N = 54, 30% with a diagnosis of ADHD) completed a modified version of the gap-overlap task including arousal-inducing auditory warning signals. Parents rated symptoms at the time of testing and at 2 years follow-up. Phasic alerting  is an index of arousal. Here, larger phasic alerting effects predicted higher ADHD-symptom levels 2 years later. Blunted pupil-dilation responses predicted externalizing symptoms at T2, controlling for ADHD and externalizing at T1. Our results support the theory that ADHD is associated with altered arousal. Blunted arousal reactivity may be a longitudinal risk factor for externalizing problems in children with ADHD symptoms.Attention-deficit/hyperactivity disorder (ADHD) follows a variable course across childhood. Disrupted arousal has been hypothesized to underlie core symptoms as well as comorbid internalizing and externalizing conditions. The current study examined eye-movement and pupil-dilation metrics indexing arousal as longitudinal predictors of ADHD, externalizing, and internalizing symptoms over a 2-year period. Participants aged 8–13 years ( About 1/3 of diagnosed children no longer fulfil diagnostic criteria by early adulthood7 . In this group, remission is particularly likely to occur in adolescence8 . The longitudinal development of ADHD is heterogeneous not only in terms of binary outcome (whether diagnostic criteria are fulfilled or not), but also in terms of symptom dimensions. A decrease in symptoms from childhood to adolescence is most likely to occur in the hyperactive/impulsive domain whereas levels of inattentive symptoms follow a more variable course9 . ADHD symptoms are continuously distributed in the general population10 . Higher symptom levels predict impairments across the symptom spectrum, with little evidence for a qualitative difference between individuals with a diagnosis and those with elevated symptom levels11 . Studies of symptom dimensions may therefore capture variation in symptomatology better than categorical differences between clinical and non-clinical groups.The developmental trajectories of ADHD from childhood to adolescence are highly heterogeneous12 . Many, but not all, children with ADHD show signs of impaired attention and executive functions on neuropsychological tests13 .Temperamental patterns characterized by either increased negative affect and irritability or strong approach motivation and dysregulated positive affect known as exuberance are also common6 . Comorbid symptoms of internalizing disorders  and externalizing disorders  are highly prevalent, and as such likely to affect the longitudinal course of the disorder15 . For example, a recent study found that an increase in symptoms of GAD over a two-year period predicted worse outcome in terms of core ADHD symptoms16 . Internalizing symptoms are also a longitudinal predictor of cognitive impairment, beyond the influence of core ADHD symptoms17 . A better understanding of the factors that predict the longitudinal development of ADHD and comorbid symptoms during late childhood could potentially guide intervention and has therefore been identified as an important research priority14 .The core symptoms of ADHD are inattention, hyperactivity, and impulsivity18 . These are further divided into tonic and short-term (phasic) arousal18 . Behaviorally, short-term increases in phasic arousal enhances sensitivity to sensory information and response preparedness. Reduced baseline arousal and impaired arousal regulation have been reported in ADHD, although results are heterogeneous19 . Altered arousal may represent an underlying mechanism of core ADHD symptoms and associated cognitive impairments in areas such as sustained attention20 . As predicted by the dimensional perspective, individual differences in arousal were linked to concurrent variability in ADHD symptoms and cognitive impairments22 .Arousal refers to a set of interacting behavioral and neural systems24. This spatially non-informative warning signal effect is known as phasic alerting25 . The effect is believed to be modulated by short-term increases in cortical arousal and activity in neurotransmitter systems such as norepinephrine and dopamine27. Phasic alerting can therefore be seen as a modulation of attention by arousal induced through external sensory stimulation. In populations with reduced tonic arousal due to brain lesions28 or pharmacological manipulations27 , phasic alerting effects on attention tend to be increased. Phasic alerting could therefore be seen as a compensatory process, where externally triggered arousal responses compensate for a lack of endogenously generated phasic arousal.The view that arousal alterations underlie some of the attention impairments seen in ADHD are further supported by recent studies showing that at least some of these can be ameliorated by brief auditory warning signals, which increase arousal but do not provide information about the location or direction of visual targetsIn the current study, we tested whether phasic arousal alterations are not only concurrently, but also longitudinally linked to symptoms of ADHD and co-occurring internalizing and externalizing disorders over a two-year interval. Arousal was studied using two measures derived from eye tracking: warning signal modulation of gaze shifts  and pupil dilation.29, and eye movement alterations are therefore likely to bias information processing and learning. Since eye movements require very little or no verbal ability, they can be measured in individuals that vary widely in cognitive ability and age and are less vulnerable than self-report measures to memory biases and difficulties with introspection. The brain mechanisms underlying eye movements are partly overlapping with those involved in orienting of attention and are modulated by arousal30 . Therefore, eye movement studies may be informative about the attentional characteristics of ADHD and its underlying brain mechanisms32 .The fixated area of the visual field is highly prioritized for further cortical processing36 , increased variability of saccadic latencies38, and difficulties to inhibit stimulus-driven gaze shifts40. Together, these eye-movement atypicalities point to possible alterations in brain networks involved in eye movement generation including a frontostriatal network involved in top-down control of eye movements and a widely distributed network including subcortical, cortical, and cerebellar regions involved in stimulus-driven eye movement generation29 .Previous research has documented alterations in both voluntary generated and reflexive eye movements in children with either a diagnosis of ADHD or elevated ADHD symptoms, including increased gaze-shift reaction time41. LC-NE arousal amplifies neural reactivity to salient stimuli and dampens reactivity to non-relevant sensory input and is therefore a potential mechanism underlying the effects of arousal on attention43. Task-related phasic LC-NE arousal has a non-linear, inverted u-shaped relationship to tonic (baseline) activity44. That is, the largest phasic arousal responses are hypothesized to occur at intermediate levels of tonic arousal, whereas either too high or too low baseline arousal leads to small phasic responses.Pupil dilation is an index of arousal closely linked to activity in the locus coeruleus-noradrenergic (LC-NE) system23 examined the latency of stimulus-driven gaze shifts in a group of 8–12-years old children (N = 71) of which 1/3 had a clinical diagnosis of ADHD. As expected, ADHD symptoms were associated with longer gaze-shift reaction times in the absence of warning cues. Auditory warning signals decreased gaze-shift reaction times in children with ADHD, and the strength of this effect was linearly related to the level of ADHD symptoms. Consistent with the theory that the effect of alerting cues was driven by changes in arousal, alerting cues induced strong pupil-dilation responses, an index of arousal modulated by LC-NE activity45 . However, at the individual level, no correlation was found between pupil-dilation amplitude and the reduction in gaze-shift reaction times after warning cues.A previous study in the current sample45. Previous studies have indicated abnormal pupil dilation in ADHD, including increased reactivity to smiling faces46 and decreased tonic pupil size, indicating hypoarousal47 . Wainstein and colleagues47 also found blunted phasic pupil dilation during a working memory task in individuals with ADHD of predominantly inattentive presentation compared to neurotypical controls. Pupil dilation was normalized by methylphenidate, which increases LC-NE and dopaminergic activity.Pupil dilation is a relatively quick response sensitive to novelty and emotional valence48 and internalizing49 disorders as well as other neurodevelopmental disorders such as autism50. Blunted autonomic nervous system (ANS) reactivity is seen in children with externalizing symptoms53, but it is debated whether this is best explained by the externalizing symptoms per se or by their overlap with ADHD19 . Increased autonomic reactivity and hyperarousal has been reported in internalizing disorders, although results are heterogeneous and differ between methods and tasks54 .Research on the relation between arousal and ADHD symptoms is complicated by the fact that atypicalities within this domain have also been found in externalizing1 for a discussion about trait liability and compensatory mechanisms in ADHD). For example, children who are more responsive to phasic alerting cues may have a better ability to take advantage of external cues or sensory input to regulate their arousal, which could in turn predict a better functional outcome. A third possibility is that individual differences in phasic alerting do not predict ADHD symptoms per se but mediate the risk for comorbid externalizing or internalizing symptoms. Here, we tested these possibilities in a follow-up study of the sample reported in23 .There are at least three potential roles that phasic alerting could play in the maintenance and developmental change of symptoms. First, it is possible that increased phasic alerting and symptoms have shared underlying mechanisms. In this case, increased phasic alerting should be associated with relatively higher symptom levels at follow-up. A second possibility is that phasic alerting can have a compensatory function for children with high levels of ADHD symptoms, and therefore predict a better longitudinal outcome of core or associated symptoms  would be predicted by (1) larger alerting effects, (2) relatively longer gaze-shift reaction times in the absence of warning cues, and (3) larger gaze-shift reaction-time variability at time point 1 (T1).Are eye movements predictive of change in ADHD symptoms? We hypothesized that larger phasic alerting effects, longer gaze-shift reaction times, and higher gaze-shift reaction-time variability would also predict a smaller symptom reduction from T1 to T2.Is pupil dilation predictive of ADHD symptoms at 2-years follow up? We hypothesized that the pupil dilation to alerting cues at T1 and would be linked to ADHD symptoms at T2. This hypothesis was left undirected since no relation between pupil dilation and symptoms of ADHD was found at T123 .Is pupil dilation predictive of change in ADHD symptoms? In an undirected analysis, we examined longitudinal links between pupil dilation to alerting cues at T1 and ADHD symptoms at T2.The following registered hypotheses were tested:Are eye movements and pupil dilation predictive of comorbid symptoms and change in comorbid symptoms? In exploratory (not registered) analyses, we examined whether eye-movement and pupil dilation metrics predicted longitudinal change in comorbid internalizing and externalizing symptoms, beyond effects attributable to ADHD symptoms.https://osf.io/9mh58/).The analysis plan and hypotheses were registered on Open Science Framework prior to analyses (link: 23). Of these, four were excluded because of lack of valid data, one because no symptom measures were available, and ten because they had taken stimulant medication  for ADHD at the day of testing. Families of thirteen children (five with ADHD) did not return the questionnaires or declined to participate at T2. The total sample at T2 therefore consisted of 54 children (16 with an ADHD diagnosis). Of these, three had missing data from the cued overlap condition, two from the uncued overlap condition, and one from the uncued gap condition, resulting in slightly different sample sizes in the various analyses. Demographic characteristics, symptom levels and the average number of valid trials are shown in Table p > 0.14, see Supplement, The final sample included N = 54 with valid eye-tracking data and symptom measures from T1 and T2. The initial sample at T1 included N = 82 children  of Uppsala, Sweden and research was performed according to the relevant regulations and guidelines. Informed consent was obtained from participants and their legal guardians. The research was conducted in accordance with the Declaration of Helsinki.55. Here, parents rate the degree to which their child shows each of the 18 ADHD symptoms listed in the Diagnostic and Statistical Manual for Mental Disorders, 5th Ed.  on a four-graded Likert scale with scores for each item ranging from 0 to 3. Cronbach’s alpha for this measure was α = 0.95. Parental ratings of ODD using the Swanson, Nolan, and Pelham–IV (SNAP-IV) scale (eight items) were used as a measure of externalizing symptoms56 . ODD is the most common comorbid externalizing disorder in ADHD and is highly correlated with other symptom dimensions such as conduct disorder. Cronbach’s alpha was α = 0.93. Since symptoms of GAD are highly correlated with a common internalizing symptoms factor57 , the six-item generalized anxiety subscale of Spence Children’s Anxiety Scale  was used as a measure of internalizing symptoms. Cronbach’s alpha was α = 0.77 at T1 and α = 0.86 at T2. Internalizing and externalizing symptoms were moderately correlated at T2  whereas no significant correlation was seen at T1 .Symptom measures were collected from parents at T1 and T2. Parents rated symptoms of ADHD using the ADHD Rating Scale-5 for Children and Adolescents59. There was a near-significant reduction in ODD symptoms. Parents of children with ADHD (n = 16) confirmed that their children had received a diagnosis of ADHD in regular care by a psychologist or physician. At T1, nine participants were treated with stimulant or non-stimulant medication and at T2, five children were on medication according to parent report. Two children had received other forms of treatment by a psychologist (n = 1) or counsellor (n = 1), and one child was treated for Hashimoto’s disease. Two participants with ADHD had a comorbid autism spectrum disorder.All clinical data are expressed as mean scores and described in Table The experimental task was an adapted version of the gap-overlap task see Fig. . In this23. Gaze shifts were identified using an I-VT filter algorithm with the following steps. First, gaze velocity was smoothed using a moving average filter covering 50 ms. Subsequently, the start of a gaze shift was detected when gaze velocity exceeded 30°/s. Gaze shift latency was defined as the time from the onset of the peripheral stimulus to the time when the point of gaze first left the central stimulus  at a sample rate of 60 HZ. Testing took place in a university lab facility or in an outpatient pediatric clinic. Data were processed as described in a previous study reporting results from this sample at T160, baseline correction was conducted through subtraction. The baseline period was defined as the 1000–666 ms interval before stimulus onset, i.e., before the onset of the alerting cues. Trials with < 50% valid samples were discarded.Pupil data were processed using the following steps. First, gaps shorter than 100 ms were covered by linear interpolation. Secondly, pupil-size was filtered using a moving median filter with a window size corresponding to 80 ms .We aimed to keep ambient illuminance constant at around 500 lx. This was not possible in all cases due to room changes. Of the final data set, 5 participants were tested at ambient illuminance < 450 lx and 7 > 550 lx. No relation between ambient lux level and pupil-dilation response was found . Violin plots showing the distribution of the experimental variables are shown in Supplement, 62 . Analyses testing relations between experimental variables and symptom measures were conducted after averaging data at the level of the individual. Outlier observations defined as standardized residuals ± 3.25 were removed . Residual plots of all statistical models indicated that the data conformed to the assumptions of normality. However, because gaze-shift reaction times often deviate from a normal distribution, we validated the analyses by repeating all analyses using individual medians rather than means as measures of central tendency. This did not change any of the results. In an additional validation analysis, data were reanalyzed using general linear models with a log link .Effect sizes are reported as standardized Are eye movements predictive of ADHD symptoms at 2-years follow up? Larger alerting effects predicted higher symptom levels of ADHD at T2 . ADHD symptoms at T2 were also predicted by gaze-shift reaction times at T1 in the uncued overlap condition , but not in the uncued gap condition . Gaze shift reaction-time variability in the uncued conditions was also linked to ADHD at T2 .Are eye movements predictive of change in ADHD symptoms from T1 to 2-years follow up? Change in ADHD symptoms was uniquely predicted by longer gaze-shift latency in the uncued overlap condition  and by higher gaze shift variability condition  after controlling for the same symptom dimension at T1. No other relations between eye movements and change in ADHD symptoms were found  = 0.96, p > 0.900].Is pupil dilation predictive of change in ADHD symptoms from T1 to 2-years follow up? Pupil dilation at T1 did not predict ADHD symptoms at T2 after controlling for ADHD symptoms at T1, .Are eye movements predictive of comorbid symptoms at 2-years follow up? Eye movements at T1 did not predict internalizing or externalizing symptoms at T2  = −2.94, p = 0.026]. No relation was found between pupil dilation at T1 and internalizing symptoms at T2, controlling for ADHD symptoms at T2 .Is pupil dilation predictive of change in comorbid symptoms from T1 to 2-years follow up? Smaller pupil-dilation responses at T1 predicted higher externalizing symptom levels at T2, after controlling for externalizing symptoms at T1 and ADHD symptoms at T1 and T2 [β = −1.00, t (44) = −3.72, p = 0.003, see Table t (44) = −0.34, p > 0.900, see Table phasic alerting effect is defined as a decrease in response time after warning signals, caused by a temporary increase in arousal. The magnitude of this effect was previously found to be associated with higher levels of ADHD symptoms)23. In the current study, we show that this measure also predicts symptom levels two years later. However, phasic alerting effects did not predict the degree of change between these time points, suggesting that altered arousal is a trait-like vulnerability factor for ADHD symptoms during late childhood and early adolescence rather than a mechanism of change. Our results therefore support the theory that arousal alterations underlie symptoms of ADHD20.Disrupted arousal is hypothesized to be a mechanism underlying ADHD symptoms and related visual attention and eye-movement alterations. Disrupted arousal may also underlie associated symptoms of externalizing and internalizing disorders. In this study, we examined whether individual differences in eye-movement metrics sensitive to arousal predict change in ADHD symptoms and comorbid internalizing and externalizing disorders over a 2-year period in a group of children oversampled for a diagnosis of ADHD. The 41. Tentatively, our results therefore suggest alterations in these systems that are relatively specific for externalizing disorders as compared to ADHD symptoms.Contrary to our expectations, the pupil-dilation response to auditory cues was not linked to ADHD symptoms, but instead predicted externalizing symptoms at T2 beyond what could be explained by core ADHD symptoms. Pupil-dilation responses at T2 were also linked to the degree of change in externalizing symptoms from T1 to T2, so that children with lower pupil-dilation responses were more likely to have higher levels of externalizing symptoms at T2, even after controlling for the same symptom dimension at T1 as well as for ADHD symptoms at T1 and T2. To our knowledge, this is the first study to examine longitudinal links between pupil dilation in mid childhood and subsequent externalizing symptoms. Pupil dilation is modulated by activity in the cholinergic and noradrenergic systems64, and thereby affect learning. Phasic alerting effects could therefore potentially have a compensatory role for children with ADHD. Consistent with this theory, stimulant medication for ADHD, such as methylphenidate (MPH) is known to increase arousal and has dose-dependent beneficial effects on several neurocognitive functions closely linked to arousal, such as vigilance and response-time variability in children with ADHD65. Sustained periods of background white noise can be beneficial for cognitive performance in children with ADHD, possibly through increased arousal24. Our results indicate that beneficial effects of increased arousal on visual attention in children with high levels of ADHD symptoms are seen already during a very short time scale. An interesting question for future studies is to examine whether phasic alerting either predicts response to medication and other treatments or changes after treatment.Studies in typically developing populations show that phasic alerting can improve attention and perception64, and could therefore potentially compensate for risk factors for development of internalizing symptoms associated with ADHD, such as peer and learning problems.Stronger alerting effects  predicted higher ADHD symptom levels at 2-years follow up. Phasic alerting can have beneficial effects on perception and learning14. Here, reduced pupil-dilation responses to auditory cues predicted a relative increase in externalizing symptoms over two years, after control for ADHD and externalizing symptoms at T1. This suggests that blunted pupillary reactivity may contribute to the maintenance or increase of externalizing symptoms during mid childhood. As noted in the introduction, previous studies have reported reduced arousal reactivity to negative emotional stimuli or unexpected sensory information as measured by electrodermal activity, electrocardiography and electroencephalogram (EEG) in children with conduct problems52 . However, the symptomatic specificity and longitudinal links of these measures remains debated48 . Our results also suggest that the link between blunted arousal reactivity and externalizing symptoms extends beyond emotional stimuli. Pupil dilation is elicited by novelty and unexpected stimuli and is closely linked to activity in the brains noradrenergic system45 .Externalizing symptoms tend to decrease from childhood to adolescence, but the longitudinal course is variable and a subset of children with ADHD show a progressive course33. As predicted, longer gaze-shift reaction times in the absence of warning signals predicted ADHD symptoms at 2-years follow up. This link was found when gaze had to be disengaged from a previously fixated location , but not as predicted in the gap condition . This suggests that impaired disengagement of visual attention rather than slow gaze-shift reaction time per se may a risk-factor for continuing ADHD symptoms. This result is consistent with previous reports of reduced gaze-shift reaction times in ADHD. Previous studies have shown that ADHD symptoms are associated with increased gaze-shift reaction time variability38. Here, we found that this measure also predicted the longitudinal development of ADHD, so that children with increased response-time variability at T1 had higher ADHD symptoms at T2, even after controlling for T1 symptoms.As described in the introduction, the phasic alerting effect is defined as the difference between gaze-shift latency with and without warning cues. The latter of these components—gaze-shift reaction times without warning signals—has consistently been linked to concurrent ADHD symptomsSome limitations should be mentioned. First, the sample size was relatively small. The naturalistic study design means that the type of treatments received between T1 and T2 was not controlled for. The study did not include a measure of tonic arousal, which could have provided valuable information about potential interactions between tonic and phasic arousal. Due to the relatively low sample rate, we were not able to analyze potentially informatively eye-movement metrics such as microsaccade rate or saccadic velocity. Importantly, all significant effects were considerably larger than the sampling error , meaning that temporal imprecision in the equipment is unlikely to have influenced the results. Inclusion of cued gap trials may have provided a more complete picture of the effects of alerting on gaze shift reaction times. It should also be noted that, since the current study examined the longitudinal course of ADHD symptoms across the whole spectrum, more research is needed to determine whether the findings also hold for classification of remission versus persistence of categorical diagnoses in clinical settings. Findings from the current study point to a potential mechanism underlying externalizing disorders, which is not shared with ADHD or internalizing symptoms. However, it should be noted that both externalizing and internalizing symptoms were measured using relatively brief instruments. Studies using broader measures of internalizing and externalizing symptoms may therefore give a more complete picture of their relationship with altered arousal processes.Supplementary Information.


Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most prevalent neurodevelopmental disorders. Interestingly, children with ADHD seem to experience more ophthalmologic abnormalities, and the impact of methylphenidate (MPH) use on retinal physiology remains unclear. Thus, we aimed to unravel the retina’s structural, functional, and cellular alterations and the impact of MPH in ADHD versus the control conditions. For that, spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were used as animal models of ADHD and the controls, respectively. Animals were divided into four experimental groups as follows: WKY vehicle , WKY MPH (1.5 mg/kg/day), SHR Veh, SHR MPH. Individual administration was performed by gavage between P28-P55. Retinal physiology and structure were evaluated at P56 followed by tissue collection and analysis. The ADHD animal model presents the retinal structural, functional, and neuronal deficits, as well as the microglial reactivity, astrogliosis, blood-retinal barrier (BRB) hyperpermeability and a pro-inflammatory status. In this model, MPH had a beneficial effect on reducing microgliosis, BRB dysfunction, and inflammatory response, but did not correct the neuronal and functional alterations in the retina. Curiously, in the control animals, MPH showed an opposite effect since it impaired the retinal function, neuronal cells, and BRB integrity, and also promoted both microglia reactivity and upregulation of pro-inflammatory mediators. This study unveils the retinal alterations in ADHD and the opposite effects induced by MPH in the retina of ADHD and the control animal models.  Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental condition with a mean worldwide prevalence of 2.2% that often manifests in childhood but can persist in adulthood. This impairing condition creates a substantial burden for the individual, their family, and the community . ADHD isMethylphenidate (MPH) is the first-line pharmacological treatment for ADHD and has been used worldwide based on its ability to relieve core target symptoms of this disorder . The priThe retina is a thin layer of tissue lining the back of the eye and is considered part of the CNS. In recent years, the concept of the retina as a possible mirror of brain changes has been claimed in several studies showing retinal alterations in both neurodegenerative diseases ,9 and psRegarding preclinical studies, in two different animal models of ADHD, the superior colliculus exhibited structural alterations and increased collicular visual responsiveness that were associated with increased distractible behavior in these animals ,18. IndeDespite its prevalence, a lack of clear understanding of ADHD pathogenesis with implications on its proper treatment still exists. Since some features commonly associated with ADHD can also be mimicked by visual impairment , the chaAnimals were purchased from Charles River Laboratories  and housed under controlled environmental conditions  with food and water ad libitum. The spontaneously hypertensive rat (SHR) is the most well-characterized and commonly used animal model of ADHD , since i2) was used to evaluate the scotopic threshold response (STR), and white light flashes (0.0095 to 9.49 cd·s/m2) were applied under scotopic and photopic conditions. The amplitude (μV) of positive STR (pSTR) and negative STR (nSTR) was evaluated to infer on retinal ganglion cells’ (RGCs) response. Moreover, a-wave and b-wave were extracted under scotopic conditions (reflecting rod photoreceptors and bipolar cells response), as well as the amplitude of b-wave under photopic conditions. A photopic flicker test (reflecting cone photoreceptors response) was also performed with bright white flashes (3.00 and 9.49 cd·s/m2). An off-line digital filter was applied on STR and b-wave (high-frequency cut-off of 50 Hz) with the RETIport 4.9.8.7 software .ERG recordings were performed after overnight dark adaptation to evaluate retinal physiology using corneal gold wire electrodes as previously described by our group ,25. UndeAfter ERG recordings, while the animals were still under anesthesia, cross-sectional images of the retina were acquired using a Micron IV   per total microglia (Iba-1+) per image was normalized to the respective retinal section’s length. From each animal, 10 images were obtained and analyzed using Fiji 1.53c software. For rhodopsin and VGlut1 immunoreactivity, the results are expressed as the mean fluorescence intensity (arbitrary units) and were determined in the entire retinal section normalized to the respective length by applying the following formula: corrected total fluorescence = (integrated intensity) − (area of picture × mean background) . The arrAfter transcardiac perfusion of animals, eyes were enucleated and retinas were dissected as flattened wholemounts, as previously reported . BrieflyRetinal protein extracts were prepared as previously described . Brieflyp < 0.05 and the n “n” represents the total number of animals used in each experimental condition. Statistical analysis was performed with the Prism 8.0.1 Software for Windows .The normality of the data was assessed with Shapiro-Wilk and Kolmogorov-Smirnov normality tests. Accordingly, data were analyzed using two-way ANOVA followed by Tukey’s test except for the quantification of the GFAP immunostaining in retinal wholemounts that were analyzed by Kruskal-Wallis test followed by Dunn’s post-test, as indicated in figure legends. The results are presented as mean ± standard error of the mean (SEM). Differences were considered significant if To assess possible structural and functional changes in the retina of the ADHD model, we used OCT  and ERG 2, Since thickness alterations reflect neural changes and these can have an impact on retinal function, we next used ERG to assess the retinal function of the SHR rats, compared to the controls, both subjected, or not, to the MPH treatment. Considering the maximum light intensity  and IPL (3.0 × 104 ± 2.3 × 103 vs. 2.5 × 104 ± 2.5 × 104 arbitrary units) as compared to WKY Veh  and inner plexiform layer (IPL). For that, the retinal cryosections were immunolabelled for the vesicular glutamate transporter 1 VGlut1; A–C. The  WKY Veh B,C. The  WKY Veh D and the WKY Veh E, and no+ Iba-1+ cells) was significantly increased in the ADHD model . The MPH treatment of the SHR animals suppressed the increase in the number of reactive microglia (5.7 ± 2.7% CD68+ of Iba-1+ cells) (+ in Iba-1+ cells (23.2 ± 5.5% CD68+ of Iba-1+ cells).It has been demonstrated that in retinal degenerative disorders or after an insult, microglial cells that are considered the resident immune cell population, become pathologically activated releasing abnormal amounts of inflammatory mediators that will contribute to tissue damage and disease exacerbation . Thus, w+ cells) A,B. On tIt has been previously shown that neuronal fractalkine (CX3CL1) acts as a regulator of microglia activation in response to tissue injury or inflammation, since CX3CL1 signals through its unique receptor, CX3CR1, that is expressed in microglia . Thus, cHaving found significant changes in microglia reactivity and knowing that these cells are the main source of inflammatory mediators, we further assessed the levels of different cytokines in the retinal tissue. Regarding the pro-inflammatory cytokine IL-1β, no differences were observed between the experimental groups A. NevertConcerning anti-inflammatory cytokines, the ADHD animal model also presented a significant increase in the IL-4 levels in the retina F. Treatm7 ± 1.2 × 106 arbitrary units; 7 ± 1.0 × 106 arbitrary units). The total protein levels of GFAP in the retina also showed a significant astrogliosis in the SHR animals (205.9 ± 28.7% of the control)  C comparecontrol) A–C. The The vessel coverage by astrocytes, which has an important role in vessel integrity and is affected by the astrocyte reactivity , was evaThe integrity of the blood-retinal barrier (BRB) is essential for normal visual function . The reaAdditionally, to explain the BRB hyperpermeability observed, we hypothesized that the retinal endothelial junction proteins, tight (TJs) and adherens junctions (AJs), that are responsible for controlling the paracellular transport pathway across BRB might be altered. Thus, we assessed the protein levels of TJs D–F and AThe present study provides new evidence on both ADHD pathophysiology and the impact of MPH, the first-line pharmacological treatment for this condition. Specifically, we demonstrated that the ADHD animal model presents retinal alterations and that MPH was able to rescue some of these features. Conversely, MPH had a negative impact in non-pathological conditions. In detail, our results show that the ADHD animal model presented retinal thinning. These results are in agreement with a previous study by Li et al., 2020 , where tOne of the hypotheses for ADHD etiology relies on the impairment in the dopaminergic system ,42, and Recent studies also demonstrated that the downregulation of the glutamate receptors (GluR) might contribute to ADHD pathogenesis . It is dAnother key objective of this study was the evaluation of the effect of MPH per se on the retina, in the control conditions. It is also important to evaluate the consequences of MPH misuse. Although MPH did not significantly alter the retinal structure, at the cellular level, there were differences. The number of cone and rod photoreceptors and RGCs decreased considerably. Moreover, MPH impaired the scotopic photoreceptor/bipolar cells pathway, and the synaptic protein synapsin and PSD95 levels. On the other hand, no alterations were found in the bipolar cell circuits under photopic conditions and on the RGCs’ function, suggesting that misuse of MPH may impact differently the dark versus light synaptic processing between the outer and the inner retina. Interestingly, an acute intraperitoneal administration of MPH (2 mg/kg) was shown to improve sensory processing and visual performance in rats ,55, demoBesides neurons, the retina is constituted by other cell types that also play an important role in its (dys)function. Microglial-mediated neuroinflammation is known to be involved in several retinal degenerative diseases , and micAstrocyte distribution in the retina is mainly associated with blood vessels. Astrocytes send processes to wrap both blood vessels and axons . In the The impact of stimulant misuse on astrocytes behavior and BRB integrity has been described. Indeed, astrocytes have been considered active players promoting drug-induced synaptic and circuit remodelling in the formation of cocaine memories , and METSince significant alterations were observed in microglia and astrocytes, and these glial cells are an important source of inflammatory mediators, we further characterized the inflammatory status of the retina in ADHD and the control conditions after the MPH treatment. Our results demonstrate that the ADHD model presents an increase in pro-inflammatory cytokines in the retina and an increase in the anti-inflammatory cytokine IL-4. It has been described that the increase in the IL-4 release by glial cells may be a mechanism to suppress the exaggerated pro-inflammatory response ,69, nameOur findings demonstrate that in the ADHD animal model, there are structural and functional alterations in the retina, which seem to be aligned with the visual alterations described in ADHD children and adolescents. The MPH treatment did not improve the retinal structural and functional impairment nor the decrease in neuronal cells observed, but partially rescued the inner BRB disruption, glial cells reactivity, and the pro-inflammatory profile in the ADHD model. Despite these significant protective effects of MPH in the ADHD model, under the control conditions, exposure to MPH had deleterious effects on the retinal physiology and on the integrity of the inner BRB, promoted glial cells reactivity, and induced a pro-inflammatory status. In sum, this study gives significant and new contribution to a better understanding of ADHD pathophysiology, specifically in an organ that has been overlooked in the field. Thus, our findings highlight the importance of the diagnosis and the consequences of the MPH treatment in ADHD, as well as the misuse of MPH in non-ADHD conditions regarding retina physiology.


Antegrade dissection and reentry (ADR) is an effective technique for wire passage in chronic total occlusion (CTO), and in recent years, the effectiveness of intravascular ultrasound (IVUS)–guided tip detection (TD)-ADR has been reported. However, the expansion of the subintimal space serves as a significant obstacle to the success of ADR, posing a limitation to the procedure.We present the first case of using IVUS-guided TD-ADR with the subintimal transcatheter withdrawal (STRAW) technique. The patient was a 68-year-old Asian female with effort angina pectoris and a CTO in the middle section of the right coronary artery (RCA). Two previous attempts at percutaneous coronary intervention (PCI) for the RCA at another hospital were unsuccessful. During the third attempt PCI, the antegrade wire migrated into the subintimal space. To address this, we performed IVUS-guided TD-ADR using the Conquest Pro 12 Sharpened Tip  wire. However, due to the expansion of the subintimal space, we were unable to puncture the true lumen. To reduce the subintimal space, we employed the STRAW technique, which allowed successful puncture of the true lumen using the CP12ST wire. Finally, stenting was performed, resulting in satisfactory antegrade blood flow.Intravascular ultrasound–guided TD provides accurate guidance for puncturing in ADR procedures, but the expansion of the subintimal space remains a significant challenge. The STRAW technique offers a solution by reducing the subintimal space and enabling successful puncture of the true lumen during IVUS-guided TD-ADR. Intravascular ultrasound–guided tip detection and antegrade dissection and reentry is a reliable technique that has emerged recently for chronic total occlusion, but enlargement of the subintimal space is still a major limitation.1 although failures still occur, particularly in complex cases.2 As a method of guidewire passage for the CTO lesion, antegrade dissection and reentry (ADR) techniques have been reported with several manners, and Stingray balloon  is most commonly employed.4 Intravascular ultrasound (IVUS)–guided tip detection (TD)-ADR has been reported as an effective method for CTO PCI.5 However, the expanded subintimal space hinders successful ADR penetration. This issue is still one of the limitations of ADR procedure. Some case reports highlighted the utility of the subintimal transcatheter withdrawal (STRAW) technique during ADR procedures using a Stingray balloon.6 Here, we present the first reported case of successful IVUS-guided TD-ADR with intraoperative expansion of the subintimal space, overcome by the utilization of the STRAW technique.Over the past decade, the success rate of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) has improved,Figure 1A). She had undergone successful PCI for the LAD, but the RCA CTO had been unsuccessfully treated in two prior attempts at another hospital. As a result, she was admitted to our hospital for retreatment of the RCA CTO. The patient was mobile and receiving antianginal therapy, including bisoprolol fumarate, amlodipine, nicorandil, and isosorbide dinitrate, along with dual antiplatelet therapy. Physical examination revealed blood pressure of 121/51 mmHg and a heart rate of 69 b.p.m. No cardiac murmurs or pulmonary rales were detected. The electrocardiogram showed no significant changes. Echocardiography indicated a 65% ejection fraction and normal left ventricular contraction, with no significant valvular disease. Blood tests showed a creatinine level of 0.63 mg/dL  and a brain natriuretic peptide level of 34.0 pg/dL . Technetium-99m tetrofosmin scintigraphy demonstrated reduced uptake in the inferior wall during pharmacological stress, while uptake at rest was normal. Effort angina pectoris due to the CTO lesion was diagnosed based on echocardiography and scintigraphy, which confirmed the viability of the inferior wall. The J-CTO score for this patient was 3 points, attributed to factors including calcification, blunt occlusion, and being a retry case.7A 68-year-old Asian female with a medical history of hypertension, diabetes mellitus, and dyslipidaemia presented with effort angina pectoris caused by a CTO in the middle portion of the RCA .Due to the anatomical complexity and it being the third attempt, we initially started with a retrograde approach. However, it was evident that the septal channels were the primary conduits for collateral circulation, which unfortunately did not appear promising due to their misty appearance. We diligently attempted to access several of these misty septal channels, hoping to establish a retrograde strategy. Unfortunately, at the distal segments of these channels, we encountered significant tight bends, and we were regrettably unable to achieve successful passage through any of them. Consequently, we opted to transition to an antegrade approach for this RCA CTO PCI procedure. An 8-French AL1.5SH catheter was engaged in the RCA through the femoral artery. AnteOwl WR  IVUS (AO-IVUS) examination revealed a heavily calcified cap at the CTO entry. We attempted to advance the Ultimate Bros 3  wire with the Corsair Pro microcatheter  into the CTO lesion, but it appeared to have migrated into the subintimal lumen. Despite attempting the balloon anchor technique, we were unable to advance the AO-IVUS catheter into the CTO lesion. After expanding the CTO lesion using a 1.5 mm ZINRAI  balloon, we introduced the AO-IVUS catheter, confirming its positioning within the subintimal space. The IVUS examination, which was performed in the subintimal space, provided information that the penetration difficulty from the subintima to the true lumen emerged due to extensive calcification. However, in a part of the vessel, which had relatively lower calcification, there appeared to be a potential for penetration from the subintima to the true lumen . Despite our efforts, penetration into the distal true lumen was unsuccessful due to the expanded subintimal space . Recognizing the difficulty of penetration with an enlarged subintimal space, we decided to perform the STRAW technique.Subsequently, we performed TD-ADR using the Conquest Pro 12 Sharpened Tip (CP12ST) wire  in combination with the FinecrossGT microcatheter . To facilitate IVUS-guided TD-ADR while continuing aspiration, a double-guiding catheter system was constructed using a 6-French AL1SH catheter engaged with the RCA. Using this 6-French guiding catheter, the Corsair Pro microcatheter was advanced to the subintimal space. Subsequently, TD-ADR was performed using the CP12ST wire with the Corsair Pro microcatheter while continuing the STRAW technique and IVUS examination through the 8-French guiding catheter . We successfully penetrated the tip of the CP12ST wire toward the true lumen under IVUS observation . The Corsair Pro microcatheter followed CP12ST into the distal true lumen. We then switched from the CP12ST wire to the Sion wire  and successfully passed through the distal RCA. After dilation with a 2.5 mm balloon, two drug-eluting stents were deployed, resulting in the successful revascularization of the RCA CTO . The procedure took 336 min, with a radiation dose of 4.7 Gy. The prolonged duration was primarily due to the initial pursuit of a time-consuming retrograde approach and challenges in wire control caused by subintimal space expansion during the IVUS-guided TD-ADR procedure. However, the introduction of the STRAW technique under IVUS-guided TD-ADR facilitated successful wire passage in ∼25 min.The subintimal aspiration using the FinecrossGT microcatheter was performed to decompress the enlarged subintimal space, which was confirmed with IVUS .Several reentry techniques have already been reported, and Stingray-ADR is the standard technique used worldwide for reentry procedures in CTO PCI.10 In our case, we utilized the STRAW technique, involving the placement of a microcatheter within the subintimal space and continuous aspiration, to effectively manage the expanding subintimal space. This approach not only enabled real-time monitoring of the reduction in subintimal space using IVUS during TD-ADR but also improved the manoeuvrability and control of the guidewire, facilitating more precise puncture into the distal target true lumen .The STRAW technique offers a viable option for decompressing the distal true lumen and enabling successful reentry in CTO cases.This approach has several limitations. A double-guiding catheter system is necessary to perform this procedure due to the inability to insert the AO-IVUS and two microcatheters even through an 8-French guiding catheter. It is important to note that this procedure becomes difficult when there is a lesion at the ostium of the coronary artery. One potential limitation is that the current approach does not effectively inhibit the in-flow, which could result in an insufficient reduction of the subintimal space with microcatheter aspiration alone. In such a scenario, instead of using a microcatheter, employing an over-the-wire balloon to expand and control inflow while simultaneously performing aspiration through the wire lumen of the balloon may offer a potential solution to reduce subintimal expansion.Intravascular ultrasound–guided TD-ADR is a reliable technique for CTO PCI; however, the expansion of the subintimal space still presents a limitation. The addition of the STRAW technique to IVUS-guided TD-ADR suggests a potential benefit in facilitating guidewire penetration into the distal true lumen when subintimal expansion occurs. Further research and experience are needed to investigate the effectiveness of this combined approach in a general CTO lesion.ytad580_Supplementary_DataClick here for additional data file.


Amphetamines are a preferred treatment for attention-deficit/hyperactivity disorder (ADHD), with the dextroamphetamine transdermal system (d-ATS) providing an alternative to oral formulations. A pivotal trial of d-ATS in children and adolescents with ADHD met primary and key secondary endpoints. This analysis reports additional endpoints and safety findings from the pivotal trial and evaluates effect size and number needed to treat (NNT) for d-ATS.In this study, a 5-week, open-label dose-optimization period (DOP) preceded a 2-week, randomized, crossover double-blind treatment period (DBP). Eligible patients received d-ATS 5 mg during the DOP, with weekly evaluations for increase to 10, 15, and 20 mg  until reaching and maintaining the optimal dose, which was utilized for the DBP. Secondary endpoints included assessment of Attention-Deficit/Hyperactivity Disorder Rating Scale IV (ADHD-RS-IV), Conners' Parent Rating Scale Revised Short Form (CPRS-R:S), and Clinical Global Impression (CGI) scores. NNT was calculated for ADHD-RS-IV and CGI-Improvement (CGI-I). Safety assessments included treatment-emergent adverse events (TEAEs) and dermal safety.p < 0.001), with effect size of 1.1 and NNT of 3 for ADHD-RS-IV remission, ≥30% improvement, and ≥50% improvement. Significant differences between placebo and d-ATS were also observed for CPRS-R:S and CGI-I scales (p < 0.001), with NNT of 2 for CGI-I response. Most TEAEs were mild or moderate, with three leading to study discontinuation in the DOP and none in the DBP. No patients discontinued due to dermal reactions.In total, 110 patients entered the DOP, with 106 patients randomized (DBP). During the DBP, the least-squares mean  difference for d-ATS versus placebo in ADHD-RS-IV total score was −13.1 (−16.2 to −10.0; d-ATS was effective in treating ADHD in children and adolescents, meeting all secondary endpoints, with a large effect size and NNT of 2–3 to achieve a clinically meaningful response. d-ATS was safe and well tolerated, with minimal dermal reactions.NCT01711021. Attention-deficit/hyperactivity disorder (ADHD) is a chronic neurodevelopmental disorder characterized by persistent inattention and/or hyperactivity/impulsivity that interferes with function or development and negatively impacts social, academic, and occupational activities  was developed as an alternative to oral amphetamine formulations and is the first and only transdermal amphetamine patch approved by the Food and Drug Administration (FDA) for use in adults and pediatric patients (children and adolescents) 6 years and older .p < 0.001) over 12 hours. Improvements in total SKAMP score were observed from 2 through 12 hours after application . Significant improvements in Permanent Product Measure of Performance (PERMP)-A and PERMP-C scores were also observed from 2 to 12 hours after d-ATS treatment compared with placebo . d-ATS was observed to be safe and well tolerated, with a systemic safety profile similar to that observed with oral amphetamines and no application-site reactions leading to discontinuation  and key secondary endpoints were met  can be used to quantify these potential benefits by providing clinical meaningfulness to changes in rating scale scores. Effect sizes estimate the efficacy of an active intervention to quantify and communicate the clinical relevance of statistically significant trial results. Cohen's effect size for comparison between two groups (active vs. placebo) is classified as small (0.20), medium (0.50), or large (0.80) Cohen, . NNT despost hoc analysis, the ADHD-RS-IV endpoint was also used to evaluate effect size, and the ADHD-RS-IV and CGI-I endpoints were used to evaluate NNT for d-ATS.To further assess the efficacy and safety of d-ATS in the treatment of 6–17-year-old children and adolescents with ADHD in a randomized laboratory classroom study, we report additional secondary endpoints from the pivotal study, including Attention Deficit/Hyperactivity Disorder Rating Scale IV (ADHD-RS-IV), Conners' Parent Rating Scale Revised Short Form (CPRS-R:S), and Clinical Global Impression-Improvement (CGI-I) and -Severity (CGI-S) scores. In a The study described herein was approved by the Western Institutional Review Board and the UC Irvine Institutional Review Board. This study included a 5-week, open-label dose-optimization period (DOP) followed by a 2-week, randomized, crossover double-blind treatment period (DBP), with details reported previously   and dermal safety, were based on the Safety Population, which included all subjects who took at least one dose of study medication and had at least one postdose safety measurement .p-values were calculated from a linear mixed model that included ADHD-RS-IV or CPRS-R:S scores for each time point as dependent variables, sequence, and treatment as fixed effects, and subject as the random effect.LS means, 95% CIs, and post hoc. Analyses were performed on the full study population and subgroups of children (6–12 years of age) and adolescents (13–17 years of age). Effect size was defined as the difference in LS mean score between treatment arms divided by the square root of the mean square error obtained from the model  of patients optimized to each d-ATS dose was 7 (7) for 5 mg, 35 (33) for 10 mg, 42 (39) for 15 mg, and 23 (21) for 20 mg  baseline ADHD-RS-IV total score for the Safety Population  was 38.3 (8.6), assessed before the start of the DOP . ADHD-RS-IV total scores from baseline through Visit 6/7 are shown in Throughout the DOP, mean ADHD-RS-IV total scores progressively improved from baseline, with mean (SD) changes from baseline of −7.4 (8.6) at Visit 1, −14.5 (9.0) at Visit 2, −20.3 (9.4) at Visit 3, −23.6 (9.3) at Visit 4, and −25.6 (9.2) at Visit 5. For both classroom days during the DBP (Visits 6 and 7 combined), ADHD-RS-IV total scores showed improvement from baseline after treatment with d-ATS compared with placebo, with a mean (SD) change from baseline of −23.4 (11.1) with d-ATS and −10.4 (11.1) with placebo. A significant difference (d-ATS vs. placebo) in LS mean (95% CI) change from baseline in ADHD-RS-IV total score was observed: −13.1  than for adolescents (0.7), who started with a lower baseline score on this subscale  in LS means of ADHD-RS-IV total scores and the inattention and hyperactivity–impulsivity subscales were observed for the full study population and subgroups of children and adolescents . Effect ectively . Effect subscale . d-ATS hsubscale .p < 0.001)  scores of 43.9 (17.1) at Visit 1, 36.6 (15.9) at Visit 2, 30.6 (15.8) at Visit 3, 26.3 (13.6) at Visit 4, and 22.7 (13.1) at Visit 5 . During < 0.001) .p < 0.001) (post hoc) NNT of 2 for CGI-I response.With respect to CGI scores, according to CGI-S assessments, most patients were moderately ill (33%) or markedly ill (61%) at baseline. During the DOP, the proportion of patients considered CGI-I responders (assessed by investigators as “very much improved” or “much improved”) increased from Visit 1 (17% [18/106]) to Visit 5  . d-ATS hSafety outcomes have been reported in detail previously  and likelihood to be helped or harmed (LHH) were not calculated from the pivotal study data. These measures would offer a more complete picture of the clinical utility of d-ATS; however, analysis of NNH and LHH would require a study that begins with a DBP rather than an open-label DOP. In addition, calculating NNH and LHH is complicated by the fact that there were no discontinuations due to TEAEs during the DBP. Lastly, there is no consensus in the literature on what change in ADHD-RS-IV score should be used to define improvement or remission.One limitation of this study is its relatively short duration. Furthermore, the classroom setting does not perfectly replicate a typical elementary or secondary classroom, which limits the generalizability of the results. Although a carryover effect was investigated for the primary endpoint   for ADHD in children and adolescents is currently available.


Attention Deficit Hyperactivity Disorder (ADHD) is a common developmental disorder affecting 5-7% of adults and children. We surveyed the literature to examine ADHD through three pillars: developmental characteristics, symptomatology, and treatment strategies. Firstly, in terms of developmental characterstics, early life stress may increase the risk of developing ADHD symptoms according to animal models’ research. Secondly, the current core symptoms of ADHD are comprised of inattention, hyperactivity, and impulsivity. However, the up-to-date literature indicates individuals with ADHD experience emotional and sensory dysregulation as well, which early-life stress may also increase the risk of. Finally, we discuss the therapeutic benefits of methylphenidate on both the current core ADHD symptoms and the sensory and emotional dysregulation found in those with ADHD. In summation, we surveyed the recent literature to analyze (i) the potential role of early-life stress in ADHD development, (ii) the involvement of emotional and sensory dysregulation in ADHD symptomatology and finally, (iii) the therapeutic intervention with methylphenidate, aiming to reduce the potential effect of early life stress in ADHD, and mainly emotional and sensory dysregulation. The apparent but currently less recognized additional symptoms of emotional and sensory dysregulation in ADHD call for further investigation of these possible causes and thus increasing treatments efficacy in individuals with ADHD. Attention Deficit Hyperactivity Disorder (ADHD) is a common developmental disorder suggested to be caused by a delay in brain maturation , affectiThe core symptomatology of ADHD comprises of inattention, hyperactivity, and impulsivity, thus forming three subtypes of ADHD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) . While iPharmacological strategies for treating ADHD symptoms include methylphenidate (MPH), being one of the most common treatments. This review focuses on MPH specifically since it has a low addictive potential , a shortTherefore, this review (August 2019 through August 2022) is conducted on three pillars: Developmental characteristics, Symptomatology, and Treatment strategies. Within developmental characteristics, the role of early life stress on the development of ADHD-like symptoms will be discussed. The specific symptoms themselves will then be discussed in the Symptomatology sections, exploring the currently accepted ADHD symptoms , as well as the proposed additional symptoms of ADHD . Finally, after discussing the way these symptoms develop and the subtleties of the Symptomatology, we will discuss how MPH mitigates ADHD symptoms, including the potential additional ADHD symptoms of sensory and emotional dysregulation. Through this discussion of Developmental Characteristics, Symptomatology, and Treatment strategies, we can follow the line of how early life stress may increase the risk of ADHD symptoms, and how certain ADHD symptoms, particularly emotional and sensory dysregulation, may give rise to the higher order symptoms of impulsivity, hyperactivity, and inattention, and how application of MPH can prevent these symptoms, respectively.Thus, we aim to understand nuances of the developmental risks, the role of sensory and emotional dysregulation on symptoms, and treatments of ADHD by considering the National Institute of Health’s call to approach a more dimensional diagnostic process for mental health concerns. Furthermore, by understanding the neurophysiological and behavioral symptoms of the disorder and determining the dis-/similarities between ADHD and emotional and sensory dysregulation, we may suggest differential and more accurate treatments of the disorders.In regards to methodology, we conducted the review between August 2017 through August 2022, as mentioned, through Google Scholar and Pubmed. A limitation worth mentioning is a large subset of these publications were gathered prior to the recent Pubmed website update, meaning some of our citations may not be available through Pubmed currently.Neglect, deprivation, abuse, and trauma may intensify the risk of ADHD development . The strThe potential role of early-life trauma on the development of inattention and hyperactivity, symptoms typically associated with ADHD, may be mitigated through environmental factors. Environmental factors like emotionally supportive parenting in childhood and early adolescence correlated with a reduction of emotional dysregulation symptoms in individuals with ADHD . ParentsEarly life stress not only correlates with the typical ADHD symptoms of impulsivity, hyperactivity, and inattention, as above-mentioned, but, according to animal models research, may intensify anxiety-like behaviors in a rat model of “emotional dysregulation” . EmotionADHD has a high comorbidity with a variety of mental disorders, like the previously mentioned generalized anxiety disorder, leading to the exclusion of emotional dysregulation as a direct feature of ADHD, and consider it a manifestation of a comorbid disorder. However, emotional dysregulation has been found in ADHD with no comorbidities, and emotional dysregulation symptoms respond to common ADHD treatments . EmotionHypothetically, emotional dysregulation may induce some of the classical symptoms of ADHD, like impulsivity and inattention. The dysregulation of emotion would make paying attention more difficult and would also increase the likelihood of impulsive behavior. This hypothesis is mirrored in data of ADHD individuals, as impulsivity and hyperactivity predict the severity of emotional dysregulation in ADHD individuals . Some stSimilar to emotional dysregulation, early life stress correlates with increased sensory dysregulation in adolescents as well . Both chThis sensory dysregulation, found in those with ADHD, seems to be tied to emotional dysregulation as well, as pain sensitivity in children with ADHD was recently shown to be associated with the severity of emotional dysregulation . This reNeurologically, these disruptions in the processing of somatosensation found in ADHD may be related to disruptions in disruptions in central nervous system inhibitory systems . The senMethylphenidate (MPH) is one of most commonly prescribed ADHD medications and is highly efficacious in treating ADHD symptoms . MPH preBoth acute and chronic MPH administration increases activity in the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC) . Acute MAcute MPH has a higher efficacy in treating attentional symptoms than impulsive symptoms . ThroughChronic MPH administration increases neuroplasticity in a variety of brain regions  and upreAfter MPH treatment, adolescents with ADHD report improved quality of life . RegardiThe current core ADHD symptoms are comprised of inattention, hyperactivity, and impulsivity . DespiteEmotional and sensory dysregulation may give rise to the current core symptoms of ADHD, which in turn induce higher order, classical symptoms. For instance, dysregulated emotions and improperly processed sensory input is quite distracting of itself. Sensorimotor gating is a preconscious regulator of attention, and is why the pre-pulse inhibition of startle is often impaired in disorders with attentional abnormalities , and visOn the treatment end, we conclude that MPH mitigates ADHD symptoms once they have developed. Acute and chronic MPH administration has beneficial effects on ADHD symptomatology . Acute aThese results lead us to conclude that: (i) developmental stress may correlate with an increased risk of ADHD development, potentially mediated by the increase of sensory and emotional dysregulation; (ii) This increased sensory and emotional dysregulation give rise to the ‘classical’ ADHD symptoms of inattention, impulsivity, and hyperactivity. These symptoms may be diagnosed through (iii) objective, physiological means, and finally (iv) mitigated with MPH application, and, following further research, possibly also by treatments aimed at these dysregulation processes. To properly further aid ADHD individuals, further research is required to better define causes, symptoms and preventative techniques. Further research is also needed to determine the role of early-life stress effects on the development of ADHD, particularly as there is limited clinical evidence for the causative role of early-life stress on ADHD development. This creates a limitation within our review paper, as the lack of research limits the potential number of references. A deeper investigation into the additional suggested symptoms of ADHD  beyond the current core symptoms of impulsivity, hyperactivity, and inattention, bares an important potential in improving both ADHD diagnosis  and treatment.In sum, better ADHD treatment strategies may be achieved by the inclusion of further research on (i) developmental stress on ADHD development, (ii) emotional and sensory dysregulation as potential ADHD additional symptoms, and finally (iii) the effects MPH on emotional and sensory dysregulation in ADHD populations. This new and comprehensive prism could lead to new tools for diagnosing and treating individuals with ADHD.AG surveyed the literature, drafted the manuscript, and co-wrote the conclusion. AA conceptualized the manuscript, co-wrote the conclusion, and edited previous versions into the final one. Both authors contributed to the article and approved the submitted version.


Perioperative neurocognitive disorder (PND) is a key complication affecting older individuals after anesthesia and surgery. Failure to translate multiple pharmacological therapies for PND from preclinical studies to clinical settings has necessitated the exploration of novel therapeutic strategies. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) treatment has emerged as a promising therapeutic strategy for treating neurodegenerative diseases and has the potential to translate basic science into clinical practice. In this study, we investigated the effects and underlying mechanism of hUC-MSCs on PND in aged mice.hUC-MSCs were isolated from an infant umbilical cord and identified using flow cytometry and differentiation assays. We established PND model by undergoing aseptic laparotomy under isoflurane anesthesia maintaining spontaneous ventilation in eighteen-month-old male C57BL/6 mice. hUC-MSCs were slowly injected into mice by coccygeal vein before anesthesia. Cognitive function, systemic and neuroinflammatory responses, neuroplasticity, endogenous neurogenesis, and brain-derived neurotrophic factor (BDNF) were assessed. To determine the brain mechanisms underlying by which hUC-MSCs mediate their neuroprotective effects in PND, K252a, an antagonist of BDNF receptor, was administered intraperitoneally before surgery. Hippocampal BDNF/TrkB/CREB signaling pathway and metabolomic signatures were evaluated.hUC-MSC treatment ameliorated the learning and memory impairment in aged mice with PND. The downstream effects were the suppression of systemic and hippocampal inflammation and restoration of neurogenesis and neuroplasticity dysregulation. Interestingly, the level of mature BDNF, but not that of proBDNF, was increased in the hippocampus after hUC-MSC treatment. Further analysis revealed that the improved cognitive recovery and the restoration of neurogenesis and neuroplasticity dysregulation elicited by exposure to hUC-MSCs were, at least partially, mediated by the activation of the BDNF/TrkB/CREB signaling pathway. Untargeted metabolomic further identified lipid metabolism dysfunction as potential downstream of the BDNF/TrkB/CREB signaling pathway in hUC-MSC-mediated neuroprotection for PND.Our study highlights the beneficial effects of hUC-MSC treatment on PND and provides a justification to consider the potential use of hUC-MSCs in the perioperative period.The online version contains supplementary material available at 10.1186/s13287-023-03499-x. With the expansion of the older population worldwide, the number of surgical procedures requiring anesthesia is increasing each year. While surgery can cure disease, prolong life, and improve quality of life, many older patients develop perioperative neurocognitive disorder (PND) following major surgery , 2. The Stem-cell therapy can overcome the deficits of traditional pharmacological therapies. It is being extensively explored as a novel treatment for neurodegenerative diseases because of its multi-target potential to alleviate cognitive impairment . MesenchThe role of inflammatory responses in PND has been extensively studied. The innate immune system activation induced by surgery is essential in PND’s onset and progression. Tissue damage following anesthesia and surgery activates peripheral immune cells and promotes release of systemic inflammatory cytokines that can directly enter the brain parenchyma across the damaged blood–brain barrier and triggers a cascade of neuroinflammatory responses , 13. ImmEighteen-month-old male C57BL/6 mice weighing 28–32 g were obtained from the Jinan Pengyue Animal Company. All animals were housed (five mice per cage) in an air-conditioned room kept at 21 ± 2 °C with a relative humidity of 45 ± 5% and 12-h light/dark cycle. They were fed standard rodent food and water ad libitum and acclimated to the environment for one week before commencing the experimental procedures. All efforts were made to minimize the number and suffering of the animals used in the study. A total of 200 aged mice were used. The number of mice that contributed data for analysis in each experiment was indicated in figure legends. All animal experiments adhere to the ARRIVE guidelines. The experimental protocol was approved by the Animal Care and Use Committee of Qilu Hospital (Qingdao), Shandong University , and was conducted in accordance with the guidelines for experimental animal use established by the Institutional Animal Care and Use Committee of Shandong University.2. The medium was changed every 3–4 days. After reaching 90% confluence, the cells were trypsinized using 0.25% trypsin and passaged at 1 × 104 cells/cm2 to passage 5 used in the study.An umbilical cord for MSC isolation and culture was obtained at Qilu hospital from a full-term healthy infant delivered after a cesarean section, whose mother had signed an informed consent form and the procedure was approved by the Qilu hospital’s human research ethics committee -086). The isolation and culture of the hUC-MSCs were conducted according to protocols published by our group and previously described procedures , 20. The6 cells were resuspended in 100 μL of cold phosphate-buffered saline (PBS) per sample and then stained with APC anti-human CD166 antibody , APC anti-human CD29 antibody , FITC anti-human CD90 (Thy1) antibody , FITC anti-human HLA-DR antibody , FITC anti-human CD73 (Ecto-5’-nucleotidase) antibody , FITC anti-human CD105 antibody , FITC anti-human CD45 antibody , respectively. After 15 min, the cells were washed and resuspended in 100 μL PBS. The cells were analyzed using a flow cytometer . In total, 50,000 events were collected per sample. The data were analyzed using FlowJo software .The characteristics of hUC-MSCs were evaluated using flow cytometry. Passage 5 hUC-MSCs were trypsinized and centrifuged. Next, 1 × 10The hUC-MSC differentiation potentials to adipocytes, osteocytes and chondrocytes were confirmed in accordance with the manufacturer’s instructions. For these assays, the cells were cultured and induced with adipogenic differentiation medium , osteogenic differentiation medium  and chondrogenic differentiation medium , and stained with Oil Red O, Alizarin Red and Alcian Blue.A modified aseptic exploratory laparotomy was performed under isoflurane anesthesia as described in our previous studies , 22. Mic7 cells/kg in 100 µL normal saline per mouse was slowly injected into mice by coccygeal vein using a 31-G needle 1 h before anesthesia. The number of hUC-MSCs used in the study was based on previous reports  or 24 h  after the initial training sessions, suggesting that control and MSC-treated mice had stronger recognition memory  = 10.043, P = 0.004] and 5   = 6.202, P = 0.006] or 24 h  after the spatial acquisition trials  = 83.69, P < 0.0001]) and IL-6  = 40.703, P < 0.0001]); however, both responses were prevented by intravenous transplantation of hUC-MSCs. To determine whether hUC-MSC-mediated cognitive improvement on PND was accompanied by changes in neuroinflammatory responses in the hippocampus, we assessed Iba-1 and GFAP by immunostaining and IL-1β and IL-6 by ELISA 24 h after surgery. Microglial and astroglial activation were significantly increased in the hippocampus after anesthesia and surgery, as evidenced by the remarkable upregulation of Iba-1  and GFAP . hUC-MSCs strongly inhibited microglial and astroglial activation  = 31.709, P < 0.0001] and IL-6  expression levels in the hippocampus and hUC-MSC treatment reversed these increases  = 44.235, P < 0.0001] and Nestin  expression, two markers of neural stem cells, in the hippocampus. hUC-MSC treatment promoted the upregulation of Sox2 and Nestin expression  = 10.683, P = 0.004], Sox2+/GFAP+  and DCX+ (a marker of immature neurons)  in the dentate gyrus (DG) region of the hippocampus in mice with PND, compared with those in control and MSC-treated mice, suggesting that hUC-MSCs reversed the reduction in neurogenesis  = 38.307, P < 0.0001] and synaptophysin (Syn) , which were effectively reversed by hUC-MSC treatment  = 20.597, P < 0.0001]  = 8.428, P = 0.009] and 15 days  after anesthesia and surgery. However, treatment with hUC-MSCs alleviated this effect  = 7.787, P = 0.011] or 15 days , and hUC-MSCs blocked the alterations  = 8.901, P < 0.0001] or 24 h  after the initial training sessions. However, the intraperitoneal injection of K252a abolished the protective effects of hUC-MSCs  = 9.483, P < 0.0001], and this effect was reversed by intraperitoneal injections of K252a , although mice in the MSC-treated group showed a significantly shorter escape latency than mice in the anesthesia and surgery group (P = 0.016). Typical escape routes on day 5 of the spatial acquisition trials are shown in Fig. F = 6.632, P = 0.001] or 24 h  after the spatial acquisition trials, while K252a decreased the number of mice treated with hUC-MSCs crossing the platform location  = 8.448, P = 0.003] and DCX  expression in the hippocampus. However, intraperitoneal injections of K252a abolished the therapeutic effects of hUC-MSCs  = 7.846, P = 0.004], Sox2+/GFAP+  and DCX  in the DG region of the hippocampus in MSC-treated mice compared with that in mice with PND. However, K252a treatment reversed the protective effects of hUC-MSCs on neural progenitor cells and immature neurons  = 12.052, P = 0.001], which were blocked by intraperitoneal injections of K252a  = 25.668, P < 0.0001]  = 7.432, P = 0.004] and p-CREB . K252a abolished the hUC-MSC-induced increases in p-TrkB and p-CREB, indicating that hUC-MSC treatment evoked the activation of the BDNF/TrkB/CREB signaling pathway  = 20.975, P < 0.0001] and increased the number of DCX+ and p-CREB+ cells  in the DG, suggesting that the BDNF/TrkB/CREB signaling pathway may mediate the neurogenesis and neuroprotective effects of hUC-MSCs in PND mice. Furthermore, blocking TrkB receptor activation with K252a altered the profiles of metabolites in the hippocampus at 15 days after surgery  in the hippocampus. β-actin was included as a loading control. Figure 8A: Original western blot gels of DCX and Sox-2 in the hippocampus. α-Tubulin was included as a loading control. Figure 8G: Original western blot gels of PSD95 in the hippocampus. α-Tubulin was included as a loading control. Figure 9A: Original western blot gels of pTrkB/TrkB and pCREB/CREB in the hippocampus. α-Tubulin was included as a loading control.


Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders. It was once thought to be a disorder affecting only children, but in those undiagnosed in childhood, symptoms do not disappear with age. There is now a growing recognition of the late diagnosis and treatment of adults with ADHD. The first-line drug in pharmacotherapy is methylphenidate, and information about its adverse effects, when used by adults, has not been as extensively described as in children. The aim of this article was to review the literature describing the risks of methylphenidate therapy for adults with ADHD. A total of 19 articles—15 clinical trials and 4 case reports presenting rare side effects resulting from methylphenidate therapy, such as reversible ischemic stroke, myocardial infarction, and psychotic episodes, were analyzed. The analysis from clinical trials included 3458 adult patients with ADHD and described the most common side effects, psychiatric adverse events, effects of methylphenidate treatment on sleep, laboratory results, body mass, and cardiovascular symptoms. Methylphenidate treatment is well tolerated, with side effects described, according to severity, as mild to moderate. We conclude that pharmacotherapy is not risk-free and methylphenidate, due to its side effects, may not be the first drug of choice for every patient. Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders . ADHD ofFor patients, immediate-release methylphenidate (IR MPH) formulations are available, although they have relatively short durations of action. Effective alleviation of ADHD symptoms lasts for about 4–6 h, necessitating multiple daily administrations. Such a regimen can be inconvenient for patients, potentially leading to noncompliance with treatment recommendations. Extended-release counterparts are available, including OROS-MPH , d-MPH-ER (extended-release d-threo MPH formulation utilizing SODAS technology), MPH-ER, MPH-LA (racemic mixture of d- and l-threo MPH), and PCR-063. Extended-release formulations offer greater convenience as they only require once-daily dosing. These formulations are designed to provide a swift onset of action and sustained relief from ADHD symptoms throughout the day. Upon administration, the first peak concentration of MPH in the bloodstream occurs at around 1.6 h, followed by a higher peak concentration after 12.5 h [The use of stimulants in the treatment of ADHD in different age groups has been confirmed to have an effect in reducing ADHD symptoms . This thExcessive ADHD diagnosis is currently a significant concern, as reflected by the increasing prevalence rates of this disorder. There is a growing worry that some of this rise is attributed to unwarranted diagnoses rather than a genuine increase in symptom severity among patients. Particularly in North America, the diagnostic criteria for ADHD and clinical practices appear to influence the escalating diagnoses . ConsideMisuse of methylphenidate often occurs with the aim of increasing concentration during studying. There are reports of recreational use, which can lead to numerous serious health and psychological consequences discussed in the results section of this article . MethylpCurrently, the growing recognition of the diagnosis and treatment of ADHD in adults is in opposition to knowledge of the side effects and safety of methylphenidate pharmacotherapy. Long-term studies on the safety of methylphenidate are an unmet medical need. Therefore, we conducted a systematic review of clinical trials in adults with ADHD and used data from published reports in this publication. Specifically, our aim was to compare the risk of methylphenidate intake in adults with ADHD compared to a group of patients taking placebo.The systematic review was conducted in accordance with PRISMA  guidelines . The revWe included adult patients, aged 18 years or older, of both sexes, who underwent a diagnosis of ADHD (any subtype) according to international diagnostic criteria such as the Diagnostic and Statistical Manual of Mental Disorders fourth or fifth edition . In addition, the diagnosis of ADHD could be confirmed by the Conners Diagnostic Interview for Adult ADHD for DSM-IV (CAADID). The coexistence of other psychiatric disorders among the subjects was not an exclusion criterion for this review in order not to limit the representativeness of the study population. We only included studies in which patients were randomly allocated drugs. Patients had to take methylphenidate at pharmacological doses and not for abuse or intentional overdose (intoxication). Publications had to be full-text, available in English. We included double-blind, randomized controlled trials comparing methylphenidate with placebo. The authors decided to also consider case series and case reports. Open-label studies could also be included, only if they were an extension of a randomized, double-blinded control trial, and only patients previously participating in a randomized trial were included. We only included study groups in which patients were randomly allocated drugs at the approved dose. Both fixed-dose and flexible-dose designs were allowed.The authors analyzed the current literature considering clinical trials, case series, and case reports. Articles published between 1988 and 2023 in the PubMed database were searched. The following terms were used to search for articles: “safety”, “side effects”, “harms”, “withdrawal symptoms”, and “addiction” in combination with “methylphenidate AND ADHD AND Adults”. The selection of articles involved conducting an initial screening based on abstracts to select studies that might be related to the topic. Article references were used to identify articles missed during database searches.We searched the database according to the search strategy and initially obtained 249 potentially eligible studies. A review of the bibliography of the initially retrieved articles revealed an additional 2 studies. After initial screening of titles and abstracts, 56 studies that were repetitive were excluded, and 149 articles were excluded after applying the inclusion criteria. Of the remaining 46 studies, 27 were excluded due to a lack of valuable results or, after reading, were found to contain information unrelated to the topic of the review. The flow diagram of the analysis is shown in The following basic information was obtained from the included studies: first author, year of publication, type of study, duration of study, type of drug preparation used, age of subjects, number of subjects, intervention, and the study’s rating on the QATQS Global Rating questionnaire. While collecting data from each specified publication, we emphasized their qualitative analysis.To evaluate potential bias and the quality of quantitative studies, the Quality Assessment Tool for Quantitative Studies (QATQS) from the Effective Public Health Practice Project (EPHPP) was employed ,27. ThisThe data extracted from analyzed studies are presented in detail in A total of 3458 adult patients with ADHD from clinical trials were randomly assigned to receive either methylphenidate or placebo. Among them, 2318 received methylphenidate, and 1140 received placebo  reporting rates were comparable between the MPH and placebo groups. The majority of reports in the investigators’ assessment were not treatment-related. Reported cases included uterine cancer, ulcerative colitis, hypovolemic shock, appendicitis, and rupture of the shoulder and collar joint. One man in the MPH group was reported with an onset of depressive disorder following a relationship breakdown; he continued treatment and received additional counselling, and this did not affect his exclusion from the analysis. The researchers considered this case as possibly treatment-related . One advAccording to the recommendations of the European Adult ADHD Network  and the This review demonstrated that the use of methylphenidate in adults with ADHD does not appear to be associated with changes in symptoms of anxiety and depression in patients with concurrent psychiatric disorders. Among patients without prior psychiatric disorders, alterations in mood, nervousness, and decreased libido were observed. Additionally, the potential for the emergence of psychotic and manic symptoms because of MPH usage was identified. Notably, one of the most frequently reported adverse effects of methylphenidate (MPH) is a loss of appetite, accompanied by corresponding reductions in body weight, relative to MPH dosage.It was shown that psychostimulants, such as methylphenidate (MPH), may elevate blood pressure and heart rate, raising concerns about cardiovascular safety. Research indicated a significant increase in blood pressure within the first week of MPH treatment. Patients receiving MPH exhibited a higher risk of clinically significant elevated heart rates compared to those on a placebo. While no changes in electrocardiogram (ECG) results were observed, instances were documented where methylphenidate could influence elevated cardiac troponin levels. Furthermore, a case of acute myocardial infarction with ST-segment elevation was described, with coronary vasospasm suspected to be linked to MPH. A reversible case of cerebral ischemic attack associated with methylphenidate usage was also reported. The effect of stimulant treatment on sleep in adults with ADHD is multifaceted and variable, resulting in either worsening or improvement of sleep patterns, contingent upon individual cases.This systematic review was large in scope and provided many summaries, but it remains an incomplete picture of the risk of methylphenidate use by adults with ADHD. An important limitation of this study pertains to the lack of distinction made between the manifestations of methylphenidate administration in female and male participants. This omission restricts the researchers’ ability to draw meaningful conclusions regarding potential differences in tolerance and responses to the medication between genders. A deeper analysis considering gender-specific responses to methylphenidate could provide valuable insights into the pharmacological effects and potential side effects of the medication within distinct demographic groups. Another consideration is the decided paucity of long-term studies. There are animal studies in the literature that warn of the potential neurotoxic effects of methylphenidate ,52,53. HADHD exerts a substantial and enduring impact on individuals throughout their lives, underscoring the importance of identifying and addressing this condition in adults. Effective management of ADHD symptoms in adults can mitigate the disorder’s detrimental effects on various aspects of daily functioning and mental well-being. Methylphenidate has emerged as a viable treatment option; however, it is imperative to acknowledge that its utilization comes with potential risks and considerations. Consequently, individuals undergoing methylphenidate treatment must remain under the vigilant supervision of healthcare professionals. These clinicians play a pivotal role in tailoring treatment plans to the individual’s specific needs, considering preexisting health conditions and potential risks associated with the medication. By maintaining a comprehensive approach that balances the benefits and potential drawbacks, clinicians can ensure that patients receive the optimal treatment while minimizing any adverse effects.


After the molecular characterization of vLDX and the comorbidities , we created a protein-protein interaction human network to which we applied artificial neural networks (ANN) algorithms. We also generated virtual populations of adults and children-adolescents totaling 2,600 individuals and obtained the predicted protein activity from Therapeutic Performance Mapping System models. The latter showed that ADHD molecular description shared 53% of its protein effectors with at least one studied psychiatric comorbidity. According to the ANN analysis, proteins targeted by vLDX are predicted to have a high probability of being related to BED and depression. In BED, vLDX was modeled to act upon neurotransmission and neuroplasticity regulators, and, in depression, vLDX regulated the hypothalamic-pituitary-adrenal axis, neuroinflammation, oxidative stress, and glutamatergic excitotoxicity. In conclusion, our modeling results, despite their limitations and although requiring in vitro or in vivo validation, could supplement the design of preclinical and potentially clinical studies that investigate treatment for patients with ADHD with psychiatric comorbidities, especially from a molecular point of view.Attention-deficit/hyperactivity disorder (ADHD) is a psychiatric condition well recognized in the pediatric population that can persist into adulthood. The vast majority of patients with ADHD present psychiatric comorbidities that have been suggested to share, to some extent, the pathophysiological mechanism of ADHD. Lisdexamfetamine (LDX) is a stimulant prodrug approved for treating ADHD and, in the US, also for binge eating disorder (BED). Herein, we evaluated, through a systems biology-based  Attention-deficit/hyperactivity disorder (ADHD) is an impairing psychiatric condition affecting children and adults and is characterized by symptoms of inattention, hyperactivity, and impulsivity . Noteworin silico head-to-head trials  and methylphenidate to treat ADHD in adults and children  are treatment options in the clinic for children, adolescents and adults patients with ADHD . CBT hasd trials . We recechildren . As an ein silico method, the efficacy of vLDX as ADHD treatment to improve some common ADHD psychiatric comorbidities. In addition, we explored the molecular mechanisms behind vLDX’s predicted efficacy in virtual adult and pediatric-adolescent comorbid patients with ADHD.The main objective of our study was to evaluate, through a systems biology-based in silico approaches to evaluate the potential relationship of LDX with psychiatric comorbidities by applying Therapeutic Performance Mapping System (TPMS) technology  that the drug (group of protein targets) and the pathology  being evaluated are functionally connected. Therefore, scores greater than 92% indicate a very strong relationship with a p-value below 0.01; scores between 77 and 92% imply a strong relationship with a p-value between 0.01 and 0.05; scores between 37 and 77% have a medium relationship and a p-value in the range 0.05–0.25; and scores lower than 37% indicate a poor relationship with p-values above 0.25. Finally, we modeled the mechanisms of vLDX for the most relevant comorbidities by using sampling methods after QSP modeling. These models provide mechanisms of action that explain how a stimulus  produces a response   . The quaenotype) .in silico clinical trial from which this detailed mechanistic study derives is described in detail elsewhere  from each QSP model . We analAccording to the bibliography-based molecular characterization of the pathologies under study , the ADHp < 0.01) of being related to BED and depression (p < 0.25) for anxiety and tic disorders and very low (p ≥ 0.25) for bipolar disorder. Although the ANN values for anxiety (76%) and tic disorders (71%) were very close to the p-value < 0.05 threshold (77%), only the results for BED and depression were considered statistically significant and subjected to detailed mechanistic evaluation.Artificial neural networks showed that the proteins targeted by vLDX had a high probability  and neuronal survival and plasticity (BDNF-NTRK2) . In bothVirtual model of Lisdexamfetamine modulated depression by regulating glutamatergic excitotoxicity, neurotransmission, neuroplasticity, the hypothalamic-pituitary-adrenal (HPA) axis, neuroinflammation, and oxidative stress . Accordiin silico method, we found that vLDX’s target proteins were related to BED and depression. Considering LDX use as treatment in patients with ADHD and taking a previously described virtual population of patients with ADHD and psychiatric comorbidities . In adults, ADHD is highly comorbid with other psychiatric disorders; 65–89% of patients with ADHD present at least one psychiatric comorbidity, mainly anxiety and mood and substance use disorders . In chilThe association between ADHD and BED has been established by several clinical studies in adults and children . In our Monoamine transporters NET1, DAT1, and SERT, which are reported LDX targets , were prOur ANN analysis also showed a highly probable relation between proteins targeted by vLDX and depression. In this sense, current available evidence in the literature points toward a slight improvement in depressive symptoms when using LDX as antidepressant augmentation therapy . In our in silico clinical trial approach are described in detail elsewhere (in vivo research on polydrug reactions in ADHD is needed to fully understand these complex interactions and how it impacts on ADHD and its psychiatric comorbidities. Lastly, although this in silico modeling allowed us to get more insight into vLDX activity on BED and depression in patients with ADHD disease, which may be helpful to guide further research, the results obtained in this study must be validated or refuted with in vitro and/or in vivo data.Notwithstanding the abovementioned, our study presented some limitations that should also be considered. Firstly, the main constraint was the available information, at the moment of the study, on the drug and diseases under study upon which our models were built. In the current study, this factor might be aggravated by the difficulty of studying psychiatric diseases molecularly and by the high genetic and signaling overlap between ADHD and its psychiatric comorbidities , which mlsewhere . Third, lsewhere . Althouglsewhere . Regardilsewhere . Therefoin silico modeling study, vLDX’s targets were predicted to be related with high probability to proteins involved in BED as a comorbidity in patients with ADHD, for which LDX is currently indicated in the US adult population. In addition, vLDX was modeled to impact the pathophysiology of an ADHD comorbidity highly prevalent in adults and adolescents, namely, depression. Reasoned mechanisms were presented to explain our results other than neurotransmitter regulation, including effects on neuroplasticity, neuroinflammation, oxidative stress, and the HPA axis. These results could inform preclinical and even clinical future investigations treatments for patients with ADHD and psychiatric comorbidities.According to our The original contributions presented in the study are included in the article/JG-C, JQ, and CM designed the study. CS-V and MC performed the investigation and formal analyses. JG-C, JQ, JS, and TP-R contributed to data interpretation. JG-C and JQ drafted the first version of the manuscript. All authors critically revised all drafts of the manuscript for intellectual content.