Introduction

Neuroinflammation is thought to play a role in many neuropsychiatric and neurological conditions [1,2,3,4]; however, its exact contribution remains poorly understood [5,6,7]. This is partially due to limited diagnostic tools to measure inflammation in the central nervous system (CNS) [8]. Positron emission tomography (PET) enables non-invasive visualization and quantification of molecular targets within CNS [9]. Development of specific PET ligands for key neuroinflammatory targets would allow assessing the role of these targets in different brain diseases and aid selection of appropriate anti-neuroinflammatory treatment [10].

Recently, several targets have been investigated for PET imaging of neuroinflammation [11,12,13,14,15,16,17,18]. Specifically, multiple PET tracers binding the 18-kDa translocator protein (TSPO) have been evaluated in psychiatric [1] and neurodegenerative disorders [2, 19, 20]. There was limited success [21,22,23,24], but also failures [25,26,27,28,29,30]. Some failures were due to existing genetic polymorphisms affecting TSPO binding in humans [31,32,33]. While this limitation can be corrected [34,35,36,37], the non-specific role of TSPO in neuroinflammation are more difficult to address [9, 38, 39], as TSPO is involved in many cellular functions not specific to central inflammation [40,41,42,43,44].

In contrast, P2X7 receptor activation lies upstream of a cascade of events leading to microglial activation [45,46,47]. P2X7 receptor activation is causally linked to the production of proinflammatory cytokines within the brain [48,49,50]. Therefore, P2X7 receptor PET tracers is a promising tool to study central neuroinflammation.

Currently, a few P2X7 receptor PET tracers have been reported [46, 51,52,53,54,55]. We previously demonstrated that [18F]JNJ-64413739 is suitable to quantify central target engagement of P2X7 therapeutic compounds [56]. Here, we evaluate [18F]JNJ-64413739 as a tool to measure neuroinflammation.

To achieve this goal, we used a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide (LPS). We selected this model because (a) priming of Toll-like receptors (TLR) by LPS is one of the hallmarks of P2X7-mediated release of pro-inflammatory cytokines [57,58,59] and (b) local LPS injection results in a spatially confined region of neuroinflammation, while other, less affected regions can serve as reference [60].

We studied [18F]JNJ-64413739 uptake in the LPS injected, control (PBS-injected), and reference brain regions by in vivo PET imaging. We compared PET imaging results to the results from ex vivo ARG, immunohistochemistry (IHC), and gene expression (qPCR) analysis. We confirmed the specificity of binding of [18F]JNJ-64413739 to P2X7 receptors by performing competition studies with the P2X7 antagonist JNJ-54175446.

Materials and Methods

Details are provided in the electronic supplementary material (ESM).

Results

Selection of LPS Dose and Time Points: Longitudinal Pilot PET Imaging Studies with [18F]JNJ-64413739

Since P2X7 tracers have not been evaluated in a rat LPS local neuroinflammation model, we first conducted a pilot imaging study with [18F]JNJ-64413739 to select the optimal time points and LPS dose for subsequent imaging experiments. Details are provided in the ESM. Based on the results of this pilot experiment, 20 μg of LPS and day 2 post-LPS were selected for all subsequent experiments. Representative examples of PET images of rats injected with 0 μg of LPS (PBS control) and 20 μg of LPS, respectively, at day 1, 2, 3, and 4 post-injections are shown in Fig. 1.

Fig. 1
figure 1

Examples of in vivo PET imaging with [18F]JNJ-64413739: PET images (summed over the entire scan period) of the rat brain (coronal view, left; horizontal view, right) injected with a PBS and a rat injected with b 20 μg of LPS. Pet images are overlaid with corresponding CT images (grayscale, − 1000 to 2500 HU). Images are centered on the injection site (the corresponding craniotomies are indicated by red arrows). Images are acquired 1–4 days post-injection.

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Verification of Induced Neuroinflammatory Markers at Day 2 Post-LPS (20 μg): a qPCR Study

To confirm neuroinflammation induced by LPS in brain tissue, we used quantitative RT-PCR to measure messenger RNA (mRNA) levels of neuroinflammatory marker genes Tspo, Aif1, and P2rx7 in both LPS- and PBS-injected brains. The results of this experiment are summarized in Fig. 2. Expression levels of Tspo and Aif1 were more than 7 and 5 times higher in LPS-injected hemisphere than that in PBS-injected hemisphere, respectively; both genes had statistically significant main effects of treatment and hemisphere, and interaction effect involving both factors (Tspo gene, p (interaction) = 0.0019; p (LPS vs. PBS) = 0.0001; p (injected vs. contralateral) = 0.0019; Aif1 gene, p (interaction) = 0.0018; p (LPS vs. PBS) < 0.0001; p (injected vs. contralateral) = 0.0021). The mRNA level of P2rx7 was assessed, and its expression level was also found to be enhanced in the LPS-injected hemisphere compared with the PBS-injected hemisphere (significant effect of treatment, p (LPS vs. PBS) = 0.0002; non-significant effect of interaction or hemisphere, p = 0.18 and p = 0.40, correspondingly). The significant effect of LPS treatment found on P2X7 receptor expression indicates that this target may be a promising biomarker to detect neuroinflammation in LPS model. It should be noted that an increase of mRNA levels of all genes studied (but especially P2rx7) was detected in the contralateral hemisphere of LPS-injected compared to PBS-injected brains. This “spreading of the inflammation” may be due to the high and non-physiological level of inflammatory insult produced by local injection of relatively high dose of LPS.

Fig. 2
figure 2

Neuroinflammatory markers detected in rat brains at day 2 post-LPS (20 μg) or PBS administration: fold changes of mRNA expression of a P2rx7, b Tspo, and c Aif1 in LPS-injected hemisphere (± s.e.m. indicated by the error bar) compared with contralateral hemisphere in LPS-injected brains, as well as in PBS-injected brains. Data were normalized to mRNA levels of each gene from contralateral hemisphere in the PBS-injected mice. Data were analyzed by a two-way ANOVA followed by Bonferroni’s post hoc test.

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Concordance Between In vivo [18F]JNJ-64413739 PET Imaging, [18F]JNJ-64413739 Ex vivo ARG, and Immunohistochemistry of Iba1

To study similarities between in vivo [18F]JNJ-64413739 uptake, ex vivo ARG, and Iba1 IHC staining, all three assessments were performed in the same animal. First, rats were PET scanned with [18F]JNJ-64413739 2 days after LPS/PBS injection. Immediately after the scan, the animals were euthanized, brains extracted, and ex vivo ARG was performed on 20-μm brain sections as described in the “Materials and Methods.” Adjacent 10-μm sections were used for the IHC staining. The in vivo PET scan showed markedly increased uptake (29 %, SUVs measured 20 min post-tracer injection) of [18F]JNJ-64413739 at the LPS-injected site relative to the contralateral site in the LPS-injected rat (Fig. 3a), but not in the PBS-injected rat (Fig. 3e) (4 % higher uptake in the contralateral site than in the injected site, SUVs measured 20 min post-tracer injection). The ex vivo ARG showed a strong signal in the cortex and striatum injected with LPS (Fig. 3b), whereas a much weaker signal was detected in the corresponding site in the control (PBS-injected) animal (Fig. 3f). The difference in signals between the injected and contralateral side was 26 % in the LPS-injected rat (Fig. 3b); while in the PBS injected rat, the difference between the injected and the contralateral site constituted only 0.9 % (Fig. 3f). A similar trend was observed in the adjacent brain sections stained for Iba1 (brain section from LPS-injected rat: Fig. 3c; brain section from PBS-injected rat: Fig. 3g; the corresponding striatal regions at the center of the injections visualized with the higher magnifications are shown in Fig. 3d and h). Because IHC and ex vivo ARG procedures were performed on the same tissue sections, decreased resolution of Iba1 staining in fresh frozen brain sections prevented visualization of morphology of individual microglial cells. Nevertheless, higher magnification images showed marked difference in the intensity of Iba1 staining between LPS- (Fig. 3d) and PBS (Fig. 3h)-injected rats, suggesting an increased number of microglial cells which corresponds to the increased uptake of [18F]JNJ-64413739 both in vivo (PET scans) and ex vivo (ARG). Interestingly, in vitro ARG, in which [18F]JNJ-64413739 was directly applied to brain sections, consistently failed to detect any signal increase at the injection site (data not shown), indicating potential difference in binding in vivo vs. in vitro.

Fig. 3
figure 3

Examples of in vivo PET/CT images with [18F]JNJ-64413739 after local injection of a LPS and e PBS: coronal view centered on the center of the injection site shown by a green cursor. PET shown in color on 0 to 1.7 SUV scale, summed over the entire scan period; overlaid with corresponding CT images shown in grayscale, − 1000 to 2500 HU). Ex vivo ARG (b injected with LPS and f injection with PBS) and Iba1 immunostaining (low magnification, c and g; higher magnification, d and h; scale bar = 100 μm) on the same rat brain 2 days after local injection of LPS or PBS.

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In vivo PET Imaging Study with [18F]JNJ-64413739 in Naïve, PBS-Injected and LPS-Injected Rats

To evaluate the uptake of the [18F]JNJ-64413739 by in vivo PET scans, we statistically compared normalized uptake (SUVR, cerebellum as a reference area) of [18F]JNJ-64413739 between the injected and control sites and found significantly higher uptake of [18F]JNJ-64413739 in the LPS-injected site (37 % increase in SUVRs measured over entire scan period, n = 5, p = 0.0152, t test). In contrast, there was no statistical difference between the injected and control sites in PBS-injected group (3 % increase in SUVRs in the injected site measured over entire scan period, n = 5, p = 0.79, t test) and corresponding sites in the naïve group (1.4 % difference in SUVRs measured over entire scan period, n = 4, p = 0.22, t test).

We then compared [18F]JNJ-64413739 uptake at the injection site and control site across experimental groups (naïve, PBS-injected, and LPS-injected). Time-activity curves (TACs) in three VOIs (injection site, control site, and cerebellum as a reference area) in the naïve (n = 4), PBS-injected (n = 5), and LPS-injected (n = 5) groups are shown in Fig. 4a through c. We found no differences in [18F]JNJ-64413739 uptake between injection site of the rats injected with PBS and the corresponding site in the naïve rats (p = 0.27, t test, SUV measured over the entire scanning period). In contrast, injection sites of rats injected with LPS showed a significantly increased uptake of [18F]JNJ-64413739 relative to those of control animals injected with PBS (34 % increase in SUV measured over entire scanning period, p = 0.036, t test). Uptake in contralateral hemispheres and cerebellums were not significantly different (p = 0.80 and p = 0.96, correspondingly) between these groups of animals.

Fig. 4
figure 4

The time-activity curves (TACs) of the uptake (SUV values) of [18F]JNJ-64413739 in three volumes of interest (VOIs): injected site (or corresponding VOI in the naïve group), red line; control site, blue line; and cerebellum as a reference area, green line; the shading around the lines indicates s.e.m. in three groups of rats (a: naïve, n = 4; b: PBS-injected, n = 5; c: LPS-injected, 20 μg, n = 5). The imaging session was conducted 2 days after injection (for the PBS- and LPS-injected groups).

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Effect of JNJ-54175446 on PET Imaging with [18F]JNJ-64413739

To demonstrate specificity of increased uptake of [18F]JNJ-64413739 at the LPS-injected site, we performed blocking studies with the previously reported P2X7 antagonist JNJ-54175446 [61]. The results of the displacement study are provided in the ESM.

In pre-treatment study, 10 min before starting the PET scan, JNJ-54175446 (n = 5) or vehicle (n = 5) was administered intravenously (2.25 mg/kg) to LPS-injected rats. TACs for both groups are shown in Fig. 5a and b, while Fig. 5c shows SUVR (reference: cerebellum) values (measured 30–60 min from the scan start) at each site (“LPS-injected site” and “control site”) in each animal. The SUVR values are grouped by treatment condition (JNJ-54175446 or vehicle). Administration of JNJ-54175446 (2.25 mg/kg) significantly (p < 0.05, t test) lowered uptake (SUV) of [18F]JNJ-64413739 in both injected and non-injected hemispheres relative to the corresponding hemispheres in the vehicle-control animals; however, there was a higher decrease at the injected site than at the control site (Fig.5a–c). Correspondingly, SUVR values were significantly lower (p = 0.04, one-sided t test) at the LPS-injected site in the animals treated with JNJ-54175446 than at the LPS-injected site in vehicle-dosed animals.

Fig. 5
figure 5

TACs of SUV values of [18F]JNJ-64413739 in three VOIs (LPS-injected site, red line; control site, blue line; and cerebellum as a reference area, green line; the shading around the lines indicates s.e.m.) in two groups of LPS-injected rats (n = 5 in each group) plotted over time. Intravenous injection of JNJ-54175446 a 2.25 mg/kg, or b vehicle (blank formulation, 0 mg/kg, B) was administered 10 min prior to the scan. c SUVR (reference: cerebellum) values measured at each site (“LPS-injected site” and “control site”) in each animal (indicated by “o” marker) during a period of 30–60 min after starting the scan; the SUVR values are grouped by treatment condition (JNJ-54175446 or vehicle).

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The results of experiments with JNJ-54175446 support the finding that the [18F]JNJ-64413739 signal at the LPS-injected site is likely specific to P2X7.

Availability of Data and Materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Discussion

We evaluated the novel P2X7 antagonist PET ligand, [18F]JNJ-64413739, in a preclinical neuroinflammatory rat model. We demonstrated increased uptake of the PET ligand at the neuroinflammatory site. These findings show promise for [18F]JNJ-64413739 as a PET ligand to study P2X7 receptors in a variety of brain disorders involving neuroinflammation [3, 4]. We acknowledge that local LPS-induced changes in neuroinflammation, as described in our paper, are extreme and most likely do not represent neuroinflammatory changes in chronic CNS diseases; detailed discussion is provided in the ESM. Nonetheless, this model was set up to address some key scientific questions described here. We plan to extend these findings in more physiologically relevant models of chronic neuroinflammation.

In recent years, a few PET tracer candidates to image P2X7 receptors have been proposed [62], and some were evaluated in preclinical models of neuroinflammation [47, 63, 64]. For example, [11C]GSK1482160 showed increased tracer uptake in both systemic LPS (5 mg/kg) model in mice [47] and in rats with experimental autoimmune encephalomyelitis (EAE) [63]. These and other recent results [55, 56] are a promising demonstration of the potential of a P2X7 receptor tracer. However, short half-life of carbon-11 may restrict the clinical application of C-11-labeled PET tracers; it would be preferable to use F-18-labeled tracers. Prior to our work, only one F-18-labeled P2X7 receptor tracer, [18F]EFB [46], has been tested in preclinical models of neuroinflammation. The preliminary evaluation of [18F] EFB was promising; however, limited brain penetration and suboptimal imaging quality may limit the use of this tracer.

We conducted PET scans with [18F]JNJ-64413739 in naïve animals and animals injected either with PBS or LPS (20 μg, 2 days post-LPS). The TACs in cerebellum and contralateral site were the same in all groups of animals (Fig. 4, blue and green colors). PBS-injected sites were indistinguishable from uptake in naïve animals, indicating that the surgery did not lead to increased [18F]JNJ-64413739 uptake. In contrast, we found a statistically significant increase of [18F]JNJ-64413739 uptake in the LPS-injected site relative to the control site (37 % increase in SUVRs measured over entire scan period, n = 5, p = 0.0152, t test). To further confirm target specificity, we showed a significant blocking effect by using P2X7 antagonist JNJ-54175446 [61]. These findings support our proposal that the observed increase of [18F]JNJ-64413739 signal at the LPS-injected site is likely specific to P2X7 receptors. The increased uptake of [18F]JNJ-64413739 is most likely a consequence of LPS-induced activation of microglial cells that express P2X7 receptors, as supported by increased staining with Iba1.

To relate increased uptake of [18F]JNJ-64413739 to neuroinflammation induced by LPS in brain tissue, we measured mRNA levels of neuroinflammatory marker genes Tspo, Aif1, and P2rx7 in both LPS- and PBS-injected brains. As expected, expression levels of all genes were elevated in LPS-injected hemisphere relative to PBS-injected hemisphere (more than 7, 5, 2 times for Tspo, Aif1, and P2rx7, respectively, Fig. 2). Interestingly, we also detected increased mRNA levels of all genes studied, but especially P2rx7, in the contralateral hemisphere of LPS-injected brains. This “spreading of the inflammation” represents a novel observation, at least, for the genes studied. Further studies are needed to determine whether this effect is specific to LPS or is a general neuroinflammatory phenomenon [65].

To investigate whether increased [18F]JNJ-64413739 uptake corresponds to an increased number of microglial cells post-LPS administration, we compared [18F]JNJ-64413739 uptake and intensity of IHC staining for the microglial marker Iba1. We performed in vivo PET scans, ex vivo ARG, and IHC staining in the same animal and used adjacent brain sections for ex vivo ARG and IHC staining. We found a similar increase in signals at the LPS-injected site relative to the contralateral site: PET scans, ARG, and IHC showed a 29 %, 26 %, and 25 % increase, respectively (Fig. 3, top row). The difference between injected and contralateral site in PBS-injected control rats was smaller: PET scans, ARG, and IHC showed a 4 %, 1 %, and 12 % increase, respectively (Fig. 3, bottom row). Overall, the in vivo PET imaging, ex vivo ARG and Iba1 IHC staining showed concordant results (Fig. 3).

Conclusion

The work presented in this paper demonstrates that (1) the brain regions of rats injected with LPS had a significantly increased uptake of [18F]JNJ-64413739 relative to the PBS-injected controls; (2) the specificity of increased uptake at LPS-injected sites was confirmed by pre-treatment and displacement studies with specific P2X7 antagonist JNJ-54175446; and (3) the observed by in vivo PET increase in uptake of [18F]JNJ-64413739 at the LPS-injected site was concordant with ex vivo autoradiography (ARG), upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. While further work is needed to study [18F]JNJ-64413739 in other models of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential imaging biomarker of central neuroinflammation and is an important step toward developing PET tracer to image neuroinflammation in the clinic.