Preservation of spatial memory and neuroprotection by the fatty acid amide hydrolase inhibitor URB597 in a rat model of vascular dementia

Abstract

Background: Chronic cerebral hypoperfusion (CCH) is a major risk factor for vascular dementia (VaD). There are currently no broadly effective prevention or treatment strategies for VaD, but recent studies have reported promising results following vascular bypass surgery and pharmacomodulation of the brain endocannabinoid system (ECS). In this study, early effects of encephalomyosynangiosis (EMS) bypass surgery and augmented endocannabinoid signaling on CCH-induced cognitive dysfunction and neuronal damage were investigated.

Methods: An animal model of VaD was established by bilateral common carotid artery occlusion (BCCAO). Cannabinoid signaling was upregulated by treatment with the fatty acid amide hydrolase inhibitor URB597 (URB). Spatial learning and memory, cerebral blood flow (CBF), revascularization, brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling, and apoptosis were compared among Sham, BCCAO, BCCAO + EMS, BCCAO + URB, and BCCAO + URB + EMS groups. Spatial learning and memory were evaluated using the Morris water maze (MWM). The CBF in cortex and hippocampus was evaluated by 3-dimensional arterial spin labeling. The neovascularization was visualized by CD34 immunofluorescence staining, and BDNF-TrkB signaling protein expression levels were assessed by Western blotting.

Results: Treatment with URB597 but not EMS alone reversed the spatial learning and memory deficits induced by BCCAO. Neovascularization was enhanced after EMS surgery but not by URB597. Alternatively, there were no significant differences in CBF among treatment groups. Expression levels of BDNF and TrkB were significantly reduced by CCH compared to Sham treatment, and downregulation of both proteins was reversed by URB597 treatment but not EMS. BCCAO enhanced neuronal apoptosis, which was also reversed by URB597.

Conclusions: Augmentation of endogenous cannabinoid signaling but not EMS protects against CCH-induced neurodegeneration and preserves spatial learning and memory, possibly by activating BDNF-TrkB signaling.

Keywords: BDNF; chronic cerebral hypoperfusion (CCH); cognitive dysfunction; encephalomyosynangiosis (EMS); endocannabinoid system (ECS).

Figure 1

Encephalomyosynangiosis (EMS) surgery. (A) Schematic diagram of EMS. (B,C,D) Surgical procedure of EMS. C, cortex, T, temporalis.

Figure 2

Effects of EMS and URB597 on chronic cerebral hypoperfusion (CCH)-induced spatial learning and memory deficits. (A) Representative swimming paths of rats in the probe trial. (B) Escape latencies during the training trials for the Sham, bilateral common carotid artery occlusion (BCCAO), BCCAO+EMS, BCAAO+URB, and BCAAO+URB+EMS groups. CCH markedly suppressed spatial learning (indicated by decreased escape latency), an effect reversed by URB597 (with or without EMS surgery) but not by EMS alone. (C,D) Numbers of platform location crossings and time spent in the target quadrant. These measures indicated significant impairment of spatial memory by CCH and reversal by URB597 but not EMS. (E) Swimming speed during the test. *P<0.05 vs. Sham; ^#P<0.05 vs. BCCAO; ^△P<0.05 vs. BCCAO+EMS. (B: F_4,160 =66.72, P<0.001; C: F_4,35 =9.324, P<0.001; D: F_4,35 =7.216, P<0.01; E: F_4,35 =1.343, P>0.05). EMS, encephalomyosynangiosis.

Figure 3

Effects of BCCAO, EMS, and URB597 on cortical and hippocampal neovascularization. (A) Immunopositivity to the endothelial cell marker CD34, indicative of neovascularization (green), was found at the cortical-temporal muscle interface following EMS surgery (red line) (magnification: 200×); (B) compared with the BCCAO group, the number of CD-34-positive microvessels (MVNs) was increased in the surgical area of cortex in EMS group rats (magnification: 200×). (C,D) Histogram showing MVN for each group. EMS but not URB957 enhanced MVN. ^#P<0.05 vs. BCCAO; ^▲P<0.05 vs. BCCAO+URB. (C: F_4,35 =4.287, P<0.05; D: F_3,28 =2.713, P>0.05). EMS, encephalomyosynangiosis; BCCAO, bilateral common carotid artery occlusion.

Figure 4

Effects of EMS and URB957 on the BCCAO-induced reduction in cerebral blood flow. (A) Sagittal images with anatomic coordinates. The red line is 2 mm caudal to bregma; (B) the level of T2 and 3D-ASL is selected (2 mm from bregma). Green represents the weakest and red the strongest CBF signal. (C,D) Quantitative analysis of CBF in the cortex and hippocampus. BCCAO significantly reduced CBF compared with the Sham group. Neither EMS nor URB597 reversed the effect of BCCAO. *P<0.05 vs. Sham. (C: F_3,28 =1.229, P>0.05; D: F_3,28 =1.308, P>0.05). EMS, encephalomyosynangiosis; BCCAO, bilateral common carotid artery occlusion; CBF, cerebral blood flow.

Figure 5

Effects of CCH, EMS, and URB597 on BDNF and TrkB expression levels. Lane numbers indicate protein samples from [1] Sham, [2] BCCAO, [3] BCCAO+EMS, [4] BCCAO+URB, and [5] BCCAO+EMS+URB groups. (A,B) Representative western blot of BDNF and TrkB; (C,D) quantitative analysis of protein expression. CCH reduced BDNF and TrkB expression compared with Sham surgery, effects reversed by URB597 (with or without EMS) but not by EMS alone. *P<0.05 vs. Sham; ^#P<0.05 vs. BCCAO; ^△P<0.05 vs. BCCAO+EMS. (C: BDNF, F_4,35 =11.527, P<0.001, TrkB, F_4,35 =4.413, P<0.05; D: BDNF, F_4,35 =9.126, P<0.001, TrkB, F_4,35 =7.466, P<0.01). EMS, encephalomyosynangiosis; BCCAO, bilateral common carotid artery occlusion; CCH, chronic cerebral hypoperfusion; BDNF, brain-derived neurotrophic factor.

Figure 6

Effects of EMS and URB597 on neuronal apoptosis. Apoptosis was estimated by expression of PARP and caspase-3. Numbers 1, 2, 3, 4, and 5 indicate the Sham, BCCAO, BCCAO+EMS, BCCAO+URB, and BCCAO+URB+EMS groups, respectively. (A,B) Representative western blot of PARP and caspase-3. (C,D) Quantitative analysis of protein expression. URB597 (with or without EMS) but not EMS alone reversed the reduction in expression induced by CCH. *P<0.05 vs. Sham; ^#P<0.05 vs. BCCAO; ^△P<0.05 vs. BCCAO+EMS. (C: PARP, F_4,35 =7.516, P<0.01, Caspase-3, F_4,35 =7.375, P<0.01; D: PARP, F_4,35 =4.338, P<0.05, caspase-3, F_4,35 =8.015, P<0.01). EMS, encephalomyosynangiosis; BCCAO, bilateral common carotid artery occlusion; PARP, poly-ADP ribose polymerase.