Attention-deficit/hyperactivity disorder (ADHD), a neurodevelopmental disorder characterised by inattention, impulsivity and hyperactivity, is commonly associated with challenges in academic, social and psychological functioning [1, 2, 3]. Although the aetiology and pathophysiology remain unclear, there is increasing evidence suggesting that the disorder is attributable to dysfunctional dopaminergic neurotransmission [4, 5, 6, 7]. Additionally, there is evidence suggesting that oxidative stress, neuroinflammation and mitochondrial dysfunction participate in the pathophysiology of ADHD [8, 9, 10]. The central nervous system is particularly susceptible to oxidative damage due to its inadequate antioxidant capacity, high energy demand and high polyunsaturated fatty acid content. Oxidative stress can cause tissue and neuronal damage, leading to impaired cellular function and alterations in the properties of cell membranes, which in turn disrupt crucial functions [11]. Antioxidant enzymes can protect against reactive oxygen species (ROS), and the most important constitutive antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [12, 13]. Consequently, the extent of oxidative stress is likely to depend in part on both the activity of antioxidant enzymes and the ROS content in a target tissue. Thus, increased oxidative stress has been recognised as a causative factor in the pathogenesis of several neurodegenerative and neuropsychiatric disorders [14, 15]. Additionally, the ADHD medication methylphenidate (MPH) has been demonstrated to induce oxidative stress by increasing ROS levels and inflammation and altering antioxidant defences in animal models, and these effects are related to alterations in mitochondrial bioenergetics [16, 17, 18]. Furthermore, various studies have reported increases in oxidative stress and alterations of antioxidant defences in children and adolescents with ADHD [19, 20]. Moreover, clinical variables such as stress, anxiety and neuroinflammation in ADHD patients are crucial in ADHD pathophysiology [21]. Thus, neuroinflammation and oxidative stress are coexisting factors, causing a worsening in clinical symptoms and triggering a vicious circle.

The ketogenic diet (KD) is a high-fat and low-carbohydrate diet that has been used globally in recent decades as an effective treatment for epilepsy [22, 23]. The medium-chain triglyceride (MCT)-based KD is an effective version of the classic KD that permits the consumption of better proportions of carbohydrates and proteins compared with the classical version [24]. MCTs are saturated fatty acids comprising 6–12 carbon atoms. Among MCTs, caprylic acid (C8) is the most abundant (50%–75% content), followed by the 10-carbon decanoic acid [capric acid (C10), 23%–45% content] and slight amounts of caproic acid (1%–3% content) and lauric acid (1%–5%). Natural dietary sources of MCTs include coconut oil, palm oil and fats [25, 26]. MCTs are easily absorbed and metabolised within liver mitochondria through $\beta$-oxidation, resulting in the generation of three major ketones, namely, acetoacetic acid, $\beta$-hydroxybutyrate and acetone (ketone bodies). MCTs can also cross the blood–brain barrier to reach the brain and serve as alternative energy sources for neurons and astrocytes [27, 28]. Additionally, C10 is a ligand for peroxisome proliferator-activated receptor gamma (PPAR$\gamma$), which is known to be involved in mitochondrial biogenesis and antioxidant defences [29]. The PPAR$\gamma$ antagonist, bisphenol A diglycidyl ether (BADGE) prevents the C10-mediated increase in CAT and citrate synthase levels [30]. Meanwhile, the activation of PPAR$\gamma$ leads to an increase in the expression of SOD, GPx and CAT [31, 32, 33, 34]; this activation is also associated with the upregulation of the phase II transcription factor—nuclear factor erythroid 2-related factor 2 (Nrf2) [34, 35, 36]. Previously, the MCT diet provoked great interest owing to its indirect or direct effects on epilepsy and energy metabolism [27]. Thenceforth, the MCT diet has been used as a potential treatment for several neurodegenerative and neuropsychiatric disorders; however, the mechanisms underlying its effects remain elusive [27, 37]. MCT diet consumption has been reported to reduce anxiety-like related behaviours in rats [38]. Moreover, oral C10 administration in mice resulted in acute anti-convulsant effects at doses that produced plasma exposures similar to those reported using an MCT-based KD in patients with epilepsy [39]. Furthermore, C10 treatment increased SOD, GPx and CAT activities [30, 40, 41]. However, the contribution and efficacy of the MCT C10 have not been extensively studied. In light of the suggestion that C10 enhances cellular antioxidant defences and alters behavioural activity, this study established an experimental animal model of ADHD-like symptoms. This was achieved by inducing unilateral striatal lesions in neonatal rats using 6-hydroxydopamine (6-OHDA). The primary aim was to investigate the effects of C10 administration on locomotor hyperactivity and detoxifying enzymes within the brains of the rats.

The MCT diet has been widely used for the management of epilepsy [24]. Nevertheless, the effectiveness of the MCT diet and the response biochemical mechanisms remain poorly understood. The potential therapeutic outcomes of MCTs such as C10 on neuropsychiatric disorders or ADHD have not been extensively studied. Therefore, we sought to determine the relative effects of C10 administration on behavioural activity and antioxidant defences in the striatum, prefrontal cortex, cerebellum and hippocampus in an experimental rat model of ADHD.

Experimental rodent models lesioned with 6-OHDA have been used in the last decade to study different aspects of ADHD-like symptoms, focusing on the destruction of dopaminergic projections in the brain [44, 45, 46, 47]. This is because deficits in catecholaminergic transmission in the brain represent a pathophysiological cause of ADHD [5, 6]. Previously, the neonatal rats lesioned unilaterally into the striatum with 6-OHDA exhibited a reduction in tyrosine hydroxylase (TH) expression in the striatum [42], which is also consistent with our previously reported finding that neonatal rats lesioned unilaterally in the striatum with 6-OHDA exhibited 33% lower expression of TH [43]. The loss of dopaminergic innervation in the ipsilateral striatum of 6-OHDA–lesioned rats resulted in increased locomotor activity (PN39) but no alterations in spatial memory. These data are also consistent with our previous findings [43]. However, several studies analysing the behavioural activity of animals obtained different results because of differences in the sites of 6-OHDA injection, age and evaluation protocols. Previous studies found that lesioning with 6-OHDA in neonatal rodents resulted in increased hyperactivity in young stages, but this hyperactivity disappeared in adulthood (PN36 or PN60) [46, 47]. Meanwhile, we observed hyperactivity in our model until PN39. Moreover, C10 administration at a dose of 250 mg/kg for 14 days had no significant effect on locomotor activity, suggesting that the dose or treatment duration of C10 was insufficient to cause a direct effect on behavioural activity. However, there are contradictory results regarding the effects of similar or high doses of C10, KD or MCT diet consumption, either acutely or chronically, on behavioural phenotypes in other experimental animal models. For example, a single oral C10 dose of 1.7–8.6 g/kg had anti-convulsant effects in mice [39]. C10 administered intraperitoneally at a dose of 175 mg/kg in mice 15 min before convulsions delayed the onset of clonic convulsions induced by picrotoxin and increased survival following exposure to a lethal dose of pentylenetetrazole [48]. Diabetic mice that were subcutaneously injected with 250 mg/kg C10 daily for 2–4 weeks exhibited significantly decreased glucose levels and improved insulin sensitivity without inducing weight gain [29]. Feeding treatment (35E% tridecanoin) for 10 days before acute seizure tests exerted anti-convulsant effects in two acute CD1 mouse seizure models [49]. Conversely, 3 months of KD consumption (10% protein and 90% fat) had no effects on locomotor activity, spatial learning and memory, depression-like behaviour and anxiety in naïve adult mice [50]. Moreover, rats consuming an MCT diet (C8 and C10 at a ratio of 40:60) for 15 days did not show improved performance in forced swim tests and social exploration; however, they did exhibit reduced anxiety-like behaviours and improved social competitiveness [38]. Recently, it was demonstrated that oral C10 administration at a dose of 525 mg/kg in adult male mice for at least 21 days did not result in behavioural differences [51]. In the same study, a single oral dose of C10 at a high dose of 5.25 g/kg decreased the distance travelled and increased anxiety-like behaviours in elevated plus maze, open-field and light/dark transition tests. Additionally, a single oral dose of 17.5–175 mg/kg C10 slightly increased the distance travelled in the open-field test. However, none of the results reached study-wide statistical significance [51]. Finally, a prospective open-label study assessed dietary intervention with K.Vita (C10 and C8 at a ratio of 80:20), a medicinal food used for patients with drug-resistant epilepsy, for 12 weeks (median intake of 240 mL in adults and 120 mL in children, representing 19% of the daily energy requirement). While the study was not intended to demonstrate a clinical response, it is worth noting that the mean frequency of seizures or paroxysmal events was significantly reduced, although only measured from the time of their introduction [52].

Additionally, oral consumption of the KD or MCT diet has been associated with several disadvantages, mainly gastrointestinal disorders, and these symptoms have been reported in several studies. Thus, the use of the KD in people with epilepsy has been associated with vomiting, diarrhoea, body weight loss and constipation [53]. A randomised trial of children with drug-resistant epilepsy treated with the classical KD or MCT diet recorded side effects such as a lack of energy, constipation after 3 months and vomiting after 12 months and found that the MCT diet does not display any advantage over the classical KD in terms of efficacy [54]. In an open-label trial involving patients with epilepsy and the use of K.Vita, the most commonly reported gastrointestinal symptoms included sensations of abdominal bloating or fullness, increased flatulence and constipation; these symptoms were most pronounced during the initial introduction period and gradually diminished over time [52]. Furthermore, mice administered 525 mg/kg C10 orally exhibited a slight but significant decrease in body weights after 2 weeks, and treatment with the highest dose of C10 resulted in marked decreases in body weights after 1 day [51]. These symptoms might be attributable to acidity; thus, C10 should be administered orally suspended in methylcellulose or separately as its respective triglycerides to avoid sodium or acid overload, especially during longer periods of administration. Moreover, alternative administration approaches, such as subcutaneous or intraperitoneal, could be employed in experimental animal models to avoid gastrointestinal side effects.

Oxidative stress, neuroinflammation and alterations in neuronal antioxidant capacity can contribute to the pathophysiology of ADHD [9]. Changes in the activities of antioxidant enzymes such as CAT, GPx, glutathione (GSH) and SOD have been observed in children with ADHD [19, 20, 55]. The excessive increases in dopamine and noradrenaline levels caused by drugs used to treat ADHD might also induce their autoxidation, increasing oxidative stress and further leading to mitochondrial dysfunction and neuronal damage [9, 16, 56]. For example, MPH induced oxidative stress and alterations in GPx, GSH and SOD activities, causing neurodegeneration in the brain of rats [17, 57].

C10 has been shown to regulate the activity of different antioxidant enzymes, playing an important role in reducing oxidative stress in vitro. Thus, C10 treatment at 10 µM for 18 h reduced oxidative stress, thereby attenuating hydrogen peroxide (H${}_2$O${}_2$)-induced cell death in two different neuroblastoma cell lines, and increased CAT activity independent of changes in CAT gene expression [40]. C10 treatment (250 µM, a similar concentration reported in the plasma of patients using the MCT diet) for 6 days increased citrate synthase, CAT and complex I activities in SH-SY5Y neuroblastoma cells. In the same study, a PPAR$\gamma$ antagonist prevented C10-mediated increases in citrate synthase and CAT activities in cells [30]. Notably, C10 bound and activated PPAR$\gamma$ in COS-7 cells, and it modulated PPAR$\gamma$ and increased the expression of PPAR$\gamma$-dependent genes without changing the expression of the PPAR$\gamma$ gene in a diabetic mouse model [29]. The effect of a diet containing C10-enriched mustard oil (200 g/kg during 30 days) was demonstrated by increased activities of SOD, GSH, CAT and GPx in brain and liver tissue and decreased lipid peroxidation in brain or liver tissue or plasma in rats [41]. GPxs are selenoproteins that participate in redox reactions via detoxifying H${}_2$O${}_2$ by reducing it to water [58]. GPx isozymes exhibit different tissue-specific expression and subcellular localisation patterns. For instance, GPx-2 has low or no expression in the human brain. The most highly expressed GPx is GPx-4, followed GPx-1 [59]. In HT22 murine hippocampal cells, treatment with C10 (250 µM for 1 week) resulted in a significant increase of Sirtuin 1 activity, upregulation of mitochondrial respiratory chain complexes and increases in the activities of mitochondrial complexes and citrate synthase [60]. Sirtuins have been shown to regulate key processes such as ROS detoxification, mitochondrial biogenesis and lipid metabolism [61]. Moreover, knockdown of Sirtuin 1 prevented the expression of SOD1, Nrf2 and haem oxygenase-1 (HO-1), the transcription of which is activated by Nrf2, and knockdown abolished the neuroprotective effects of Sirtuin 1 induced by hyperbaric oxygen preconditioning against cerebral ischaemia in rats [62]. In cultured cortical astrocytes, 200 µM C10 increased basal respiration and ATP turnover, whereas in isolated hippocampal mitochondria, tridecanoin treatment increased respiration linked to ATP synthesis and plasma antioxidant capacity in the ferric reducing ability of plasma (FRAP) assay. This treatment additionally increased the hippocampal mRNA expression of HO-1 and forkhead box protein O1(FOXO1) [49]. C10 treatment (200 µM) in mouse brain slices promoted astrocyte glutamine synthesis and stimulated mitochondrial respiration in cortical astrocytes in mouse primary cultures [63]. However, repeated C10 administration for 14 days at 250 mg/kg had no significant effects on the expression of CAT, GPx-1/2 or Nrf2 in any of the brain regions analysed. Considering the acute behavioural effects of C10, these results suggest that the utilised dose of C10 has no long-lasting or cumulative effects on antioxidant defences. Nevertheless, it is vital to assess the impact of C10 on remaining antioxidant activity and its influence on oxidative stress because the standard activity or expression of antioxidant enzymes in brain tissue samples may not accurately represent neuroprotective and intracellular antioxidant capacities. Another possibility is that oxidative stress may be more effectively assessed by measuring parameters other than the levels of antioxidant enzymes. These parameters could include lipid peroxidation in urine or diacron reactive oxygen metabolites (d-ROMs) in serum or plasma, among others. It is also possible that the benefits of C10 administration may have only a marginal impact on oxidative stress. For instance, in most of the aforementioned studies, in vitro C10 treatment increased antioxidant activity, but no data regarding related protein expression were reported.

As an energy source and naturally occurring dietary compound, MCTs have demonstrated various properties and functions in metabolism, including the modulation of lipid metabolism, glucose regulation, heat production and hormone secretion [64, 65]. Consequently, the cognitive benefits of MCTs as an energy supply for humans have led to their clinical application in treating various neurological and metabolic disorders. They have also been considered for use in healthy individuals as a source of ketone bodies and for their physiological functions [66]. Although the clinical applications of MCTs are promising, there have been inconsistent conclusions in clinical trials, with numerous unresolved questions, such as their impact on the risk of heart disease [65]. The crucial components of MCTs are C8 and C10, and their varying percentages may influence the desired outcomes. Therefore, further studies are needed to understand the underlying mechanisms distinguishing the effects of $\beta$-hydroxybutyrate and different MCT types. Given the increasing public awareness and popularity of MCT dietary supplementation for its potential in neurological and metabolic disorders, additional research is necessary to explore how MCTs are used outside of clinical contexts. Moreover, we need to elucidate the long-term effects of early dietary interventions with MCTs on health and disease.

To serve as a valid experimental model for ADHD, several criteria must be met, including face, construct and predictive validity [67, 68]. Consequently, confirming the validity of experimental ADHD models is challenging, and no ideal model exists that can encompass all the core symptoms of ADHD. In recent years, many researchers have developed experimental ADHD models via neurotoxin-induced dopaminergic pathway lesions [47]. Neonatal rodents with dopaminergic pathway lesions induced by 6-OHDA closely replicate numerous fundamental characteristics of ADHD in humans [44, 45, 46]. Differences in the dose, volume or injection site of 6-OHDA (whether unilateral into the striatum, intracisternal or into the lateral ventricle) affect the rate of 6-OHDA absorption, which is fundamental for predictive validity. Moreover, the choice of administration route may lead to differences in behavioural outcomes and responses to treatments [42, 45, 46]. In a previous study, neonate rats with unilateral striatal 6-OHDA-induced lesions were employed as an experimental model to study ADHD-like symptoms [42]. This model demonstrated both face validity (reduction in spatial attention and an increase in locomotor hyperactivity) and construct validity (decrease in TH expression in the striatum) [42, 43]. An experimental model can be considered viable even if it does not meet all predictive and face validity criteria. However, it cannot be deemed valid unless construct validity is achieved [68]. Achieving construct validity can be challenging in ADHD due to its complex aetiology and high heterogeneity, which make its neurobiology not fully understood. Despite these challenges, experimental ADHD models, while not perfect reflections of ADHD in humans, can help elucidate the probable underlying mechanisms of ADHD pathogenesis. They can also shed light on the molecular, genetic and cellular mechanisms in various tissues, brain areas, circuits and neurotransmitter systems, which may help identify new therapeutic targets. The limitations of this study include the absence of measurements of dopamine levels associated with 6-OHDA lesion and dopaminergic innervation. However, the previously observed reduction in TH immunoreactivity [42, 43] could be regarded as an indirect indicator of dopamine depletion in the striatum. Furthermore, the study identified a decreased count of dopaminergic neurons in the substantia nigra, which resulted from retrograde degeneration induced by the 6-OHDA lesion [69]. This indicates that the 6-OHDA lesion may lead to dopamine depletion significant enough to produce ADHD-like behaviour, contributing to the study of ADHD [47, 70]. Additionally, the potential influence of sex differences in the animal subjects was not found to be significant.

In conclusion, this study demonstrated that C10 administration for 14 days in 6-OHDA–lesioned rats did not induce changes in locomotor activity, probably because the brain concentration of C10 was insufficient to alter behavioural activity. This study also showed that C10 administration failed to substantially alter the expression of detoxifying enzymes in any of the brain regions analysed. Taken together, these findings suggest that further comprehensive studies are needed to evaluate the expression of antioxidant defences in brain tissue under treatment with C10 at high doses and for longer periods and examine its potential behavioural effects. Although the use of naturally found dietary compounds for various disorders has been considered a healthier and safer approach for patients, they are still far from being considered standard treatments owing to the lack of controlled clinical studies that may well validate both their high efficacy and safety as well their anti-antioxidant or inflammatory properties. Well-designed prospective studies and clinical trials are indispensable for confirming the promise of C10 as an adjunct to standard pharmacological treatments for neuropsychiatric disorders, such as ADHD.

The data that support the findings of this study are included in the article; further inquiries can be directed to the corresponding author.

SC-T performed the experiments and analysed the data. JCC designed the study and wrote the manuscript. Both authors contributed to editorial changes in the manuscript. Both authors read and approved the final manuscript. Both authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The animal study was reviewed and approved by Hospital Infantil de México Federico Gómez, Institutional ethical, Animal Care and Use Committees (HIM2018-030).

Not applicable.

This work was supported by Fondos Federales HIM 2018-030 SSA 1497. SC-T is recipient of a fellowship from CONACYT.

The authors declare no conflict of interest.