Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XFe24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP+ ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance.

Background: Glaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects. Methods: The COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release. Results: We found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis. Conclusions: TRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.

Serotonin (5-hydroxytryptamine, 5-HT) receptor agonists are neuroprotective in CNS injury models. However, the neuroprotective functional implications and synaptic mechanism of 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), a serotonin receptor (5-HT1A) agonist, in an adult male Wistar rat model of chronic glaucoma model remain unknown. We found that ocular hypertension decreased 5-HT1A receptor expression in rat retinas because the number of retinal ganglion cells (RGCs) was significantly reduced in rats with induced ocular hypertension relative to that in control retinas and 8-OH-DPAT enhanced the RGC viability. The protective effects of 8-OH-DPAT were blocked by intravitreal administration of the selective 5-HT1A antagonist WAY-100635 or the selective GABA _A receptor antagonist SR95531. Using patch-clamp techniques, spontaneous and miniature GABAergic IPSCs (sIPSCs and mIPSCs, respectively) of RGCs in rat retinal slices were recorded. 8-OH-DPAT significantly increased the frequency and amplitude of GABAergic sIPSCs and mIPSCs in ON- and OFF-type RGCs. Among the signaling cascades mediated by the 5-HT1A receptor, the role of cAMP-protein kinase A (PKA) signaling was investigated. The 8-OH-DPAT-induced changes at the synaptic level were enhanced by PKA inhibition by H-89 and blocked by PKA activation with bucladesine. Furthermore, the density of phosphorylated PKA (p-PKA)/PKA was significantly increased in glaucomatous retinas and 8-OH-DPAT significantly decreased p-PKA/PKA expression, which led to the inhibition of PKA phosphorylation upon relieving neurotransmitter GABA release. These results showed that the activation of 5-HT1A receptors in retinas facilitated presynaptic GABA release functions by suppressing cAMP-PKA signaling and decreasing PKA phosphorylation, which could lead to the de-excitation of RGC circuits and suppress excitotoxic processes in glaucoma. SIGNIFICANCE STATEMENT We found that serotonin (5-HT) receptors in the retina (5-HT1A receptors) were downregulated after intraocular pressure elevation. Patch-clamp recordings demonstrated differences in the frequencies of miniature GABAergic IPSCs (mIPSCs) in ON- and OFF-type retinal ganglion cells (RGCs) and RGCs in normal and glaucomatous retinal slices. Therefore, phosphorylated protein kinase A (PKA) inhibition upon release of the neurotransmitter GABA was eliminated by 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), which led to increased levels of GABAergic mIPSCs in ON- and OFF-type RGCs, thus enhancing RGC viability and function. These protective effects were blocked by the GABA _A receptor antagonist SR95531 or the 5-HT1A antagonist WAY-100635. This study identified a novel mechanism by which activation of 5-HT1A receptors protects damaged RGCs via the cAMP-PKA signaling pathway that modulates GABAergic presynaptic activity.