Background
Microglia change morphologically and functionally from their resting state to their activated state in response to several neuroinflammatory and neurodegenerative diseases. In other words, microglial activation accompanies any damage to the brain environment. This is one reason why activated microglia represent an important marker of neuroinflammation [
1]. Specifically, non-invasive imaging of activated microglia is a useful tool to detect in vivo neuroinflammatory disease. To date, the first-generation translocator protein 18 kDa (TSPO) marker [
11C](
R)PK11195 has been widely used as a PET radioligand for that purpose. PET studies using [
11C](
R)PK11195 have been carried out on patients with several neuroinflammatory diseases such as Alzheimer’s disease (AD) [
2], Parkinson’s disease (PD) [
3], and Huntington’s disease [
4]. In addition, ischemic stroke causes direct insults to brain tissue and the immediate activation of microglia, which can also be visualized on PET with [
11C](
R)PK11195 [
5]. This is an experimental advantage in studies on time course changes in microglial activation, unlike animal models for chronically developed neurodegenerative brain disorders. Specifically, since stroke is a sudden onset disease, an early depiction of the extent at which the brain is compromised is important for treatment to delay the disease progression. In this context, TSPO imaging may be preferable. To date, it is well documented that the accumulation of [
11C](
R)PK11195 or the second-generation tracer [
18F]DPA714 is reported to occur relatively later after brain injury resulting from toxin injection [
6] or traumatic insults [
7,
8].
The imaging of endocannabinoid receptor type 2 (CB2) has recently been considered to be an alternative method for targeting activated microglia using PET imaging. The endocannabinoid system in the central nervous system has been identified to provide neuroprotective effects following brain injury. In particular, CB2 is upregulated in microglia during neurodegenerative and neuroinflammatory diseases such as AD [
9‐
11], multiple sclerosis [
12], PD [
13] and ischemia [
14‐
17], and is associated with microglial activity [
18]. Moreover, the administration of a selective CB2 agonist reduced infarct volume and improved motor function scores [
16]. Recently, a PET tracer that binds specifically to CB2 was developed to illustrate CB2 availability in vivo [
19]. Although it was reported that the binding of [
11C]NE40 24 h after ischemic injury using a photochemically induced thrombosis (PIT) technique failed to increase [
20], six out of nine PET data were not corrected for the attenuation of radioactivity, and the correction is critical for the quantification of PET data. Considering the early-developed neuroprotective role of microglia, a failure to detect the elevation of [
11C]NE40 uptake in AD patients [
21] is understandable because the activated microglia during the chronic state of neurodegeneration are considered to be acting as proinflammatory agents [
22]. Therefore, the stroke model might be suitable for evaluating the early response of microglia because the insult can be controlled purposefully.
In neuroinflammation, microglia are considered to play a dual role in their function. Classically activated M1 phenotype microglia release proinflammatory mediators, whereas alternatively activated M2 phenotype microglia enhance phagocytic activity and reduce the production of inflammatory mediators [
23,
24]. In our previous study, we demonstrated that neuronal damage occurred on days 1, 3, 7, and 14 after PIT using [
11C](
R)PK11195 [
25]. The uptake peak of [
11C](
R)PK11195 was found on day 7 after PIT treatment and overlapped with the high immunoreactivity area of microglial marker Iba1, suggesting that the uptake of [
11C](
R)PK11195 reflects microglial activity. However, it is difficult to distinguish between neurotoxic M1 and neuroprotective M2 microglia phenotypes on PET imaging with [
11C](
R)PK11195. Then, we focused on [
11C]NE40, which targets CB2, with the goal of observing neuroprotective microglia at an acute stage of brain injury.
Therefore, the purpose of the present study was to focus on the early phase of stroke and to elucidate the different binding patterns of CB2 and TSPO tracers in a very acute stage of brain insult, such as stroke, by comparing the levels of [11C]NE40 binding with those of [11C](R)PK11195 binding in rats after PIT surgery. These in vivo data were evaluated with immunohistochemistry for CB2, TSPO, and several cell type-specific markers after completion of the PET measurements, resulting in cessation of the in vivo study with the same animals.
Methods
Animals
Eight 8-week-old male Sprague-Dawley rats (250–300 g) purchased from the SLC Company (Hamamatsu, Japan) were used in this study. They were housed in cages with free access to food and water. All animal protocols and the following experiments were approved by the Ethics Committees of the Central Research Laboratory at Hamamatsu Photonics and Hamamatsu University School of Medicine. In addition, all applicable institutional and/or national guidelines for the care and use of animals were followed.
The PIT procedure was performed as reported previously [
26]. Briefly, the rats were anesthetized and maintained with 2% halothane in a mixture of 70% room air and 30% O
2 throughout the following procedure. After the insertion of an infusion line into the tail vein, the scalp and temporal muscle were flipped, and then a subtemporal craniotomy was performed. The main trunk of the left middle cerebral artery (MCA) was observed through the dura mater under an operating microscope through a window that was anterior to the foramen of the mandibular nerve. After the intravenous infusion of 20 mg/kg of rose bengal (Wako Pure Chemical Industry, Osaka, Japan), photoillumination was performed utilizing a green light at 540 nm (model L4887, Hamamatsu Photonics, Hamamatsu, Japan), which was delivered to the MCA through the dura mater for 10 min using a 3-mm optic fiber placed onto the window within the skull base. After confirming thrombotic occlusion of the MCA, the incision was closed. The animals were then allowed to awake from anesthesia and were returned to their cages.
PET measurements
We utilized a high-resolution animal PET scanner (SHR-38000, Hamamatsu Photonics, Japan) under an axial field of view (FOV) of 330 mm, a transaxial FOV of 108 mm, and a transaxial spatial resolution of 2.3 mm in the center. Eight animals were scanned twice a day using PET with [11C](R)PK11195 and [11C]NE40 beginning 24 h after PIT. The interval between the two scans was set to 2 h, and the order of the scans was counterbalanced. The animals were anesthetized using an initial dose of chloral hydrate (400 mg/kg, i.p.) followed by a continuous infusion of chloral hydrate (100 mg/kg/h, i.v.) during the entire imaging experiment. They were placed in the prone position on a fixation plate and then set within the gantry hole of the PET scanner. After a 15-min transmission scan utilizing an external 68Ge/68Ga rod source (67 MBq) for attenuation correction, a serial emission scan that lasted for 60 min was performed immediately following each tracer injection of [11C](R)PK11195 or [11C]NE40 at a dose of 48 MBq/kg; tracers were injected intravenously through the cannula that was inserted into the tail vein. The specific activity of each tracer used was above 50 GBq/μmol. No arterial sampling was conducted. The PET data were reconstructed using 3D DRAMA (iteration 2, gamma 0.1) with a Gauss filter of 1.0 mm in full width at half maximum (FWHM), yielding a voxel size of 0.65 × 0.65 × 1.0167 mm for the reconstructed image. To obtain the anatomical information, X-CT scans were performed immediately following PET measurement using a ClairvivoCT (Shimadzu Corporation, Kyoto, Japan).
Data analysis and statistics
Using an image analysis software (PMOD, version 3.1; PMOD Technologies Ltd, Zurich, Switzerland), we estimated the levels of BP
ND (an availability of TSPO and CB2) and R1 (a tracer influx index according to blood flow: a ratio of K1/K′1, or an influx in target of interest/an influx in reference of interest) for [
11C](
R)PK11195 and [
11C]NE40 based on a simplified reference tissue model [
27,
28] and then created the parametric brain images of BP
ND and R1 (a tracer uptake index). During this process, the time-activity curve from the bilateral cerebellar cortex was used as a reference input function because the infratentorial brain region might be less affected by the MCA occlusion event; however, functional connection emerging as diaschisis could be a possibility of the confounding factor. The selection of the intact contralateral cortical region as a reference region as conducted elsewhere [
20] seems inadequate because the numbers of the CD11b+/CD3+ cells (for microglia) were comparable in ischemia-affected and non-affected hemispheres 24 h after stroke [
29].
As described elsewhere [
30,
31], the elliptical regions of interest (ROIs), ranging from 12 to 24 mm
2 wide, were symmetrically placed in the bilateral brain regions covering the peri-infarct area mainly in the frontal and parietal cortices by referring to the X-CT images, on which the coronal slices of the frontal and parietal cortices were determined according to the Paxinos rat brain stereotactic atlas [
32] (see Additional file
1: Figure S1).
Student’s t test statistics were used to compare the conditions and the significance level was set at p < 0.05 with a correction for multiple comparisons because multiple loci were chosen.
Immunohistochemistry
The rats were anesthetized with chloral hydrate (400 mg/kg) and then transcardially perfused with saline followed by 4% paraformaldehyde (PFA) (pH 7.4). The brains were removed, post-fixed in 4% PFA, and immersed in cryoprotectant solution (30% sucrose in 0.1 M phosphate buffer) until the tissue sank. Tissues were frozen in dry ice and stored at −80 °C until they were used. Twenty-micrometer frozen coronal sections were cut using a cryostat. The slides were blocked with 5% goat serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature (RT) and then incubated with primary antibodies for 2 h at RT. After being washed, the slides were then incubated for 2 h at RT with secondary antibodies. The following primary antibodies were used in this study: mouse anti-GFAP (1:500, Millipore), rabbit anti-Iba1 (1:1000, Wako), mouse anti-CD11b/c (1:500, DB Pharmingen), mouse anti-NG2 (1:200, Millipore), rabbit anti-cannabinoid receptor 2 (1:100, Abcam), and rabbit anti-rat TSPO (1:200, Biobyt). The following secondary antibodies were used in this study: Alexa Fluor 488 anti-rabbit-IgG and Alexa Fluor 594 anti-mouse-IgG (1:500, Invitrogen).
Conclusions
The present increase in [
11C]NE40 binding, but not [
11C](
R)PK11195 binding concomitant with a higher CB2 immunochemical expression within the microglia over the peri-infarct region early after PIT injury, indicates that acutely activated microglia in early-stage ischemic stroke might be involved in the neuroprotective process of neuroinflammation. While a recent new CB2 tracer [
11C]A836339 has been reported to exceed the sensitivity of [
11C]NE40 to bind to CB2 under the chronic state of neurodegeneration in vivo [
9], the present result suggests that [
11C]NE40 might be adequate for depicting activated microglia at a very early stage of brain disorders.
Acknowledgements
The authors would like to thank Dr. Norihiro Harada (Hamamatsu Photonics KK) and Drs. Sumiko Mikawa and Hiromu Furukawa (Department of Neuroanatomy and Neuroscience, Hamamatsu University School of Medicine) for their excellent support.