In the present work, the administration of the cannabinoid URB447 after HI in neonatal rats revealed strong neuroprotective and neurorestorative effects, as it reduced brain infarct and global and regional neuropathological damage, together with increased cell proliferation and neuroblast number in the dentate gyrus. We also assessed whether sex would influence the therapeutic effect of the treatment: our results suggest that the cannabinoid was neuroprotective for both sexes, with stronger neurogenic benefit in females.

In the last few years, several authors have noted that HI might affect males and females differently [6,31,32,33,34], which prompted us to separate our data by sex to evaluate a possible dimorphic response in brain damage after HI and subsequent URB447 treatment. Clinically, male sex is nowadays considered a risk factor in perinatal brain injury [23]. Here, non-treated HI males and females revealed similar post mortem neuropathology, despite demonstrating a trend towards more histological damage in males. In preclinical models, other authors have reported similar results to ours, with no sex differences in lesion size or tissue atrophy, where histological brain damage associated with HI was comparable across sex [20,35,36,37]. The pathophysiology of neonatal HI is complex and not fully understood yet, and so are the mechanisms leading to potential sexual differences; further investigation is needed to understand the basis of sexual dimorphism after HI.

The need for novel and effective therapies to treat neonatal hypoxic–ischemic encephalopathy is urgent. However, research works do not usually take into consideration sex dimorphism in their experimental designing, despite sex possibly being a crucial factor when evaluating the therapeutic effectiveness of neuroprotectants. We previously reported that the cannabinoid URB447 helped mitigate brain injury and white matter demyelination following neonatal HI [19], so we further wanted to characterize sex-specific outcomes in response to URB447 treatment. The neuroprotection parameters (hemispheric and hippocampal ratios and global and regional neuropathological scores) revealed increased benefit in females, but both treated males and females recovered the histological values observed for sham. Very few studies have evaluated the therapeutic effect of cannabinoids based on sex after acute brain damage. In a traumatic brain injury model, ∆9-tetrahydrocannabinol treatment impaired motor function and increased levels of cytokine interleukin-6 in males only [38], thus suggesting sex-specific cannabinoid effects. Together with the hormonal modulation of the ECS [39], different responses to cannabinoid administration on males and females have been described [40], and also sex differences in CB1 and CB2 receptor expression: female rats showed higher levels of CB1 and CB2 mRNA in the amygdala, hypothalamus, hippocampus and cortex compared to males under control conditions [41], the last two being regions affected by HI in our model and evaluated here.

Most of the studies assessing the therapeutic effect of the ECS after neonatal brain injury have focused on neuroprotection [19,42,43], but little is known about the potential of cannabinoids as brain-tissue-repairing therapies after HI. Moreover, the neurogenic response of the neonatal brain after damage is still not fully clear, with works suggesting that HI can enhance neurogenesis [44,45], while others report reduced cell proliferation [46,47].

Here, we wanted to explore the potential of URB447 as a neurogenic therapy, and showed both enhanced cell proliferation and an increased number of neuroblasts, thus suggesting a double neuroprotective/neurorestorative effect of the cannabinoid after HI. Different interactions with CB1 and CB2 receptors have previously been reported to modulate neurogenesis, including the increased proliferation of neural progenitors [48] and migration [49]. In the context of neonatal HI, we and others have described increased cell proliferation in the hippocampal dentate gyrus [46] and subventricular zone [50] by CB1 agonists 2-AG and WIN55,212-2, respectively. However, CB1 agonism may not be neuroprotective in certain types of neurons (like GABAergic) [51], so caution must be taken when choosing the cannabinoid to test. In this work, we opted for a CB1 antagonist to discard possible non-desirable neurotoxic or side effects. Further, CB2 receptor activation is also related to the enhancement of neurogenesis [48,49], as CB2-deficient mice showed impaired neural progenitor proliferation [48]. URB447 is also a CB2 agonist, so this cannabinoid may be a promising therapy targeting both neuroprotection and neuroregeneration after HI with a single molecule. In line with our results, Avraham and collaborators observed that the inhibition of neurogenesis was reverted in rodents after using a CB2 agonist [52]. CB2 expression levels are higher in less-differentiated cells [53,54], and CB2 receptor inhibition impaired neuroblast migration after stroke in mice [55].

We also wanted to test whether sex could affect neurogenesis after injury and subsequent URB447 administration. In non-pathological situations, some authors have reported that males generate more new cells in the hippocampus in the first postnatal week [56,57], whereas others have noted higher proliferation levels in females [58,59]. Here, we first showed that HI generated a similar reduction in cell proliferation and neuroblast counts for both sexes. When evaluating the neurogenic effect of the cannabinoid, females showed increased cell proliferation and neuroblast counts when comparing to non-treated pups, a response not observed for males. During brain development, sex may account for brain dimorphism [60,61,62], thus becoming a potential factor influencing different responses to damage and ulterior neurorestorative outcomes to cannabinoids or to other treatments.

In our work, we observed augmented Ki67+ proliferating cells in the contralateral hemisphere of non-treated HI animals, a possible compensatory mechanism of the non-damaged hemisphere in response to injury, as previously reported [63]. In our model, animals are exposed to systemic hypoxia, which also affects the contralateral hemisphere. At low oxygen concentrations, hypoxia-inducible factor-1α (HIF-1α) may promote neural stem cell proliferation [64], which may account for the observed increase in Ki67+ cells.

Based on the results presented, some study limitations should be considered. The present work revealed important differences in the responses of males and females to HI and URB447 treatment, but the pathways involved could not be reported by the histological methods used. To better understand the mechanisms that follow HI and the treatment with the cannabinoid, further experiments should be performed in future works, which may include the analysis of HIF-1α expression. Also, long-term studies evaluating the effect of URB447 on brain neuroprotection/neuro-regeneration after HI must be considered, as similarly reported for 2-AG [46].

In conclusion, URB447 showed robust neuroprotective effects in male and female rats subjected to HI, as both sexes showed reduced brain infarct areas and improved total and regional neuropathological scores. Not only that, URB447 also enhanced neurogenesis in female rats, based on increased proliferation and neuroblast cell counts after treatment with the cannabinoid. Thus, our results support the role of URB447 as a double neuroprotective and neuroregenerative treatment, although further investigation is needed to fully understand the mechanisms behind these effects.