In the present study, we found that repeated CBD treatment prevented memory impairments and decreased hippocampal CA1 neurodegeneration that were caused by ischemia. Cannabidiol also increased hippocampal BDNF levels, attenuated the TGCI-induced decreases in SYN and PSD-95 levels, and elevated dendritic spine number and arborization in the hippocampus in ischemic animals.
The effects of TGCI on spatial memory performance have been well documented [29–32]. Studies from our group showed deleterious effects of TGCI on retrograde memory in rats in the AvRM up to 39 days after the ischemic insult [33–35]. In the present study, memory impairments were detected up to 14 days after TGCI in rats in the AvRM and OLT. Cannabidiol attenuated these effects of TGCI, reflected by decreases in latency and the number of errors in the AvRM, indicating improvements in spatial memory performance. Cannabidiol also ameliorated memory deficits in the AvRM paradigm in middle-aged diabetic rats that underwent chronic cerebral hypoperfusion [36]. Spatial memory recovery in the Y-maze, OLT, and Morris water maze was also observed in BCCAO mice that were treated with CBD [17, 18]. The beneficial effects of CBD on memory function may extend to other conditions that are associated with deficits in memory processing. For example, single or repeated (14 days) injections of CBD (10 mg/kg, i.p.) ameliorated memory function in the object recognition test in rats that were subjected to iron overload [37]. Moreover, accumulating evidence indicates that CBD reduces learned fear in paradigms that are translationally relevant to phobias and posttraumatic stress disorder [38].
Selective hippocampal CA1 damage is known to impair hippocampus-dependent memory, such as spatial learning and memory performance [29, 39–41]. In the present study, we found the significant loss of CA1 neurons (detected by NeuN immunohistochemistry) in TGCI animals, which paralleled memory impairments in those animals in the AvRM. Cannabidiol treatment attenuated hippocampal CA1 neuronal loss that was induced by TGCI in rats. These results extend previous studies that reported histological neuroprotection in the CA1 subfield after CBD administration in mice [18] and gerbils [42] that were subjected to TGCI.
The extent to which the modest reduction of CA1 pyramidal loss that was elicited by CBD treatment contributes to memory preservation (or recovery) is uncertain. Fish oil treatment was reported to restore memory loss that was caused by TGCI without rescuing hippocampal CA1 pyramidal cells [24, 43–45]. Alterations at the electrophysiological, synaptic, and subcellular levels and morphological changes that extend beyond certain structures can result in the dysfunction of complex behaviors and their recovery [46]. Hippocampal damage contributes to ischemia-induced cognitive deficits, and the extent of such deficits depends on whether such damage is intra- or extra-hippocampal, the task that is applied to assess such deficits, and the specific memory process that is measured [47]. Interestingly, TGCI rats that were treated with CBD exhibited an increase in NeuN immunoreactivity in the DG of the hippocampus compared with respective controls. However, the implications of this finding are unclear. Neurogenesis in the DG has been shown to reflect a compensatory mechanism that is triggered by TGCI in rats [48] and mice [49]. Moreover, CBD increases doublecortin expression, a marker of newborn neurons, in the DG in ischemic mice. The increase in NeuN immunoreactivity in the DG may reflect hippocampal neurogenesis, but further studies are necessary to confirm this possibility.
Several possible mechanisms may underlie the neuroprotective effects of CBD in GCI, such as CBD-induced neurogenesis, the rescue of CA1 neuronal death, and improvements in synaptic plasticity [18, 44, 50–51]. BDNF is a neurotrophin that regulates activity-dependent synaptic plasticity and contributes to learning and memory processes [52]. Favorable effects of BDNF on functional recovery and neuroplasticity after CI have been reported [28, 53–55]. In the present study, hippocampal BDNF levels did not significantly decrease 14 days after TGCI. However, CBD treatment increased BDNF in the hippocampus, suggesting that this effect may be related to functional recovery in ischemic rats. Indeed, evidence indicates that CBD interacts with serotonin 5-hydroxytryptamine-1A (5-HT1A) receptors, which may positively regulate BDNF levels. Mishima et al. (2005) reported that CBD reduced the infarct size in rats with middle cerebral artery occlusion, a model of focal cerebral ischemia. The effect of CBD was inhibited by the 5-HT1A receptor antagonist WAY100135 [56]. Notably, the direct activation of postsynaptic 5-HT1A receptors in the hippocampus resulted in higher BDNF levels and an increase in neuroplasticity in mice with BCCAO [55]. Moreover, the antidepressant-like effects of CBD have been associated with higher levels of SYN and PSD-95 in the medial prefrontal cortex and higher levels of BDNF in the prefrontal cortex and hippocampus. An intracerebroventricular injection of the TrkB receptor antagonist K252a and mammalian/mechanistic target of rapamycin inhibitor rapamycin abolished the antidepressant-like effects of CBD. The positive behavioral effects of CBD were related to plastic changes through activation of the BDNF-TrkB signaling pathway [57].
Both SYN and PSD-95 are the main proteins that participate in structural synaptic plasticity [58]. Synapsin is a marker of presynapse development and activity [59], and PSD-95 is an essential factor for synaptic plasticity and postsynaptic membrane stabilization [60]. Experimental evidence indicates that SYN and PSD-95 levels markedly decrease in the hippocampus after CI [61–63]. In the present study, TGCI significantly decreased hippocampal levels of SYN and PSD-95. However, CBD at the tested dose (10 mg/kg) only slightly attenuated these effects of TGCI. Supporting our data, Sales et al. (2019) did not observe any difference in SYN or PSD-95 levels in the hippocampus in healthy mice that were treated with 10 mg/kg CBD for 7 days [57]. In contrast, treatment with 10 mg/kg CBD for 14 consecutive days reversed iron-induced reductions of hippocampal SYN levels in rats [64]. Moreover, CBD reversed the decrease in hippocampal PSD-95 levels in mice that were exposed to a model of chronic unpredictable stress [19].
Under some conditions, modifications of the structure of dendritic spines are strongly associated with synaptic plasticity, which is critical for cognitive flexibility [65]. Cerebral ischemia can cause poor spatial memory performance as a result of hippocampal damage and a decrease in synaptic function [66, 67]. In the present study, TGCI significantly decreased the number of dendritic spines in neurons that survived the ischemic insult in the DG (granular neurons), CA1, and CA3. These effects of TGCI on dendritic deterioration were prevented by CBD treatment. Various pharmacological interventions can stimulate neuroplastic changes. For example, acute melatonin administration attenuated dendritic spine loss in the hippocampus in TGCI rats [50]. Treatment with progesterone resulted in a similar recovery of dendritic spines and mitigated ischemia-induced learning and memory deficits [68]. Acetyl-L-carnitine treatment before and after 2-vessel occlusion (i.e., a model of ischemia) prevented ischemia-induced dendritic spine loss in the hippocampus, paralleled by the normalization of long-term potentiation in the hippocampus [69]. The biased 5-HT1A receptor agonist NLX-101 was recently reported to reverse dendritic spine loss in mice that were subjected to BCCAO [55]. The ability of CBD to increase hippocampal BDNF levels and reverse or prevent dendritic spine loss in the hippocampus suggests its influence on neuroplasticity and consequently cognitive recovery (or preservation) under conditions of GCI.
One limitation of the present study was that we did not investigate the pharmacological mechanism of action of CBD in rats with TGCI. We recently demonstrated the involvement of cannabinoid CB1, CB2, 5-HT1A, and peroxisome proliferator-activated receptor-γ (PPAR-γ) receptors in functional recovery that is elicited by CBD in BCCAO mice [70]. Whether similar mechanisms are engaged in TGCI rats remains to be determined. The pharmacological profile of CBD is complex. It has potent antiinflammatory and antioxidant properties [71, 72] and exerts its actions throughout both the endocannabinoid system and other neurotransmitter systems. Treatment with CBD increases anandamide levels, in turn further activating cannabinoid CB1 receptors [73, 74]. Cannabidiol has been reported to act as a CB1/CB2 receptor inverse agonist [75]. Other effects of CBD have been reported to be mediated by PPAR-γ [76] and G-protein-coupled receptors [77]. Moreover, CBD was reported to increase adenosine A2A receptor signaling [78] and modulate energy metabolism through elevations of the mitochondrial complex and creatine kinase activity [79].
Overall, the present findings suggest that the beneficial effects of CBD on spatial memory recovery in TGCI rats at least partially occur through molecular mechanisms that underlie synaptic plasticity and dendritic remodeling, suggesting that CBD may be useful for functional improvement after CI.