The present study revealed distinct alterations in the volume and neuronal density of motor cortex areas in an animal model of ADHD development. Furthermore, morphometric abnormalities in this region of the brain were accompanied by impaired immunological, oxidoreductive and metabolic homeostasis in the PFC.
-
Motor cortex alternations
The current study showed some morphological abnormalities in M1 and M2 of SHRs. For example, the volume of both M1 and M2 have been significantly reduced in SHRs, at almost all age stages. Interestingly, the volume reduction in adult SHRs was the most notable in M1 in the right hemisphere. Although detailed volumetric measurements of specific motor cortex areas in SHRs have not been carried out yet. Similar changes have been observed in our previous studies among other brain regions involved in motor activity, including the prefrontal cortex and striatum 15,16. In addition, our results align with the majority of earlier reports with the most extensive ADHD patient sample. Namely, detailed studies carried out with MRI structural data analysis techniques according to VBM (voxel-based morphometry) and ROI (region-of-interest) approaches suggest a significant total volume reduction of the motor cortex areas (regions equivalent to M1 and M2 in rodents) in children with ADHD which is consistent with our results 77–81. Furthermore, analogous studies conducted on male adults with ADHD reported reduced grey matter volume in the cortex areas in question 82. It is noteworthy that Batty et al. 81 reported a developmental trajectory of cortical thickness in the ADHD and control groups that appears to be similar to the observed in the presented study pattern of motor cortex volume changes in the rat (SHRs/WKYs) development considering the rats vs. human age comparison83. Interestingly in the present study, we found that in adult SHRs, the volume reduction was the most notable in M1 of the right hemisphere. This finding was consistent with previous research showing a right-sided reduction in the brain structures involved in the movement in SHRs 15,16,84. Similar observations were also found in motor cortex areas of children and adolescents with ADHD 18,78,85. In addition to motor cortex volume alternations in ADHD, our developmental study suggests a different pattern of neuronal distribution in the SHRs motor cortex compared to the control group across the lifespan studied. Namely, we observed a decreased neuronal density in most of the M1 and M2 layers in juvenile SHRs (between 4 and 6 weeks of age) compared to control peers. At the outset, it is essential to emphasize that our study is the first report showing detailed changes in neuronal density in M1 and M2 in SHRs. Therefore, it is difficult to compare with previously published ones. At the same time, existing studies could support our findings that suggested a reduction in grey matter density in cortical structures involved in motor control in juvenile SHRs, which might result from ongoing inflammation and oxidative stress 15,86,87. Moreover, the study provided by Ashtari et al. 88, who used the diffusion tensor imaging (DTI) technique, showed lower FA (Fractional Anisotropy) in the motor cortex area of children with ADHD, which also confirms the reduced grey matter density in this part of the brain. It was also recently reported that reduced neuronal density and grey matter deficits reflect a more systematic disruption to the anatomical organization of large-scale brain networks 89,90. Interestingly, children with ADHD have shown significantly decreased functional connectivity compared to the controls, which was especially noted to be impaired in prefrontal areas, including the motor cortex 89,91,92. In addition, the current study could be supported by previous reports presenting delayed maturation of cortical and subcortical areas in children with ADHD 93,94.
The present work has also clearly shown that during subsequent life stages of SHRs, neuronal density gradually increased and became significantly higher in adult animals compared to age-matched controls. Tajima et al. 95 have also shown an elevated neuronal density in the motor areas in adult SHRs compared to the WKYs peers, which affirms our findings. In addition, changes in FA and MD during the early stages of ventricular enlargement may explain the increase in motor cortex volume observed in our study, which was accompanied by a decrease in neuronal density in this area in 9-week-old SHRs. Moreover, previous studies have also shown specific repair mechanisms that were activated aftermath of rapid brain ventricles enlargement. Namely, it has been confirmed that intracranial pressure reduction led to rapid recovery of corticobasal pathways (an increase in FA and a decrease in MD), which was probably related to the restoration of axoplasmic flow 96,97. If so, it is possible that in the present study, these mechanisms were responsible for an increase in the number of neurons in 10-week-old animals. Furthermore, it should be noted here that the dynamics of the volume alterations were not parallel to the observed changes in neuronal density. The explanation for this phenomenon could be differences in the method of volumetric measurements versus neuronal density measurements. The first analysis looked at the white and grey matter, whereas the second analysis was conducted only in the GM area. However, further research on this issue is needed.
It is noteworthy that, in our study, the timing of the most significant motor cortex morphometric abnormalities was found to be at the prepuberty period between 4 and 6 weeks of age in SHRs, approximately equivalent to an age of 6–10 years in children 83. This time range aligns with our previous behavioral analyses when we observed the most severe ADHD symptoms in SHRs 48. Furthermore, our observations correspond with the analysis of mental health among children, where the greatest manifestation of the condition in question occurs before puberty 98.
A puzzling result of our analysis is the sharp drop in neuronal density in 5-week-old animals across WKYs development. Postnatal rat brain development is known to show intense alterations in the cellular composition of the brain, the growth of which is modulated by different combinations of programmed cell proliferation and apoptosis during the early postnatal stages 99. Regrettably, the current literature lacks detailed analyses of neuronal density in motor cortex areas, considering the same developmental intervals of the WKYs as presented in this paper. However, the observed phenomenon could be explained by the reorganization and adaptation alterations of the CNS associated with the period of gradual transmission from childhood to adulthood. Namely, it has been suggested that during puberty (approximately 28–42 days postpartum), there are phases of decreased as well as increased neuroplasticity in subcortical and cortical regions 100. The dynamics of these changes are determined by the elevated levels of gonadal steroid hormones (oestradiol, testosterone, dehydroepiandrosterone) appearing during this period 101. More precisely, previous studies demonstrated that exposure to oestradiol initiating early puberty might be associated with reduced brain plasticity 102,103. Furthermore, Kolb and Gibb 104 suggested that pubertal hormones contribute to a reduction of GM volume associated with neuronal loss and synaptic pruning. At the same time, it has been observed that testosterone affects the increase in white matter volume 105, which could explain the lack of apparent reductions in motor cortex volumes during puberty presented in our results.
Finally, it is worth noting that the brain areas we have investigated correspond directly to the regions depicted in the Rat Brain Atlas 50, which is a significant advantage of our findings over MRI studies because the image contrast in structural MRI might not be sufficiently high to detect the boundaries between different compartments 106.
-
Inflammatory markers
The present results demonstrate that the PFC levels of almost all inflammatory markers assessed were significantly elevated in 5-week-old SHRs compared to age-matched WKYs (except IL-1β). Although data concerning the PFC cytokine levels in SHRs are scarce, some of them confirm the results of the present study. For example, elevated levels of IL-1β, IL-6, and TNF-α in the PFC were recently reported in juvenile SHRs 107. Unfortunately, in contrast to the content of proteins described above, in the available literature, there is a complete lack of data concerning the content of IL-1β in the brain tissue of both SHRs and ADHD patients. Nevertheless, as is well known, both IL-1α and IL-1β belong to the IL-1 family, bind the same IL-1 receptor, and are highly pro-inflammatory cytokines 108. Interestingly, in the present study, the PFC content of IL-1β was unchanged in 5-week-old SHRs, while in the serum of these strain, its level increased 15. Similarly, elevated serum levels of IL-1β have previously been observed in ADHD children but not adults 109. This phenomenon, i.e., the lack of differences in this cytokine content in PFC between SHRs and WKYs in both age groups, is difficult to explain and very intriguing. This may be due to the fact that both IL-1β and IL-α have different bioavailability relative to each other depending on the tissue under study 110,111. In addition, it has been reported that in stroke mice, IL-1α preceded the IL-1β expression 112. Interestingly, it was previously observed that intra-arterial administration of IL-α in mice with experimental cerebral ischemic stroke played a neuroprotective and neurorestorative role 113. Moreover, we observed significantly elevated IL-6 levels in juvenile SHRs, which has previously been reported in serum of SHRs and ADHD children 15,114. However, not much is known about this cytokine's mechanisms of action. So far, it has been shown that administration of IL-1β and IL-6 in rodents reduced brain dopamine levels 115 a decrease of which is observed in patients with ADHD 116.
Furthermore, our outcomes indicate elevated levels of mTOR and AKT-1 in the PFC of 5-week-old SHRs vs. their control peers. Explaining the increased content of these inflammatory markers is challenging, as there requires to be more data concerning them in the available literature. However, the PI3K/AKT/mTOR pathway has been implicated in the pathophysiology of ADHD, given that this signaling is specifically involved in neuron development and synapse formation 117. Furthermore, it has been mentioned that the activation of this pathway by growth factors is a fundamental pathway that regulates neuron proliferation, its maturation, and fusion into mature brain circuits 118. It is important to recall that dysregulation of this intracellular signaling system in neurons has resulted in several detrimental effects, such as oxidative imbalance, membrane depolarisation, mitochondrial damage, and energy disruption 119,120. In addition, it has been inferred that several mutations of proteins that participate in this pathway, for example, phosphatase and tensin homolog deleted on chromosome 10, neurofibromin 1, tuberous sclerosis complex 1, and tuberous sclerosis complex 2, are related to the symptoms observed in ADHD patients 121,122. Likewise, it has also been reported that upregulation of the microglial PI3K/AKT pathway triggers the production of pro-inflammatory cytokines 123. Under these conditions, microglia adopt a neurotoxic phenotype and is able to hasten neuronal damage 123,124. It should be noted that inflammation-activated microglia also release regulatory proteins such as IL-10 and TGF-β 125.
Notably, chronically elevated levels of pro-inflammatory cytokines such as IL-1α and TNF-α impair GCsR activity by increasing glucocorticosteroid resistance 126. In the submitted study, we observed a significantly elevated level of GCsRs in the PFC of juvenile ADHD individuals, which aligns with previous reports 127,128. Furthermore, a recent study led by van der Meer et al. 127 showed overexpression of the NR3C1 9β gene encoding GCsR in children with ADHD. It was found that this NR3C1 gene polymorphism stabilizes the mRNA of the splice variant GCsR-9𝛽, which may lead to increased expression of the GCsR𝛽 receptor 129. Of note, there is growing scientific interest in hypothalamic-pituitary-adrenal (HPA) axis dysfunction in psychiatric disorders 130. It appears to be an essential target for investigations of the pathophysiology of ADHD since HPA axis (Hypothalamic–Pituitary–Adrenal axis) dysfunction has previously been linked to impairments in attention, arousal, perception, memory, and emotional processing- dysfunctions often attributed to ADHD 126,131. Additionally, recent data underline a significant association between GR𝛽 overexpression and GM volume reduction in the cerebral cortex of ADHD children 127.
Interestingly, elevated levels of the inflammatory markers under study have not been observed in adult SHRs and reached values similar to matched controls, which remains consistent with previous literature reports 40. This can be caused by the significantly elevated serum cortisol and corticosterone levels that we previously observed in adult SHRs 49, which exhibit immunosuppressive effects 132. However, we observed a significant increase in IL-6 and AKT-1 levels in 10-week-old WKYs compared to their 5-week-old counterparts which is consistent with previous findings 38,133,134. Previous studies have been proposed that brain ageing is associated with upregulation of a number of cytokines including those mentioned above 133,134.
-
Oxidative stress markers
Numerous literature data indicate that the pathophysiology of ADHD is related to disturbances in oxidative stress balance 35,135,136. However, the causal relationship has not yet been undoubtedly determined. Interestingly, the present results demonstrate that the MDA as a biomarker for oxidative stress (a product of lipid peroxidation 137) levels in the PFC were significantly higher in 5-week-old SHRs than in age-matched WKYs. The present data are thus consistent with the previous studies reporting higher levels of MDA in the PFC of SHRs 138 and also in the plasma of children with ADHD 139,140. However, these results are sometimes contradictory. For example, Oztop et al. 141 reported lower plasma MDA levels in ADHD children. These discrepancies may arise from the different availability of this biomarker in a specific tissue and the different sensitivity and specificity between assay methods. In addition, in order to accurately compare our results with the available literature, other fundamental aspects would have to be taken into account, such as diet, physical activity, intestinal absorption of vitamins (antioxidants), i.e., B6, C, D, and unsaturated fatty acids, among others 142,143. Therefore, it is especially challenging to confront our results. Nonetheless, we hypothesized that elevated brain MDA levels in juvenile SHRs might be involved in the ADHD symptoms that we have previously observed in the animals of this strain 48. Moreover, it was also reported that oxidative stress might disturb DA synthesis 144. Importantly, DA-positive neurons constitute the brain population of neuronal cells that help control normal cerebral functions (movements and emotions) 145, and any alterations in the activity of this population may lead to occur symptoms similar to those observed in ADHD patients 116. Moreover, previous studies provided that increased oxidative stress in PFC was associated with higher motor activity in SHRs 146, strengthening our described above hypothesis. In addition, oxidative stress might also affect changes in neuronal cell morphology, migration, and plasticity 144,147 and, in this way, induce ADHD symptoms in SHRs and patients.
We also have found that the PFC activity of SOD and POD in 5-week-old SHRs significantly increased compared to age-matched controls. Explaining the changes observed in the activity of these antioxidant enzymes in the brain tissue is difficult due to the lack of available data from other studies concerning this issue. As of yet, it has been only reported that there were no significant differences in SOD levels in the blood of pediatric patients with ADHD 144. The discrepancy in the activity of SOD between the present results and Ceylan et al. 144 may be caused by the difference in its activity between serum and brain tissue that was previously observed in the rats 148. In reference to POD, it is generally known that it is an important antioxidant enzyme that plays an essential role in maintaining oxidative balance 149 which is disturbed in ADHD subjects 35. It should also be underlined that recent reports have shown a significant increase in the total antioxidant status (TAS) of children and adolescents with ADHD. TAS represents the cumulative effect of all antioxidants in the analyzed plasma, supporting our results 150. In the present study, an increase of oxidants and antioxidants in the PFC of juvenile SHRs might suggest an oxidative imbalance in juvenile SHRs, and the activation of compensatory mechanisms 151. Moreover, the present findings might indicate that 5-week-old SHRs demonstrate an adaptive response to oxidative stress in the PFC also through a significant increase in -SH, GSR, and GST levels compared to controls. Unfortunately, there is no detailed data describing these antioxidant levels in the PFC of SHRs and/or ADHD children/adults. In the case of -SH, it has only been reported that the levels of these groups were higher in the salivary and plasma of children with ADHD 141,152 and also in the spleen of juvenile SHRs 15. However, these results are sometimes contradictory. For example, some authors have reported that children with ADHD had lowered serum -SH levels when compared to non-affected individuals 153. This discrepancy between the results in our study and those of Öğütlü et al. (2020) 153 could probably be explained by the different availability of these groups in the specific tissue and/or species-specific differences. Previous investigations have also shown that dynamic thiol/disulfide homeostasis plays a very important role in antioxidant mechanisms, apoptosis, detoxification, cellular signal transduction, and regulation of enzyme and transcription factor activity 154. In addition, it has been previously reported that elevated levels of -SH are associated with the pathogenesis of several neuronal diseases, i.e., Parkinson's, Alzheimer's disease, Friedreich's ataxia, multiple sclerosis, and amyotrophic lateral sclerosis 155. In reference to GST, it has only been found that, in the plasma of ADHD children, the level of this enzyme was significantly elevated 156, which coincides with the present results. On the other hand, Ceylan et al. 157 found the reduction in the level of GST in the serum of ADHD children. The discrepancies may be attributed to the differences in the used method and to tissue specificity 158. For GSR, it is only known that the level of this enzyme might increase in response to oxidative stress induced by hypoxia which was previously observed in the cerebral cortex of rodents 159. It is worth noting that some previous studies also demonstrated a relationship between elevated GSR levels and oxidative stress caused by hypertension in SHRs 160. With age in SHRs, the levels of all oxidative markers undergo a significant decrease and reach statistically similar values to WKYs at 10 weeks of age, which is consistent with the previous evidence 40. This may be the result of compensatory-adaptive mechanisms against oxidative stress 161.
After all it should be noted that the prefrontal cortex is an area of the brain that is particularly susceptible to oxidative stress-induced damage 162. Therefore, more detailed studies on oxidoreductive balance in brain areas involved in ADHD are needed to help understand the pathomechanism of the disorder.
-
Metabolism markers
The spectrophotometric determination showed a significant increase in glucose (G) and fructosamine (FrAm) content in the PFC of 5-week-old SHRs compared to controls. These findings support previous studies reporting reduced global G metabolism in the brain of children with ADHD using PET (positron emission tomography) analysis 163. The changes in the G metabolism during brain development may result from mitochondrial dysfunction (mitochondrial DNA mutation or deletion) reported in childhood neurodevelopmental disorders such as ADHD and ASD 164,165. Furthermore, the altered G homeostasis in the prefrontal cortex in ADHD may also be indicated by elevated levels of FrAm in this structure, as reported in the present study in juvenile SHRs. FrAm is a glycoprotein formed by a non-enzymatic mechanism of protein glycation via a polyol pathway that allows the assessment of long-term sugar (G and fructose) control 166. An elevated FrAm level has previously been reported in several disorders often co-occurring with ADHD, such as psychosis, bipolar disorder, and depression 167. Most likely, the increase in G and FrAm content in the PFC observed during the present study in juvenile SHRs was associated with an impaired oxidative stress balance and activation of inflammatory cascades. This is supported by the fact that it has been observed that elevated brain G levels in diabetes result in the activation of key molecular pathways-NF-κB (nuclear factor kappa light chain enhancer of activated B cells) and PI3K/AKT - involved in pathological brain inflammation and increased oxidative stress 168. Furthermore, it has been shown that under these conditions, cellular stress triggers mitochondrial oxidative damage, which can result in the inhibition of neurogenesis, neurodegeneration, neuronal dysfunction, and grey matter deficits 169,170. Furthermore, alterations in these metabolic markers might also be associated with insulin resistance due to HPA dysregulation, as previously observed in many psychiatric disorders, including ADHD 15,130,171.
On the other hand, the hyperglycemia, mitochondrial dysfunction, and decreased cerebral blood flow observed in ADHD impair aerobic respiration 163–165, 172. When aerobic respiration is insufficient to meet the cells' energy demands, anaerobic metabolism increases. Under the conditions mentioned above, the cellular concentration of pyruvate and LA increases 173 Additionally, there is also an increase in LDH activity, which catalyzes the conversion of pyruvate to lactate 173. Similarly, in the present study, we observed elevated levels of LDH and lactate in the PFC of 5-week-old SHRs compared to controls. Our analyses are consistent with previous studies that have found increased expression of the LA transporter-MCT1 in brain microvessels and increased transport of LA from the periphery to the brain via the blood-brain barrier (BBB) in juvenile SHRs 174. Furthermore, previous studies suggest that the primary neurotransmitters, including NA, DA, 5-HT, and GABA, contribute to LA production, especially during stress 175. The LA produced with LDH was long thought to be a by-product of the glycolytic process. LA is considered a primary energy substrate when glucose metabolism is impaired and, in this way, plays an important role in brain energetics 176. On the one hand, LA is able to fuel the energy production in nerve cells and induce synaptic remodeling, axon excitability, and memory formation 177. On the other hand, it has been reported that there are many adverse effects of elevated LA levels in the brain. For example, the elevated level of LA in PFC might cause an increase in the formation of lactic acid–calcium complexes, which decreases calcium ion levels and contributes to the upregulation of the GABAergic system that has been previously reported in ADHD 177,178. Moreover, increased LDH and LA level was observed during mouse brain aging and also in Alzheimer's disease 179,180. Nevertheless, the exact roles of LDH and LA in ADHD brain function remain a topic of discussion and deserve further molecular and systematic studies. After all, increased production of LA and elevated LDH activity confirms previous outcomes reported mitochondrial dysfunction and neuronal energy metabolism disturbances in ADHD pathogenesis 153,164.
It is generally known that mitochondrial dysfunction causes loss of cellular integrity and leads to apoptosis/necrosis 181. ALT and AST are the most commonly used biochemical markers of necrosis, cell damage, and mitochondrial dysfunction 182,183. Our study presents significantly elevated ALT and AST levels in the PFC of juvenile SHRs compared to age-matched controls. These results agree with previous studies that observed elevated serum ALT levels in these animals 184. Although there are no studies presenting levels of these markers in ADHD patients, the study conducted by Skalny et al. 185 outcomes demonstrated an elevated level of alanine and asparagine amino acids in the serum of ADHD children, that which might be a result of increased ASP and ALT activity 185. Thus, the significant increase in ALT and AST activity in juvenile SHRs might confirm serious metabolic alterations in ADHD 186.
As is widely known, iron (Fe) metabolism is closely related to mitochondrial function and plays an essential role in cellular metabolism. Significantly elevated Fe levels in juvenile SHRs compared to peer controls observed in the present study might suggest a disruption of mitochondrial metabolism of this microelement since the extracellular iron is uptaken by cells and transported to the mitochondria, where it is used to synthesize co-factors essential for the enzyme's function involved in oxidoreductive reactions, DNA synthesis and repair, and many other cellular processes 187. It should be emphasized that this is the first study reporting Fe levels in the PFC in SHRs. Moreover, this result is fully coincided with a recent study reporting that in the SHRs, Fe and ferritin (Fe storage protein) levels increased following hypertension or ischemic-induced brain damage 188,189. Interestingly, previous studies have investigated the association between serum Fe level and ADHD disease, but the results are inconsistent. For example, Chen et al. 190 observed significantly elevated serum levels of this microelement in ADHD children compared to controls, while meta-analyzes suggested a lack of any differences 191,192.
Ambiguity in human studies may be caused by not considering differences in Fe supply and other substances significantly affecting Fe availability in children's diets 193. Therefore, using a standardized diet is necessary to obtain reliable results. In addition, differences in iron levels in pathologically altered tissues could be higher than in peripheral tissues such as blood, plasma, and serum 194. It is most possible that, in the present study, elevated Fe levels in the PFC juvenile SHRs may have induced/prompted neurodegenerative changes in this part of the brain 195. This supposition was strongly supported by data showing that redundancy of this microelement in the brain is able to induce oxidative stress, and lipid peroxidation, causing blood-brain barrier destruction and neuronal death 196,197.
Finally, it should be mentioned that we did not observe differences in the level of metabolic markers in adult SHRs in comparison to controls, might be a result of the suppression of the inflammation and oxidative stress in adult animals following the compensatory mechanisms mentioned before 15,161.
-
Protein–Protein Interaction Analysis
The additional in-silico analysis using STRING software provides strong support for our hypothesis. Namely, it confirmed a significant association between the studied immunological, oxidative stress and metabolic markers on neurodevelopmental and behavioral processes. More specifically, the string database enabled the setting up of a protein-protein interaction (PPI) network for all protein markers analyzed in the presented study exhibiting strong interactions between them. In addition, the use of GO molecular function enrichment analysis allowed the detection of fourteen terms (biological processes) involved in the neurodevelopment and behavior of Rattus norvegicus in general, mediated by the protein markers studied. The results of STRING of PPI analysis demonstrate that morphometric changes in PFC, M1 and M2 in SHRs may be related to changes in immune, oxidoreductive and metabolic balances. Nevertheless, this speculation needs to be confronted with further experimental studies to provide reliable findings. However, it is noteworthy that the PPI analysis presented here may provide an important jumping-off point for further research into the involvement of immune oxidoreductive and metabolic factors in the pathogenesis of ADHD.