In vivo imaging of Α7 nicotinic receptors as a novel method to monitor neuroinflammation after cerebral ischemia

In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [11C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [18F]DPA‐714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [11C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [18F]DPA‐714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non‐treated MCAO rats, PHA‐treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.

NS14492 is a selective orthosteric a7 nAChR agonist PET radioligand that showed a cerebral binding accordingly to the distribution of these receptors in the healthy human brain (Breese et al., 1997;Ettrup et al., 2011). Nevertheless, although these receptors have shown major implications in brain disorders, the role of a7 receptors after brain diseases such as cerebral ischemia and its involvement on neuroinflammatory reaction with PET imaging is still largely unknown. For this reason, the purpose of this study was to investigate changes in the expression of a7 nAChRs in the rat brain after cerebral ischemia using PET with [ 11 C] NS14492 and immunohistochemistry. In particular, we were interested in studying the role of a7 receptors on inflammation after cerebral ischemia in rats. Thus, ischemic rats treated with the a7 nAChRs agonist PHA 568487 were subjected to in vivo PET imaging of neuroinflammation with [ 18 F]DPA-714, a specific radioligand for the translocator protein (18KDa) (TSPO), to image brain inflammation Martin et al., 2010) and to magnetic resonance imaging (MRI) for the evaluation of brain damage and blood brain barrier (BBB) permeability. Finally, real-time polymerase chain reaction (qPCR) was used to evaluate the role of a7 receptors on the expression of markers of microglia/macrophages activation and leukocyte infiltration. Hence, the results reported here provide novel information about the activity of a7 nAChRs on neuroinflammation after cerebral ischemia and might ultimately contribute to a better design of novel strategies for the treatment of neurologic diseases such as ischemic stroke.

| Animal models
Adult male Sprague-Dawley rats (n 5 70; 297 6 8.3 g body weight; Janvier, France) were used for both in vivo and ex vivo studies. Animal experimental protocols and relevant details regarding welfare and drug side effects were approved by the animal ethics committee of CIC bio-maGUNE and were conducted in accordance with the ARRIVE guidelines and Directives of the European Union on animal ethics and welfare.

| Magnetic resonance imaging
T 2 -weighted (T 2 W) MRI scans were performed in all ischemic animals at 24 hr after reperfusion to select rats presenting cortico-striatal lesions for inclusion in the PET and gadolinium enhanced T 1 -weighted (T 1 W) MRI studies of BBB permeability. Furthermore, T 2 W-MRI scans were used to evaluate the infarction volume in treated and control rats before (day 1) and after the treatment (day 7). T 1 W-MRI scans were performed in ischemic rats after the treatment to determine changes in signal intensity (expressed in %) 30 min after injection of gadolinium based contrast agent (Gadovist). MRI scans were performed under 2%-3% of isoflurane in 30/70% of O 2 /N 2 . For the evaluation of BBB integrity, the tail vein was catheterized with a 24-gauge catheter for intravenous administration of Gadovist (0.2 mmol/ml, 1 ml/kg body weight).
MRI experiments were performed on a 7 Tesla horizontal bore Bruker Biospec 70/30 MRI system (Bruker Biospin GmbH, Ettlingen, Germany). T 2 W images were acquired with a Bruker's RARE (Rapid Acquisition with Relaxation Enhancement) sequence with the following parameters: TE 5 75 ms; TR 5 4,500 ms; Rarefactor 5 8; Nav 5 2; Matrix averages; Image matrix 5 256 3 256; FOV 5 25.6 3 25.6 mm (giving an isotropic in-plane resolution of 100 mm) and covering the whole brain by 18 consecutive slices of 1 mm thickness. T 1 W images were acquired with same spatial location, orientation and resolution using also a RARE sequence with the following parameters: TE 5 20 ms; TR 5 1,000 ms; Rarefactor 5 4; Nav 5 2 averages.

| Magnetic resonance imaging image analysis
T 2 W MRI images were used to calculate brain infarction volumes.
Regions of interest (ROIs) were manually defined using Paravision 6 software for each rat on the region of increased signal in the injured hemisphere. The total lesion volume was calculated by summing the area of the infarcted regions of all slices affected by the lesion and multiplying it by the slice thickness. T 1 W-MRI images were used to calculate changes in signal intensity (in %) upon contrast agent leakage in through the affected BBB using Image J software. T 1 W-MRI images before and after contrast agent injection were co-registered and subtracted to measure pixel-by pixel any signal changes (expressed in %).
Total volume as well as mean and standard deviation signal intensity changes were measured in regions of increased signal intensity (upon a threshold of 4% considered as noise level). Incidence maps of permeability were later calculated as % incidence 5N8 Permeable pixels 3 100/Total Cerebral Volume (in pixels).

| Radiochemistry
The radiotracer [ 11 C]NS14492 was synthesized as previously described (Ettrup et al., 2011; with minor modifications) by 11 C-methylation of 4- Healthcare, Waukesha, WI, USA), based on a one-step procedure, as previously described (Damont et al., 2008;Kuhnast et al., 2012). Yields and specific activity values were equivalent to those previously reported. Radiochemical purity was above 95% at injection time.

| Positron emission tomography scans and data acquisition
PET scans were performed using a General Electric eXplore Vista CT camera (GE, Healthcare, Waukesha, WI, USA). Scans were performed in rats anaesthetized with 4% isoflurane and maintained by 2-2.5% of isoflurane in 100% O 2 . The tail vein was catheterized with a 24-gauge catheter for intravenous administration of the radiotracer. For longitudinal assessment of a7 receptors (radiotracer: [ 11 C]NS14492), animals were scanned before and during the following month after ischemia. The radioactivity (70 MBq) was injected and dynamic brain images were acquired (30 frames: 3 3 5, 3 3 10, 3 3 15, 3 3 30, 3 3 60, 6 3 120, 8 3 300 s) in the 400-700 keV energetic window, with a total acquisition time of 60 min. For evaluation of PHA 568487 treatment efficacy after ischemia, 70 MBq of [ 18 F]DPA-714 were injected at the start of the PET acquisition and dynamic brain images were acquired for a total of 30 min (23 frames 3 3 5, 3 3 15, 4 3 30, 4 3 60, 4 3 120, 5 3 180 s). After each PET scan, CT acquisitions were also performed (140 mA intensity, 40 kV voltage), to provide anatomical information of each animal as well as the attenuation map for the later PET image reconstruction. Dynamic acquisitions were reconstructed (decay and CT-based attenuation corrected) with filtered back projection (FBP) using a Ramp filter with a cutoff frequency of 0.5 mm 21 .

| Positron emission tomography image analysis
PET images were analyzed using PMOD image analysis software (PMOD Technologies Ltd, Z€ urich, Switzerland). To verify the anatomical location of the signal, PET images were co-registered to the anatomical data of a MRI rat brain template. Two type of Volumes of Interest (VOIs) were established as follows: (1) A first set of VOIs was defined to study the whole brain [ 11 C]NS14492 and [ 18 F]DPA-714 PET signal. Whole brain VOIs were manually drawn in both the entire ipsilateral and contralateral hemispheres on slices of a MRI (T 2 W) rat brain template from the PMOD software. (2) A second set of VOIs was automatically generated in the cortex and the striatum by using the regions proposed by the PMOD rat brain template, to study the evolution of

| Immunohistochemistry and cell counts
Immunohistochemistry staining was carried out before ischemia (day 0), and at days 7 and 28 after cerebral ischemia as described previously (Martin et al., 2015). The first set of sections was stained for a7 with rabbit anti-rat a7 (1:300, AbCam, Cambridge, UK), for CD11b with mouse anti-rat CD11b (1:300; Serotec, Raleigh, NC, USA) and for the

Madrid). Standardized image acquisition was performed with an Axio
Observer Z1 (Zeiss, Le Pecq, France). The number of microglia, macrophages and other infiltrated leukocytes and astrocytes immunopositive for a7 within the ischemic area was assessed at 0, 1, 3, 7, 14, 21 and 28 days after ischemia (a7 1 /CD11b 1 and a7 1 /GFAP 1 ) and at day 7 to evaluate the inflammatory effect of a7 activation (TSPO 1 /CD11b 1 and TSPO 1 /GFAP 1 ). Cells were manually counted in ten representative and different fields at 100x magnification by using Image J (NIH) software.

| Real-time quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from frozen brain tissue and cultured cells using Trizol (Invitrogen). Subsequently, cDNA synthesis was conducted using SuperScript III retrotranscriptase (200 U/ml; Invitrogen) and random hexamers as primers (Promega). Primers specific for the rat proinflammatory and anti-inflammatory markers and for chemokines were designed using Primer Express software (Applied Biosystems) at exon junctions to avoid genomic DNA amplification (see Table 1). Data was normalized to HPRT gene (best M coefficient using GeNorm Software v3.5; Vandesompele et al., 2002). qPCR reactions were performed with SYBR-Green using a BioRad CFX96 qPCR detection system as

| Statistical analyses
For PET, the percentage of injected dose per cubic centimeter (%ID/cc) within each region and time point following cerebral ischemia were averaged and compared with the averaged baseline control values (day 0) using one-way ANOVA followed by Dunnet's multiple-comparison tests for post-hoc analysis. The effect of PHA in ischemic rats was compared with control infarcted rats using an unpaired t test and the comparison within time points before and after the treatment was performed using a paired t test. Likewise, cellular expression of both microglial/a7 and astrocytic/a7 receptors before (day 0) and at days 7 and 28 after ischemia and microglial/TSPO and astrocytic/TSPO after treatment were compared using the same statistical analysis than that for PET imaging. For neurological outcome, results before (1day after MCAO) and after treatment (7 days after MCAO) were averaged and compared with baseline average values using Mann-Whitney U tests.
The effect of treatments in the mRNA levels for pro-inflammatory/antiinflammatory and leukocyte infiltration markers in sham, treated and non-treated ischemic rats were compared using a one-way ANOVA followed by Bonferroni's multiple-comparison tests for post hoc analysis.
The level of significance was regularly set at P < 0.05. Statistical analyses were performed with GraphPad Prism version 6 software.

| R E SU LTS
The levels and distribution of nicotinic a7 receptors were explored by [ 11 C]NS14492 PET imaging after 90 min MCAO and 1-28 days after reperfusion in rats. Coronal images with normalized color scale illustrate the evolution of the PET signals at control and at 1, 3, 7, 14, and 28 days after reperfusion ( Figure 1). The extent of brain damage after cerebral ischemia was assessed using T 2 W MRI at 1 day after ischemia onset. Hyperintense regions of T 2 W images represented similar infarct extents as well as locations affected. All ischemic rats subjected to nuclear studies showed cortical and striatal MRI alterations (mean 6 SD 5 301.07 6 52.75 mm 3 , n 5 8).

| [ 11 C]NS14492 after cerebral ischemia
The time course of nicotinic receptor a7 distribution was evaluated using [ 11 C]NS14492 in the ipsilateral and contralateral cerebral cortex, in the striatum and the whole brain at control (day 0) and 1, 3, 7, 14, 21, and 28 days after MCAO (Figure 1, n 5 8). All brain regions evaluated in the injured hemisphere showed a similar [ 11 C]NS14492 binding evolution after focal cerebral ischemia. In the ipsilateral whole brain (cerebrum), PET signal for [ 11 C]NS14492 showed a non-significant increase at day 1 followed by a significant PET signal increase from days 3 to 7 after ischemia in comparison to control (day 0) values (p < .05; p < .001, Figure 1f). In fact, the highest uptake was reached at day 7 following MCAO. Subsequently, the PET signal evidenced a progressive decrease from day 14 to day 28 after ischemia. In the contralateral hemisphere, [ 11 C]NS14492 PET signal at day 1 displayed a mild increase followed by a progressive increase during the first week after ischemia onset and by a decline later on (Figure 1g). The cerebral cortex in the ipsilateral hemisphere displayed a statistically significant [ 11 C]NS14492 PET signal progressive increase from day 3 to days 7-14 followed by a sharp decrease from 21 days after MCAO (p < .05; p < .01; p < .001, with respect to control animals, Figure 1h). In contrast, non-statistically significant differences were observed in the contralateral hemisphere despite the weak increase of PET signal observed after ischemia onset ( Figure 1i). The ischemic striatum showed a similar [ 11 C]NS14492 PET signal uptake over the first month after reperfusion than that observed in the cerebral cortex (p < .05; p < .001 with respect to control animals, Figure 1j). Finally, the contralateral striatum displayed a non-significant [ 11 C]NS14492 binding at day 7 after MCAO ( Figure 1k).
3.2 | Cellular expression and immunoreactivity of nicotinic a7 receptors in microglia, infiltrated leukocytes and astrocytes after MCAO Immunofluorescence staining exhibited a7 expression in a heterogeneous population of microglia/macrophages, other leukocytes and astrocytes after cerebral ischemia (Figure 2). At day 7, cells with the morphology of amoeboid reactive microglia/macrophages showed intense CD11b immunoreactivity in the lesion (in red; Figure 2b) followed by a decrease at day 28 (in red; Figure 2b). The over-reactivity of microglia/macrophages co-localized with the cellular expression of nicotinic a7 receptors (in blue and red; Figure 2d) at day 7 after ischemia. The number of a7 1 /CD11b 1 cells displayed a significant progressive increase from days 1-7 in relation to day 0 followed by a sharp decrease during the following weeks after reperfusion (p < .001, Figure   2e). Likewise, astrocytes displayed an increase of the GFAP immunoreactivity at day 28 (in white; Figure 2a) after ischemia, forming a thin astrocytic rim in the vicinity of the lesion. In addition, reactive astrocytes showed co-localization with a7 expression (in blue and white; Figure 2d) after MCAO. The number of a7 1 /GFAP 1 cells showed a significant sharp increase from days 7-28 relative to day 0 (p < .001, Figure 2f). In the contralateral hemisphere both a7 1 /CD11b 1 and a7 1 /GFAP 1 cells displayed non-significant changes at different days after cerebral ischemia (Figure 2g,h).

| Effect of nicotinic a7 receptor activation on lesion outcome and neuroinflammation after ischemia
The infarct volume and the levels and distribution of TSPO receptors were explored by MRI and PET imaging after the chronic treatment with the a7 agonist PHA 568487 (PHA) and vehicle at 7 days after MCAO (Figure 3a-f). The infarct volume measured with T 2 W-MRI at day 1 after ischemia showed similar values before the start of the treatment with PHA to avoid bias (Figure 3g). At day 7, the infarct volume showed a significant decrease in comparison to day 1 (before the treatment) in both non-treated and PHA treated ischemic rats (p < .05; p < .01, Figure 3g). Besides, the pharmacological activation of a7 receptors after ischemia displayed a significant reduction of the lesion volume relative to non-treated ischemic animals at day 7 after MCAO showed a significant decrease of [ 18 F]DPA-714 binding in the ischemic cerebral hemisphere in comparison to non-treated ischemic rats (p < .05, Figure 3h). Neurological score at day 1 after ischemia showed similar values reflecting that the groups presented similar neurological impairment before the start of the treatments (Figure 3i). At day 7, the neurological score showed an improvement in relation to day 1 and treated ischemic rats with PHA displayed a major neurofunctional improvement in relation to that showed by non-treated ischemic rats (p < .05; p < .001, Figure 3i). Immunofluorescence staining exhibited TSPO expression in CD11b and GFAP positive cells after ischemia in vehicle and PHA-treated rats (Figure 4). At day 7, cells with the morphology of amoeboid reactive microglia/macrophages and others leukocytes showed intense CD11b immunoreactivity (in red; Figure 4a) in the lesion that co-localized with TSPO receptor expression (in green and red; Figure 4c). The number of TSPO 1 /CD11b 1 cells displayed a significant decrease in treated ischemic rats with PHA at day 7 after ischemia in comparison with control ischemic rats (p < .01, Figure 4d).

| Effect of the PHA treatment on the expression of pro-inflammatory and anti-inflammatory microglia/ macrophages and leukocyte infiltration markers
We next investigated the impact of a7 receptors activation on microglia and infiltrated macrophages phenotypes after stroke. Polarized  Figure 6i). The activation of a7 receptors with the treatment displayed a significant reduction of T cell infiltration compared with non-treated ischemic rats (p < .05, Figure 6i).
3.5 | Permeability of the blood brain barrier after the activation of a7 receptor in ischemic rats Finally, the BBB permeability was explored by gadolinium contrast enhanced T 1 W-MRI at day 7 after MCAO following the daily treatment with PHA and vehicle (Figure 7). Averaged MRI images of permeability maps for all the rats included in the study (MCAO, n 5 10 and MCAO 1 PHA, n 5 9) showed that brain regions irrigated by the MCA and affected by the brain infarction become permeable to the contrast agent to some extent (Figure 7a,b). The infarct volume measured with T 2 W-MRI at day 1 after ischemia showed similar values before the start of the treatment with PHA to avoid bias ( Figure 7c). The % of permeability day 7 after reperfusion showed non-significant changes between PHA-treated and control (non-treated) ischemic rats ( Figure   7d). Overall, these results showed that the activation of the a7 nAChRs did not significantly reduce the BBB disruption after cerebral ischemia.

| D ISC USSION
The physiological mechanism termed the "cholinergic antiinflammatory pathway" described the attenuation of the systemic inflammatory response through the stimulation of the vagus nerve (Borovikova et al., 2000). Indeed, the activation of a7 nAChRs following brain injury has displayed the reduction of inflammatory cytokines release by macrophages inhibiting the inflammatory process (Wang et al., 2003;. Therefore, the in vivo imaging PET of these receptors is of vital importance since they have shown major potential implications in therapeutics of inflammation of cerebral diseases such as cerebral ischemia (Han, Li, et al., 2014;Han, Shen, et al., 2014;Kalappa, Sun, Johnson, Jin, & Uteshev, 2013;. Because of this, we have assessed the in vivo expression of a7 receptors and TSPO in reactive glial cells using PET imaging procedures, in combination with MRI, immunohistochemistry, qPCR and neurofunctional evaluation after cerebral ischemia in rats.

| PET imaging of a7 nAChRs with [ 11 C]NS14492
A previous study has shown the high BBB penetration of the a7 receptor PET radioligand [ 11 C]NS14492, an indispensable requirement for an useful PET brain tracer that is influenced by its lipophilicity (Ettrup et al., 2011). Such study also showed that the highest [ 11 C]NS14492 binding in the pig brain was in the cortical areas and thalamus, followed by a moderate binding in striatum and low binding in the cerebellum (Ettrup et al., 2011). Indeed, the biodistribution of [ 11 C]NS14492 in the pig brain was in accordance with the ex vivo distribution of a7 nAChRs in the human brain as measured with [ 125 I]-a-bungarotoxin, a selective antagonist for these receptors (Breese et al., 1997). Hence, all these findings are consistent with the [ 11 C]NS14492 distribution in the healthy rat brain before the induction of the cerebral ischemia showed in the present study (Figure 1).

| In vivo and ex vivo overexpression of a7 receptors after experimental stroke
Recently, we have demonstrated the role played by a4b2 nAChRs on neuroinflammation after cerebral ischemia in rats using in vivo PET imaging. Indeed, this study provided novel information that might contribute to a better design of anti-inflammatory strategies (Martin et al., 2015). Similarly, a7 nAChRs have been proposed as pharmacological targets with major clinical implications in the control of the inflammatory response (de Jonge & Ulloa, 2007). In the present study, a7 receptor expression was evaluated with [ 11 C]NS14492 from day 1-28 after MCAO in rats, to show its relationship to the neuroinflammatory reaction after cerebral ischemia. In the infarcted brain hemisphere, [ 11 C] NS14492 binding experienced a progressive increase at days 1-3 followed by a maximum increase at day 7 and a progressive decline from days 14-28 after reperfusion (Figure 1). These results stand in agreement with the PET imaging distribution of TSPO, a well-known marker for neuroinflammation following cerebral ischemia Martin et al., 2010;Martin et al., 2015). Hence, taking into FIG URE 5 a7 receptors activation after cerebral ischemia induces changes in mRNA expression of pro-inflammatory and anti-inflammatory microglial markers. qPCR was performed using RNA extracted from the region of the infarction at sham (control; n 5 4) and at day 7 after MCAO in vehicle (n 5 8) and PHA-treated rats (n 5 7). Expression of mRNA for pro-inflammatory markers iNOS (a), TNF (b) and CCL2 (c) and for anti-inflammatory markers Arginase (d), Mannose receptor (e), and TGFb (f) were performed after ischemia. **p < .01 and ***p < .001 compared with sham account that the TSPO overexpression is principally due to the activation of microglia/infiltrated macrophages and astrocytes (Martin et al., 2010), our results could suggest that [ 11 C]NS14492 binding increase is linked to the inflammatory response. The first step to verify this hypothesis was the characterization of the immunohistochemical characterization at days 7-28 after cerebral ischemia (Figure 2). These results confirmed the overexpression of a7 receptors in microglia/macrophages and infiltrated leukocytes at day 7 and in astrocytes at day 28 after MCAO. Therefore, these findings supported the evidence that the increase of [ 11 C]NS14492 uptake after cerebral ischemia was mainly promoted by the a7 nAChRs expression on CD11b positive cells.

| a7 receptor activation reduces tissue damage effects and attenuates inflammation after ischemia
Pharmacological activation of a7 nAChRs with the agonist PHA 568487 has previously shown the reduction of brain edema, ischemic stroke injury, neuroinflammation and oxidative stress following ischemic stroke in mice (Han, Li, et al., 2014;Han, Shen, et al., 2014;Zou et al., 2016). In our study, we explored the effect of PHA on brain edema with T 2 W-MRI and neuroinflammation by using in vivo

| Effect of PHA on pro-inflammatory/antiinflammatory microglial/macrophages
The activation of a7 receptors with PHA following cerebral ischemia in mice has been previously related to the reduction of proinflammatory and the increase of anti-inflammatory markers gene expression suggesting the direct influence of these receptors on the microglial function through the control of microglial polarization state following stroke (Han, Shen, et al., 2014). For this reason, we evaluated the mRNA expression of typical microglial markers in ischemic rats after treatment with PHA. In our study, the expression FIG URE 6 a7 receptors activation after in vivo and in vitro ischemia induce changes in RNA expression of cell-adhesion markers. qPCR for in vivo ischemia was performed using RNA extracted from the region of the infarction at sham (control; n 5 4), at day 7 after MCAO in vehicle (n 5 8) and PHA-treated rats (n 5 7). qPCR for ex vivo ischemia was performed using RNA extracted from bEnd.3 cell culture at control (n 5 3), control with PHA (n 5 3), ischemia (n 5 3) and ischemia with PHA (n 5 3). Expression of mRNA for leukocyte infiltration markers E-selectin (a and b), P-selectin (b and f), ICAM (c and g), VCAM (d and h) and CD3 (i) were performed after ischemia. *p < .05, **p < .01, and ***p < .001 compared with control, # p < .05, ## p < .01 and ### p < .001 compared with treatment COL AS ET AL.

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of known pro-inflammatory markers (iNOS, TNF and CCL2) together with anti-inflammatory markers (arginase, mannose and TFGb) showed a significant increase in relation to sham-operated rats ( Figure 5). After treatment with PHA, both pro-inflammatory and anti-inflammatory marker levels experienced a similar nonsignificant decrease in relation to non-treated ischemic rats with the exception of arginase that displayed similar gene expression after a7 receptor activation ( Figure 5D). Our results stand in contrast with the alteration of microglia/macrophage polarization by the administration of PHA immediately after permanent MCAO observed by Han and collaborators (Han, Shen, et al., 2014) that might be argued by the use of a different experimental design, MCAO model and rodent specie. Despite this, it must be pointed out that the chemokine (C-C motif) ligand 2 (CCL2) displayed the most noticeable expression decrease after a7 nAChRs activation.
CCL2 is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system inflammation (Selenica et al., 2013). Therefore, these results motivated the evaluation of the potential role of a7 receptor on the expression of leukocyte infiltration markers in the injured brain after cerebral ischemia.

| Effect of a7 receptor activation on leukocyte infiltration markers and permeability of the BBB
To tackle this concern we studied the effect of PHA on the mRNA expression of cell adhesion molecules as E-selectin, P-selectin, ICAM and VCAM following in vivo and in vitro cerebral ischemia ( Figure 6).
Following MCAO, all cell adhesion molecules showed an expression increase that was severely reduced after the treatment with PHA for all the receptors studied. Similarly, in vitro ischemia on bEnd.3 cells, a cerebral endothelial cell line with characteristics of the BBB (Brown et al., 2007), showed an increase of selectines and CAMs after the induction of in vitro ischemia that was reverted after the activation of a7 receptors ( Figure 6). In addition, the activation of a7 receptors with PHA promoted the decrease of the expression of CD3 marker, a component of the T lymphocyte co-receptor commonly expressed in all T cells ( Figure 6). These results stand in agreement with the control of a7 Serial cerebral images of permeability maps using Gadovist contrast agent following cerebral ischemia. Permeability maps were co-registered with MRI-T 1 W to localize anatomically the permeability of the BBB in vehicle (a) and PHA-treated (b) ischemic rats at day 7 after cerebral ischemia. Infarct volume measured with T 2 W-MRI at day 1 after ischemia (c). The % of permeability was evaluated at day 7 after PHA treatment (n 5 9) in relation to vehicle (n 5 10) treated ischemic rats (d) [Color figure can be viewed at wileyonlinelibrary.com] properties (Jones et al., 2018). Therefore, the reduction of infiltrated T cells after treatment with PHA could promote the improvement of the neurological outcome after cerebral ischemia. Likewise, TSPO expression has been described in other inflammatory cells such as T lymphocytes (Zheng et al., 2011), hence, the decrease of TSPO levels after PHA treatment observed in this study could be also influenced by the decrease of T cell infiltration. Furthermore, the reduction of the expression of different leukocyte infiltration markers might also promote the decrease of both macrophage infiltration and activation of microglia as previously showed with in vivo and ex vivo studies (Figures 4 and 5).
Despite this, other mechanisms such as the leakage of the BBB can enhance the infiltration of leukocytes into the brain parenchyma following stroke. Indeed, after ischemic stroke BBB is usually damaged due to the complex process driven by the cascade of mediating factors (Merali, Huang, Mikulis, Silver, & Kassner, 2017). For this reason, we evaluated the effect of PHA on BBB integrity by MRI and the contrast agent Gadovist at day 7 after cerebral ischemia (Figure 7). MRI images showed similar percentage of permeability values in both non-treated and PHA-treated ischemic rats ( Figure 7d). Therefore, these results confirmed that the activation of the a7 receptors did not reduce the BBB disruption after cerebral ischemia evidencing the role of these receptors on the recruitment of leukocytes through the control on adhesion molecules expression of the cerebrovascular endothelium.
In summary, PET imaging with [ 11 C]NS14492 was carried out to evaluate the role of a7 nAChRs on neuroinflammatory response after experimental ischemic stroke in rats. The present findings showed [ 11 C] NS14492 binding increase in the injured hemisphere during the first week after MCAO followed by a progressive decrease later on. a7 receptor overexpression was identified in microglia and infiltrated macrophages at day 7 after ischemia. Besides, the activation of a7 nAChRs was able to promote a decrease of inflammation, volume of infarction, expression of cell adhesion molecules, decrease of T cell infiltration and the improvement of the neurological outcome after MCAO in rats.
These results provide valuable knowledge regarding the role of nicotinic a7 receptors on inflammatory reaction after brain ischemia that might contribute to the discovery of a novel target for theranostics of ischemic stroke.