Cannabinoid receptor subtypes 1 and 2 mediate long-lasting neuroprotection and improve motor behavior deficits after transient focal cerebral ischemia
- W. Schmidta, b,
- F. Schäfera,
- V. Striggowa,
- K. Fröhlicha,
- F. Striggowa, b,
, 
- a KeyNeurotek Pharmaceuticals AG, ZENIT Technology Park, Leipziger Straße 44, D-39120 Magdeburg, Germany
- b AG Neurodegeneration and Intervention Strategies, German Center for Neurodegenerative Diseases (DZNE), Research Site Magdeburg, Leipziger Straße 44, D-39120 Magdeburg, Germany
Abstract
The endocannabinoid system is crucially involved in the regulation of brain activity and inflammation. We have investigated the localization of cannabinoid CB1 and CB2 receptors in adult rat brains before and after focal cerebral ischemia due to endothelin-induced transient occlusion of the middle cerebral artery (eMCAO). Using immunohistochemistry, both receptor subtypes were identified in cortical neurons. After eMCAO, neuronal cell death was accompanied by reduced neuronal CB1 and CB2 receptor-linked immunofluorescence. In parallel, CB1 receptor was found in activated microglia/macrophages 3 days post eMCAO and in astroglia cells at days 3 and 7. CB2 receptor labeling was identified in activated microglia/macrophages or astroglia 3 and 7 days post ischemia, respectively. In addition, immune competent CD45-positive cells were characterized by pronounced CB2 receptor staining 3 and 7 days post eMCAO. KN38-72717, a potent and selective CB1 and CB2 receptor agonist, revealed a significant, dose-dependent and long-lasting reduction of cortical lesion sizes due to eMCAO, when applied consecutively before, during and after eMCAO. In addition, severe motor deficits of animals suffering from eMCAO were significantly improved by KN38-7271. KN38-7271 remained effective, even if its application was delayed up to 6 h post eMCAO. Finally, we show that the endocannabinoid system assembles a comprehensive machinery to defend the brain against the devastating consequences of cerebral ischemia. In summary, this study underlines the therapeutic potential of CB1 and/or CB2 receptor agonists against neurodegenerative diseases or injuries involving acute or chronic imbalances of cerebral blood flow and energy consumption.
Highlights
Abbreviations
- 2AG, 2-arachidonylglycerol;
- anandamide, N-arachidonyl ethanolamide;
- DAGL, diacylglycerol lipase;
- eMCAO, endothelin-induced transient occlusion of the middle cerebral artery;
- FAAH, fatty acid amide hydrolase;
- MAGL, monoacylglycerol lipase;
- PBS, phosphate-buffered saline
Key words
- cerebral ischemia;
- endocannabinoid system;
- CB1 receptor;
- CB2 receptor;
- neuroprotection
Figures and tables from this article:
- Fig. 1. Effect of eMCAO-induced transient cerebral ischemia on the localization of CB1 receptor in the frontal cortex layer II/III. Co-staining of CB1 receptors (green fluorescence) and cell-specific marker proteins of (A–D) mature neurons (NeuN, red fluorescence), (E–H) astroglia (GFAP, red fluorescence), (I–L) microglia/macrophages (ED1, red fluorescence) or (M–P) immune competent cells (CD45, red fluorescence). (A, E, I, M) Contralateral control cortices without exposure to eMCAO and (B, F, J, N) ipsilateral cortices 24 h post eMCAO, (C, G, K, O) 3 days post eMCAO or (D, H, L, P) 7 days post eMCAO are shown. CB1 receptor-positive neurons are indicated by arrows (A–D). Astroglia was CB1 receptor-positive 3 and 7 days post eMCAO (H, arrow). Co-labeling of microglia/macrophages and CB1 receptors was detected only 3 days post eMCAO (K, arrow). CB1 receptor and CD45 staining of microglia/macrophages did not reveal any co-localization (M–P). Immunohistochemical specificity control of CB1 receptor antibody (Q, R). Brain sections were either regularly immunostained with the CB1 receptor antibody (Q) or exposed to the CB1 receptor antibody after antibody pre-adsorption with the corresponding immunizing peptide (R). Scale bars = 10 μm.
- Fig. 2. Effect of eMCAO on the cellular localization patterrn of CB2 receptors in the frontal cortex layer II/III. Co-staining of CB2 receptors (green fluorescence) and specific cell markers of (A–D) mature neurons (NeuN, red fluorecence), (E–H) astroglia (GFAP, red fluorescence), (I–L) microglia/macrophages (ED1, red fluorescence) or (M–P) immune competent cells (CD45, red fluorescence) is shown. (A, E, I, M) Contralateral control cortices without exposure to eMCAO and (B, F, J, N) ipsilateral cortices either 24 h, (C, G, K, O) 3 days or (D, H, L, P) 7 days post eMCAO were analyzed. CB2 receptors on mature neurons are indicated by arrows (A–D). CB2 receptors and astroglia were co-localized 7 days post eMCAO (H, arrow), whereas microglia/macrophages were CB2 receptor-positive 3 days after MCAO (K, arrow). CB2 receptors and CD45-positive immune competent cells were co-localized at each time point after eMCAO (M–P). Immunohistochemical specificity control of CB2 receptor antibody (Q, R). Brain sections were either regularly immunostained with the CB2 receptor antibody (Q) or exposed to the CB2 receptor antibody after antibody pre-adsorption with the corresponding immunizing peptide (R). Scale bars = 10 μm.
- Fig. 3. Dose-dependent effect of KN38-7271 on cortical infarct volumes after transient focal cerebral ischemia. KN38-7271 was consecutively administered (i.p.) 2 h prior, during eMCAO as well as 30 min, 4 h, on days 3 and 6 post eMCAO. Final drug doses as indicated. Cortical (black bars) and striatal (gray bars) infarct volumes were determined 7 days post eMCAO. Data are given as mean volume ± SEM of n = 8–24 animals per treatment group. Infarct volumes after eMCAO without or with vehicle administration were used as separate controls. Values on top of bars indicate% of infarct volume reduction compared to vehicle treatment. ∗∗∗p < 0.001 between KN38-7271- and vehicle-treated groups.
- Fig. 4. Therapeutic time window of KN38-7271 after eMCAO. KN38-7271 effects on (A) eMCAO-induced infarct volumes and (B) skilled motor behavior deficits in the ladder rung walking test were determined 7 days post eMCAO. KN38-7271 (0.1 mg/kg) was administered i.p. Time points of consecutive drug applications per treatment group: (I) 2 h before eMCAO, during eMCAO and 30 min, 4 h, 3 days, 6 days after eMCAO, (II) 30 min, 4 h, 3 days and 6 days after eMCAO, (III) 2 h, 4 h, 3 days and 6 days after eMCAO, (IV) 4 h, 3 and 6 days after eMCAO or (V) 6 h, 3 and 6 days after eMCAO. Data are given as mean ± SEM (n = 8–24 animals per treatment group). Values above bars indicate relative changes compared to vehicle administration. ∗∗∗p ⩽ 0.001 between KN38-7271- and vehicle-treated groups.
- Fig. 5. Long-term effects of KN38-7271 on ischemia-dependent motor deficits and infarct volumes. Time points of analysis after eMCAO as indicated. Both, the animal’s skilled motor behavior in the ladder rung walking test (A–C) and ipsilateral lesion sizes (D) were analyzed in the same animal cohorts throughout the experiment. KN38-7271 was administered (i.p.) 2 h before, during and 30 min, 4 h, on days 3 and 6 post eMCAO. (D) Cortical (black bars) and striatal (gray bars) infarct volumes were analyzed individually. Drug doses as indicated. Data are means ± SEM withn = 8–16 animals per treatment group. Values above bars reflect relative changes between KN38-7271- and vehicle-treated groups. ∗∗∗p ⩽ 0.001, ∗∗p ⩽ 0.01.
- Fig. 6. The endocannabinoid system mediates neuronal protection after cerebral ischemia. The following drugs were investigated: KN38-7271 (dual CB1/CB2 receptor agonist), ACEA (specific CB1 receptor agonist), JWH133 (specific CB2 receptor agonist), MAFP, a non-selective inhibitor of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), URB597 (specific FAAH inhibitor), URB602 (specific MAGL inhibitor) and RHC-80267, a specific diacylglycerol lipase (DAGL) inhibitor. Inhibition of FAAH and MAGL blocks the degradation of endogenous cannabinoids, whereas inhibition of DAGL attenuates their synthesis (see text for details). All drugs (final intracerebral concentration 10 μM) were consecutively injected (i.c.v.) during eMCAO as well as 6 h and 24 h post eMCAO. Cortical (black bars) and striatal (gray bars) infarct volumes were analyzed individually 7 days post eMCAO. Data are given as mean ± SEM, derived from 8–24 animals per treatment group. Values above bars indicate relative changes compared to vehicle treatment. ∗∗∗p < 0.001.
- Correspondence to: F. Striggow, AG Neurodegeneration and Intervention Strategies, German Center for Neurodegenerative Diseases (DZNE), Leipziger Straße 44, D-39120 Magdeburg, Germany. Tel: +49-(0)391-6117-224; fax: +49-(0)391-67-24532.
Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
