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

Most antiepileptic therapies are symptomatic and adversely suppress normal brain function by nonspecic inhibition of neuronal evidence that neuroinammation Although the potential effectiveness of anti-inammatory for curing epilepsy has been extensively discussed, the limited quantitative data regarding spatiotemporal characteristics of neuroinammation after epileptic seizures makes it dicult to be realized. We quantitatively analyzed the spatiotemporal changes in neuroinammation 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 neuroinammation in several animal models of CNS disease. acidic protein; IL-1β, interleukin 1beta; MRI, Magnetic Resonance Imaging; PET, positron emission tomography; ROI, region of interest; SRTM, simplied reference tissue model; SUV, standardized uptake value; TAC, time-activity curve; TNF-alpha: tumor necrosis factor-alpha; TSPO, translocator protein.


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 majority of patients with epilepsy; however, most of the treatments are symptomatic and adversely affect normal brain function by nonspeci c 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 neuroin ammation induced by epileptic seizures is involved in the pathogenesis of epilepsy [2,6,7]. Clinical and preclinical studies have demonstrated that various proin ammatory mediators, such as IL-1β and TNF-α, are upregulated in the epileptic tissues [8,9], and antiin ammatory manipulations effectively suppress epileptic seizures [10,11]. In ammation in the brain involves activation of glial cells, such as microglia and astrocytes, which have been reported to modulate neuronal excitability in epileptic foci [2,8,12,13]. The pro-in ammatory mediators released by the activated microglia have been reported to promote neuronal excitability and seizures in preclinical experiments [14]. 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 [13]. 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-in ammatory 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 neuroin ammation in the brain could be quantitatively assessed by noninvasive positron emission tomography (PET) imaging using radiolabeled molecular probes targeting speci c 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 neuroin ammation [15,16]. Several chemical compounds have been designed and synthesized as speci c PET probes for targeting the TSPO, including carbon-11 labeled PK11195 ([ 11 C]PK11195), the rst radiolabeled non-benzodiazepine-type PET probe for TSPO [17][18][19][20]. 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 [21][22][23][24][25][26]. In line, attempts have also been made to reveal the neuroin ammation induced by epileptic seizures in animals and humans using [ 11 C]PK11195 or other second-generation TSPO ligands, including [27][28][29]. However, because of limited challenges and varied investigation designs, the detailed spatiotemporal dynamics of in ammatory glial cells in the pathophysiology of epilepsy are not fully understood. In this study, we quantitatively analyzed the early spatiotemporal changes in neuroin ammation 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 speci c accumulation of [ 18 F]DPA-714 was focused in the epileptogenic regions from 3 days after epileptic seizures and activated microglia were predominantly responsible for the [ 18 F]DPA-714 accumulation in the 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 temperature-controlled 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).

Status epilepticus model
To assess the early spatiotemporal changes in neuroin ammation after epileptic seizures, pharmacological status epilepticus was induced by systemic administration of kainic acid, the most extensively used and well-validated animal model of human temporal lobe epilepsy [31]. Rats were subcutaneously injected with kainic acid (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 [32]. 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% iso urane 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-ltered back projection (ramp lter, cutoff frequency at 0.5 cycles per pixel) after Fourier rebinning for quanti cation 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 de ned according to the MRI template. PET images were expressed as standardized uptake values (SUVs). The decaycorrected mean value in each ROI was used to generate regional time-activity curves (TACs). For the quantitative analysis, the cerebellar white matter was de ned as the reference region, and the binding potential (BP) was calculated using the simpli ed reference tissue model (SRTM) [33] in the PMOD software package.

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. Statistical signi cance was set at P < 0.05.

Results
Accumulation of [ 18 F]DPA-714 in the brain 3 days after status epilepticus To assess brain-wide changes in neuroin ammation 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 kainic acid (15 mg/kg). As shown in Fig. 1, apparent accumulation of [ 18 F]DPA-714 was observed in several 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 epileptogenic regions were similar (Fig. 2). The brain uptake of [ 18 F]DPA-714 reached a peak within the rst minute after the bolus injection of radiotracer and decreased gradually thereafter in the amygdala, ventral hippocampus, and entorhinal cortex. Meanwhile, the brain uptake of 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 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 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 con rmed by immunohistochemistry at 7 days after status epilepticus. As shown in Fig. 6, abundant CD11b-positive activated microglia were observed in the 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, in which the highest level of [ 18 F]DPA-714 accumulation was observed. These results suggest that activated microglia could be the main source of [ 18 F]DPA-714 accumulation at least for 1 week after status epilepticus.

Discussion
In this study, we performed brain-wide quantitative evaluation of the early spatiotemporal changes in neuroin ammation induced by status epilepticus using [ 18 F]DPA-714 PET imaging and found that neuroin ammation was predominantly restricted to the epileptogenic regions in the early phase after epileptic seizures. Moreover, activated microglia were predominantly responsible for the speci c Over 50 chemical compounds have been designed and synthesized as TSPO PET probes for the quantitative assessment of in ammatory processes in the brain [17][18][19][20][21][22][23][24][25]. [ 11 C]PK11195, the rst radiolabeled non-benzodiazepine-type PET probe for TSPO has been extensively used as the gold standard for TSPO imaging in preclinical and clinical studies [21,22,25,26]. Nevertheless, several disadvantages have restricted its practical application in the quantitative estimation of neuroin ammation, such as poor signal-to-noise ratio, low brain uptake, and high levels of nonspeci c binding [34]. The pyrazolopyrimidine compound [ 18 F]DPA-714, which has higher a nity and better bloodbrain barrier permeability, was developed by Kassiou's group and has been used for the quantitative assessment of neuroin ammation in CNS diseases, including stroke [24], trauma [35], Alzheimer's disease [36], and amyotrophic lateral sclerosis [37]. Using an acute focal neuroin ammation model,  (Figs. 1 and 4). Furthermore, the accumulation of [ 18 F]DPA-714 in the epileptogenic regions was successfully displaced by unlabeled PK11195 (Fig. 3), suggesting that [ 18 F]DPA-714 was speci cally 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 neuroin ammatory processes throughout the brain in the early phase of epileptic seizures.
In the present study, we successfully evaluated the early spatiotemporal changes in neuroin ammation following status epilepticus induced by systemic injection of kainic acid, using [ 18 F]DPA-714 PET imaging. Elucidation of the spatiotemporal changes in neuroin ammation during different stages 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 neuroin ammation following epileptic seizures [27][28][29]. Brackhan et al. [27] reported that accumulation of [ 11 C]PK11195 was observed in most brain areas until 5-7 days after status epilepticus, and was subsequently more concentrated in epileptogenic regions thereafter. In the present study, we demonstrated that signi cant accumulation of In the present study, we also demonstrated that activated microglia were predominantly responsible for the speci c accumulation of [ 18 F]DPA-714 in the epileptogenic regions, at least within the rst week.
Although TSPO was initially recognized as a speci c biomarker for activated microglia [15,16], some results have also demonstrated that TSPO might also be upregulated in reactive astrocytes [23,39]. Using CNS neurodegenerative disease animal models, Ji et al. [23] 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 [27,40]. Consistent with these observations, we found that CD11b-positive activated microglia, but not GFAP-positive reactive astrocytes, were concentrated in the 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 [2,8,13]. Activated microglia might contribute to neuronal hyperexcitability and susceptibility to seizures through the release of in ammatory mediators, such as IL-1β and TNF-α [2,41]. Pharmacological interventions that inhibit microglial activation or block IL-1 signaling can suppress epileptic seizures [10,11]. Reactive astrocytes induced by status epilepticus have been reported to lose homeostatic buffering capabilities, which might facilitate spontaneous seizures [42,43]. Recent studies demonstrated that microglia are activated immediately after status epilepticus [41,44] and might contribute to astrocyte reactivation [45].

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.    DPA-714 in epileptogenic regions were estimated with simpli ed reference tissue model using cerebellar white matter as the reference region, at 1, 3, 7, and 15 days after status epilepticus. Data are expressed as means ± SE (n = 4 for day 1, n = 7 for day 3, n = 10 for day 7, and n = 4 for day 15, respectively, from 14 animals).

Figure 6
Immunostaining of CD11b (A) and GFAP (B) in the coronal brain sections at 7 days after status epilepticus. Dashed lines in B indicate the area shows sparse GFAP-immunoreactivity. Scale bars in the left panels of A and B indicate 1 mm, and those in the small panels of 3 in A and 2 in B indicate 100 µm, respectively. Ctr, control; SE, status epilepticus; Hi, hippocampus; Th, midline thalamus; Amg, amygdala; Pir, piriform cortex.