Monoacylglycerol Lipase Inhibition Using ABX-1431 Attenuates Cerebral Ischaemia Early After Traumatic Brain Injury

Abstract

An early event in the pathology of traumatic brain injury (TBI) is a reduction in cerebral blood flow (CBF), which exacerbates secondary injury development and inhibits brain recovery. The endogenous cannabinoid system signalling (eCBs) might be critical in TBI recovery due to modulating synaptic activity and exerting neuroprotective and anti-inflammatory effects. In the brain, eCBs predominantly occur at cannabinoid receptor type 1 via the eCB 2-arachidonoylglycerol (2-AG). The aim of this work was to test the efficacy of potentiating 2-AG signalling by monoacylglycerol lipase (MAGL) inhibition using ABX-1431 immediately following TBI. Laser speckle contrast imaging (LSCI) was used to create a high-resolution map of regional cerebral blood flow (CBF) over the pericontusion cortical surface. In-vivo two-photon laser scanning microscopy (2PLSM) was used to monitor cerebral microcirculation (i.v. fluorescein isothiocyanate dextran, FITC) and mitochondrial respiration and brain tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence, NADH) during 4 hours after CHI. After baseline imaging, male C57BL/6 J mice (10–12 weeks, >28 g) were subjected to a modified moderate Shohami weight-drop closed-head injury (CHI) followed by i.p. injection of ABX-1431 (5 mg/kg) or vehicle 30 min after the insult (10 mice per group). Differences between groups and between time points were determined using two-way repeated measures (ANOVA) for multiple comparisons and post hoc testing with the statistical significance level set at p < 0.05. Optical imaging revealed that CHI caused a decrease in regional CBF, arteriole diameters (vasospasm), and blood flow volume, leading to capillary microthrombosis and a reduction in capillary flow velocity. Compromised cerebral microcirculation led to the development of tissue hypoxia. ABX-1431 application, in a ~30-minute delay, mitigated the development of microvascular dysfunction, microthrombosis formation, and tissue hypoxia compared to the saline control group (p < 0.05, starting 1 hour after CHI). Therefore, MAGL inhibition by ABX-1431 attenuates cerebral ischaemia early after TBI. The observed 2-AG-mediated cerebrovascular relaxation might involve both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium.

1. Introduction

An early event in the pathology of traumatic brain injury (TBI) is a reduction in cerebral blood flow (CBF), which exacerbates secondary injury and inhibits brain recovery by causing dampened oxygen and metabolic delivery to injured brain tissue, which is vital for its proper function and survival. A critical therapeutic strategy is to identify treatments that block the transition from primary to secondary injury in an attempt to prevent the progressive decline in brain function and the manifestation of neurologic disorder before it starts. The endogenous cannabinoid system signalling (eCBs) might be critical in TBI recovery due to its role in modulating synaptic activity and exerting neuroprotective and anti-inflammatory effects [1]. In the brain, eCB signalling predominantly occurs at cannabinoid type 1 receptor via the eCB 2-arachidonoylglycerol (2-AG). Potentiating 2-AG signalling by inhibiting its enzymatic degradation by monoacylglycerol lipase (MAGL) immediately following mTBI injury might attenuate pathological sequelae of TBI [2]. In this work, we tested the efficacy of potentiating 2-AG signalling using a novel potent and selective MAGL inhibitor ABX-1431 immediately following TBI.

2. Methods

The animal protocol was approved by the Institutional Animal Care and Use Committee of the Lovelace Biomedical Research Institute. Twenty laboratory-acclimated male wild-type C57BL/6 J mice (10–12 weeks, >28 g) were randomly assigned to the following groups: (1) TBI+ABX-1431; (2) TBI+vehicle (saline). Laser speckle contrast imaging (LSCI) was used to create a high-resolution map of regional cerebral blood flow on the cortical surface using an RFLSI III System (RWD Life Science, Shenzhen, China) [3]. In-vivo two-photon laser scanning microscopy (2PLSM) using a Prairie View Ultima System (Bruker, Billerica, MA) was utilised to monitor cerebral microcirculation (i.v. fluorescein isothiocyanate dextran, FITC), and mitochondrial respiration and brain tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence, NADH) at a baseline and up to 4 hours after TBI [4]. During the study, the mice were maintained on isoflurane (1%), nitrous oxide (69%), and oxygen (30%) face-mask anaesthesia. For imaging, the mouse’s head was secured in a stereotactic frame, the skull was exposed through a midline scalp incision, and the incised skin was glued around the edges of the frontal and parietal bones using dental cement. To improve spatial resolution, the skull optical clearing treatment was performed in two steps before the experiment. Solution 1 (saturated supernatant solution of 75% (vol/vol) ethanol and urea) was applied to the exposed skull for about 10 min to allow the skull to turn transparent gradually. Then, Solution 1 was removed, and Solution 2 (0.7 M NaOH solution with dodecylbenzene sulfonic acid at a volume-mass ratio of 24:5, pH 7.2) was added to the same area for further clearing within 5 min [5]. After baseline imaging, the mice were subjected to a modified moderate Shohami’s [6] weight-drop closed-head injury (CHI) followed by i.p. injection of ABX-1431 (5 mg/kg) or vehicle 30 min after the insult. This model induces a focal blunt injury over an intact skull that triggers a profound neuroinflammatory response within the intrathecal compartment with high consistency and reproducibility, leading to neurological impairment and breakdown of the blood–brain barrier [6]. Where appropriate, statistical analyses were done using GraphPad Prism by Student’s t-test or Kolmogorov-Smirnov test. Differences between groups were determined using a two-way analysis of variance (ANOVA) for multiple comparisons and post-hoc testing. Data are presented as a mean ± s.e.m., and the statistical significance level was p < 0.05.

3. Results

CHI led to cerebrovascular vasospasm, microcirculation dysfunction, and microthrombosis, associated with reduced mitochondrial respiration/tissue oxygen supply. The maximal effect was observed ~1 hour after the insult, with slight recovery by the end of the imaging. In the vehicle group, by the end of the monitoring period, LSCI revealed a significant decrease in regional cerebral blood flow (Fig. 1a, 0.81 ± 0.09 a.u., p < 0.05 from baseline). The diameters of the penetrating and precapillary arterioles (vasospasm) decreased from 17.4 ± 0.3 to 15.6 ± 0.2 μm (Fig. 1b), and blood flow volume reduced by 0.79 ± 0.05 a.u. (Fig. 1c, p < 0.05 from baseline). This led to a reduction in capillary red blood cell flow velocity from 1.12 ± 0.16 to 0.77 ± 0.15 mm/s in the contusion area of the cerebral cortex (Fig. 2a, p < 0.05 from baseline). Due to microthrombosis, the number of functioning capillaries decreased from 1009 ± 51 per mm³ at a baseline to 811 ± 49 per mm³ in the contusion area of the brain cortex (Fig. 2b, p < 0.05 from baseline). Microcirculation impairment reduced mitochondrial respiration and tissue oxygen supply, inversely reflected by an increase in NADH autofluorescence (Fig. 2c, 1.21 ± 0.06 normalised units, p < 0.05 from baseline). ABX-1431 application, in a ~30-minute delay, mitigated the development of vasospasm, microvascular dysfunction, microthrombosis formation, and tissue hypoxia compared to the saline control group (p < 0.05, 1 hour after CHI from the vehicle group). By the end of the monitoring period, LSCI revealed a better preserved regional cerebral blood flow (Fig. 1a, 0.94 ± 0.01 a.u., p < 0.05 from baseline). The diameters of the penetrating and precapillary arterioles (vasospasm) decreased from 16.9 ± 0.4 μm (Fig. 1b), and blood flow volume reduced by 0.92 ± 0.4 a.u. (Fig. 1c, p < 0.05 from the vehicle group). Capillary red blood cell flow velocity was 0.97 ± 0.13 mm/s in the peri-contusion area of the cerebral cortex (Fig. 2a, p < 0.05 from the vehicle group). The number of functioning capillaries decreased only to 956 ± 42 per mm³ in the peri-contusion area of the brain cortex (Fig. 2b, p < 0.05 from the vehicle group). Mitochondrial respiration and tissue oxygen supply were also better preserved than in the vehicle group (Fig. 2c, 1.09 ± 0.04 normalised units, p < 0.05).

Fig. 1.

ABX-1431, delivered 30 min after TBI, (a) mitigated a decrease in regional cerebral blood flow, (b) alleviated arteriolar vasospasm, and (c) restored blood flow volume through arterioles in the pericontusional area compared to the saline control group, n = 10, * = p < 0.05. Red arrow—TBI, black arrow—ABX-1431 application

Fig. 2.

ABX-1431, delivered 30 min after TBI, (a) restored capillary flow velocity, (b) re-recruited collapsed capillaries, thereby (c) preserving tissue oxygen supply in pericontusional area compared to saline-treated animals, n = 10, * = p < 0.05. Red arrow—TBI, black arrow—ABX-1431 application

4. Discussion

ABX-1431, delivered 30 min after TBI, mitigated the development of cerebrovascular vasospasm, microvascular dysfunction, microthrombosis formation, and tissue hypoxia compared to the saline control group. The observed 2-AG-mediated cerebrovascular relaxation might involve both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium [7].

5. Conclusion

MAGL inhibition by ABX-1431 attenuates cerebral ischaemia early after TBI.