P < 0.001) and multivariable analysis . An independent external cohort validated the significance of p-AMPK low expression. In addition, in vivo experiments demonstrated that AMPK activation status was negatively related to HCC occurrence and blocking autophagy by chloroquine counteracted the protective effect of AMPK phosphorylation. These results present novel insight into a critical predictive role of AMPK activation in hepatocarcinogenesis and AMPK activation seems to be a potential target for the prevention of hepatocellular carcinoma in patients with liver cirrhosis.Metformin has been demonstrated to prevent hepatocellular carcinoma (HCC). Metformin acts mainly by phosphorylation of AMPK. However, the phosphorylation status of AMPK and its role in the prediction and prevention of HCC in cirrhotic patients remains unclear. The phosphorylation status of AMPK (Thr172) was determined by immunostaining in tissue microarrays of 426 cirrhotic liver tissues. Low expression of p-AMPK was observed in 94 (22.1%) cases. The median follow-up time was 87 months. HCC occurrence probability at 1/3/5/10 years after Hassab procedure was 3.1/9.6/13.8/30.6% in patients with p-AMPK low expression and 0/0.3/0.3/8% in patients with p-AMPK high expression, respectively. HCC occurrence risk was significantly higher in patients with p-AMPK low expression in univariable analysis (HR, 6.25; 95% CI: 3.36–11.60;  Cirrhosis is a milieu that is conducive to development of hepatocellular carcinoma (HCC), the second most lethal cancer worldwide2. Therefore, it is of vital importance to identify novel effective prediction and prevention strategies for the management of liver cirrhosis. Metformin is a first-line antidiabetic drug. In recent years, metformin has been suggested to play a role in the suppression of various cancers in patients with diabetes, including HCC, gastric cancer, colorectal cancer, etc5.Liver cirrhosis is the 13th leading cause of death globally, with worldwide mortality having increased by 45.6% from 1990 to 20137. In response to certain stressful conditions, such as energy deprivation, AMPK is activated by phosphorylation at Thr172 in α subunit. Once activated, AMPK regulates various molecules and signaling pathways to modulate adaptive changes and maintain metabolic homeostasis9. Recently, emerging evidence has indicated the potential role of AMPK signaling in tumor initiation and progression11. An early finding showed that AMPK pathway activation by metformin was involved in metformin-induced growth inhibition of cancer cells12. Further, AMPK activation not only downregulated mTOR but also suppressed the excess aerobic glycolysis (Warburg effect) characteristic of most transformed cells and genetic ablation of the a1 catalytic subunit of AMPK accelerated Myc-induced lymphomagenesis13. This line of investigation suggested a tumor suppressor role for AMPK activation. AMPK is also a promising target for treating fibrotic diseases. AMPK activation reduced thioacetamide-induced liver fibrosis in mice and inhibited the activation of cultured hepatic stellate cells and macrophages14. These data suggested that AMPK activation was an important factor regulating both fibrosis and cancer. Therefore, it is intriguing to understand the phosphorylation status of AMPK in cirrhotic livers and its role in the process from cirrhosis to HCC.Metformin exerts pharmacological effects mainly through the phosphorylation of the AMP-activated protein kinase (AMPK), a highly conserved heterotrimeric serine/threonine kinaseIn the present study, we use population study and in vivo experiments to address the role of p-AMPK in the pathogenesis of HCC.P > 0.05). Patients in low p-AMPK expression group had a significantly higher proportion of patients with Child-pugh B or C , diabetes mellitus , and GI bleeding  compared with patients in high p-AMPK expression group  and low p-AMPK expression group . Typical images were shown in Fig. , 77.7% aP < 0.001). As shown in Table P < 0.001). Because diabetes and alcohol are known risk factors for HCC and Child-pugh grade indicates different cirrhosis degree, stratified analysis was conducted according to diabetes mellitus, alcohol, and Child-pugh grade. The results showed there was a consistent trend toward higher HCC occurrence risk in low p-AMPK expression group irrespective of DM, alcohol history or Child-pugh grade . Patients in West China hospital cohort did not differ significantly from patients in Chongqing cohort regarding age, AST, GGT, TBIL, ALB, AFP, ascite, hepatic encephalopathy, DM, alcohol history, and GI bleeding (P > 0.05). Patients in West China cohort had a significantly lower ALT and lower proportion of male and Child-pugh B or C patients. In addition, patients in West China cohort were all HBV-related. As shown in Table P < 0.001). As shown in Table P < 0.001).The predictive value of p-AMPK expression was also evaluated in an independent cohort of 131 HBV-related cirrhosis patients from West China Hospital of Sichuan University. The median follow-up time was 84.5 months. As shown in Table n = 8) or PBS (n = 9) were used to inhibit AMPK activation or as control. In p-AMPK low-expression group, metformin (n = 7) or AICAR (n = 6) were used to activate AMPK, PBS (n = 7) was used as control. The experimental flow chart was shown in Fig. To verify if AMPK activation can reduce HCC occurrence, we performed liver biopsy in mice at 8 weeks in the model process. The expression of p-AMPK was detected by immunohistochemical staining. The same grouping criteria with human liver samples were adopted. The typical images were shown in Fig. 15. The protein levels of ACC, the rate limiting enzyme involved in fatty acid synthesis, or serum triglycerides levels were not different significantly in neither different p-AMPK expression level mice treated with PBS nor different p-AMPK modulators . As expected, CQ decreased numbers of autophagosomes in the liver compared with metformin alone and promoted accumulation of p62 without suppressed LC3B-II production . In total 215 patients lacked integrated clinic data were excluded. Approximately 147 patients were excluded with insufficient tissue samples for investigation. Another 158 patients who were not following up regularly or without follow-up data were excluded. All human sample collection procedures were approved by the Ethical Review Committee of the Hospital. Informed consent was obtained in all cases before surgery. The diagnosis of liver cirrhosis was confirmed by pathology.All patients were followed up until HCC detection or the end of the study in December 2016, with a median follow-up time of 87 months. The patients were monitored by measurement of serum alpha fetoprotein level and abdominal ultrasound every 3–6 months. When HCC was suspected, enhanced CT or MRI scanning was used to confirm the HCC diagnosis. The time from surgery to the first detection of HCC was recorded. The follow-up data were obtained through medical record system if the patients follow up in our hospital regularly. If the patients did not follow up in our hospital, the time interval of follow-up and the examination items were first checked. Only the patients followed up with the stated time interval and examination items were included in this study.Type 2 Diabetes was diagnosed as a fasting plasma glucose level of >7.0 mmol/L (126 mg/ dL), or a plasma glucose level of >11.1 mmol/L (200 mg/dL) at 2 h in a 75-g oral glucose tolerance test, or typical diabetes mellitus symptoms together with a casual plasma glucose level of >11.1 mmol/L (200 mg/dL).Past and current alcohol intake was assessed at the time of the first visit and at each following visit. The assessment of alcohol consumption was made according to patient declarations. We defined alcohol drinkers as subjects who still consumed alcohol at least one time each week after surgery.Gastrointestinal bleeding (GI bleeding) was defined as at least one upper gastrointestinal bleeding occurred before or after operation.20. In brief, from 1995 to 2009, 152 HBV-positive cirrhotic liver tissues with complete clinical and follow up data were included in these tissue microarrays. All patients were followed up until May 2014, with a median observation time of 84.5 months. The same follow-up protocol with Chongqing cohort was adopted. In total 21 patients were excluded in this study because they were not treated with Hassab procedure .Futher more, tissue microarrays of 152 cirrhotic liver tissues were gifted from the West China Hospital with complete patients’ authorization and full ethical approval of the Institutional Clinical Ethics Review Board of Sichuan University. The detail clinical information has been described in a previous articleAll human studies presented in our manuscript have been approved by the appropriate ethics committee and performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments.22. α-SMA+ activated hepatic stellate cells, collagen stain area, F4/80+ macrophages, and p62 positive cells were quantified using Image J software by applying the appropriate pixel threshold equally on all selected pictures and using measure function to calculate the covered area. Immunohistochemical staining quantification was performed on three randomly selected high-power fields per liver section. Data are represented as % of covered area for each cell type over total tissue area23.Immunohistochemical staining of tissue microarrays was carried out using a two-step protocol. Following antigen retrieval, the slices were incubated with primary antibody overnight at 4 °C. Negative controls were prepared by replacing the primary antibodies with PBS. To evaluate the expression of p-AMPK, tissue microarrays were assessed independently by two experienced pathologists with minimal inter-observer variability, and we used a semi-quantitative assessment method wherein the scoring parameters included staining intensity  and the percentage of positive cells . We added the percentage of positive cells to the intensity score to determine the final staining scores. Total score <4 was defined as low p-AMPK expression, while a score ≥4 was defined as high p-AMPK expressionMale C57BL/6 mice were obtained from Chongqing Medical University. To induce hepatic carcinogenesis, 4-week-old C57BL/6 male mice received 75 mg/kg DEN  in two divided doses and CCL4  twice a week until 20 weeks. After 8 weeks, fresh liver tissues were obtained through liver biopsy. Immunohistochemical staining was used to detect the level of AMPK phosphorylation. The same criteria with human tissue microarrays were adopted to distinguish the level of p-AMPK expression. In mice with p-AMPK high expression, dorsomorphin  was used to inhibit p-AMPK, or PBS as a control. In mice with p-AMPK low expression, metformin  or AICAR  were used to activate AMPK. For autophagy inhibition, mice received 50 mg/kg of chloroquine  via intraperitoneal injection once every 3 days along with a concurrent application of metformin. All mice were killed at 32 weeks. All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.Liver tissues were used for harvesting total protein extraction and estimation. Polyacrylamide gel electrophoresis (PAGE) was performed, and PAGE gels were transferred to nitrocellulose membranes. Following transfer, membranes were blocked with 5% nonfat milk, incubated with antibodies for AMPK , p-AMPK , ACC , mTOR, p-mTOR, RAGE, CD44(ab157107), DLK1, LC3B , p62 , and antibody for GAPDH  was used as a loading control. The PVDF membranes were visualized using the Chemico-EQ system . The targeted protein bands were analyzed and quantified using Image Lab 3 software .The levels of alanine aminotransferase (ALT), total bilirubin (TBIL), albumin (ALB), and triglycerides were detected by an automatic biochemical analyzer .The liver tissues were fixed with 2.5% glutaraldehyde Ultrathin sections were cut and doubly stained with uranyl acetate and lead citrate. For autophagic vacuole quantification, 10 micrographs, primary magnification ×2000, were randomly taken from each sample and the total amount of autophagic vacuoles was counted.t test or Mann–Whitney U test, while that of differences in categorical data were assessed using the chi-squared test or Fisher’s exact test (two-tailed). Survival analysis was performed using the Fine and Gray model by accounting for the competing risk of death24.The primary outcome was HCC occurrence after Hassab procedure. The significance of intergroup differences in continuous data were assessed using the Student's P values were two-sided and P < 0.05 was considered significant. Statistical analyses were performed using SPSS 22.0 or R 3.4.0 (http://www.R-project.org/).Continuous variables are described as mean±standard deviation, and categorical variables are expressed as frequency (%). The results of survival analysis are described as hazard ratio (HR) and 95% confidence interval (CI). The multivariable model was generated by using the backward selection method. All reported Supplementary Information


The inflammatory biomarker pro-adrenomedullin (ProADM) provides additional prognostic information for the risk stratification of general medical emergency department (ED) patients. The aim of this analysis was to develop a triage algorithm for improved prognostication and later use in an interventional trial.We used data from the multi-national, prospective, observational TRIAGE trial including consecutive medical ED patients from Switzerland, France and the United States. We investigated triage effects when adding ProADM at two established cut-offs to a five-level ED triage score with respect to adverse clinical outcome.Mortality in the 6586 ED patients showed a step-wise, 25-fold increase from 0.6% to 4.5% and 15.4%, respectively, at the two ProADM cut-offs . Risk stratification by combining ProADM within cut-off groups and the triage score resulted in the identification of 1662 patients (25.2% of the population) at a very low risk of mortality  and 425 patients (6.5% of the population) at very high risk of mortality . Risk estimation by using ProADM and the triage score from a logistic regression model allowed for a more accurate risk estimation in the whole population with a classification of 3255 patients (49.4% of the population) in the low risk group  and 1673 (25.4% of the population) in the high-risk group .Within this large international multicenter study, a combined triage score based on ProADM and established triage scores allowed a more accurate mortality risk discrimination. The TRIAGE-ProADM score improved identification of both patients at the highest risk of mortality who may benefit from early therapeutic interventions (rule in), and low risk patients where deferred treatment without negatively affecting outcome may be possible (rule out). Timely and an accurate risk stratification of patients in the emergency department (ED) supports the clinician in reducing time to effective treatment—a key predictor for patient outcome. A shorter time to effective treatment is an important outcome predictor for patients with septicemia , pneumonAccordingly, several triage scores in the ED have been consequently proposed , 10. TheThe aim of this analysis was to develop a clinical algorithm based on a combination of a clinical triage score and ProADM levels for an improved prognostic assessment in unselected medical ED patients. The effects of using these scores on timeliness and patient outcomes will later be investigated in an intervention trial.The TRIAGE study is a multi-national, prospective, observational cohort study. From March 2013 to October 2014, we included consecutive medical patients presenting with a medical urgency at three participating tertiary care hospitals in Aarau (Switzerland), Paris (France), and Clearwater , respectively. As an observational quality control study, the Institutional Review Boards (IRB) of the three hospitals approved the study and waived the need for individual informed consent (main Swiss IRB: Ethikkommission Kanton Aargau (EK 2012/059); French IRB: CCTIRS—Le Comité consultatif sur le traitement de l'information en matière de recherche (C.C.T.I.R.S.) (CPP ID RCB: 2013-A00129-36); US IRB MPM-SAH Institutional Review Board, Clearwater Florida [IRB number 2013_005]).http://www.clinicaltrials.gov/ct2/show/NCT01768494) and the study protocol, as well as more detailed information about the study design, has been published previously [The study was registered at the “ClinicalTrials.gov” registration website  was assigned based on the routine hospital algorithm . All parThroughout the hospital stay, physicians, nurses and social care workers managed patients in accordance to local hospital guidelines according to the underlying medical condition and independently of the research team. All patients were contacted by telephone interview 30 days after hospital admission using a predefined questionnaire to assess vital and functional status, and unplanned hospital readmission among other outcomes.The main research aim of this secondary analysis was to derive a triage algorithm based on ProADM and triage risk classes using already established ED triage scores for an improved prognostic assessment in unselected medical ED patients. ProADM was first used within cut-off ranges based on previous studies  and in aIn accordance with the initial study, our primary endpoint was 30-day mortality following ED admission. To verify survival status, we followed all patients during hospital stay and contacted them 30 days after inclusion by telephone.Left over blood samples of routinely collect blood tubes on admission were immediately centrifuged, aliquoted and frozen at -20° C for later batch analysis of blood biomarkers. The results of these analyses were not available at the time of hospitalization of the patients. Thus, physicians, patients and outcome adjudicators were blinded to their results. ProADM was batch-measured with a sandwich immunoassay with an analytical detection limit of 0.08nmol/L as described elsewhere .We used descriptive statistics including mean with standard deviation (SD), median with interquartile range (IQR) and frequencies to describe the populations, as appropriate. We displayed observed and expected probabilities for all adverse outcomes within deciles of ProADM and goodness-of-fit assessed with the Hosmer-Lemeshow test. Based on these analyses and previous research , we defiTests were two-tailed and carried out at 5% significance levels. Analyses were performed with STATA 12.1 .Table 1. Overall ProADM distribution is skewed right as shown by a histogram .From a total of 7342 ED patients initially included in participating Swiss, French, and United States (USA) hospitals, 6586 patients  had complete follow-up information, as well as biomarker and triage score data and were included in the final analysis . The most prevalent principal diagnoses were cardiovascular diseases (25.4%), neurological diseases (20.9%), gastrointestinal diseases (13.2%), and acute infections (13.1%). We observed a high prevalence of comorbidities including hypertension, coronary heart disease, diabetes, and cancer. Regarding adverse outcomes within 30 days of ED admission, 4.8% of patients died and 6.8% of patients were admitted to ICU. Additional patient characteristics are shown in Fig 1, we estimated associations between admission ProADM levels divided into deciles and 30-day mortality. There was a very low observed mortality in the lowest four ProADM deciles  with an exponential increase thereafter, particularly in the highest 2 ProADM deciles . We therefore used ProADM cut-off levels of ≤0.75nmol/L indicating low risk, >0.75–1.5nmol/L indicating moderate risk, and >1.5nmol/L indicating high risk. The overall risk for 30-day mortality showed an almost 25-fold (from 0.6% to 4.5% and 15.4%) increase within these ProADM groups . For patients in the low risk ProADM group (i.e. <0.75nmol/L), mortality could be excluded with a high negative predictive value (NPV) of 99.4% (95% CI: 99.0 to 99.6) and a sensitivity of 94.0% (95% CI: 90.7 to 96.3). Conversely, in patients with high ProADM levels  specificity was 83.4% (95% CI: 82.4 to 84.3) and positive predictive value (PPV) was 15.4% (95% CI: 13.4 to 17.6) (Table 3).As demonstrated in Fig 2). The proportion of 30-day non-survivors increased from 0.3% in patients classified as “not urgent” and ProADM ≤0.75nmol/L to 19.3% in patients classified as “very urgent” and ProADM >1.5nmol/L. In patients classified as “not urgent” according to the triage score, we found a strong increase in the risk of 30-day mortality with increasing ProADM levels (from 0.3% to 13.6%). Table 3 shows sensitivity, specificity and positive and negative predictive values at different triage score as well as ProADM cut-offs.In a next step, we combined the three ProADM categories  with triage score risk subgroups . The addition of ProADM further improved prognostication of the triage score for all patient groups . When only using ProADM to classify patients, 45.1% (n = 2972) of patients had biomarker levels ≤0.75nmol/L and had thus a low risk of adverse outcomes. A total of 36.2% (n = 2382) were at an intermediate risk (ProADM >0.75–1.5nmol/L) and 18.7% (n = 1232) at a high risk .Using the above analyses, we developed a simplified easy to use clinical algorithm based on the initial triage score and the ProADM category and used it virtually within our patient cohort. Based on the triage score, 21.2% (n = 1397) of patients were initially classified as very low urgent (“blue”), 19.5% (n = 1285) as low urgent (“green”), 34.9% (n = 2301) as intermediately urgent (“yellow”), 18.5% (n = 1220) as highly urgent (“orange”), and 5.8% (n = 383) as very urgent (“red”). These patients were further classified in three categories as shown in Fig 3C shows patients after reclassification based on ProADM cut-off groups and the triage score group. Thereby, “not urgent” and “moderate urgent” patients were upgraded to the next higher category if ProADM levels were >1.5nmol/L. Conversely, “moderate urgent” and “very urgent” patients were downgraded to the next lower category if ProADM levels were ≤0.75nmol/L.Fig 4 shows that the combination of ProADM cut-offs with triage score information thereby resulted in an improvement in sensitivity and specificity with respect to observed mortality. Results were also robust in subgroup analyses based on main medical disciplines or symptoms on ED admission . These results were also confirmed in category-based reclassification statistics (Table 4). The addition of ProADM within cut-off groups resulted in a net reclassification improvement (NRI) of 0.39 for predicting mortality.Fig 4). In doing so, the proportion of non-survivors in the high-risk category was further increased , whereas rule out was also improved (9/3255). The relative risk reduction from the simplified to the logistic model was 74.3%. This effect was robust throughout the main medical disciplines with a strong risk reduction in the low risk population and a stable or increased prediction rate in higher risk categories. Stratified for main symptoms and diagnoses on admission, the logistic regression prediction model was superior in ruling out patients than the simplified model. The relative risk reduction ranged between 65.4% and 100%. The performance of the regression model is provided in S1 File.Finally, we also investigated the predictive ability of using ProADM and triage scores as continuous variables within a logistic full regression model  of non-survivors can be identified, the benefit may outweigh costs associated with marker measurement. At this point, it is unclear whether such an algorithm will result in improved outcomes of patients. There is therefore a need for an interventional trial looking at outcomes and also costs associated with an improved ED triage.In summary, combining a traditional triage score with clinically relevant ProADM cut-off levels allowed a more objective triage and risk stratification of consecutively entering ED patients, independent of their main complaint or disease. It highly improved prediction of 30-day mortality in lower triage classes which might enforce physician`s compliance with triage decisions. This will likely lead to more overruling decisions in favor of an outpatient  strategy in subjects without a severe illness. Often, these patients are hospitalized based on a high preference on the part of relatives due to logistical reasons. Adherence to the biomarker enhanced algorithm would also improve patients`safety who formerly would have been discharged home based on a lower triage score but in fact carried a highly significant risk of short-term mortality .Apart from that, a further main strength of this study is the prospective multinational, multicenter design with >6000 unselected representative patients. Thus, these results will be largely generalizable to many international ED settings.There are some limitations of this study. Firstly, we did not evaluate prediction power of the combined triage algorithm for the primary task of a triage score, the treatment priority. Rather, we investigated a more objective, but not less relevant “hard” endpoint that should be considered in early risk and site of care assessment. Secondly, participating ED personnel were not blinded to the triage score levels, and thus may adapt their priority recommendation accordingly, possibly overestimating the triage score`s performance. Thirdly, we had no validation cohort. Thus, an internal and external validation of the proposed triage approach will be fundamental to prove its effectiveness. Fourthly, within this observational cohort, we were not able to definitively demonstrate whether an improved triage of patients translates into improved outcomes; for this reason, a randomized controlled trial ultimately testing this strong hypothesis is warranted. Fifthly, implementation of the suggested logistic regression based combined algorithm must be available in an electronical form, as required for a pragmatic use. Sixthly, due to missing values, we did not address to smoking and body mass index in the full logistic regression model, as two important confounders of ProADM. However, we included initial diagnoses involving obesity to make a pass at the missing body mass index. Seventhly, we only focused on ProADM based on its performance in previous research , but othThe combination of established triage scores and ProADM firstly allowed the identification of a higher proportion of ED patients at a high risk of short-term mortality, and secondly improved risk stratification in this heterogeneous patient cohort. Whether the hypothesized benefits will translate into a safe “real-life” improvement of patients`outcome has to be assessed in an interventional trial. Optimized resource allocation will increase patient safety and has high relevance in current health care discussion.S1 FigProADM, pro-adrenomedullin.(PPTX)Click here for additional data file.S2 Fig(A-C) stratified for main medical disciplines on admission, (D-G) stratified for main symptoms on admission. ProADM, pro-adrenomedullin.(PPTX)Click here for additional data file.S1 File(A) odds ratios; (B) intercept and calibration slopes(DOCX)Click here for additional data file.


The diagnosis of chronic rhinosinusitis (CRS) based on clinical presentation alone remains challenging. To improve the accuracy of clinical diagnosis, the Canadian Rhinosinusitis Guidelines recommend the use of specific symptom and endoscopic criteria. Our study objective was to determine whether symptom and endoscopic criteria, as defined by the Canadian Rhinosinusitis Guidelines, accurately predict CT-confirmed CRS diagnosis.A retrospective cohort study of 126 patients who underwent CT sinuses based on clinical suspicion of possible CRS. The presence of symptom and endoscopic criteria, as defined by the Canadian Rhinosinusitis Guidelines, were compared between patients with and without a CT-confirmed CRS diagnosis using two-tailed Fisher’s exact tests. Positive predictive values and likelihood ratios were determined for each symptom and endoscopic finding.Overall, 56.3% of patients had a CT-confirmed diagnosis of CRS. With the exception of nasal polyps, none of the symptom or endoscopic criteria had a statistically significant correlation with positive CT sinuses. For symptom criteria, positive predictive values ranged from 52.4% to 63.4%; likelihood ratios ranged from 0.85 to 1.34. For endoscopic criteria, positive predictive values and likelihood ratios were 71.4% and 1.94 (edema); 63.0% and 1.32 (discharge); and 92.9% and 10.1 . 35.2% of patients with CT-confirmed CRS had normal endoscopic exams.The Canadian Rhinosinusitis Guidelines’ symptom and endoscopic criteria for CRS, with the exception of nasal polyps on endoscopy, do not accurately predict CT-confirmed disease. In addition, a normal endoscopic exam does not rule out CRS. Chronic rhinosinusitis (CRS) is one of the most prevalent chronic diseases in the developed world . The estThe diagnosis of CRS based on symptoms alone has been a longstanding challenge. Despite multiple revisions to diagnostic criteria over the past 20 years, the symptom-based diagnostic criteria still have a specificity of only 2–12%, and a positive predictive value (PPV) of 35–54% . For thiThe Canadian Clinical Practice Guidelines for acute and chronic rhinosinusitis  define fThis was a retrospective cohort study. We identified all CT sinuses imaging studies ordered for evaluation of possible CRS through a tertiary level rhinology clinic between January 2014 and March 2016. To achieve this, we used a comprehensive imaging database to view all CT sinuses ordered in that time frame. Patients were excluded if there was evidence of previous endoscopic sinus surgery (ESS), sinonasal mass, symptom duration <8 weeks, age < 18 years, or existing diagnosis of CRS. A chart review was performed for each of these patients The following variables were extracted from routine questionnaires for past medical history: age, gender, smoking status (yes/no), history of asthma (yes/no), and history of nasal allergy (yes/no). Symptoms of CRS , and findings on nasal endoscopy  were documented during the patient interview and physical exam. Patients were considered symptom positive based on the Canadian guidelines if 2 or more of these symptoms were present for >8–12 weeks.t-test was applied. The significance level was 0.05.The CT studies were assigned a Lund-Mackay score , with a n = 3); nasal mass (n = 10); previous endoscopic sinus surgery (n = 47); and duration <12 weeks or diagnosis of recurrent acute bacterial sinusitis (n = 4); resulting in 126 patients included. 86% of patients met the Canadian symptom criteria for CRS. Overall, 56.3% of patients had CT-confirmed diagnosis of CRS.A total of 190 CT sinuses were identified. After reviewing CT scans and charts, the following patients were excluded: existing diagnosis of CRS , nor did they demonstrate a statistically significant difference between groups with and without CRS. The Canadian symptom criteria collectively (i.e. two or more CPODS symptoms) showed high sensitivity (87.3%) and low specificity (16.4%), but did not show a statistically significant difference between the CT+ and CT- groups.Table p = 0.003, LR = 10.07). Findings of discharge or middle meatal edema were less strongly predictive  and showed no statistically significant difference between groups. A substantial number of patients with CRS had a normal endoscopic exam (35.2%).Endoscopic evidence of polyps was highly specific and predictive of disease, and showed a statistically significant difference between CT+ and CT- groups , comparable to other international CRS guidelines  , 10, 11,p = 0.02). Conversely, edema and discharge were relatively non-predictive, and were not significantly different between groups. We found that 35.2% of patients with CRS have no endoscopic findings at all. This finding supports the current International Consensus Statement [Endoscopic examination is a routine and important step in evaluating patients for CRS. The presence of nasal polyps was highly predictive of disease (PPV 92.9%), and was the only variable in our study that demonstrated statistically significant difference between CT+ and CT- groups  variables were extracted by patient-filled questionnaire, and were not formally assessed with pulmonary function tests, skin prick testing, or other objective measures. We therefore did not perform in-depth analyses of these variables as they pertain to CRS. The designation of Lund-Mckay ≥4 as diagnostic for CRS also presents some limitations. Firstly, although this cutoff is widely accepted in rhinologic literature, it does not account for patients with marked single-sinus or unilateral disease. Second, we have no way of documenting symptomatology at the time of CT scan. The extent of disease could be over- or under-reported, depending on the patient’s condition at the time of scan . Similarly, their condition may have improved by the time of their CT scan if treatment was initiated at their first visit. However, we feel that the cohort of patients converting from a positive to a negative scan (ie Lund-Mackay <4) after therapy initiation  would be exceedingly small. This could be addressed in future prospective studies by withholding treatment until baseline imaging is obtained.CRS continues to present a diagnostic challenge. Although certain symptoms (such as hyposmia and purulent rhinorrhea) have been ascribed high predictive values in the literature , 10, thiThe Canadian Clinical Practice Guidelines for acute and chronic rhinosinusitis define symptom and endoscopic criteria for CRS, which apart from nasal polyps on endoscopy, are not highly predictive of CT-confirmed disease. In addition, negative endoscopy does not reliably rule out CRS. These results highlight the ongoing diagnostic challenge presented by CRS.


ABSTRACTCASE STUDYTHA is a 40-year-old immigrant from Myanmar who has been living in the United States for about 6 years. He has a history of hepatitis B virus (HBV) infection for many years, for which he has never received any treatment. He is negative for hepatitis B ’e’ antigen (HBeAG), with normal liver enzymes on his routine primary care visits. THA was referred to a hepatology clinic when recent laboratory studies revealed a slowly increasing tumor marker—alpha-fetoprotein level (AFP). His AFP increased from a normal level of 8 ng/mL  in December 2014 to 44 ng/mL in June 2015. His HBV DNA level was 30,409 IU/mL. Because of his history of chronic hepatitis B infection and the rising AFP level, a computed tomography (CT) scan was done and showed a 4 cm × 3.5 cm liver mass in segment 8, accompanied by cirrhotic morphology to the liver  A  with an Eastern Cooperative Oncology Group (ECOG) performance status of 0, Child-Turcotte-Pugh A cirrhosis.THA was determined to be a good candidate for transplant, based on the BCLC and Milan criteria . The modTHA was then referred to interventional radiology for consideration of locoregional liver-directed therapies. This would offer disease control and stability while he underwent the additional testing necessary before being placed on the transplant list. After a patient is formally placed on the list for transplant, there is at least a 6-month wait to receive a liver due to the organ allocation process and medical necessity. Bridging liver-directed therapies protect a patient’s candidacy for curative transplant and may help to decrease the dropout rate from the transplant waiting list, thereby having a positive effect on posttransplant survival and tumor recurrence rates .During his transarterial chemoembolization procedure, THA underwent an arteriogram, which revealed the solitary hypervascular mass in the right lobe. Selective transarterial chemoembolization was then done by injecting 1 vial of 100 to 300 µm drug-eluting LC Bead particles loaded with 75 mg of doxorubicin into the target artery supplying the tumor. A postembolization arteriogram showed no residual tumor blush see  below.THA experienced mild nausea and several days of minimal fatigue. He recovered well, with no residual effects. A follow-up magnetic resonance imaging performed 6 weeks after his procedure revealed greater than 95% tumor response see  below. TARTICLEPrimary liver cancer is one of the most common malignancies occurring worldwide as well as one of the most common causes of cancer-related deaths . HepatocWorldwide, chronic infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) is the primary risk factors for HCC. As was the case with our patient THA, as many as one in eight individuals are chronically infected with HBV in countries such as Myanmar . ObesityA separate risk factor, genetic hemochromatosis, is due to excess iron absorption related to mutations in the high iron (HFE) gene . Althougp < .001), respectively. The 10-year actuarial survival for patients treated with resection was 33%, compared with 49% for patients who received a liver transplant (p = .002). Systemic therapy with sorafenib (Nexavar) in patients with advanced disease yields a 1-year survival rate of approximately 44%  categorizes patients into four categories based on the potential for success with various treatment strategies. They include: patients with disease that may be treated with surgery (either resection or transplantation) and whose performance status (PS) and comorbidities allow for such surgery; patients for whom surgery is not an option due to PS and/or comorbidities; patients with unresectable disease; and patients with metastatic disease .For patients with unresectable disease who are being treated with TACE, the NCCN refers to two staging systems . One of p = .001, p = .001, p = .004, p = .000, respectively). When survival statistics for each stage of disease according to the various systems were compared, the CLIP system had distinct survival periods without overlap, as well as a statistical difference between adjacent prognostic scores. These outcomes indicate the validity of this particular staging system.The CLIP system was compared with six other staging systems in patients who had undergone TACE to determine which was the most useful in this population . MultivaThe second staging system mentioned by the NCCN with regard to TACE for HCC is the Barcelona Clinic Liver Cancer (BCLC) staging system . The BCLp < .001; B2 subgroup [22.1 months] vs. B3 subgroup [14.1 months], p = .001; there was no difference between B3 [14.1 months] and B4 subgroups [17.2 months], p = .48; Because of the heterogeneity of patients considered BCLC stage B, researchers have worked to further subdivide the class, with the goal of selecting an appropriate population for treatment with TACE . These sOther groups have advocated offering treatment with TACE to an even broader population of patients with HCC. One such group is the Expert Panel Opinion on Interventions in Hepatocellular Carcinoma (EPOIHCC), which recommends expanding the appropriate patient population to include those with BCLC A, B, or C disease, Eastern Cooperative Oncology Group (ECOG) PS < 2, and Child-Pugh < C . TACE haThe BCLC staging system see  links thConventional TACEConventional TACE is not a new technology. Use of embolizing agents mixed with chemotherapy or given alone (bland) for the treatment of HCC dates back to the late 1970s and 1980s . MateriaLimitations to the use of some of these agents include large particle size or irregular shape, making it more likely larger vessels will be occluded instead of the smaller targeted distal vasculature feeding the tumor. Incomplete vascular occlusion is another issue with some of these materials, as well as problems related to clogging the catheter used to deliver the agent to the intended area of the vasculature . EmbolizEthiodized oil has also been used in combination with various chemotherapy agents, such as doxorubicin, epirubicin, mitoxantrone, fluorouracil (5-FU), mitomycin, and cisplatin, for the treatment of HCC. It allows for the delivery of the chemotherapy to the site of the tumor with subsequent prolonged, local release of the chemotherapy drug . One issTo overcome compatibility issues, the chemotherapy drug must first be dissolved in other substances, such as water-soluble x-ray contrast agents. The mixture is then added to the ethiodized oil. This combination of chemotherapy, ethiodized oil and an embolizing agent, had been viewed as the standard of care for patients with intermediate-stage HCC until the introduction of drug-eluting beads (DEBs).Drug-Eluting BeadsMore recently, DEBs have emerged for the treatment of HCC. There currently are two DEB preparations available: LC Bead  and QuadraSphere Microspheres . The LC Beads are made of polyvinyl alcohol modified by sulfonate sodium salt, and QuadraSphere is made of vinyl alcohol and a sodium acrylate co-polymer. Both preparations consist of uniform-sized beads that can be loaded with various chemotherapeutic agents, most commonly doxorubicin, irinotecan, or oxaliplatin .Pharmacokinetic studies of both preparations have been done in animal models to evaluate doxorubicin plasma concentrations, release rate, and concentration at the tumor site. The study that evaluated doxorubicin-loaded DEBs in a rabbit model of liver cancer demonstrated a significantly lower plasma concentration of doxorubicin compared with intra-arterial administration. A peak intratumor doxorubicin concentration of 413.5 nmol/g was observed at 3 days, compared with a peak tumor concentration of 0.09 nmol/g in the group receiving doxorubicin intra-arterially. Intratumor doxorubicin concentrations remained high through days 7 (116.7 nmol/g) and 14 (41.76 nmol/g) in the DEB group compared with the intra-arterial route (0 nmol/g within 1 hour of injection), intra-arterial followed by bland beads (5–25 nmol/g), or a combination of ethiodized oil, doxorubicin, and bland beads (12–36 nmol/g). Plasma concentrations in the group treated with DEBs were minimal at all measured time points .Similar results were reported with the QuadraSphere preparation loaded with doxorubicin, with peak tumor concentrations being observed at 3 days (40.632–50.052 nmol/g) and detectable levels throughout the 7-day study period. Pronounced tumor necrosis was observed at 3 days and continued through the 7-day study period . PharmacBoth bead preparations are available in various sizes to accommodate vessels of various sizes. LC Beads are available in 70–150 µm, 100–300 µm, 300–500 µm, and 500–700 µm sizes. QuadraSphere microspheres are supplied as dry particles, which swell when hydrated. The particle sizes of available QuadraSphere dry (hydrated) microspheres are 30–60 µm (120–240 µm), 50–100 µm(200–400 µm), 100–150 µm (400–600 µm), and 150–200 µm (600–800 µm). Studies have been conducted comparing various bead sizes to see whether there are differences in outcome depending on the bead size used.One retrospective study conducted in patients with a diagnosis of HCC and Child-Pugh A/B, absence of portal vein thrombosis, no extrahepatic disease, and an ECOG PS of 0 to 2 compared a single TACE treatment using doxorubicin-loaded LC Beads with a particle size of 100–300 µm (n = 39) and 300–500 µm n = 22; . Choice There was found to be a lower incidence of postembolization syndrome and fatigue after treatment in the 100–300 µm group (8% and 36%) compared with the 300–500 µm group . Complete response tended to be higher in the 100–300 µm group  and partial response was higher in the 300–500 µm group . The authors noted that the 300–500 µm size was likely chosen in some earlier clinical trials because it was similar to that of the embolization materials used in conventional TACE . Smallerp = .04 and 0% vs. 14.3%, p = .001, respectively). Again, there was found to be an advantage in the use of smaller-sized beads for DEB-TACE.A second retrospective study showed similar results . This stOur patient (THA) was treated with LC Bead 100–300 µm loaded with 75 mg of doxorubicin. He suffered minimal adverse events, primarily several days of mild fatigue and some mild nausea. Our institution uses only LC Bead 100–300 µm, although the other sizes are on the formulary.When the QuadraSphere microsphere preparation was first marketed, our institution considered adding the product to our formulary. Because of the absence of studies directly comparing the two bead preparations and existence at that time of extended stability data only for the doxorubicin-loaded LC Bead preparation , the QuaClinical Evidence on Drug-Eluting Bead Transarterial ChemoembolizationBoth prospective and retrospective studies have compared conventional TACE and DEB-TACE. Results of these studies demonstrated an advantage to DEB-TACE with regard to efficacy, toxicity, or both see .p = .11; DCR: 63.4% vs. 51.9%, p = .11).One randomized, prospective, multicenter trial compared treatment with doxorubicin-loaded DC Bead (n = 93) to treatment with conventional TACE, which consisted of doxorubicin in ethiodized oil followed by an embolic agent administered every 2 months for a maximum of 3 doses . At 6 mop = .012, p < .001, respectively), despite a higher mean total dose of doxorubicin in the DEB-TACE group (295 vs. 223 mg).Supplementary analyses demonstrated differences in OR and DCR among patients with advanced disease (66.7% of patients in both groups), defined as Child-Pugh B, ECOG PS of 1, bilobar or recurrent disease . No difference between groups in overall incidence of treatment-related serious adverse events was observed within 30 days of treatment (≤ = .34). However, there were fewer doxorubicin-related adverse events and less liver toxicity in the group treated with DEB-TACE compared with conventional TACE .A complete response was observed in four of the tumors treated with DEB-TACE, and six patients had a partial response. Five patients were reported to have grade 3/4 aminotransferase elevations, and one patient was reported to have grade 3 hyperbilirubinemia, all of which returned to baseline within several days of treatment. Seven patients were reported to have grade 1/2 postembolization syndrome. The authors hypothesized that not all tumors retain ethiodized oil–doxorubicin, likely making conventional TACE less effective than the prolonged release of doxorubicin when given as DEB .p < .0001); bilirubin > 2.0 mg/dL (p = .0007); and Child-Pugh A vs. B/C (p = .037).Use of DEB-TACE has also been evaluated in patients with advanced disease. One retrospective study reported outcomes of DEB-TACE in patients with BCLC stage C (n = 30)/D (n = 13) disease . Median p = .025) and patients who had more than two DEB-TACE procedures  is used after the procedure for pain control, with the goal of switching the patient to oral medications prior to discharge.When HCC is confined to the liver, it can be successfully treated with DEB-TACE. Current recommendations have expanded the use of TACE to patients with more advanced disease, including those with BCLC C disease and a good performance status and liver function. Patients with HCC rarely present without underlying liver disease, which poses its own mortality risk and can affect patients’ ability to tolerate the TACE procedure. We do not perform TACE on patients with an ECOG PS > 2. Patients with encephalopathy and poorly controlled ascites related to underlying liver disease are not candidates for TACE at our institution.The TACE procedure is also being evaluated in combination with sorafenib, where ischemic damage caused by TACE and the subsequent release of angiogenic growth factors may be addressed by use of this kinase inhibitor.Chemoembolization is considered palliative therapy. Although a cure for unresectable HCC is possible after TACE, it rarely occurs. Repeated segmental selective TACE tailored to tumor response has been shown to significantly improve survival in patients with unresectable HCC .The rationale for TACE in our patient (THA) was to provide bridging therapy prior to liver transplantation. It provided a means to control tumor growth while the patient awaited organ allocation and resulted in tumor necrosis, which had the added benefit of reducing the risk for tumor dissemination during transplant surgery. Approximately 20% of patients drop off the transplant list due to tumor progression . The TACOverall, chemoembolization provides localized therapy to isolated malignant lesions. Adverse events due to the chemotherapy are limited, and the procedure can be performed with a minimal hospital stay. Treatment can provide benefit to patients by keeping the tumor in check, either for palliation or as a bridge to transplantation. We look forward to the addition of other agents, such as tyrosine kinase inhibitors, to this treatment to further control tumor growth and spread.


Systemic lupus erythematosus (SLE) is a chronic autoimmune and inflammatory disease affecting multiorgans of human body. Independent studies show that SLE patients had higher caries prevalence compared to non-SLE individuals. However, the underlying mechanisms remain unclear. In present study, we enrolled SLE patients to explore potential factors contributing to the susceptibility of SLE patients to dental caries . Dental examination confirmed SLE patients were more vulnerable to caries. Although subjects in both groups announced similar oral hygiene habits, more dental plaque was found on tooth surfaces of SLE patents as revealed by plaque index. In addition, the salivary function was impaired in SLE group as salivary flow rate, buffering capacity, and pH were lower among SLE subjects compared to healthy controls. Importantly, disturbed microbial community with lower richness and diversity was observed in SLE group, as well as disequilibrium between acidogenic/aciduric pathogens and alkali-generating commensal bacteria. Our data suggest that SLE increases patients' sensitivity to dental caries through imposing stress to both host and oral microbes. Systemic lupus erythematosus (SLE) is a chronic disease affecting several organs of the human body. It is a heterogeneous disease with autoimmune origin and is characterized by the presence of autoantibodies against nuclear antigens . The worOral manifestations have been found frequently in SLE patients, including recurrent infections or mouth ulcers, severe gingivitis, temporomandibular joint disorder, osteonecrosis of the mandible, hyposalivation , and excessive dental caries –6. Among Streptococcus mutans and Streptococcus sobrinus species were enriched in SLE patients' salivary microbiome, while a decrease in salivary flow rate, pH, and buffer capacity was observed compared to healthy subjects [ Candida spp., Staphylococcus spp., Enterobacteria, and Pseudomonas spp. between SLE patients and healthy individuals [Although some investigations have revealed the association between SLE and dental caries, the study aimed at exploring reasons why SLE patients are more vulnerable to dental caries is still in its infancy, and the existent findings seem to be controversial. For example, Loyola Rodriguez et al. 2016) found that found thividuals . Apart fividuals , 11. ThuConsidering saliva function and oral microbial community are both involved in the pathogenesis of dental caries, we hypothesized that SLE might increase the susceptibility of SLE patients to dental caries through impairing saliva function and introducing disequilibrium into oral microbial community. To test our hypothesis, treatment-naïve SLE patients and healthy controls were recruited in present clinic study. The caries frequency was checked; then oral hygiene habit, saliva variables , and characteristics of salivary microbial community  were investigated.This study is conducted in accordance with the Declaration of Helsinki (1964) and is approved by the ethical committee of the First Affiliated Hospital of Zhengzhou University. Informed and voluntary written consent from subjects were obtained. The overall design of present study was shown in Subject Enrollment. Patients were enrolled when they visited the hospital searching for medical help due to SLE. The diagnosis was performed by a rheumatologist using the criteria of the American College of Rheumatology [N = 347) completed a health questionnaire regarding their systemic health. Exclusion criteria included pregnancy, occurrence of other systemic diseases , and receiving antidepressants treatment in the last 3 months or taking antibiotics/antifungal drugs in the past 1 week. Following the systemic health questionnaire, the oral health was screened by a dentist. Noninclusion criteria included detectable oral diseases other than dental caries, harmful behaviors , and use of removable dentures. Finally, 20 SLE patients meet the inclusion and exclusion criteria and were included in present clinical study, and majority of enrolled subjects (2 out of 18) were women. Moreover, another group of age- and sex-matched healthy people (N = 20) were recruited as controls.matology  first, aOral Hygiene Survey. The frequency of tooth brush per day, use of fluoride products, and artificial saliva of each subject were reported and recorded.Caries Examination. Dental examination was carried out by a dentist with the WHO criteria for the diagnosis and coding of caries [f caries . The WHOPlaque Index Assay. The plaque index (PI) was recorded as described previously [eviously . PI indeSaliva Collection for Microbial Assay. Volunteers were asked to not eat or drink for 8 hours and to refrain from oral hygiene (such as brushing or flossing of teeth) for 12 h before sampling, and each subject was instructed to expectorate about 5 ml spontaneous, unstimulated whole saliva into a sterile cryogenic vial put on ice  in the morning [ morning . SpecimeSaliva Flow Rate and Buffer Capacity Analysis. The saliva flow rate and buffer capacity assay were conducted 1 h after microbial sampling. After resting for 5 minutes, volunteers chewed a piece of paraffin wax (30 seconds) and expectorated into a 50 mL cryogenic vial on ice within 5 minutes [ minutes . The salDNA Extraction and PCR-DGGE. About 1 ml of saliva was used for genomic DNA isolation with QIAamp DNA micro Kit , and an extra lysozyme treatment was added to lyse cell wall as described by Wang et al. [g et al. . The conμL, containing 100 ng genomic DNA, 40 pmol of forward and reverse primer, 200 μmol/L dNTPs, 4 mmol/L MgCl2, 5 μL 10x PCR buffer, and 2.5 U Taq DNA polymerase  [The universal primers targeting the V4-V5 hypervariable region (~300 bp) of the 16S rRNA gene locus were used in the PCR amplification, and the sequence of primers was Bac1 (5′-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGACTACGT-GCCAGCAGCC-3′) and Bac2 (5′-GGACTACCAGGGTATCTACTAATCC-3′) . The volia, USA) . Cyclingia, USA) . The resμg/mL ethidium bromides prepared with 1x TAE buffer for 15 min, and destained in 1x TAE buffer for 10 min. DGGE images were captured through Molecular Imager Gel Documentation system (Bio-Rad Laboratories).Polyacrylamide gels (8% (w/v)) were prepared with a denaturing urea/formamide gradient between 40% (containing 2.8 mol/L urea and 16% (v/v) formamide) and 60% (containing 4.2 mol/L urea and 24% (v/v) formamide). About 300 ng of amplicons was loaded into each well. The gels with PCR products were submerged in 1x TAE buffer, and the loaded DNA fragments were separated by electrophoresis at 58°C using a fixed voltage of 60 V in the Bio-Rad DCode System for 17 h . Immediately after electrophoresis, gels were rinsed once, stained using 0.5 qPCR Analysis of Oral Samples. S. mutans, S. sobrinus, S. sanguinis, S. gordonii, Actinomyces naeslundii, and all bacterial counts were quantified using qPCR as previously described [escribed . PrimersStatistical Analysis. Mann-Whitney U test was used to compare age, DMFT scores, tooth brushing frequency, plaque index, and qPCR data between groups.DGGE images were normalized and analyzed with Quantity One Software . Bands detected from each sample were taken as microbial richness, and alpha diversity comparison was based on Shannon index taking both gel tracks and band intensity into consideration . The unwt-test was then used.For intergroup comparison of salivary parameters, richness, and diversity, the quantitative data was first analyzed using Levene's test for homogeneity of variances to assess the equality of variances. One-way ANOVA with Dunnett N = 20) and healthy volunteers (N = 20) were enrolled to investigate their dental health, oral hygiene, saliva function, and oral microbiome, all of which are highly associated with caries pathogenicity [Although independent clinical investigations indicate the higher prevalence of dental caries in SLE patients, the factors contributing to the susceptibility of SLE sufferers to tooth decay was still unclear. In present study, SLE patients  and ammonia producing bacteria (S. sanguinis and S. gordonii). These four bacteria were chosen since the pH drop caused by organic acid produced by acidic and aciduric bacteria is among the most important virulence of cariogenic bacteria while S. sanguinis and S. gordonii in the biofilm can also generate alkali to antagonize the pH drop through arginine metabolism [ S. sanguinis and S. gordonii in the saliva of SLE patients was statistically higher than that of control group (p < 0.05). However, S. sanguinis and S. gordonii showed a decreasing tendency in SLE group (p > 0.05). These data suggested that microbial disequilibrium between acidogenic/aciduric pathogens and alkali-generating commensal bacteria colonized in the oral cavity might be involved in the caries risk of SLE patients.The DGGE assay shows that oral microbiome of SLE changed a lot compared to healthy control without explaining how it. To give a detailed example how SLE changed the oral microbial community, we further used qPCR to determine the relative abundance of some specific taxa including acidogenic bacteria (tabolism , 30. As In conclusion, our data further confirmed that SLE patients were more vulnerable to caries infection. The poor plaque control, dysfunction of saliva gland, and disturbed microbial community with microbial disequilibrium increase patients' sensitivity to dental caries. However, we admit that the conclusion drawn from present study should be taken carefully due to the small sample size, and more investigations are needed to confirm it. Furthermore, studies are necessary to reveal through which pathways SLE plays its influence on saliva function and oral microbial community.


The characteristics of all four ditches are presented in Macrophyte cutting was last conducted in 1993 (Neuenkirchen ditch) and 1999 (Estebrügge and Jork ditches), prior to the monitoring. Due to the short distance between the water body and the first apple tree line of the Neuenkirchen orchard, the use of pesticide application machines between the ditch and first tree line was prevented at this site, leading to pesticide application of the first tree line directly in the path to the water body (the pesticide application machines were operated between the first and second tree lines).2.2Water samples were taken from April to October over three years (2001–2003). Event-driven water samples were collected manually as soon as possible after pesticide application to measure the peak pesticide concentrations. Five samples of 0.5 L were taken from the upper 20 cm of the water layer at evenly dispersed intervals along a stretch of 100 m and combined to make a 2.5-L water sample. All samples were stored in glass flasks, except for samples collected after the application of copper-bearing products, which were stored in polyethylene flasks. Weekly water samples were collected during periods without pesticide application to assess the background contamination of pesticide residues. In parallel, 30-mL samples were taken automatically at hourly intervals, providing the basis for the weekly integrated samples . Samples were stored in the dark at 4 °C and analysed as soon as possible.2.3Water samples were filtered (4–7 µm folded filter), and a surrogate standard was added (acetochlor for liquid chromatography (LC) analysis and tetrachlorvinphos for gas chromatography (GC) analysis). The surrogate standard was used to check the analytical measurement procedures and to evaluate the results, but not for result corrections. The samples were then subjected to solid-phase extraction . Subsequently, the samples were evaporated to dryness under a nitrogen atmosphere and dissolved in acetonitrile and an internal standard solution using an ultrasonic device LC and GC coupled to mass spectrometry – LC-MS/MS (PE Sciex API 2000) and GC–MS (Finnigan GCQ) – were used for identification and quantification of the target substances in the samples. The pesticides were identified by their retention time and multiple reaction monitoring transitions (LC-MS/MS) or full-scan spectra (GC–MS), respectively. The pesticides were quantified with reference standards in the solvent, and quantification followed the internal standard method. All results are presented as averages of duplicate injections of the sample extracts. The pesticide data were not corrected by the respective recovery rates. Fenoxycarb, indoxacarb, methoxyfenozide and thiacloprid were only analysed in 2003. Benomyl, dithianon, glyphosate, metiram, naphthylacetamide and sulphur were not analysed. The following 19 substances were analysed but not detected at levels above the limit of detection (LOD): azoxystrobin, azinphos-methyl, cyanazine, dimefuron, dichlofluanid, epoxiconazole, flufenacet, linuron, metazachlor, metobromuron, metconazole, metoxuron, propaquizafop, prochloraz, quinoxyfen, spiroxamine, tetradifon, tolylfluanid and vinclozolin.2.4Copper and the pyrethroid insecticide beta-cyfluthrin were analysed separately with methods suitable for these active substances. Beta-cyfluthrin was analysed using GC coupled with an electron capture detector and GC–MS/MS following immediate filtration after sampling. The recovery rate for beta-cyfluthrin, analysed separately, was 83%, on average (relative standard deviation of 13%).Copper residues were analysed using inductively coupled plasma optical emission spectrometry in 2001 and 2002. Therefore, the water samples were filtered through a 0.45-µm cellulose acetate membrane filter and acidified. The LOD for this method was 20 µg/L. Starting in 2003, the water samples were analysed for copper residues using atomic adsorption spectrometry to lower the LOD with this method to 0.3 µg/L.2.5Method validation was performed for all substances (except for beta-cyfluthrin and copper) according to European Union Commission Directive 96/46/EC The mean recoveries of the active substances found in the water samples ranged from 70% to 110% with relative standard deviations < 10%, meeting the requirements of European Union Commission Directive 96/46/EC The recovery rate of the surrogate standards was 93%, on average (relative standard deviation of 13%), meeting the requirements of European Union Commission Directive 96/46/EC 2.6Storage stability tests were performed for all substances (except for beta-cyfluthrin and copper) using surface water from the control Estebrügge ditch (grassland), according to European Union Commission Directive 96/46/EC


N=12 per group) from postnatal day 25  until PD95 (young adult), and imaged one day before and one day after the 70-day course of treatment. Our LDX dosing regimen yielded blood levels of dextroamphetamine comparable to those documented in patients. Longitudinal analysis of striatal volume revealed significant hypertrophy in LDX-treated animals when compared to vehicle-treated controls, with a significant treatment by time point interaction. These findings confirm a causal link between long-term stimulant treatment and striatal hypertrophy, and support utility of longitudinal MRI in rodents as a translational approach for bridging preclinical and clinical research. Having demonstrated comparable morphological effects in both humans and rodents using the same imaging technology, future studies may now use this rodent model to identify the underlying cellular mechanisms and behavioral consequences of stimulant-induced striatal hypertrophy.Stimulant treatment is highly effective in mitigating symptoms associated with attention-deficit/hyperactivity disorder (ADHD), though the neurobiological underpinnings of this effect have not been established. Studies using anatomical magnetic resonance imaging (MRI) in children with ADHD have suggested that long-term stimulant treatment may improve symptoms of ADHD in part by stimulating striatal hypertrophy. This conclusion is limited, however, as these studies have either used cross-sectional sampling or did not assess the impact of treatment length on their dependent measures. We therefore used longitudinal anatomical MRI in a vehicle-controlled study design to confirm causality regarding stimulant effects on striatal morphology in a rodent model of clinically relevant long-term stimulant treatment. Sprague Dawley rats were orally administered either lisdexamfetamine  or vehicle ( Acutely, stimulants are thought to confer their therapeutic effects by altering monoaminergic neuromodulation of circuits intrinsic to the prefrontal cortex, sensorimotor cortex, and the basal ganglia.8 as well as the basal ganglia, particularly of nuclei comprising the striatum.11 The striatum is the main input region of the basal ganglia that plays a role in habit learning, motivation, goal-directed behaviors, reward processing and motor control,16 functions that have been shown to be disturbed in ADHD.18 Some studies indicate that striatal volume shows evidence of normalization in patients with a history of psychostimulant treatment relative to untreated patients.20 Moreover, Sobel et al.9 found that localized increases in striatal volume in treated patients paralleled symptom improvement. These findings suggest that long-term stimulant treatment may intervene in the disease process and reduce symptom severity by altering striatal morphology .Studies using anatomical magnetic resonance imaging (MRI) suggest that individuals with ADHD exhibit reduced volumes of various cortical regions,21 These limitations have precluded causal links from being drawn between stimulant treatment and changes in morphology and behavior. Furthermore, little is known about the molecular mechanisms of stimulant-induced changes in striatal morphology. As MRI analyses in humans are limited to detecting macroscopic changes in the brain, animal models are needed to determine the underlying cellular and molecular nature of stimulant-induced alterations in striatal morphology. Ultimately, understanding how these changes relate to performance on behavioral tasks probing impulsivity, attention, and other cognitive domains affected in ADHD,22 will provide a crucial step towards developing novel, more targeted treatments with fewer side effects than currently available medications.23Studies investigating striatal morphology in ADHD have in general been limited by the use of cross-sectional designs. The two longitudinal studies that have been performed did not assess the impact of treatment length on dependent outcomes.in vivo rodent MRI techniques to allow for repeated brain measurements in each animal across treatment course . In addition, we set out to confirm our study findings by calculating regional volumes, by an independent rater, in ex vivo gadolinium-contrasted high-resolution MRI scans acquired on the same animals that completed the longitudinal study. Given the prominent use of MRI as a research tool in clinical populations, the use of this technology in rodents confers high translational value as findings could be readily compared with clinical samples.The primary aim of the current investigation was to establish causality regarding the effects of long-term stimulant treatment on striatal morphology in a rodent model using comparable MRI technologies to those used in human studies. To test the hypothesis that long-term stimulant treatment induces striatal hypertrophy, we used a vehicle-controlled, longitudinal study design with a rodent model of clinically relevant stimulant administration. In order to track changes in regional volume as a function of treatment and length of exposure, we made novel use of longitudinal ad libitum. The Institutional Animal Care and Use Committee at the Nathan Kline Institute for Psychiatric Research approved of all the animal protocols used in this study.Male, Sprague Dawley rats between the ages of postnatal day (PD) 23–94 were used in the present study. To habituate the animals to oral deposition of drug vehicle before study initiation, we acquired all our experimental animals through timed-pregnant female rats purchased from Charles River Laboratories . At birth, males were culled and remained with the mother until weaning (PD21), after which they were pair-housed with siblings receiving the opposite treatment until the end of the study. All rats were housed under a 12 h light/dark cycle in a temperature-controlled environment with food and water available −1), which was slowly deposited per os using a 1000 μl pipette inserted towards the back of the mouth. One day following the first MRI scan, the animals were treated with either stimulant (N=12) or vehicle (N=12) for 10 weeks (PD24–94), encompassing the young juvenile period until early adulthood.24 Animals were pseudo-randomly assigned to either treatment group, and the sample size was determined on the basis of a prior publication where stimulant-induced increases in striatal volume in adult rats were detected via post-mortem MRI.25 The stimulant group received twice daily  5 mg ml−1 kg−1 lisdexamfetamine , a prodrug which is metabolized to dextroamphetamine (d-AMPH) once in circulation.26 The control group received the same dosing regimen of 1 ml kg−1 vehicle only. The 10-week duration of treatment was chosen on the basis of results from the MTA  study where methylphenidate, another stimulant, was found to have maximal effects on symptom improvement in the first 10–12 weeks of the 14-month treatment trial.6 On the basis of prior pharmacokinetic assessments of oral LDX in rodents,27 the dosing regimen was chosen to approximate peak circulating levels of d-AMPH in humans given a mid-range, 50 mg dose of LDX (93.3±19.5 ng ml−1 at the 3 h Tmax)28 and to mirror the pharmacokinetics of long-release LDX formulations in humans by accommodating for the faster elimination rate of this drug in rodents. Pharmaceutical grade LDX dimesylate was kindly provided by Shire US  and solutions were freshly prepared on a daily basis.All animals were weighed daily from PD15 until study completion, with treatment adjusted to account for increases in body weight over the course of development. Before the first MRI scan at PD23 (see below), all animals were habituated to the oral delivery of drug vehicle from PD15 until PD22. The vehicle consisted of apple juice (5 ml kgn=5 per group) underwent blood extraction at PD30 to measure plasma levels of d-AMPH following the first daily dose of either LDX or vehicle. Blood was extracted at the Tmax for LDX-derived d-AMPH (3 h in Sprague Dawley rats27) through the retro-orbital plexus by heparinized capillary tubing following anesthesia with 50 mg kg−1 ketamine (intramuscular) under isofluorane (3% isoflurane in 75% NO2 and 25% O2). Samples were centrifuged at 1000 g to recover plasma, which was then mixed 1:10 in phosphate-buffered saline  before analysis using gas chromatographic-mass spectrometric (GC/MS) instrumentation with deuterated d-AMPH as the internal standard. The system was operated in the negative chemical ionization (NCI) mode with 5% ammonia/methane as the reactant gas. After protein precipitation of 0.5 ml of each plasma sample, the supernatant was made alkaline, derivitized with pentafluorobenzoyl chloride, and simultaneously extracted into toluene. An aliquot of the toluene extract was then injected into the GC/MS equipped with a DB-1 15 m × 0.25 mm ID, 0.25 μm film capillary column. The generated standard curve was linear with a limit of detection of 1 ng ml−1. Intra- and inter-assay precision was better than 94 and 98%, respectively.During the dosing period, a subset of rats in both the LDX and vehicle group  volume transmit coil (72 mm ID) and a two-channel (PD23) or a four-channel (PD95) receive-only surface coil were used for radiofrequency (RF) transmission and reception. A magnetization prepared rapid acquisition gradient echo sequence was used for image acquisition.29 The isotropic image resolution for PD23 animals was 0.175 × 0.175 × 0.175 mm3, and for PD95 animals was 0.2 × 0.2 × 0.3 mm3. The inversion time was 1.1 s, the segment repetition time 3.4 s, and total acquisition time 1 h using four averages. To prevent head motion and anxiety during the scan, all animals were anesthetized with isofluorane gas. Oxygen was used as the carrier gas and delivered at low flow rate (⩽0.5 l min−1) to a cone positioned before the bite bar. Before each scan, animals were exposed to 3% isofluorane for induction and then secured in the RF coil with an integrated bite bar, stereotaxic holder, and anesthesia mask. During the scans, isofluorane anesthesia was reduced to 1.5%. Monitoring of the heart rate, respiratory rate and O2 was performed using an SA Instruments monitoring unit  during the whole procedure. Body temperature was monitored through a rectal probe, and was maintained at 37±0.2 °C by a forced warm air unit interfaced to the SA Instruments unit.All study animals had two anatomical MRI scans: first, one day before treatment initiation (PD23), and second, one day following treatment completion (PD95). Scanning was performed on a 7.0 Tesla Agilent  40 cm bore system. The gradient coil insert had an internal diameter of 12 cm, a maximum gradient strength of 600 mT m−1, 20 mg kg−1) delivered by intraperitoneal injection. Animals were then euthanized through transcardial perfusion with 4% paraformaldehyde in phosphate buffer , after which the head was removed and processed for gadolinium-contrasted ex vivo high-resolution MRI. Briefly, the whole head was post-fixed in 4% paraformaldehyde buffer (pH 7.4) on a slow-rocking shaker overnight at 4 °C. The head was then stripped to the skull and transferred to 0.05% sodium azide in phosphate buffer for 1 week at 4 °C. The skull was then incubated in a solution of 0.3% gadolinium  in 0.05% sodium azide/phosphate buffer and gently rocked for 2 weeks at 4 °C. For imaging, the skull was placed in a solution of Fomblin Y , which has no MR signal, to eliminate any background saturation artifacts. For each sample, a high-resolution anatomical MRI scan was acquired for 9 h and 49 min overnight, using the volume transmit coil, the four-channel receive surface coil and Spin Echo three-dimensional acquisition with 80 μm isotropic resolution . Following the scan, the brains were removed from the skull, repeatedly washed in phosphate buffer to remove residual Fomblin Y, and weighed for subsequent normalization procedures .At the conclusion of the final scan, all animals were removed from isofluorane and deeply anesthetized with a ketamine/xylazine mixture (100 mg kghttp://www.itksnap.org) on the in vivo  as well as high-resolution ex vivo (PD95) MRI scans, each modality by an independent rater. Landmarks for tracing were established according to the segmented Sprague Dawley brain atlas . The whole striatum was traced along an axial plane in each hemisphere in all scans. racing of the thalamus and hippocampus was also performed but only on ex vivo MRI scans, as lower resolution and signal-to-noise ratio of the longitudinal in vivo scans prevented clear delineation of these structures .Manual delineation of regions of interest was performed using itk-SNAP software (3) of traced regions was calculated using standard itk-SNAP algorithms. For analyses of the ex vivo high-resolution MRI, regional volumes were normalized by individual brain weight. This normalization procedure was performed to appropriately scale volumes of individual structures to overall brain size, which was reduced in the LDX-treated animals . Supporting the use of body weight as a proxy measure of brain weight for these analyses in our study, (i) we found a strong, significant correlation between body weight and brain weight in our PD95 animals  comprising the approximate dorsal, mid, and ventral aspects of the striatum . FSL was then used to compute the volume of each segment, which was normalized to each respective animal's brain weight for subsequent group comparisons.For analysis of striatal subregions in the P⩽0.05 was considered statistically significant. All the data are presented as mean±s.e.m.SPSS 21  was used for all statistical comparisons. A post hoc t-tests. At the completion of the study , brain weight between the treatment groups was compared with a t-test. Pearson's correlation was used to assess the relationship between brain and body weight at PD95.Animal weight across the treatment period (PD24–94) was compared between groups by repeated-measures analysis of variance (ANOVA) with treatment as the between-subject factor, and age as the within-subject factor. Significant interactions were examined with in vivo MRI , striatal volumes (mm3) were compared between treatment groups with a 2 × 2 repeated-measures ANOVA with treatment as the between-subject factor, and time point as the within-subject factor. Significant interactions were examined with post hoc t-tests. Analyses were first conducted on raw striatal volumes and then confirmed with normalized striatal volumes.For analysis of longitudinal ex vivo MRI (PD95), normalized volumes within three regions of interest  in LDX- vs vehicle-treated animals were compared cross-sectionally with a t-test.For the t-test comparison of each subregion/hemisphere combination.Volumetric differences between striatal subregions  across both hemispheres were assessed by repeated-measures ANOVA with treatment as the independent variable, and subregions/hemisphere as the dependent variables. To estimate the magnitude of treatment effects on subregional volumes, we computed the effect size  of each between-group Tmax (ref. −1. This is comparable to peak circulating levels of d-AMPH in children with ADHD given a mid-range dose of 50 mg LDX (93.3±19.5 ng ml−1 at Tmax),28 indicating that our dosing regimen of oral LDX in rats yielded clinically relevant levels of plasma d-AMPH. d-AMPH was not detected in the plasma of vehicle-treated animals.On the basis of a blood sample draw at the 3 h max ref.  from a sf=5.315, P<0.0001), with LDX-treated animals weighing significantly less than vehicle-treated controls by the end of treatment =−2.56, P=0.02; t(22)=−0.39, P=0.70). These results are consistent with the known appetite-suppressant effects of stimulant treatment in human patients.32As revealed by repeated-measures ANOVA, body weight showed a significant treatment × time point interaction (t(22)=−3.49, P=0.002; At the completion of the study, PD95 animals in the LDX group showed a significant reduction in brain weight when compared with vehicle-treated controls =0.519, P=0.005; Across all PD95 animals (both the LDX and vehicle group), brain weight was found to be significantly correlated with body weight =4.53, P=0.04; f=2.90, P=0.10; Post hoc testing revealed no significant difference between the groups in left or right striatal volume at PD23 (left striatum: t(22)=0.81, P=0.43; right striatum: t(22)=1.70, P=0.10). At PD95, relative to controls, the LDX group showed a significant increase in volume of the left (t(22)=2.68, P=0.0014) but not right striatum (t(22)=−0.75, P=0.461; A 2 × 2 repeated-measures ANOVA of left striatal volume revealed a significant treatment × time point interaction =4.89, P=0.04). This interaction was not significant for the right striatum =0.62, P=0.42). Post hoc testing revealed no significant difference between the groups in left or right striatal volume at PD23 (left striatum: t(22)=1.00, P=0.16; right striatum: t(22)=1.34, P=0.10). At PD95, the LDX group showed a significant increase in volume of the left and right striatum, relative to controls (left striatum: t(22)=4.43, P=0.0002; right striatum: t(22)=2.34, P=0.02).All comparisons are shown in T-tests revealed significantly higher volume of the left striatum (t(22)=3.11, P=0.005) and right striatum (t(22)=2.166, P=0.04) in LDX-treated animals relative to vehicle-treated controls. Neither the thalamus nor the hippocampus showed a significant group difference in volume =1.95, P=0.06; right thalamus: t(22)=1.76, P=0.09; left hippocampus: t(22)=1.87, P=0.08; right hippocampus: t(22)=1.39, P=0.18).All comparisons are shown in f=3.842, P=0.063). To explore trends in our data, we performed between-group t-test comparisons of each subregion within each hemisphere. This exploratory analysis revealed the largest effect size of LDX treatment on volume in the mid  and dorsal  striatum, with a relatively smaller effect in the ventral aspect of this structure . This pattern of effects was consistent between both hemispheres  across the left and right hemisphere  and young adulthood  induced significantly greater striatal growth, with the left striatum showing a stronger effect and a significant treatment by time point interaction. To test the reliability and specificity of these findings, region of interest analyses were performed by an independent rater on high-resolution 35 Many of the symptoms associated with ADHD respond to acute treatment with stimulants and symptom improvement is often noted within 30–60 min of oral drug administration. With maintenance of treatment, continued symptom reductions are reported over the subsequent 10–14 months.6 Although the acute phase of these therapeutic effects is thought to result from altered monoaminergic modulation of circuits underlying attention, impulsivity, and motivation,36 continued improvement in symptoms as a function of prolonged treatment is not well understood. Using neuroanatomical imaging of individuals with ADHD, studies have found that in comparison to youth with ADHD who are medication-naive, those with a history of stimulant therapy exhibit larger striatal volume,20 with morphometric analyses finding localized increases in volume that correlate with symptom improvement.9 Although limited by their cross-sectional design, these studies suggest that striatal hypertrophy might be a neurobiological correlate of continued improvement in ADHD symptoms stemming from long-term stimulant treatment. Other studies, however, have not reported stimulant-induced effects on striatal volumes in ADHD.38 These discrepancies in findings could potentially relate to methodological issues such as the developmental stage (childhood vs adolescence), stimulant dosing or class (methylphenidate vs amphetamines), duration of treatment, sample size, or technical approach for assessing striatal morphology.20 Moreover, it should be noted that two longitudinal studies have been performed to investigate the trajectory of volumetric changes in basal ganglia structures between medicated and unmedicated patients with ADHD and healthy controls. These studies have been discordant, with Castellanos et al.7 finding that initial differences in striatal volume between patients with ADHD and healthy controls disappear by adolescence, while Shaw et al.21 showing that these initial differences persist into adolescence/adulthood. In addition, neither study found an association between basal ganglia volume and treatment history. However, both studies treated medication status as a dichotomous variable and thus did not assess the impact of the dose or duration of exposure on striatal morphology. Future longitudinal studies emphasizing prospective, chronic treatment with stimulants are therefore needed to confirm these effects and their relationship to symptom prognosis.The striatum is a major input nucleus of the basal ganglia that is implicated in the pathophysiology of ADHD and other conditions such as addiction, schizophrenia and obsessive-compulsive disorder.6 the majority of stimulants' salutary effects occur through acute drug action. It is unlikely that volumetric changes in the striatum, which may occur gradually as a function of stimulant treatment, account for the immediate benefits of stimulant treatment on symptom improvement. However, structural changes in the striatum in medicated individuals with ADHD have been associated with improvement in clinical outcomes.9 Taken together, a potential implication of our findings is that increases in striatal volume across development, stemming from either normative neurodevelopment, or its interaction with long-term stimulant treatment, may impact the natural progression of ADHD in affected individuals. For example, over 50% of children with ADHD will remit by early adulthood.39 Future studies could test the hypothesis that striatal maturation, at least in part, accounts for this attenuation of ADHD symptoms, and whether long-term treatment with stimulants augments this progression by arcing striatal growth towards a normative neurodevelopmental trajectory. In addition, exploratory analyses suggested that the effects of LDX on striatal hypertrophy might be more pronounced in the mid/dorsal, than ventral, subregions of this structure. Subsequent research might investigate in both humans and preclinical models the behavioral correlates of pharmacologically induced hypertrophy of dorsal vs ventral striatal subregions.Despite continued improvement in ADHD symptoms with chronic stimulant treatment ,41 analysis of this region tested the specificity of stimulant effects on the striatum vs cortico–striatal–thalamic–cortical circuitry in general. The hippocampus was included as a negative control as stimulant treatment has been shown to cause nonsignificant volumetric changes in this structure in rodents.42 As we did not detect a volumetric change in either the thalamus or hippocampus in our study, our findings suggest that the effects of long-term stimulant treatment may be specific to the striatum and not cortico–striatal–thalamic–cortical loops or other brain regions more broadly. In addition, the hippocampal finding verified that our analytical procedures, such as adjusting for brain weight, did not yield biased estimates of striatal volume between groups. It has yet to be determined, however, whether stimulant-induced striatal hypertrophy impacts striatal activity and cortico–striatal–thalamic–cortical function, and what consequences this may have for behavior.In addition to measuring striatal volume in our study, we assessed the effects of long-term stimulant treatment on thalamic and hippocampal volume. As the thalamus receives indirect input from the striatum through well-established cortico–striatal–thalamic–cortical loops,43 ADHD is a highly heterogeneous disease with presumably many different etiologies and pathophysiologies.45 Thus the use of such models may limit the extrapolation of findings to only a small subset of ADHD cases.46 However, despite the heterogeneity of ADHD, up to three-quarters of patients respond well to stimulant treatment,48 implying that generalizable effects on the brain may underpin the therapeutic response to stimulants irrespective of individual pathology, thus supporting our use of a ‘typical' rat model of stimulant exposure. In addition, our tracing procedure did not allow us to discern the impact of stimulant treatment on functionally distinct areas of the striatum, such as those comprising the associative, limbic, and motor circuits.50 Future studies may address this issue by performing surface analysis of the striatal structure, which can detect localized changes in morphometry. The use of post-mortem high-resolution MRI in our study did not allow us to determine the nature of the volumetric changes. Although studies have shown that chronic stimulant exposure can lead to increased dendritic arborization and spine formation,51 an increase in volume may also result from alterations in cellular number, somatic size, myelination, or innervation.52 As MRI analysis is limited to detecting macroscopic changes in the brain, future studies will be needed to elucidate the cellular and molecular underpinnings of stimulant-induced striatal hypertrophy. A number of studies have suggested that delayed maturation of the cortex may play a major role in ADHD pathology,53 although evidence of normalization with stimulant treatment has been controversial.54 Without MRI scans at least one additional developmental period , our study design precluded us from assessing the effects of long-term stimulant treatment on nonlinear growth trajectories of cortical regions.55 Modeling nonlinear patterns of brain development requires assessments from at least three time points.57 Finally, although our finding of striatal hypertrophy in our rodent model of long-term stimulant treatment recapitulates human findings, we did not assess whether this alteration is associated with behavioral outcomes. Given the possibility that the salutary effects of stimulants on behavior in humans may in part depend on the presence of a pathological state,58 future studies may evaluate the clinical application of our rodent-stimulant model by investigating whether stimulant-induced increases in striatal volume yield behavioral changes in symptom domains relevant to ADHD such as impulsivity, attention,36 and aggression.59A general limitation of this study is that it does not use an animal model of ADHD. This is important to consider since stimulant effects on the brain may differ in children with ADHD when compared with healthy individuals. Although animal models of ADHD do exist ,in vivo MRI in rodents to measure brain changes as a function of treatment and natural brain development. Use of this technique has high translational potential to serve as a tool for bridging preclinical and clinical research. As our findings of stimulant-induced striatal hypertrophy in rodents agree with what has previously been found in humans, this suggests high translational validity of our rodent model of long-term stimulant exposure. The principal advantage of our animal model is that mechanisms underlying this effect can be simultaneously investigated at a molecular, cellular, and circuit level, which can then be related to alterations in behaviors relevant to those disrupted in ADHD. Such investigations may yield novel, more targeted approaches for ADHD therapy.The long-term goal of our study is to invoke a translational neuroscience approach towards identifying the cellular and molecular mechanisms by which clinically relevant doses of stimulants improve the symptoms of ADHD. This study is an important, initial step in that direction. To our knowledge, this is the first demonstrated use of repeated, longitudinal