- and [ 18 F]FDG-PET Study

Abstract

Focal cerebral ischemia elicits strong inflammatory responses involving activation of resident microglia and recruitment of monocytes/macrophages. These cells express peripheral benzodiazepine receptors (PBRs) and can be visualized by positron emission tomography (PET) using [¹¹C]PK11195 that selectively binds to PBRs. Earlier research suggests that transient ischemia in rats induces increased [¹¹C]PK11195 binding within the infarct core. In this study, we investigated the expression of PBRs during permanent ischemia in rats. Permanent cerebral ischemia was induced by injection of macrospheres into the middle cerebral artery. Multimodal imaging 7 days after ischemia comprised (1) magnetic resonance imaging that assessed the extent of infarcts; (2) [¹⁸F]-2-fluoro-2-deoxy-d-glucose ([¹⁸F]FDG)-PET characterizing cerebral glucose transport and metabolism; and (3) [¹¹C]PK11195-PET detecting neuroinflammation. Immunohistochemistry verified ischemic damage and neuroinflammatory processes. Contrasting with earlier data for transient ischemia, no [¹¹C]PK11195 binding was found in the infarct core. Rather, permanent ischemia caused increased [¹¹C]PK11195 binding in the normoperfused peri-infarct zone (mean standard uptake value (SUV): 1.93 ± 0.49), colocalizing with a 60% increase in the [¹⁸F]FDG metabolic rate constant with accumulated activated microglia and macrophages. These results suggest that after permanent focal ischemia, neuroinflammation occurring in the normoperfused peri-infarct zone goes along with increased energy demand, therefore extending the tissue at risk to areas adjacent to the infarct.

Introduction

Focal cerebral ischemia elicits strong inflammatory responses involving the rapid activation of glial cells (microglia, astrocytes) and recruitment of hematogenous cells (granulocytes, T cells, monocytes/macrophages) from the blood stream triggered by the upregulation of cell adhesion molecules, chemokines, and cytokines. These multifaceted responses follow specific temporospatial patterns (Hallenbeck et al, 1986; Schroeter et al, 1994; Schroeter et al, 1999; Mabuchi et al, 2000; Schroeter et al, 2001; Wang et al, 2008) and are termed ‘neuroinflammation.’ Although responses of inflammatory cells are only part of the complex process of neuroinflammation, in this study, we use them as a surrogate marker for the localization of neuroinflammatory processes overall. Different cell types involved in the neuroinflammatory response, namely activated microglia (Stephenson et al, 1995; Ji et al, 2008) and blood-borne cells of the monocyte-macrophage lineage (Zavala and Lenfant, 1987; Stephenson et al, 1995), express peripheral benzodiazepine receptors (PBRs). This expression pattern renders them detectable by the isoquinoline carboxamide PK11195 that selectively binds to PBRs (Benavides et al, 1983). In addition, reactive—but not resting—astrocytes demarcating the ischemic lesion from vital brain tissue have recently been shown to express PBRs (Chen et al, 2004; Rojas et al, 2007). Under physiologic conditions, PBR expression in the brain is undetectable by [¹¹C]PK11195-PET. A recent study showed that an approximately four-fold increase in PBR mRNA levels leads to detectable [¹¹C]PK11195 binding in vivo (Rojas et al, 2007); the actual receptor density that these mRNA levels go along with is currently unknown. Autoradiographic findings in the brains of stroke patients show localized [³H]PK11195 binding in the periphery of an infarct (Benavides et al, 1988).

¹¹C-labeled PK11195 enables in vivo imaging of the PBR status using positron emission tomography (PET). At variance with the above-mentioned autoradiographic study in human stroke, PET studies in human and animal strokes showed heterogeneous results regarding the localization and time course of [¹¹C]PK11195 uptake, with binding occurring within the ischemic core (Cremer et al, 1992), the peri-infarct region (Gerhard et al, 2005; Price et al, 2006), or remote areas (Pappata et al, 2000) at varying time points ranging from 3 to 30 days after ischemia. In a recent study using transient occlusion/reperfusion of the middle cerebral artery (MCA) in rats, [¹¹C]PK11195 was observed to bind to the core of the ischemic territory (Rojas et al, 2007), with a maximum at 4 to 7 days after focal ischemia. Histologic and immunohistochemical studies revealed a maximum macrophage influx at 6 to 7 days after focal ischemia, with the characteristic pattern of a bandlike macrophage accumulation around the infarct, with additional macrophages infiltrating the outer zones of the infarct, although the infarct core was relatively spared (Schroeter et al, 1994; Schroeter et al, 1997; Lehrmann et al, 1997). On the basis of these data, we chose the time point 7 days after ischemia for this study.

We hypothesized that in permanent ischemia, increased [¹¹C]PK11195 uptake marking an overexpression of PBRs would predominantly colocalize with activated microglia and macrophages in regions with preserved blood flow. We sought to characterize the metabolic situation in those areas that are subjected to inflammatory processes, to assess the risk of secondary damage of the respective tissue. To induce permanent ischemia, we used a macrosphere injection model of focal ischemia in rats that mimics arterio-arterial embolism, the leading etiology of human stroke (Mohr, 1993; Barnett et al, 2000; Grau et al, 2001; Prabhakaran et al, 2006).

Material and methods

Animals and Surgery

All animal procedures were carried out in accordance with the German Laws for Animal Protection and were approved by the local animal care committee and by the local governmental authorities. Spontaneously breathing male Wistar rats weighing 290 to 330 g (n = 5) were anesthetized with 5% isoflurane and maintained with 2.5% isoflurane in 65%/35% nitrous oxide/oxygen. Ischemia was produced by intraarterial injection of TiO₂ spheres into the MCA as described elsewhere (Gerriets et al, 2004). In brief, after exposure of the left common carotid artery, internal carotid artery (ICA), and external carotid artery, the external carotid artery and the pterygopalatine branch of the ICA were ligated. A nylon tube filled with saline and four TiO₂ macrospheres of 0.315 to 0.355 mm in diameter was inserted through the common carotid artery into the ICA until the tip of the tube was placed distal to the origin of the pterygopalatine artery. The macrospheres were inserted into the ICA by slow injection of ∼0.2mL of saline.

Radiochemistry

Radiosynthesis of [¹¹C]PK11195 was accomplished by N-methylation using the method of Camsonne et al (1984) with slight modifications. ¹¹C was produced through the ¹⁴N(p,α)¹¹C nuclear reaction using the MC16 cyclotron (Scanditronix, Uppsala, Sweden). [¹¹C]CO₂ was converted to [¹¹C]methyl iodide using the automatic synthesis system FX_C Pro from General Electric Medical Systems (GEMS, Uppsala, Sweden). [¹¹C]methyl iodide was trapped in 0.3 mL of dimethylformamide (DMF), containing 1 mg NaH as a base and 1 mg (R)-N-desmethyl-PK11195 (ABX, Radeberg, Germany). The mixture was heated at 80°C for 3 mins. After dilution with eluent (EtOH/H₂O 60/40 (v/v)), the product was isolated by preparative HPLC (Nucleosil 100 C18; Macherey-Nagel, Düren, Germany, 10 × 250 mm, flow: 3mL/min). The product fraction was diluted with 50mL of water, [¹¹C]PK11195 fixed on a C18 SPE Cartridge (strata-X, Phenomenex, Torrance, CA, USA) and eluted with 0.7 mL of EtOH. The final formulation for injection consisted of 10% isotonic EtOH solution. Radiochemical purity was >99%. The specific activity was in the range of 0.5 to 2.2Ci/ μmol. The radiochemical yield amounted to 8 to 15% (decay corrected).

[¹⁸F]-2-fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) was produced as described earlier (Hamacher et al, 1986).

Positron Emission Tomography

Seven days after the induction of ischemia, PET imaging was performed on a microPET Focus 220 scanner (Concorde Microsystems, Inc., Knoxville, TN, USA; 63 image planes; 1.5-mm full width at the half maximum). Animals were anesthetized with 5% isoflurane, maintained with 2% isoflurane in 65%/ 35% nitrous oxide/oxygen, and placed in the scanner. The temperature was monitored using a rectal probe and maintained at 37 ± 0.5°C by a warm heating pad. After a 10-min transmission scan for attenuation correction, rats received an intravenous bolus injection of [¹¹C]PK11195 (1.1 to 2.5 mCi per rat), and the emission data were acquired for 30 mins. After data acquisition, animals remained in the scanner under anesthesia. [¹⁸F]-2-fluoro-2-deoxy-D-glucose-PET was performed in the same session and without changing the position of the animal between scans, thus allowing for a precise coregistration of the resulting images. Independent measurements were ensured by using the ¹¹C-labeled radiotracer first and waiting for five half-lives before the next tracer injection; i.e., 100 mins after [¹¹C]PK11195 injection, animals received an intravenous bolus injection of [¹⁸F]FDG (1.8 to 2.3 mCi per rat), and the emission data were acquired for 60mins. After PET imaging, blood was collected from the tail vein to determine the blood glucose level. [¹¹C]PK11195-PET data were reconstructed using a two-dimensional filtered back projection of the total uptake over the 30-min acquisition time. The [¹⁸F]FDG-PET data were reconstructed in time frames of 6 × 30, 3 × 60, 3 × 120, and 12 × 240 secs. An image-derived input function was extracted from the [¹⁸F]FDG-PET data, and parametric images from the kinetic constants were determined by applying a two-tissue-compartment kinetic model with four rate constants and the blood volume as free parameters. Therefore, K1, the rate constant describing the transport of [¹⁸F]FDG from blood into the brain tissue, is related to cerebral blood flow and can be used to identify the severely hypoperfused zone after permanent ischemia. Ki = K1 × k3/(k2 + k3) is the rate constant describing the metabolization of [¹⁸F]FDG and is related to glucose consumption (Sokoloff, 1977).

Magnetic Resonance Imaging (MRI)

On day 7 after induction of ischemia, and immediately before PET imaging, rats were reanesthetized with isoflurane, and experiments were carried out on a 4.7 T BioSpec system (Bruker BioSpin, Ettlingen, Germany) with a 30-cm bore horizontal magnet, equipped with a self-shielded gradient system (max gradient: 100 mT/m; rise time: <250 μsecs). Radio frequency transmission was achieved using a Helmholtz coil (12-cm diameter), and the signal was detected using a 22-mm diameter surface coil. The animals were positioned prone in a dedicated cradle using a stereotactic headholder with the surface coil placed directly over the head. Twelve contiguous 1-mm-thick T₂-weighted coronal images of the brain were acquired (relaxation time/echo time = 3,000/ 14 msecs, 16 echoes, field of view = 3.0 cm, matrix 128 × 128) using a multislice multiecho Carr–Purcell–Meiboom–Gillsequence. During scanning, body temperature and respiration rate were monitored and maintained using a combination of in-house equipment and DASYLab (DasyLab, Moenchengladbach, Germany) version 9.0 software.

Image Analysis

For image coregistration, the image analysis software VINCI (MPI, Cologne, Germany) was used, allowing for a fast automated coregistration of all multimodal imaging data as described earlier (Cizek et al, 2004). Positron emission tomography images were additionally coregistered to anatomic data of a three-dimensional rat brain atlas constructed from the brain slices presented by Swanson (2003).

Quantitative data evaluation was based on a region of interest (ROI) analysis of PET images to determine local radiotracer uptake, K1 or Ki values, respectively, within defined areas. Circular ROIs were placed in the infarct core, infarct margin, peri-infarct zone, and in the homotopic contralateral region.

For the [¹¹CjPKlligB-PET data, a standard uptake value (SUV) was calculated for each ROI, dividing the mean ROI activity by the injected radioactive dose per body weight. Standard uptake values were individually determined and then averaged between animals.

Immunohistochemistry

After PET imaging, rats were deeply anesthetized and decapitated. The brains were rapidly removed, frozen in isopentane, and stored at −80°C before further histologic and immunohistochemical processing. Adjacent serial coronal brain sections 10- μm-thick were cut at 500- μm intervals and stained using (1) H&E (hematoxylin and eosin) to depict histology; (2) mAb against the CD11b antigen to identify activated microglia/macrophages (clone OX42, dilution 1:1,000, AbD Serotec, Oxford, UK, cat-#MCA275R); or (3) mAB against the CD68 antigen to identify phagocytic microglia/macrophages (clone ED1, dilution 1:1,000, AbD Serotec, cat-#MCA341). The H&E staining was performed according to standard protocols. For visualization of anti-CD11b and anti-CD68, the ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (Sigma, Munich, Germany) as the final reaction product was used.

Owing to a high cell density within the area of interest, and using cytoplasmatic stainings, a quantitative assessment of cell numbers was not feasible. To quantify cell accumulation in different regions, elliptic ROIs were placed in the infarct core, infarct rim, and in homotopic contralateral regions. The mean optical density of ROIs was assessed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA, Version 1.38x, http://rsb.info.nih.gov/ij/).

Statistical Analysis

Descriptive statistics and Student's t-tests were performed using Microsoft Excel 2003 (Microsoft Corp., Redmond, WA, USA); statistical significance was set at a level of <5% (P<0.05).

Results

Characterization of Macrosphere-Induced Focal Cerebral Ischemia by Multimodal Imaging In Vivo

In our macrosphere model of cerebral ischemia, four TiO₂ spheres were injected into the ICA after ligation of the pterygopalatine artery (Figure 1, arrows), leading to a permanent occlusion within the ICA-MCA vessel arborization. Using multimodal imaging, the individual infarct was characterized with MRI depicting the exact anatomic location and extent of the infarct, and with the K1 rate constant of [¹⁸F]FDG providing a functional correlate of local cerebral blood flow. Infarct sizes determined by MRI and metabolically disturbed regions determined by [¹⁸F]FDG-PET in vivo corresponded well to the respective H&E staining histology (Figure 1).

Figure 1

Multimodal imaging characterized the variable extent of macrosphere-induced cerebral ischemia in vivo. (A) Large and (B) small infarcts caused by intraarterial injection of the same number of spheres into the ICA (photographs on the left, arrows on spheres) were distinguished noninvasively by multimodal imaging, including MRI (upper rows, contours on infarcts) and [¹⁸F]FDG-K1 depicting glucose transport (middle rows; all images coronal sections). Infarct sizes assessed in vivo correlated with the respective histology (lower rows, H&E staining, objective × 1). The relative hyperintensity of bilateral dorsal brain regions compared with ventral areas results from an artifact caused by the surface coil. (C) Representative section map delineating the typical ischemic territory.

Neuroinflammatory Processes in the Macrosphere Model of Permanent Cerebral Ischemia

Neuroinflammatory processes were characterized immunohistochemically. All lesions showed pannecrosis within the infarct core. Some CD11b-positive microglia were found within the necrotic core of the ischemic lesion as well, but those showed signs of disintegration (Figure 2A, arrow). As a constitutive expression, resting microglia showed a ramified shape and faint CD11b immunoreactivity in the contralateral hemisphere. Activated microglia and macrophages, as identified by CD11b immunoreactivity, were detected in the zone surrounding the infarct, constituting a thick rim bordering the lesion. In this area, intensely CD11b-positive microglial cells showed stellate morphology with stout processes (Figure 2A). This hypercellular rim of resident and infiltrating immune cells showed a significantly higher optical density (0.36 ± 0.025) than homotopic contralateral regions (0.25 ± 0.025, P<0.05; Figure 2A), and was the histopathological feature of the ongoing neuroinflammation (Table 1). Phagocytic microglia and macrophages expressing the CD68 antigen were mostly detected within the necrotic infarct core, with the highest cell density close to the rim of the lesion in which neuroinflammation was still ongoing in the vital tissue (Figure 2B). CD68-positive microglial cells in the rim were of stellate shape with stout processes (Figure 2B, black arrowhead), whereas phagocytes of monocytic origin showed a round morphology (Figure 2B, white arrowhead). The rim of CD68-positive cells showed a significantly higher optical density (0.32 ± 0.038) than contralateral regions (0.13 ± 0.015, P<0.01; Figure 2B). Similar to CD11b-positive cells, phagocytes located within the necrotic tissues showed signs of disintegration (Figure 2B, arrow).

Table 1

Characterization of distinct local zones caused by cerebral ischemia

Term	Tissue characteristics	Description
Infarct core	K1 of [18F]FDG <0.08mL/cm3 per min PLUS Ki of [18F]FDG <0.013/min	Hypoperfused zone with disrupted energy metabolism, tissue primarily damaged by ischemia
Infarct margin	K1 of [18F]FDG <0.08mL/cm3 per min PLUS Ki of [18F]FDG ≥ 0.013 and <0.03/min	Hypoperfused tissue with preserved [18F]FDG metabolism
Peri-infarct zone	K1 of [18F]FDG > 0.08 mL/cm3 per min PLUS Ki of [18F]FDG> 0.03/min PLUS increased [11C]PK11195 uptake	Normoperfused tissue with increased [18F]FDG metabolization rate, colocalizing with neuroinflammation
Rim	Immunohistochemically defined zone with dense accumulation of immune cells	Neuroinflammatory processes putting tissue at risk for secondary damage

[¹⁸F]FDG, [¹⁸F]-2-fluoro-2-deoxy-d-glucose.

Figure 2

Characteristic neuroinflammation occurred in the macrosphere model of permanent cerebral ischemia. (A) Activated microglia identified by CD11b staining formed a thick rim around the necrotic infarct core. CD lib-positive cells within the infarct core showed signs of disintegration (arrow). Optical density in the rim was significantly higher than in homotopic contralateral regions. (B) CD68-positive phagocytes were mainly found at the lateral edge of the infarct core, constituting a rim bordering vital tissue. Microglial cells in this rim were morphologically characterized by their stellate morphology with stout processes (black arrowhead), whereas phagocytes of monocytic origin showed a round morphology (white arrowhead). Cells invading the core showed signs of disintegration (arrow). Optical density in the rim was significantly higher than in homotopic contralateral regions. Objectives × 1, × 40; bars represent means ± s.e.m.; *P<0.05, **P<0.01.

In Vivo Imaging of Neuroinflammation Using [¹¹C]PK11195 and [¹⁸F]FDG

As detailed in the Material and Methods section, both [¹¹C]PK11195- and [¹⁸F]FDG-PET were performed in the same session without changing the position of the animal between the scans, thus allowing a precise coregistration of both radiotracers. The [¹⁸F]FDG influx (K1) was used as a surrogate of local cerebral blood flow. Maps showed the absence of relevant reperfusion within the infarcted area and allowed to delineate areas of preserved perfusion (‘normoperfusion’) with a glucose transport rate constant, K1 > 0.08 mL/cm³ per min (outlined in Figure 3, left column).

Figure 3

In vivo imaging of neuroinflammation using [¹¹C]PK11195-PET: multimodal imaging identified and exactly localized neuroinflammatory processes in vivo. Left column: Mathematical modeling of [¹⁸F]FDG-influx (K1) was used to delineate the infarct by identifying the area of undisturbed cerebral blood flow (≥0.08mL/cm³ per min; white contour), matched on coronal sections of a standardized rat brain atlas. Accumulation of [¹¹C]PK11195 could be clearly identified adjacent to the infarct in rats with (A) large and (B) small infarcts caused by a permanent MCA occlusion. Middle and right columns: Immunohistochemical validation of neuroinflammatory processes using anti-CD11b (middle column) and anti-CD68 (right column, objective × 1).

Increased [¹¹C]PK11195 binding was observed only in areas of normoperfusion, but never within the ischemic core itself (Figure 3, left column). More specifically, [¹¹C]PK11195 uptake was restricted to the perfused tissue of the peri-infarct zone in all experimental animals independent of the variable location and size of the infarction. The localization of the [¹¹C]PK11195 signal corresponded well to the massive accumulation of activated microglia and macrophages as depicted by CD11b immunohistochemistry (Figure 3, middle column), and to CD68 + phagocytosing cells as far as they were located in perfused areas (Figure 3, right column).

Functional Characterization of a Distinct Neuroinflammatory Peri-infarct Zone

We used the rate constant K1 of [¹⁸F]FDG describing the transport of [¹⁸F]FDG from blood into the brain tissue as a surrogate marker for cerebral blood flow (Figure 4B, upper diagram), and the rate constant Ki to assess [¹⁸F]FDG metabolization (Figure 4B, middle diagram). On the basis of these two parameters, we were able to identify four distinct regions of the ischemic brain (Table 1; Figure 4A). The ‘infarct core’ was characterized by a significant decrease in [¹⁸F]FDG transport (K1 = 0.042 ± 0.011 mL/cm³ per min) compared with that in the ‘contralateral cortex’ (K1 = 0.099 ± 0.002 mL/cm³ per min, P < 0.01) as well as by a significantly decreased [¹⁸F]FDG metabolic rate constant (Ki = 0.009 ± 0.002 per min) compared with the contralateral side (Ki = 0.020 ± 0.003 per min, P<0.01). Adjacent to the core, an ‘infarct margin’ of tissue was defined as showing reduced [¹⁸F]FDG transport of K1 <0.08mL/cm³ per min, with a regular [¹⁸F]FDG metabolic rate constant (Ki = 0.015 ± 0.002 per min). The ‘peri-infarct zone’ adjacent to the infarct margin was characterized by preserved [¹⁸F]FDG transport (K1 = 0.099 ± 0.009 mL/ cm³ per min) and by an increased [¹⁸F]FDG metabolic rate constant (Ki = 0.032 ± 0.003 per min) that was 60% higher than in the contralateral hemisphere (Ki = 0.020 ± 0.003/min, P < 0.01).

Figure 4

Neuroinflammation occurring in a distinct peri-infarct zone puts the vital brain tissue at risk for secondary damage. [¹⁸F]FDG transport rate constant (K1) as surrogate marker for cerebral blood flow, and [¹⁸F]FDG metabolic rate constant (Ki), matched on horizontal sections of a standardized rat brain atlas were used to functionally characterize distinct regions in the ischemic brain. (A) Low cerebral blood flow and a decreased [¹⁸F]FDG metabolic rate constant characterized the infarct core (white arrow, K1 < 0.08 mL/cm³ per min, Ki < 0.013/min). The infarct margin (blue arrow) showed reduced blood flow with a regular [¹⁸F]FDG metabolic rate constant (blue contour outlines metabolic rate constant ≥ 0.013/min). Undisturbed cerebral blood flow (K1 ≥ 0.08 mL/cm³ per min, outlined by white contour) was found in the peri-infarct region (yellow arrow) and contralaterally (gray arrow). The peri-infarct zone was characterized by an increased [¹⁸F]FDG metabolic rate constant (Ki ≥ 0.03/min; green signal) that highly colocalized to elevated [¹¹C]PK11195 binding (SUV ≥ 1.2; red signal; overlay: yellow signal). (B) Distinct zones in the ischemic brain were identified by a quantitative assessment of [¹⁸F]FDG-transport (K1) as a surrogate marker for cerebral blood flow, the [¹⁸F]FDG metabolic rate (Ki), and [¹¹C]PK11195-binding (shown as means ± s.d.;**P <0.01).

Standard uptake values for [¹¹C]PK11195 were determined as a quantitative assessment of neuroinflammatory processes (Figure 4B, lower diagram). No relevant [¹¹C]PK11195 binding was detected in the infarct core (SUV: 0.65 ± 0.13) or in the infarct margin (1.02 ± 0.35) compared with that in homotopic contralateral regions (0.90 ± 0.28). Exclusively in the peri-infarct region, we observed significantly increased [¹¹C]PK11195 binding (1.93 ± 0.49) compared with that in homotopic contralateral regions (0.90 ± 0.28; P = 0.001). For all animals, the background noise stayed below an SUV of 0.97, showing a good signal-to-noise ratio for the area of microglia-macrophage accumulation, whereas SUV did not differ from noise levels in areas of the resting microglia. The neuroinflammatory processes in the peri-infarct zone as assessed by [¹¹C]PK11195-PET colocalized well with the focally increased [¹⁸F]FDG metabolization rate (Figure 4A).

Supplementary Table 1 provides the neuropathological extent of individual ischemic lesions and the respective functional data collected from distinct regions.

Discussion

Focal ischemia induces a neuroinflammatory response with an essential effect on infarct demarcation and tissue regeneration (Stoll et al, 1998; Dirnagl et al, 1999). This process is mediated by resident microglia and by hematogenous monocytes. The histologic differentiation between resident and invading cells is difficult, as there is no immunocytochemical marker to distinguish between the two populations (Flaris et al, 1993). Earlier studies have approached this problem using morphologic criteria along with a detailed histopathological time course to identify and track invading cells (Mabuchi et al, 2000). Others have used bone marrow chimeric mice to label hematogenous cells (Schilling et al, 2003), or depleted peripheral macrophages to identify the role of residential cells (Schroeter et al, 1997). All these studies agree that resident microglia predominate over invading monocytes in the dense cellular rim of the peri-infarct zone (Schroeter et al, 1997; Mabuchi et al, 2000; Schilling et al, 2003). Both cell populations play the same functional role in neuroinflammation, i.e., phagocytosing debris and producing pro-inflammatory cytokines, and therefore represent a target for therapeutic interventions. Novel therapeutic strategies aiming at neuroinflammatory processes hold promise for treating stroke patients in the future. However, the development and clinical implementation of such treatment critically depends on reliable tools to noninvasively monitor neuroinflammation in vivo (Becker, 2001; Jander et al, 2007). Both activated microglia and monocytes express PBRs, making them accessible for visualization using the radiotracer [¹¹C]PK11195 and PET. The PBR upregulation can be detected in the inflammation of the central nervous system irrespective of the etiology, including traumatic brain injury (Raghavendra et al, 2000) or experimental autoimmune encephalomyelitis (Mattner et al, 2005). In this study, we evaluate the ability of [¹¹C]PK11195-PET to precisely localize and quantify neuroinflammatory processes in a clinically relevant rat stroke model.

In the macrosphere model, permanent focal ischemia is induced by an MCA occlusion with a defined number of TiO₂ spheres (Gerriets et al, 2003). From a clinical point of view, the macrosphere MCA occlusion model mimics arterio-arterial embolism of ‘hard’ arteriosclerotic plaque material more closely than the classic occlusion method with an intraluminal thread. As arterio-arterial embolism is one of the most frequent causes of stroke, the macrosphere model seems to be a most relevant model for studying the pathophysiology of stroke (Mohr, 1993; Barnett et al, 2000; Grau et al, 2001; Prabhakaran et al, 2006).

In the macrosphere stroke model, depending on the exact macrosphere trapping within the MCA arborization, infarcts affect between 20% and 60% of the hemisphere (Gerriets et al, 2003). We used T₂-weighted MRI to morphologically assess the extent of individual lesions in vivo. For individual functional assessment, [¹⁸F]FDG-PET data were used to calculate [¹⁸F]FDG influx (K1) as a surrogate parameter for local cerebral blood flow, and the [¹⁸F]FDG metabolic rate constant (Ki) as an indicator for cerebral glucose consumption and tissue viability. This combined approach allowed us to characterize distinct regions in the ischemic brain on an individual basis, both structurally and functionally. We performed double tracer PET with [¹⁸F]FDG and [¹¹C]PK11195 in one session without moving the animal in the scanner. This enabled us to precisely relate the PBR status in any given voxel to the functional status and viability of the respective tissue, yielding data that have not been provided by other [¹¹C]PK11195-PET studies.

Characterizing metabolically distinct regions of the ischemic brain, we identified an ‘infarct margin’ directly adjacent to the (necrotic) infarct core that showed reduced blood flow—indicated by a reduction in the [¹⁸F]FDG transport rate constant (K1)—to ∼40% of the unaffected contralateral hemisphere, but had a regular [¹⁸F]FDG metabolic rate constant. This infarct margin represents that tissue well known to be at risk for secondary ischemic damage owing to metabolic starvation (Mies et al, 1993). Adjacent to the infarct margin, we defined the ‘peri-infarct zone’ with preserved blood flow indicated by a normal transport rate constant of [¹⁸F]FDG (K1), but an increased metabolic rate constant (Ki of [¹⁸F]FDG). Most interestingly, in our permanent ischemia model, neuroinflammatory processes as assessed by [¹¹C]PK11195-PET exclusively localized to this peri-infarct zone, and this result did not depend on the size and location of the infarct. This is in contrast to a recent PET study on a rat model of transient focal ischemia describing [¹¹C]PK11195 binding exclusively to the infarct core 7 days after induction of ischemia (Rojas et al, 2007). From other models of permanent ischemia, it is known that resident and infiltrating immune cells regularly form more of a rim around the ischemic core than is the case in reperfusion ischemia (Jander et al, 1995; Lehrmann et al, 1997; Schroeter et al, 1999; Schroeter et al, 2001). However, the complete lack of [¹¹C]PK11195 binding in the infarct core in our permanent ischemia model is most likely explained by a lack of reperfusion of the infarct core that prevents the radiotracer from entering into the respective tissue, which is in contrast to models that induce transient ischemia.

However, although there might be additional neuroinflammatory processes going on within the infarct core, as is also suggested by our immunohistochemical data, those events in the infarct core are of minor functional relevance with regard to secondary tissue damage and recovery of function, as the infarct core is likely to turn into necrosis in permanent ischemia. In contrast, with regard to recovery, the viability of the peri-infarct zone is of major importance, and secondary tissue damage occurring in this region might be detrimental to regeneration. In this study, we show for the first time that neuroinflammatory processes in this peri-infarct zone are accompanied by an increased metabolic rate constant of [¹⁸F]FDG that was 60% higher than that of the remaining brain tissue, indicating enhanced energy metabolism. At the same time, tissue perfusion was not increased to compensate for this increased metabolism, thus putting this tissue at risk for metabolic disturbance.

Several earlier studies in animals and in humans analyzed the localization and time course of stroke-induced neuroinflammation, and found inflammation predominantly within the ischemic core (Cremer et al, 1992; Garcia et al, 1994) or in peri-infarct regions (Gerhard et al, 2000; Pappata et al, 2000; Mabuchi et al, 2000; Price et al, 2006). As discussed, we attribute differences in localization to different experimental ischemia models or to the individual clinical situation, respectively. However, to the best of our knowledge, our study is the first to correlate the individual site of neuroinflammation to the functional, i.e., metabolic state of the respective tissue. Furthermore, using a surrogate parameter for cerebral blood flow, we show that the lack of reperfusion in the ischemic core excludes [¹¹C]PK11195 binding in permanent focal ischemia.

The complete functional repertoire of the inflammatory cells that are activated and/or recruited after stroke is still unknown. Besides the harmful effects, such as the production of cytotoxic agents and free radicals, beneficial functions include the demarcation of necrotic tissue and the promotion of tissue repair and regeneration. However, despite the potential beneficial effects that neuroinflammatory processes may promote, we propose that the focal metabolic disturbance we observe in the peri-infarct zone in combination with the concurrent destructive potential of neuroinflammation, e.g., the production of reactive oxygen species by inflammatory cells, primarily put the affected tissue at risk of secondary damage. Novel treatment strategies aimed at reducing these neuroinflammatory processes may, at least to some extent, aid in balancing this delicate equation between beneficial and detrimental effects of neuroinflammation.

Multiple attempts have been made in recent years to develop a radiotracer with higher affinity to PBRs than PK11195 (Zhang et al, 2003; Fujimura et al, 2006; Brown et al, 2007). Higher affinity suggested by the differences in dissociation constants may address some of the issues related to nonspecific binding reported with PK11195. However, in our model of permanent focal ischemia, nonspecific binding of [¹¹C]PK11195 did not pose a significant problem because of the intense and circumscribed signal of specific [¹¹C]PK11195 accumulation, with a mean SUV of 1.93 within the ROI and a good signal-to-noise ratio. We propose that although novel PBR ligands with higher binding affinity may be helpful in studying disease models with more subtle microglial activation like that associated with neurodegenerative disorders, the robust neuroinflammatory processes occurring with permanent focal cerebral ischemia can be detected unequivocally with the well-established [¹¹C]PK11195.

In brief, the exact colocalization of neuroinflammatory processes with an increased [¹⁸F]FDG metabolic rate constant as shown in this study for the first time identifies the peri-infarct zone to be at risk of secondary tissue damage by neuroinflammation and metabolic disturbances. Therefore, the peri-infarct zone may represent the ‘tissue-to-treat’ with any novel therapeutic strategies aiming at neuroinflammatory processes, and the evaluation of the differential effects of such treatments should include specifically the peri-infarct zone. The possibility of analyzing neuroinflammation noninvasively and repeatedly over time in the same experimental subject makes [¹¹C]PK11195 a promising radiotracer in the preclinical and clinical development of such treatments (reviewed by del Zoppo et al, 2000;Wang et al, 2007). Further studies using these imaging techniques might help in uncovering the functional role of inflammatory cells, the relationship between infarct core and peri-infarct zone, and factors that determine peri-infarct tissue to survive or to succumb to death. In addition, using [¹¹CjPKlligB-PET to stratify patients according to their individual neuroinflammatory patterns may help to select individuals who would benefit most from immunomodulatory treatments.