[18F]DPA-714 PET imaging for the quantitative evaluation of early spatiotemporal changes of neuroinflammation in rat brain following status epilepticus

Most antiepileptic drug therapies are symptomatic and adversely suppress normal brain function by nonspecific inhibition of neuronal activity. In recent times, growing evidence has suggested that neuroinflammation triggered by epileptic seizures might be involved in the pathogenesis of epilepsy. Although the potential effectiveness of anti-inflammatory treatment for curing epilepsy has been extensively discussed, the limited quantitative data regarding spatiotemporal characteristics of neuroinflammation after epileptic seizures makes it difficult to be realized. We quantitatively analyzed the spatiotemporal changes in neuroinflammation in the early phase after status epilepticus in rats, using translocator protein (TSPO) positron emission tomography (PET) imaging, which has been widely used for the quantitative evaluation of neuroinflammation in several animal models of CNS disease. The second-generation TSPO PET probe, [18F]DPA-714, was used for brain-wide quantitative analysis of neuroinflammation in the brains of rats, when the status epilepticus was induced by subcutaneous injection of kainic acid (KA, 15 mg/kg) into those rats. A series of [18F]DPA-714 PET scans were performed at 1, 3, 7, and 15 days after status epilepticus, and the corresponding histological changes, including activation of microglia and astrocytes, were confirmed by immunohistochemistry. Apparent accumulation of [18F]DPA-714 was observed in several KA-induced epileptogenic regions, such as the amygdala, piriform cortex, ventral hippocampus, mediodorsal thalamus, and cortical regions 3 days after status epilepticus, and was reversibly displaced by unlabeled PK11195 (1 mg/kg). Consecutive [18F]DPA-714 PET scans revealed that accumulation of [18F]DPA-714 was focused in the KA-induced epileptogenic regions from 3 days after status epilepticus and was further maintained in the amygdala and piriform cortex until 7 days after status epilepticus. Immunohistochemical analysis revealed that activated microglia but not reactive astrocytes were correlated with [18F]DPA-714 accumulation in the KA-induced epileptogenic regions for at least 1 week after status epilepticus. These results indicate that the early spatiotemporal characteristics of neuroinflammation quantitatively evaluated by [18F]DPA-714 PET imaging provide valuable evidence for developing new anti-inflammatory therapies for epilepsy. The predominant activation of microglia around epileptogenic regions in the early phase after status epilepticus could be a crucial therapeutic target for curing epilepsy.


Introduction
Epilepsy is a chronic neurological disorder characterized by spontaneous recurrent seizures caused by neuronal hyperexcitability and hypersynchrony in the brain, and approximately 50 million people worldwide suffer from epilepsy [1,2]. Current antiepileptic drug therapy is effective in the This article is part of the Topical Collection on Neurology majority of patients with epilepsy; however, most of the treatments are symptomatic and adversely affect normal brain function by nonspecific suppression of neuronal activity [3]. Moreover, approximately one-third of patients with epilepsy are pharmaco-resistant to the currently available antiepileptic drugs [4,5], suggesting that the development of alternative pharmacological targets for curative therapy of epilepsy is strongly desired.
Accumulating evidence suggests that neuroinflammation induced by epileptic seizures is involved in the pathogenesis of epilepsy [2,[6][7][8]. Clinical and preclinical studies have demonstrated that various pro-inflammatory mediators, such as IL-1β and TNF-α, are upregulated in the epileptic tissues [9,10], and anti-inflammatory manipulations effectively suppress epileptic seizures [11,12]. Inflammation in the brain involves activation of glial cells, such as microglia and astrocytes, which have been reported to modulate neuronal excitability in epileptic foci [2,9,13,14]. The pro-inflammatory mediators released by the activated microglia have been reported to promote neuronal excitability and seizures in preclinical experiments [15]. Changes in the morphology and membrane protein composition in activated astrocytes in the epileptic foci cause functional impairment, buffering the ions, water, and neurotransmitters, which might lead to neuronal hyperexcitability and hypersynchrony [14]. These observations suggest that activated glial cells in the epileptic foci could be a curative therapeutic target for epilepsy. Although the potential effectiveness of anti-inflammatory strategies based on activated glial cells in the epileptic foci has been extensively discussed, quantitative evaluation of the spatiotemporal characteristics of glial activation following epileptic seizures is limited.
The spatiotemporal changes in neuroinflammation in the brain could be quantitatively assessed by non-invasive positron emission tomography (PET) imaging using radiolabeled molecular probes targeting specific biomarkers expressed in the activated glial cells. A mitochondrial outer membrane protein, 18 kD translocator protein (TSPO), is generally recognized as a biomarker of activated microglia in the brain due to its unique expression property; it is expressed at low levels in the resting microglia but is markedly upregulated by activation in response to neuroinflammation [16,17]. Several chemical compounds have been designed and synthesized as specific PET probes for targeting the TSPO, including carbon-11 labeled PK11195 ([ 11 C]PK11195), the first radiolabeled non-benzodiazepine-type PET probe for TSPO [18][19][20][21][22]. Due to the development of these PET probes, TSPO-PET imaging has been used extensively for the quantitative analysis of activated microglia in various diseases of the central nervous system in humans and animals, such as stroke, multiple sclerosis, Alzheimer's disease, and Parkinson's disease [23][24][25][26][27][28][29]. In line, attempts have also been made to reveal the neuroinflammation induced by epileptic seizures in animals and humans using [ 11 C]PK11195 or other second-generation TSPO ligands, including [ 18 F] DPA-714 [30][31][32][33]. However, because of limited challenges and varied investigation designs, the detailed spatiotemporal dynamics of inflammatory glial cells in the pathophysiology of epilepsy are not fully understood. In this study, we quantitatively analyzed the early spatiotemporal changes in neuroinflammation following epileptic seizures using TSPO PET imaging with [ 18 F]DPA-714 which has improved signal-to-noise ratio and better imaging sensitivity than [ 11 C] PK11195. We found that specific accumulation of [ 18 F]DPA-714 was focused in the KA-induced epileptogenic regions from 3 days after epileptic seizures and activated microglia were predominantly responsible for the [ 18 F]DPA-714 accumulation in the KA-induced epileptogenic regions.

Animals
Thirty-three male Sprague-Dawley rats (7-10 weeks old) were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan). The rats were housed in a light-and temperaturecontrolled room (approximately 22°C, lights on at 8:00 and off at 20:00) and allowed free access to food and water. All experimental animal protocols were approved by the Animal Care and Use Committee of RIKEN, Kobe Branch, and were performed in accordance with the Principles of Laboratory Animal Care (National Institutes of Health Publication No. 85-23, revised 2011). [ 18 F]DPA-714 was synthesized as reported previously by Kassiou's group [34]. The product was identified and purified by high-performance liquid chromatography on a COS-MOSIL C18-AR-II column (10 × 250 mm, Nacalai, Kyoto, Japan). The molar activity ranged from 33 to 160 GBq/µmol. The radiochemical purity analyzed by HPLC was greater than 99%.

Status epilepticus model
To assess the early spatiotemporal changes in neuroinflammation after epileptic seizures, pharmacological status epilepticus was induced by systemic administration of kainic acid (KA), the most extensively used and well-validated animal model of human temporal lobe epilepsy [35]. Rats were subcutaneously injected with KA (15 mg/kg; Cayman Chemical, Ann Arbor, MI, USA) dissolved in saline at a concentration of 7.5 mg/mL. Status epilepticus was observed for 4 hours and scored according to the Racine scale [36]. Rats showing class V seizures (rearing with falling, tonic-clonic convulsions, bouncing, and continuous automatisms) were used in PET imaging and immunohistochemistry.

PET scans
All PET scans were performed on a microPET Focus220 (Siemens, Knoxville, TN, USA), which was designed for small laboratory animals. Rats were anesthetized with 1.5% isoflurane and nitrous oxide/oxygen (7:3) and placed in the prone position in the PET scanner gantry. During the PET scan, the body temperature was maintained at 37°C using a small animal warmer connected to a thermometer (BWT-100A; Bio Research Center, Nagoya, Japan). A 60-min emission scan was performed immediately after a bolus injection of [ 18 F]DPA-714 (≈100 MBq per animal) via a cannula inserted into the tail vein; the energy window was 400-650 keV with a coincidence time window of 6 ns. Emission data were collected in the list mode and sorted into dynamic sinograms (6×10 s, 6×30 s, 11×60 s, and 15×180 s, for a total of 38 frames). The acquired data were reconstructed by standard 2D-filtered back projection (ramp filter, cutoff frequency at 0.5 cycles per pixel) after Fourier rebinning for quantification and by a statistical maximum a posteriori probability algorithm (MAP) (12 iterations with point spread function correction) for image registration.

PET image analysis
PET images were co-registered to an MRI template using the PMOD imaging processing software (version 3.6, PMOD Technologies, Zürich, Switzerland), and each region of interest (ROI) was defined according to the MRI template. PET images were expressed as standardized uptake values (SUVs). The decay-corrected mean value in each ROI was used to generate regional time-activity curves (TACs). For the quantitative analysis, the cerebellar white matter was defined as the reference region, and the binding potential (BP) was calculated using the simplified reference tissue model (SRTM) [30,37,38] in the PMOD software package.
For the correlation analysis between the CD11b-positive activated microglia and [ 18 F]DPA-714 accumulation in the brain, semi-quantitative measurement of CD11b immunohistochemical staining was performed using ImageJ software (NIH, Bethesda, MD, USA) in 4 rats which have also examined [ 18 F]DPA-714 PET scan, as previously reported [39]. Eight square regions of interest (ROIs) were delineated on the CD11b-stained coronal brain section including the epileptogenic regions ( Fig 6C). In order to quantify CD11b immunostaining, the optical density for each ROI was calculated and normalized by an internal reference ROI (red square in Fig 6C) delineated on the same coronal brain section.

Statistical analysis
All results are expressed as mean ± SE. A two-way analysis of variance (ANOVA) with Bonferroni's multiple-comparison procedure was used to assess differences in the temporal changes in BP values between the ROIs. A Pearson's test was used for correlation analysis between the CD11b immunostaining and corresponding [ 18 F]DPA-714 accumulation in the brain. Statistical significance was set at P<0.05.

Accumulation of [ 18 F]DPA-714 in the brain 3 days after status epilepticus
To assess brain-wide changes in neuroinflammation in the early phase after epileptic seizures, we performed [ 18 F]DPA-714 PET scans in rats showing class V seizures, 3 days after the subcutaneous injection of KA (15 mg/kg). As shown in Fig 1, apparent accumulation of [ 18 F]DPA-714 was observed in several KA-induced epileptogenic regions: higher accumulation was observed in the limbic structures, such as the amygdala and piriform and entorhinal cortices, as well as in the ventral hippocampus. Moderate accumulation was seen in the widespread cerebral cortical regions and mediodorsal thalamus, while no obvious accumulation was observed in the cerebellum (Fig. 1). The brain pharmacokinetics of [ 18 F] DPA-714 in these KA-induced epileptogenic regions were similar (Fig. 2). The brain uptake of [ 18

Spatiotemporal changes in [ 18 F]DPA-714 accumulation in the brain after status epilepticus
We then examined the spatiotemporal changes in neuroinflammation following epileptic seizures through four consecutive PET scans with [ 18 F]DPA-714 on 1, 3, 7, and 15 days after status epilepticus. One day after status epilepticus, the accumulation of [ 18 F]DPA-714 was barely seen in the brain, except for the cerebral ventricles and surrounding circumventricular area, similar to that in the normal rat brain (Fig. 4). As shown in Figs. 1 and 4, the apparent accumulation of [ 18 F]DPA-714 was observed in several KA-induced epileptogenic regions from 3 days after status epilepticus. The accumulation of [ 18 F]DPA-714 was more evident in the limbic regions, including the amygdala, piriform cortex, mediodorsal thalamus, and ventral hippocampus, at 7 days after status epilepticus. Quantitative analysis revealed that the BP in the ventral hippocampus and most KA-induced epileptogenic regions peaked at 3 days after status epilepticus, whereas BP in the amygdala and piriform cortex was maintained until 7 days after status epilepticus and decreased gradually thereafter (Fig. 5).

Immunohistochemistry
Finally, the activation of the microglia and astrocytes were confirmed by immunohistochemistry at 7 days after status epilepticus. As shown in Fig. 6A, abundant CD11b-positive activated microglia were observed in the KA-induced epileptogenic regions, such as the amygdala, piriform cortex, and hippocampus. In contrast, GFAP-positive activated astrocytes were broadly observed throughout the brain. Notably, activated astrocytes were rarely observed in the central area of the amygdala and dorsal thalamus, in which the high level of [ 18 F]DPA-714 accumulation was observed ( Fig  6B). These results suggest that activated microglia could be the main source of [ 18  To further confirm whether the localized microglia activation in the epileptogenic regions is correlated with the accumulation of [ 18 F]DPA-714 at 7 days after status epilepticus, we performed a semi-quantitative measurement of CD11b immunostaining and compared that with the corresponding accumulation of [ 18 F]DPA-714 in the same rats. As shown in Fig 6C, the intensity of CD11b immunostaining was significantly correlated with the accumulation of [18F] DPA-714 in these rats at 7 days after status epilepticus.

Discussion
In this study, we performed brain-wide quantitative evaluation of the early spatiotemporal changes in neuroinflammation induced by status epilepticus using [ 18 F]DPA-714 PET imaging and found that neuroinflammation was predominantly restricted to the KA-induced epileptogenic regions in the early phase after epileptic seizures. Moreover, activated microglia were predominantly responsible for the specific accumulation of [ 18  Over 50 chemical compounds have been designed and synthesized as TSPO PET probes for the quantitative assessment of inflammatory processes in the brain [18-21, 24, 26, 27, 29, 40]. [ 11 C]PK11195, the first radiolabeled nonbenzodiazepine-type PET probe for TSPO, has been extensively used as the gold standard for TSPO imaging in preclinical and clinical studies [24,28,29,40]. Nevertheless, several disadvantages have restricted its practical application in the quantitative estimation of neuroinflammation, such as poor signal-to-noise ratio, low brain uptake, and high levels of nonspecific binding [41]. The pyrazolopyrimidine compound [ 18 F]DPA-714, which has higher affinity and better blood-brain barrier permeability, was developed by Kassiou's group and has been used for the quantitative assessment of neuroinflammation in CNS diseases, including stroke [26], trauma [42], Alzheimer's disease [43], and amyotrophic lateral sclerosis [44]. Using an acute focal neuroinflammation model, Chauveau et al. [38] (Fig. 3), suggesting that [ 18 F] DPA-714 was specifically bound to TSPO even in the early phase after epileptic seizures. Considering together, these observations suggest that PET imaging with [ 18 F]DPA-714, the second-generation TSPO radioligand, is a reliable and higher sensitive imaging tool for the quantitative evaluation of changes in neuroinflammatory processes throughout the brain in the early phase of epileptic seizures.
In the present study, we successfully evaluated the early spatiotemporal changes in neuroinflammation following status epilepticus induced by systemic injection of KA, using   of seizure, especially the early phase after epileptic seizures, could be crucial for understanding the pathophysiological mechanisms of epileptogenesis and improving the treatment methods. Several TSPO PET imaging studies have attempted to reveal spatiotemporal changes in neuroinflammation following epileptic seizures [30][31][32]. Brackhan et al. [30] reported that accumulation of [ 11 C]PK11195 was observed in most brain areas until 5-7 days after status epilepticus (rats showing stage 4 or 5 seizures according to the Racine scale) induced by systemic administration of pilocarpinelithium, and was subsequently more concentrated in epileptogenic regions thereafter. Similarly, we demonstrated that significant accumulation of [ 18 F]DPA-714 was restricted to the KA-induced epileptogenic regions from 3 days after systemic KA-injection induced status epilepticus (Figs. 1 and  4). Moreover, we also clearly demonstrated the spatiotemporal difference in [ 18 F]DPA-714 accumulation between the KA-induced epileptogenic regions, such as the BP for [ 18 F] DPA-714 in most KA-induced epileptogenic regions peaked at 3 days after status epilepticus but remained until 7 days after status epilepticus in the amygdala and piriform cortex (Fig. 5). These observations suggest that [ 18 F]DPA-714 PET imaging enables to evaluate the early phase of neuroinflammation following epileptic seizures.
In the present study, we also demonstrated that activated microglia were predominantly responsible for the specific accumulation of [ 18 F]DPA-714 in the KA-induced epileptogenic regions, at least within the first week. Although TSPO was initially recognized as a specific biomarker for activated microglia [16,17], some results have also demonstrated that TSPO might also be upregulated in reactive astrocytes [27,45]. Using CNS neurodegenerative disease animal models, Ji et al. [27] clearly demonstrated that the dominant expression of TSPO (originally called PBR, peripheral benzodiazepine receptor) in the activated astrocytes was associated with minimal or reversible neuronal injury, whereas dominant TSPO expression in the activated microglia was associated with irreversible neuronal insults. Whereas, TSPO expression during epileptogenesis and chronic phase of epileptic seizures correlated well with microglial activation rather than with reactive astrocytes [30,46]. Consistent with these observations, we found that CD11b-positive activated microglia, but not GFAP-positive reactive astrocytes, were concentrated in the KA-induced epileptogenic regions, which showed a high accumulation of [ 18 F]DPA-714 (Fig. 6). Activation of microglia and astrocytes has been implicated as a crucial process in the pathophysiology of epileptogenesis [2,9,14]. Activated microglia might contribute to neuronal hyperexcitability and susceptibility to seizures through the release of inflammatory mediators, such as IL-1β and TNF-α [2,47]. Pharmacological interventions that inhibit microglial activation or block IL-1 signaling can suppress epileptic seizures [11,12]. Reactive astrocytes induced by status epilepticus have been reported to lose homeostatic buffering capabilities, which might facilitate spontaneous seizures [48,49]. Recent studies demonstrated that microglia are activated immediately after status epilepticus [47,50] and might contribute to astrocyte reactivation [51]. In line with this finding, Sano et al. [52] demonstrated that early inhibition of microglial activation after the early phase of status epilepticus suppresses astrocyte reactivation and epileptic seizures. These observations suggest that microglial activation in the early period of epileptic seizures could be a crucial therapeutic target for pharmacological intervention, and [ 18 F]DPA-714 PET imaging allows quantitative evaluation of spatiotemporal changes in microglial activation in the early phase after epileptic seizures.
Growing evidence has demonstrated that the functional role of activated microglia in CNS diseases is dependent on the varied activation states, such as the classical M1 or alternative M2 phenotypes. The M1 and M2 phenotypes of activated microglia have been shown to dynamically change with the progression of CNS diseases, such as stroke [53], multiple sclerosis [54], and Alzheimer's disease [55], and to be involved in different pathophysiological processes of these diseases, respectively. Although the M1 and M2 phenotypes of activated microglia have also been observed in   [47,56]. Since TSPO is a general biomarker for activated microglia expressed in both M1 and M2 phenotypes [56], a phenotype-specific PET ligand for activated microglia needs to be developed in the future.
In conclusion, we demonstrated that [ 18 F]DPA-714 PET imaging is a reliable and sensitive tool for the quantitative evaluation of early spatiotemporal dynamics of neuroinflammation following status epilepticus in vivo. The brainwide [ 18 F]DPA-714 PET imaging analysis showed that neuroinflammation focused in the epileptogenic regions and activated microglia were predominantly correlated with [ 18 F]DPA-714 accumulation at least for 1 week after status epilepticus. Given that most of the antiepileptic drug therapies are symptomatic and adversely affect normal brain function by suppressing neuronal activity nonspecifically, our results suggest that anti-inflammatory treatment based on microglial activation could be a curative therapeutic strategy for epileptic seizures and that [ 18 F]DPA-714 PET imaging could be a fascinating important tool for determining therapeutic windows and monitoring the therapeutic efficacy.