Neuroprotective effects of minocycline and KML29, a potent inhibitor of monoacylglycerol lipase, in an experimental stroke model: a small-animal positron emission tomography study

Tomoteru Yamasaki, Akiko Hatori, Yiding Zhang, Wakana Mori, Yusuke Kurihara, Masanao Ogawa, Hidekatsu Wakizaka, Jian Rong, Lu Wang, Steven Liang, Ming-Rong Zhang, Tomoteru Yamasaki, Akiko Hatori, Yiding Zhang, Wakana Mori, Yusuke Kurihara, Masanao Ogawa, Hidekatsu Wakizaka, Jian Rong, Lu Wang, Steven Liang, Ming-Rong Zhang

Abstract

Hypoxia caused by ischemia induces acidosis and neuroexcitotoxicity, resulting in neuronal death in the central nervous system (CNS). Monoacylglycerol lipase (MAGL) is a modulator of 2-arachidonoylglycerol (2-AG), which is involved in retrograde inhibition of glutamate release in the endocannabinoid system. In the present study, we used positron emission tomography (PET) to monitor MAGL-positive neurons and neuroinflammation in the brains of ischemic rats. Additionally, we performed PET imaging to evaluate the neuroprotective effects of an MAGL inhibitor in an ischemic injury model. Methods: Ischemic-injury rat models were induced by intraluminal right middle cerebral artery occlusion (MCAO). PET studies of the brains of the ischemic rats were performed at several experimental time points (pre-occlusion, days 2, 4, and 7 after the MCAO surgery) using [11C]SAR127303 for MAGL and [18F]FEBMP for 18 kDa translocator protein (TSPO, a hall-mark of neuroinflammation). Medication using minocycline (a well-known neuroprotective agent) or KML29 (a potent MAGL inhibitor) was given immediately after the MCAO surgery and then daily over the subsequent three days. Results: PET imaging of the ischemic rats using [11C]SAR127303 showed an acute decline of radioactive accumulation in the ipsilateral side at two days after MCAO surgery (ratio of the area under the curve between the ipsilateral and contralateral sides: 0.49 ± 0.04 in the cortex and 0.73 ± 0.02 in the striatum). PET imaging with [18F]FEBMP, however, showed a moderate increase in accumulation of radioactivity in the ipsilateral hemisphere on day 2 (1.36 ± 0.11), and further increases on day 4 (1.72 ± 0.15) and day 7 (1.99 ± 0.06). Treatment with minocycline or KML29 eased the decline in radioactive accumulation of [11C]SAR127303 for MAGL (minocycline-treated group: 0.82 ± 0.06 in the cortex and 0.81 ± 0.05 in the striatum; KML29-treated group: 0.72 ± 0.07 in the cortex and 0.88 ± 0.04 in the striatum) and increased uptake of [18F]FEBMP for TSPO (minocycline-treated group: 1.52 ± 0.21 in the cortex and 1.56 ± 0.11 in the striatum; KML29-treated group: 1.63 ± 0.09 in the cortex and 1.50 ± 0.17 in the striatum). In MCAO rats, minocycline treatment showed a neuroprotective effect in the sensorimotor cortex suffering from severe hypoxic injury, whereas KML29 treatment saved neurons in the striatum, including bundles of myelinated axons. Conclusions: PET imaging allowed visualization of the different neuroprotective effects of minocycline and KML29, and indicated that combination pharmacotherapy using these drugs may be an effective therapy in acute ischemia.

Keywords: KML29; MAGL; Minocycline; PET; TSPO.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1
Figure 1
PET imaging for MAGL and TSPO in the brain of MCAO rats. A: Representative 0-60 minute summed PET images of [11C]SAR127303 (upper) and [18F]FEBMP (lower) in the brain pre-occlusion and days 2, 4, and 7 after MCAO surgery. The pseudo color bar represents the level of radioactive accumulation (SUV) in the brain. B-E: TACs of [11C]SAR127303 (B, C) and [18F]FEBMP (D, E) in the contralateral (B, D) and ipsilateral (C, E) cerebral hemisphere (n = 3 for each group). F and G: The ratio of AUC0-60 min in the ipsilateral to contralateral cerebral hemisphere.
Figure 2
Figure 2
Immunofluorescence staining of rat brain sections on day 2 after MCAO surgery. Representative immunofluorescence images in the whole hemisphere (×4 magnification) are shown for MAGL (A; green), NeuN (B; red), and MBP (C; pink). D shows merged images of A-C. All images (A-D) on the top include yellow squares indicating the positions of the four close-ups. E-H: Close-up images (×20) for MAGL, NeuN, MBP, and merged stains in the cortex area of the contralateral hemisphere. I-L: Close-up images of the striatum area in the contralateral hemisphere. M-P: Close-up images of the cortex area in the ipsilateral hemisphere. Q-T: Close-up images of the striatum area in the ipsilateral hemisphere. Scale bar: 1 mm in the top images (A-D) and 50 μm in the middle and bottom images (E-T). Yellow arrows indicate a remarkable decline in MAGL signals overlapping with decreased MBP signals (T).
Figure 3
Figure 3
PET imaging for MAGL in the brains of MCAO rats treated with minocycline or KML-29, or without medication. PET images were averaged between 0 and 60 minutes after injection of [11C]SAR127303 and exhibited as coronal and horizontal views for healthy control, ischemia, minocycline-treated, and KML29-treated rats. The AUC0-60 min values in regions of interests (cortex: B; striatum: C) were calculated from time-activity curves of the contralateral and ipsilateral sides in each rat. The ratio of AUC0-60 min was calculated by dividing the AUC0-60 min value of the ipsilateral side by the AUC0-60 min of the contralateral side in each region of interest (D). Radioactivity in PET images is expressed as SUV. Values are presented are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 4
Figure 4
PET imaging for TSPO in the brain of MCAO rats with or without medication treatment. PET images were summed between 0 and 60 minutes after injection of [18F]FEBMP and are shown as coronal and horizontal slices for healthy control, ischemia, minocycline-treated, and KML29-treated rats. The AUC0-60 min values in regions of interests (cortex: B; striatum: C) were calculated from time-activity curves of the contralateral and ipsilateral sides in each rat. The ratio of AUC0-60 min was calculated by dividing the AUC0-60 min value of the ipsilateral side by the AUC0-60 min of the contralateral side in each region of interest (D). Radioactivity in PET images is expressed as SUV. Values are presented are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 5
Figure 5
Histological analysis of the neuroprotective effect of medication. A-D: Immunofluorescence staining for MAGL in brain sections of control, ischemia, minocycline-treated, and KML-29-treated rats. E-H: Immunofluorescence staining for TSPO in brain sections of each rat. Cresyl violet I-L: staining for neurons in coronal brain sections of each rat. Yellow indicates neuronal injured areas. Scale bar: 1 mm.
Figure 6
Figure 6
Our proposal for combination pharmacotherapy against ischemia. Ischemia induces two main factors, oxidative stress and neuroexcitotoxicity, which both lead to neuroinflammation. Therefore, neuroprotective effects should be enhanced by preventing both factors by using an anti-oxidative agent and an MAGL inhibitor.

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