A precise diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires comprehensive knowledge of morphological analysis, with a focus on the quantity and quality of cells being examined. Some research has utilized techniques such as immunocytochemistry, flow cytometry, polymerase chain reaction (PCR), and interphase fluorescence in situ hybridization (iFISH) on cerebrospinal fluid (CSF) cytospin samples to detect any remaining leukemic cells in the CSF. To obtain reliable results using immunocytochemistry and flow cytometry, it is essential to use freshly collected specimens within a limited timeframe. At the same time, PCR requires a sufficient number of cells for DNA extraction. On the other hand, the iFISH procedure on CSF cytospin samples can be challenging and requires practice. Therefore, there is a need for a fast, easy method that will be affordable and marketable in laboratories where the above methods are not available, or the sample is insufficient to use those methods.The samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF sample was centrifuged at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. Other steps  were the same as in the standard protocol for blood samples. The BCR-ABL1 rearrangements were performed and evaluated in 200 interphase cells.90% of Ph(+) cells were found in CSF.We propose a significantly streamlined iFISH method for detecting blast/residual leukemic cells in acute lymphoblastic leukemia using CSF as a complementary test option. Accurately detecting the central nervous system (CNS) involvement in children with acute lymphoblastic leukemia (ALL) is pivotal at the initial diagnosis and follow-up after treatment. Less than 2% of pediatric patients have CNS involvement at the initial diagnosis, and CNS relapses can be reduced to less than 5% after CNS prophylaxis , 2. HoweThe patients are considered at increased risk of CNS relapse if the detection of blast cells in CSF is accompanied by a CSF-WBC count exceeding 5 cells/μl. Based on this, a specific risk score was generated: CNS1, denoting the absence of identifiable leukemic cells in CSF; CNS2, indicating the presence of blast cells in a CSF sample containing < 5 WBC/μl; and CNS3, a CSF sample that has ≥ 5WBC/μl together with identifiable blast cells, or the presence of cerebral mass, or cranial nerve palsy together with leukemic cells in the CSF .ALL is the most common childhood cancer. Approximately 25% of adults and 5% of children diagnosed with ALL have the Philadelphia chromosome, also known as the BCR-ABL1 rearrangement. This rearrangement involves the fusion of the BCR gene located on chromosome 22 with the ABL gene on chromosome 9, creating a constitutively active tyrosine kinase fusion protein. The presence of the Philadelphia chromosome in ALL is linked to a more severe form of the disease and is considered a poor prognostic factor, especially for children. As a result, patients with Ph + ALL are often treated with more aggressive methods, such as bone marrow transplantation. Despite these efforts, the relapse rate for Ph + ALL remains high, ranging from 40 to 80% .The diagnosis of CNS involvement in ALL continues to pose a challenge due to underdiagnosis. The current standard diagnostic method is the morphological examination of CSF. Accurately identifying blast cells in CSF is crucial. Still, distinguishing between blast and healthy cells can be difficult, especially when the cell count is low . CytospiWhile PCR and NGS have been suggested for detecting residual leukemic blasts in CSF, both require adequate cell numbers for nucleic acid extraction , 11–13. We performed iFISH analyses on the CSF sample to identify the blast cells. iFISH on this sample was performed using a ZytoLight ® SPEC BCR/ABL1 dual color dual fusion probe (Germany) and evaluated 200 interphase cells. The samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF-WBC count was 1300 cells/μl in positive sample and 19 cells/μl in negative sample. After centrifugation at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. The remaining steps, such as fixation, hybridization, washes, and slide preparation, followed the standard protocol for blood samples. For the iFISH procedure, the slides were soaked in 99% ethanol, rinsed with dH2O, aged at room temperature for 2 h, dehydrated in an ethanol series , and then the IVD BCR/ABL1 t fusion FISH probe was applied. We added 6 μL of probe to each slide and covered it with a glass coverslip (22 × 22 mm). Denaturation was performed in ThermoBrite (Abbott Molecular) at 75 °C for 5 min, followed by overnight hybridization at 37 °C with humidity. Post-hybridization washes included 2xSSC at 75 °C for 3 min in a water bath, followed by 0.4xSSC/Tween 0.05% at room temperature. Subsequently, dehydratation in an ethanol series  was performed, and the nuclei were stained with DAPI . The cut9/L), anemia (Hb 85 g/dL), and thrombocytopenia (PLT 47 × 109/L). Morphological examination of the blood smear revealed 93.0% of blast cells in peripheral blood. Blood chemistry and blood coagulation parameters were normal. A morphological study of bone marrow aspirate detected 91% of blast cells. According to clinical guidelines, immunophenotyping using the flow cytometry method was performed. The following phenotype was established: CD10+ (100%), CD19+ (88%), CD20+ (63%), CD34+ (100%), CD45+ (65%), CD81+ (41%), CD24+ (22%), CyCD79a+ (72%), CD99+ (92%), CD79a+ (97%), CD22+ (18%). Fluorescence in situ hybridization (FISH) for BCR-ABL1 rearrangements was performed on the marrow specimen. The 50% Ph(+) cells were found.The male patient born on 02.08.2007  came to the hospital with complaints of high-grade fever, fatigue, and bone pain. Upon admission to the hospital physically, some features of the hematological disorder, such as lymphadenopathy and splenomegaly, were revealed. The hematological analyzer showed an extremely high WBC count . After the course of treatment, the patient was discharged in remission. After a year since leukemia manifestation, no leukemic cells were observed in blood and bone marrow aspirate, and BCR-ABL fusion signal in bone marrow was not found by FISH as well.Four years after the initial diagnosis, the patient went to the doctor with neurological symptoms: complaints of headache, fatigue, strabismus, and diplopia. Symptoms of hematological disorders such as hepatosplenomegaly and lymphadenopathy were not found. Head MRI revealed nonspecific gliotic areas in cerebral white matter, idiopathic intracranial hypertension, and mild ventriculomegaly. The complete blood and bone marrow aspirate count data were within normal limits. FISH did not determine the BCR-ABL fusion signal in bone marrow and peripheral blood. The CSF was analyzed, and BCR-ABL1 rearrangements were performed on the specimen. The 90% Ph (+) cells were found in CSF  count in this sample was 19 cells/μl. Despite the limited number of cells, we were able to successfully fix the cells, and our method performed flawlessly. In this particular case, we utilized the sample from a pediatric patient diagnosed with B-cell acute lymphoblastic leukemia (B ALL). Initial diagnostic tests did not reveal any abnormalities such as BCR-ABL translocation, ETV6-RUNX1 translocation, or MLL-rearrangements. However, the patient consistently exhibited high minimal residual disease (MRD) levels in the bone marrow, and the CSF was also affected. Using an in vitro diagnostic (IVD) BCR/ABL1 t9;22) probe on the fixed CSF sample, the result was negative as expected (Fig. 2 probe oThe main difficulty in CSF analysis is the low cell amount in the sample. Though cytogenetic manuals suggest the iFISH cytospin protocol for CSF samples is cell fixation right on the slide , it is dIn conclusion, our proposed method might be a valuable tool for diagnosing and monitoring residual leukemic cells in CSF samples and could be a potential complementary test for detecting the presence of blast cells or residual leukemic cells in the case of ALL.


Spontaneous cerebrospinal fluid (CSF) leak in the lateral recess of the sphenoid (LRS) sinus in a pediatric population is rare and is surgically challenging to repair.We report a case of a 13-year-old girl with hydrocephalus and a ventriculoperitoneal shunt who presented with a two-month history of clear rhinorrhea from the right nostril. Computed tomography (CT) of the head was performed and showed CSF leak through a defect in the lateral recess of the sphenoid sinus. Defect closure was achieved using an endoscopic endonasal approach.CSF leak with meningoencephalocele from the sphenoid sinus is amongst the most difficult cases for repair. Yet the successful rate of repair is as high as 90 % if done endoscopically. Moreover, the location of the defect determines the surgical approach.Spontaneous CSF rhinorrhea from the lateral recess of the sphenoid (LRS) sinus, although rare, requires prompt diagnosis and treatment, as it may lead to significant mortality and morbidity. •Spontaneous cerebrospinal fluid (CSF) rhinorrhoea is a rare condition leading to significant mortality and morbidity if not diagnosed and treated promptly.•The success rate of endoscopic repair is as high as 90 % endoscopically, however the location of the defect determines the surgical approach.•The incidence of meningoencephaloceles is 1 in 4000–5000 and tends to present as spontaneous CSF leaks This condition is caused by the disruption of the barriers between the anterior and middle cranial fossa and the sinonasal cavity. There are many different causes that could lead to this condition, which can be classified as either traumatic or non-traumatic . In compHerein, we review our recent experience with a surgical repair in a pediatric patient that presented to our clinic with a spontaneous CSF leak in the LRS sinus.This work has been reported in line with the SCARE 2020 criteria .2A 13 year-old female with hydrocephalus and a ventriculoperitoneal (VP) shunt that was inserted 6 months prior to her presenting complaint, presented to us with spontaneous clear fluid discharge from the right nostril of two months duration. There was no previous history of trauma, fever, chronic rhinitis, sinusitis or any previous surgical procedures. The patient did not complain of any headache, visual disturbances or nasal obstruction. Nasal endoscopy showed right-sided clear rhinorrhea with no oedema or other incidental findings. CT head revealed CSF leak through the defect in the right wall of the sphenoid sinus. Incidental marked bilateral hydrocephalus was found with J-shaped flattened sella and a scalloped inner calvarial cortex. .Fig. 1CTAfter which, MRI brain was done and confirmed the CSF leak and a tectal glioma causing aqueductal stenosis with moderate supraventricular hydrocephalus. A meningoencephalocele was also noted through a defect in the roof of the right LRS sinus. .Fig. 2BiThe patient was taken for surgical repair by an otolaryngologist. Under general anaesthesia, an endoscopic endonasal approach was performed. Right sided uncinectomy, complete anterior and posterior ethmoidectomy and sphenoidotomy was done. Partial removal of the superior turbinate and lateral widening of the sphenoid ostium was performed. Portion of the medial pterygoid bone was removed with a Kerrison rounger for better access and visualization of the lateral extension of the sphenoid sinus. The defect was visualized lateral to the vidian canal, showing a high flow porous leak from the bone in the lateral recess of the sphenoid sinus.Closure of the defect was done in a multilayer fashion using a tissue patch, followed by fat harvested from the paraumbilical area, fibrin sealant (Tisseel) then absorbable hemostatic agents (Surgicel) was applied to pack the repaired area. Intra-operatively, no leak was visualized following the repair. A merocel pack was inserted at the end of the procedure.Post-operatively, the patient received intravenous ceftriaxone once daily, and was instructed complete bed rest with 10–15 degrees bed elevation for one-week duration. She was advised to avoid straining and lifting heavy objects for one month. Oral laxatives were prescribed up to three times a day. Subsequently, she was shifted to a neurosurgical center for close observation and active neurosurgical measures were taken to reduce her ICP. She had the lumbar drain removed, and a ventriculoperitoneal shut inserted successfully. Routine post-operative follow-ups up to one year revealed no CSF leak.3The sphenoid sinus anatomy is highly variable within the population, and various degree of pneumatization of the LRS has been reported in 16–54 % of adults . A spontThe sphenoid sinus (SS) is a paired paranasal sinus occurring within the body of the sphenoid bone, surrounded by important neurovascular structures. The SS invaginates into the posterior region of the nasal capsule at the sphenoethmoidal recess during the third month of intrauterine life . There iPatients tend to present with intermittent clear rhinorrhoea as their chief complaint. Other symptoms may include headaches, recurrent meningitis and seizures ,13. TherIn several cases, for instance, posttraumatic CSF leaks may resolve with conservative measures such as bed rest, head elevation, avoidance of strenuous activities and reducing CSF pressure by a spinal tap or drain for 7–10 days. Leaks that persist despite conservative measures require surgical intervention to avoid serious life-threatening complications .There have been various viewpoints in the literature with regards to the commonest locations for spontaneous CSF leaks. Although rare, some studies reported the incidence of a LRS sinus leak as high as 35 % . Those cCSF leak with meningoencephalocele from the sphenoid sinus is amongst the most difficult cases for repair . While mAfter drilling the medial pterygoid plate, the vidian nerve may be identified and preserved by exposing it and limiting the drilling to the inferior portion of the medial pterygoid plate. Although the vidian nerve may be sacrificed, it can be useful to identify the internal carotid artery when followed posteriorly . In caseA variety of graft materials can be used in repair of CSF leaks depending on the size, site and the flow rate. However, superiority of one over the other has not been clearly established in the literature. Multilayered techniques in general have shown to yield optimal results .Congenital skull base defects in pediatrics are rare and infrequently diagnosed ,16. The Repair of defects in the LRS sinus in pediatric patients has not been widely discussed in the literature. In our case, we repaired the defect by an endoscopic transphenoidal approach using 0, 30 and 70-degree scopes, followed by a multilayered closure. The porous bony defect along with a high flow of CSF leak seen intraoperatively posed a challenge during surgical repair since there was no clear osteodural defect to patch. Moreover, given our patient's history of raised ICP and a spontaneous ‘high flow’ CSF leak, a temporary lumbar drain was used as a CSF diversion technique . A perma4Spontaneous CSF rhinorrhea from the lateral recess of the sphenoid (LRS) sinus, although rare, is surgically challenging. The aim of this paper was to report our experience with CSF leak and meningoencephalocele in a pediatric patient that was repaired successfully using an endoscopic endonasal approach.Not declared.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Dr. Mashael AlKandery; data collection, data analysis, and write up.Dr. Lulwah AlSaidan; data collection, data analysis, and write up.Dr. Farhan Owayed; data collection, data analysis, and write up.Dr. Marwan AlQunaee; data analysis and contribution.Dr. Mutlaq Al-Sihan; data analysis and contribution.Dr. Abdulmohsen AlTerki; data analysis and contribution.Dr. Lulwah AlSaidanWritten informed consent was obtained from the patient's parents/legal guardian for publication and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.There is no conflict of interest to declare by any of the authors of this study.


The groups had similar T2-weighted signal intensity ratios and native T1 times. T2 relaxation times were longer in the troponin-positive group, and the difference remained significant after excluding infarct-pattern late gadolinium enhancement (LGE) from the analysis. Extracellular volume (ECV) was higher in the troponin-positive group  and correlated strongly with T2 relaxation time . Late gadolinium enhancement was 32% more prevalent in the troponin-positive group , with 29% having infarct-pattern LGE. T2 relaxation time was independently associated with troponin positivity , and both T2 relaxation time and ECV predicted troponin positivity . Left ventricular end-diastolic and left atrial volumes were the strongest predictors of troponin positivity . The increased T2 relaxation time and ECV and their significant correlation in the troponin-positive group suggest myocardial injury with oedema, while the non-ischaemic LGE could be due to myocardial fibrosis or acute necrosis. These CMR imaging biomarkers provide important clinical indices for risk stratification and prognostication in patients with hypertensive crisis.Hypertensive crisis can present with cardiac troponin elevation and unobstructed coronary arteries. We used cardiac magnetic resonance (CMR) imaging to characterize the myocardial tissue in patients with hypertensive crisis, elevated cardiac troponin, and unobstructed coronary arteries. Patients with hypertensive crisis and elevated cardiac troponin with coronary artery stenosis <50% were enrolled. Patients with troponin-negative hypertensive crisis served as controls. All participants underwent CMR imaging at 1.5 Tesla. Imaging biomarkers and tissue characteristics were compared between the groups. There were 19 patients  with elevated troponin and 24  troponin-negative controls. The troponin-positive group was older (57 ± 11 years vs. 47 ± 14 years,  Hypertensive crisis remains a common reason for emergency room visits and is associated with increased major adverse cardiovascular events (MACE) ,2. An imPositive cardiac troponin and unobstructed coronary arteries are associated with both acute myocardial infarction  and acute myocardial injury , and both can occur in patients with hypertensive crisis with or without left ventricular hypertrophy (LVH) . Acute mCardiovascular magnetic resonance (CMR) is the gold standard for evaluating cardiac structure and function but also has the unique ability of myocardial tissue characterization. Guidelines on the evaluation of myocardial infarction with unobstructive coronary arteries (MINOCA) recommend CMR for determining aetiological diagnosis ,11. The The prognostic implications of raised cardiac troponin in patients with hypertensive crisis have been established in many studies ,14. DespAdult patients aged 18 years and above with hypertensive crisis referred to Tygerberg Hospital in the Western Cape province of South Africa were enrolled in this study. Diagnosis of hypertensive crisis was based on a systolic blood pressure of ≥180 mmHg and/or a diastolic BP of ≥110 mmHg . All parAcute myocardial infarction was diagnosed as rising and/or falling plasma levels of hs cTcT with at least one value above the 99th percentile of the upper reference limit with features of myocardial ischaemia. Those without features of myocardial ischaemia were considered to have acute myocardial injury . PatientThe following exclusion criteria were applied: hypertensive disorders of pregnancy, altered level of consciousness, inability or refusal to consent, and patients under 18 years of age. This study was approved by the Health Research Ethics Committee (HREC) of Stellenbosch University, and all participants granted a written consent. The Declaration of Helsinki was adhered to.All participants underwent CMR imaging using a 1.5 T scanner  within 48 h of presentation to Tygerberg Hospital. The scanning protocol included breath-held, ECG-gated balanced steady-state free precession (bSSFP) cine; T2-weighted short-tau inversion recovery (STIR); parametric mapping (T1 and T2); extracellular volume (ECV) estimation; and late gadolinium enhancement (LGE) imaging . Native 42, a commercially available software , Calgary, AB, Canada). Enrolment into this study lasted two years, with an interval of six months from the last participant to the time the imaging data were analysed. The images were anonymised to minimise further reporting bias, and postprocessing was carried out for the group as a single cluster without being classified as cases and controls. Ventricular epicardial and endocardial contours were drawn in end-diastolic and end-systolic phases to determine cardiac end-diastolic volume (EDV), end-systolic volume (ESV), LV ejection fraction, and LV mass [The postprocessing of images was performed by M.A.T. using CVi LV mass . Papilla LV mass . Left ve LV mass .To determine the T2-weighted signal intensity (SI), T1 mapping and T2 mapping, endocardial and epicardial contours were drawn in the relevant sequence’s basal-, mid-, and apical short-axis slices. Epicardial and endocardial contours were carefully drawn to avoid contamination from blood pool and epicardial fat, and offsets of 20% and 10% were applied from the epicardial and endocardial borders, respectively. To determine the T2-weighted myocardial: skeletal SI ratio, a region of interest was drawn in the serratus anterior muscle in the same slice with myocardial contouring . To estiLate gadolinium enhancement was assessed qualitatively in conjunction with other sequences analysing the same cardiac segment and was considered present when visualized in 2 orthogonal planes or in 2 adjacent slices. The pattern of LGE was classified into infarct-pattern LGE , or non-ischaemic LGE  insertion point) ,22. The t-test, Mann–Whitney test, and 1-way ANOVA with post hoc analysis as appropriate. Categorical variables are presented as proportions and percentages and compared using Fisher’s exact test. The correlation of continuous variables was determined using Spearman’s correlation , while binary logistic regression analysis was used to determine factors associated with troponin-positive unobstructed coronary arteries. The area under the receiver operator characteristics (ROC) curve (AUC) was used to determine the predictors of troponin-positive unobstructed coronary arteries. GraphPad Prism version 6.04 for MacOS (GraphPad Software 10.0.0 (131), www.graphpad.com, accessed on 12 July 2023) was used to make scatter plots. A predetermined p value of <0.05 (two-tailed) was considered significant.Sample size and power calculation leveraged the reproducibility of measurements using CMR imaging for cardiac volumes, mass, and LV ejection fraction . Data weEighty-two patients with hypertensive crisis were screened for enrolment into the study . Fifty-ep = 0.015), and 63% were men. Blood pressure, duration of hypertension, and the rate of nonadherence to antihypertensive medication were similar in the groups. The hs cTnT, serum creatinine, haemoglobin, and NT-proBNP levels were higher in the troponin-positive group. Chronic kidney disease  and dyslipidaemia (p = 0.011) were more prevalent in the troponin-positive group; however, there were more patients with diabetes in the troponin-negative group (p = 0.034). Fifteen (79%) of the troponin-positive, unobstructed coronary artery group had a 20% change in cardiac troponin level (delta 20%) on repeat measurement compared to baseline values.The troponin-positive group with unobstructed coronary arteries were on average 10 years older than the troponin-negative group , cough (p = 0.005), shortness of breath (p = 0.009), New York Heart Association classes III and IV, and palpitations (p < 0.001) were more prevalent in the troponin-positive group. Angiotensin-converting enzyme inhibitor or receptor blocker (p = 0.034), beta-blocker (p = 0.002), platelet inhibitor (p < 0.001), and statin (p < 0.001) were more frequently prescribed in the troponin-positive group. However, there were more patients in the troponin-negative group on calcium channel blockers (p = 0.004).Chest pain (p = 0.051), but the prevalence of QTc prolongation was similar. T-wave inversion was more prevalent among the troponin-positive group than the control group . Only one patient with a neurological emergency had T-wave inversion.The corrected QT interval tended to be higher in the troponin-positive group (Findings from CMR are presented in 2 higher in the troponin-positive group than the troponin-negative group (p < 0.001). Using LV ejection fraction, impaired LV systolic function was 36% more prevalent in the troponin-positive group . The prevalence of LVH did not differ in the two groups (p = 0.221); however, indexed LV mass was 20 g/m2 higher in the troponin-positive group (p = 0.030), indicating a larger degree of LVH than the troponin-negative group.The indexed LV end-diastolic and end-systolic volumes were higher in the troponin-positive unobstructed coronary artery group. Similarly, the indexed left atrial volume was 13 mL/mp = 0.004) and mid-ventricular  T2 relaxation time. The difference in mid-ventricular T2 relaxation time was maintained after excluding patients with infarct-pattern LGE from the troponin-positive group. Global ECV  and mid-ventricular  ECV were significantly increased in the troponin-positive group when compared to the troponin-negative group. Unlike T2 relaxation time, the difference in ECV was not maintained after excluding cases with infarct-pattern LGE from the analysis. T2 relaxation time correlated significantly  and ECV  in the troponin-positive unobstructed coronary artery group. However, there was no significant correlation between native T1 and ECV . None of the imaging biomarkers correlated with hs cTnT  and mid-ventricular ECV (p = 0.020) were statistically significant. Non-ischaemic LGE occurred similarly in the troponin-negative group and the group with delta 20% (p = 0.681).p = 0.050), the difference in prevalence was not maintained after excluding participants with infarct-pattern LGE . All the troponin-negative cases with LGE had non-ischaemic LGE .A contrasted CMR study was conducted with 41 participants (95%). Two participants from the troponin-positive group were excluded from the contrasted study due to concerns about acute kidney injury. Overall, 63% of the participants displayed LGE. Among the troponin-positive group with unobstructed coronary arteries, infarct-pattern LGE was noted in 29%, while 53% had a non-ischaemic LGE ; the remaining 18% had no LGE. Although LGE was 32% more prevalent in the troponin-positive group  and end-systolic (p = 0.029) volumes were higher, while LV ejection fraction was lower in the group with non-ischaemic LGE than the group with ischaemic LGE (p = 0.029). Although hs cTnT tended to be higher in the group with infarct-pattern LGE, the difference did not reach statistical significance (p = 0.060). Similarly, indexed LV mass and maximum wall thickness were numerically higher in the group with non-ischaemic LGE, but the difference did not attain statistical significance (p = 0.083 for both). Native T1 relaxation time, T2 relaxation time, ECV, and T2-weighted SI ratio did not differ in the groups with infarct-pattern vs. non-ischaemic LGE.p = 0.026) and T2 relaxation time  maintained an independent association in the multivariable logistic regression model, with a Nagelkerke R square of 0.577. The model correctly classified 82.9% of cases (87.5% of the troponin-negative group and 76.5% of the troponin-positive group).The results of binary logistic regression for factors associated with troponin-positive unobstructed coronary arteries are presented in p = 0.001) and indexed LAV  were the best CMR imaging predictors of troponin-positive unobstructed coronary artery, followed by mid-ventricular ECV , mid-ventricular T2 relaxation time , indexed LV mass , global T2 relaxation time , and LV ejection fraction . Native T1  and global ECV  did not differentiate the troponin-positive group from troponin-negative controls. The sensitivity, specificity, likelihood ratios, and cut-offs associated with the AUC of the different parameters are presented in The receiver operator characteristic curve and corresponding area under the curve for the imaging biomarkers in predicting troponin-positive unobstructed coronary artery are presented in Acute myocardial injury and type 2 myocardial infarction occur as HMOD in hypertensive crisis and unobstructed coronary arteries. Increased T2 relaxation time and ECV, and their correlation in the troponin-positive group, could indicate myocardial injury with oedema, while non-ischaemic LGE could be interpreted as either myocardial fibrosis or acute necrosis. We used CMR to assess myocardial tissue characteristics in hypertensive crisis patients with troponin-positive, unobstructed coronary arteries. The main findings are as follows: (1) T2 relaxation time and ECV were significantly increased in the troponin-positive unobstructed coronary artery group. Although native T1 time was numerically higher in the troponin-positive unobstructed coronary artery group, this did not differ statistically. (2) Increasing indexed LV mass and LV EDV, systolic dysfunction, and T2 relaxation times were significantly associated with troponin-positive unobstructed coronary arteries. (3) There was a trend for a higher prevalence of LGE, mainly driven by infarct-pattern LGE that occurred in 29% of the troponin-positive group. (4) No infarct-pattern LGE occurred in the troponin-negative group. Furthermore, indexed LV EDV and LAV demonstrated a diagnostic utility for predicting troponin-positive unobstructed coronary arteries using the ROC curve, followed by ECV and T2 relaxation time.Although the troponin-positive group was, on average, ten years older than the troponin-negative group, our cohort was younger and with fewer comorbidities compared to previous research, and this may partly explain the observed lower prevalence of obstructive coronary disease ,28. High+/K+-ATPase, leading to increased intracellular Na+ concentration and an osmotic gradient that promotes vasogenic interstitial oedema and interstitial expansion [The increased T2 relaxation time in the troponin-positive unobstructed coronary artery group is evidence of myocardial oedema. T2 relaxation time is a sensitive indicator of intracellular and extracellular water (acute free water) and provides a reliable, pixel-wise quantitative measure of acute myocardial oedema . Althougxpansion .The ECV fraction is a nonspecific measure of interstitial expansion related to various pathologies, including diffuse interstitial fibrosis, infiltrative diseases, and interstitial myocardial oedema . The sigAlthough the increase in T2 relaxation time could be due to the oedema associated with myocardial infarction, the troponin-positive unobstructed coronary artery group maintained significantly higher T2 relaxation time and ECV after excluding cases of infarct-pattern LGE. The ischaemic imbalance associated with hypertensive crisis could cause myocardial injury and/or type 2 myocardial infarction, resulting in myocardial edema and an increased T2 relaxation time and ECV. T2 relaxation time showed an independent association with troponin-positive unobstructed coronary arteries , differeMost CMR-based studies involving patients with elevated troponin and unobstructed coronary arteries did not include patients with hypertensive crisis, making comparison with this study difficult . HoweverLate gadolinium enhancement is the gold standard imaging method for demonstrating myocardial replacement fibrosis and is instrumental in establishing aetiologic diagnoses in many cardiac pathologies . The repFurthermore, increased LV volumes and reduced ejection fraction among the group with non-ischaemic LGE, compared to those with infarct-pattern LGE, point to a chronic cardiac remodelling associated with longstanding uncontrolled hypertension.Indexed LV EDV and left atrial volume demonstrated diagnostic utility for troponin-positive unobstructed coronary artery. Other significant predictors of being troponin-positive with unobstructed coronary arteries included LV mass, LV ejection fraction, and T2 relaxation time, a sensitive marker of increased myocardial water. As alluded to earlier, an increased augmentation index in a hypertensive crisis can cause a sudden increase in LV filling pressure . PreviouOur findings provide evidence for myocardial interstitial expansion, oedema, and increased prevalence of LGE in patients with hypertensive crisis, raised troponin, and unobstructed coronary arteries. Raised troponin and unobstructed coronary arteries encompass acute myocardial injury and type 2 myocardial infarction, two seemingly separate entities with common prognostic implications. However, differentiating them can be challenging in clinical practice . The patLimitations: Our findings should be interpreted in light of the following limitations. It was a single-center study with a limited sample size with unequal distribution of males and females, resulting in a type 2 statistical error, especially in some subgroup analyses. Similarly, the number of covariates included in the regression model relative to the sample size may cause overfitting bias.Coronary angiography was obtained when clinically indicated and was not conducted in the troponin-negative control group. This limited application of coronary imaging excluded many patients with hypertensive crisis and positive troponin from the study. In addition, intravascular ultrasound, fractional flow reserve, and optical coherence tomography were not included in the study, and therefore, plaque rupture or erosion was not systematically assessed.Coronary vasospasm could result in myocardial ischaemia and increased troponin. Although a provocative assessment of the coronary arteries was not carried out as part of the study, none of the cohort demonstrated ST-segment elevation at presentation, during the hospital stay, or during coronary angiography. Also, myocardial perfusion imaging, which could have identified myocardial ischaemia and provided evidence of microvascular dysfunction, was not carried out as part of this study.Follow-up CMR was not carried out to determine the effect of treatment of hypertensive crisis on the structural and functional alterations as well as myocardial oedema, especially since myocardial stunning could complicate hypertensive crisis.Despite identifying infarct-pattern LGE, a significant overlap could be present in cases of acute myocardial injury and type 2 myocardial infarction, making the differentiation difficult.Finally, postprocessing of CMR images was conducted unblinded, which could result in some bias. These limitations notwithstanding, the key findings from this study provide insights into myocardial tissue architecture that will be useful in risk stratification and prognostication in patients with hypertensive crisis and troponin-positive unobstructed coronary arteries. Further studies will be required to confirm our findings and determine the prognostic implications of the altered structure, function, and myocardial oedema.One-third of the troponin-positive hypertensive crisis group had CMR evidence of myocardial infarction, but this does not fully explain the observed differences in T2 relaxation time and ECV. The increased T2 relaxation time and ECV and their significant correlation in the troponin-positive group suggest myocardial injury with oedema, while the non-ischaemic LGE could be due to myocardial fibrosis or acute necrosis. In addition, the increased T2 relaxation time, ECV, and presence of LGE may contribute to the MACE associated with hypertensive crisis, myocardial injury, and type 2 myocardial infarction. Our study provides evidence for the clinical utility of CMR imaging in risk stratification and prognostication of patients with hypertensive crisis. However, longitudinal research is required to corroborate these findings and determine the prognostic implications.


Linezolid is used off-label for treatment of central nervous system infections. However, its pharmacokinetics and target attainment in cranial cerebrospinal fluid (CSF) in tuberculous meningitis patients is unknown. This study aimed to predict linezolid cranial CSF concentrations and assess attainment of pharmacodynamic (PD) thresholds (AUC:MIC of >119) in plasma and cranial CSF of adults and children with tuberculous meningitis. A physiologically based pharmacokinetic (PBPK) model was developed to predict linezolid cranial CSF profiles based on reported plasma concentrations. Simulated steady-state PK curves in plasma and cranial CSF after linezolid doses of 300 mg BID, 600 mg BID, and 1200 mg QD in adults resulted in geometric mean AUC:MIC ratios in plasma of 118, 281, and 262 and mean cranial CSF AUC:MIC ratios of 74, 181, and 166, respectively. In children using ~10 mg/kg BID linezolid, AUC:MIC values at steady-state in plasma and cranial CSF were 202 and 135, respectively. Our model predicts that 1200 mg per day in adults, either 600 mg BID or 1200 mg QD, results in reasonable (87%) target attainment in cranial CSF. Target attainment in our simulated paediatric population was moderate . Our PBPK model can support linezolid dose optimization efforts by simulating target attainment close to the site of TBM disease. Linezolid is an antibiotic in the oxazolidinone class and was approved in 2000 by the US Food and Drug Administration (FDA) . The WorTBM only makes-up 1–2% of all TB cases, but it is the most severe manifestation of tuberculosis, resulting in death or disabilities in up to 50% of the affected patients. Furthermore, TBM is particularly prevalent in young children under the age of five years . LinezolIn clinical trials and practice, doses of linezolid varying from 300 and 600 mg BID to 1200 mg once daily (QD) orally are being used to treat TB ,11, contIn this study, a CNS PBPK model was developed to predict the pharmacokinetics of linezolid in the CNS of adults and children with TBM and to assess whether currently investigated linezolid doses achieve sufficient cranial CSF drug exposures.A linezolid 4-compartment permeability-limited CNS PBPK model was developed using Simcyp simulator software Version 19 Release 1 , based on a previously published brain model  which waIn order to build the PBPK model, molecular pharmacological studies were performed in-house to identify the transporters involved in linezolid efflux at the blood–brain barrier. Human embryonic kidney 293 (HEK293) cells overexpressing a single efflux transporter present at the blood–brain barrier   were useSupplementary File S2.0–12 with the reported median values in each study. Acceptance criteria for the model were defined as an AUC0–12 not deviating more than two-fold (0.5–2.0×) from the median observed AUC0–12 values reported in the literature [0–12-cranial CSF/mean predicted AUC0–12-serum.The performance of the CNS model was evaluated based on human linezolid plasma and cranial CSF exposure data after single and multiple intravenous doses (600 mg in adults and 10 mg/kg in children) that were extracted from literature. An overview of these studies can be found in terature . The per0–24 over the minimum inhibitory concentrations  was determined at steady state (≥4 doses), we assumed that the AUC0–24 was double the AUC0–12 [The developed CNS model was used to predict cranial CSF exposure based on plasma curves of actual pulmonary TB patients, as reported in the literature. An overview of these studies can be found in  100–119 ,27, and  100–119 ,30,31,32efflux,vitro values were 16 and 2.1 µL/min/mg protein, respectively. The inclusion of BCRP and P-gp in the BBB of the CNS model had only a modest effect on the calculated AUC (AUCno transporters/AUCtransporters = 1.1).Linezolid was confirmed to be a substrate for BCRP, but not for P-gp, MRP1, MRP4, or MRP5 . Due to . All steady-state cranial CSF AUC-ratios were within the two-fold range (0.71–1.37) of the ratios reported in the literature [Optimized plasma PK curves, i.e., by using the literature-based plasma clearance values, as well as predicted cranial CSF and brain concentration-time curves after single dose linezolid, can be found in Figure 2terature ,6,7,8,33Mycobacterium tuberculosis , when dosed at 600 mg BID and 1200 mg QD in adults, as well as ~10 mg/kg BID in children. However, in our simulations, 13% of the adult (600 mg BID and 1200 mg QD), and 44% of the paediatric simulated population (~10 mg/kg), still did not reach the PK–PD threshold in cranial CSF , and we subsequently used the model to predict cranial CSF linezolid concentrations in adults and paediatric TB patients with population specific plasma clearance values. Model-based predictions were able to demonstrate that mean cranial CSF exposure values reach nial CSF .0–24:MIC of >100–119 [The optimal dosage of linezolid in MDR-TB is still under debate, mainly because of toxicity related concerns, such as myelosuppression and peripheral neuropathy, after prolonged use of linezolid (>28 days). Currently, a total daily dose of 600 mg seems to offer a good balance between efficacy and toxicity in MDR-TB ,13, but >100–119 ,27.In order to build our PBPK model, we performed additional molecular pharmacological experiments to assess transporter-mediated clearance values for linezolid at the blood–brain barrier, as these were missing in the literature. By using HEK293 cells and MDCKII cells overexpressing a single efflux transporter, we showed that linezolid is a weak substrate for BCRP, and it is an even weaker substrate for P-gp. The effect of these transporter values on blood–brain barrier permeability in the PBPK model was limited, suggesting that passage of the BBB is mostly dependent on passive diffusion.Our model-based approach proved to be informative, but it still suffered from limitations. First, clearance could not be mechanistically incorporated in the model. This is because the clearance pathways of linezolid are not fully unravelled yet and appear largely independent from classic phase I/II metabolism. Plasma clearance values from the literature were used per study simulation to optimally reflect clinically observed values. In the studies reporting PK values of critically ill patients used for verification of the CNS model, clearance values ranged between 7.3 and 16.6 L/h ,6,7,8. HOur CNS PBPK model can be used to predict cranial CSF disposition of linezolid in case plasma concentrations and corresponding clearance values are available. The validity of these predictions could then be confirmed by comparing our modelled CSF disposition results with actual spinal/cranial CSF linezolid exposures in TBM patients. Unfortunately, combined plasma and total CSF exposure data are not yet available for linezolid. Recently, linezolid plasma and CSF concentrations were reported in 17 TBM patients, but only for two time points (2 and 6 h post dose), so total exposure or plasma clearance could not be calculated . At the Our CNS PBPK model predicted cranial CSF disposition and target attainment in TBM patients, using plasma clearance values observed in pulmonary TB patients. Our simulations suggest acceptable CNS exposure following a total daily dose of 1200 mg, with PK–PD thresholds in cranial CSF being reached in 87% of the simulated adult population. In our paediatric population (~10 mg/kg BID), simulated target attainment in cranial CSF was only moderate (56%). Our model can be used to predict cranial CSF disposition in case plasma concentration measurements of a patient population are available. These simulations can complement ongoing linezolid dose optimization efforts by providing target attainment estimates at difficult-to-sample sites of TBM disease.


Brucella suis as HIPDs of interest for sero-surveillance purposes. Porcine serum samples (n = 597) were collected from six Lao abattoirs in March to December of 2019. Serological enzyme-linked immunosorbent assay (ELISA) methods were chosen for their high-throughput and relatively low-costs. The true seroprevalence for CSF and PRRS seropositivity were 68.7%, 95% CI (64.8–72.3) and 39.5%, 95% CI (35.7–43.5), respectively. The results demonstrated no evidence of Brucella spp. seroconversion. Lao breed pigs were less likely to be CSF seropositive (P < 0.05), whilst pigs slaughtered at <1 year of age were less likely to be PRRS seropositive (P < 0.01). The testing methods could not differentiate between seropositivity gained from vaccine or natural infection, and investigators were unable to obtain the vaccine status of the slaughtered pigs from the abattoirs. These results demonstrate that adequate sample sizes are possible from abattoir sero-surveillance and lifetime health traceability is necessary to understand HIPDs in Lao PDR.National disease surveillance systems are essential to a healthy pig industry but can be costly and logistically complex. In 2019, Lao People's Democratic Republic (Lao PDR) piloted an abattoir disease surveillance system to assess for the presence of high impact pig diseases (HIPDs) using serological methods. The Lao Department of Livestock and Fisheries (DLF) identified Classical Swine Fever (CSF), Porcine Respiratory and Reproductive Syndrome (PRRS) and  Lao People's Democratic Republic (Lao PDR) is a landlocked nation in the Greater Mekong Subregion. Lao PDR shares borders with Thailand, Vietnam, Cambodia, China and Myanmar. Structured animal health surveillance programmes are necessary to meet World Animal Health Organisation (WOAH) member nation requirements, thereby satisfying World Trade Organisation (WTO) requirements to trade animal products on the international market. In 2019, the Lao Department of Livestock and Fisheries (DLF) of the Ministry of Agriculture and Forestry piloted an abattoir-based disease surveillance system to detect high impact pig diseases (HIPDs) and ruminant diseases [Moo laat and commercial operations utilise animals of Landrace/Large White type genetics [The Lao pig industry represents a diverse collection of animal types and management styles, from the smallholder to the commercial operator. Lao traditional breeds include small, dark-haired varieties like the genetics . Smallhogenetics , 4, 5. Dgenetics . Abattoigenetics .Brucella suis (Brucellosis) [Many HIPDs are endemic in Lao PDR  at low lellosis) , 9. In tBrucella suis is a zoonotic pathogen of pigs that spreads through contact with raw pig meat, tissues and body fluids to humans [o humans . Brucello humans . Brucello humans . Symptomo humans . CSF reco humans , 12, 13.o humans . PRRSv co humans . Howevero humans , 16, 17.o humans .The current Lao disease surveillance system relies primarily on disease outbreak reporting by village veterinary workers, as described elsewhere . At offiet al. [The sample collection method and sample size justification is detailed in Siengsanan-Lamont et al. . The proet al. . This meFrom March to December 2019 in each selected province, two PAFO and District Animal Health Office (DAFO) staff visited a local abattoir once every month to collect ten jugular blood samples from pigs being slaughtered that day. The abattoirs were chosen by local PAFO and DAFO staff and remained the same throughout the study period. The sample submission questionnaire collected provenance data, age, breed, sex and body condition score (BCS) with a column for brief antemortem health assessment of the animals [® Classical Swine Fever E2 Competition ELISA [® PRRS Indirect [® Brucellosis Serum Indirect Multi-species [All serum samples were tested using the ID Screenon ELISA , ID ScreIndirect  and ID S-species  from IDVThe sample information was stored in the Pathogen Asset Control System (PACS) at NAHL, then extracted to Microsoft Excel and RStudio  to calcuBrucella spp. were excluded from risk factor analysis. CSF and PRRS samples with complete data (n = 381) underwent univariate risk factor analysis using the glm function in RStudio [P ≤ 0.2 were included in multivariate logistic regression analysis. Multivariate logistic regression modelling used the glmer function from the lme4 package in RStudio [Due to low numbers of seropositive cases,  RStudio . Availabdata n = 81 underw RStudio .Table 2P ≤ 0.05 were retained in a forward stepwise procedure for the final model, with the best fit determined by comparing Akaike information criterion (AIC) values and selecting the model with the lowest AIC value.Variables with a The final multivariate model for CSF seropositivity included PRRS result and animal breed as fixed effects, with abattoir of slaughter as a random effect. For the final PRRS model, the region of origin was included as a random effect and fixed effects included abattoir, CSF result and age group. The random effects were chosen due to the respective variable creating a possibility for hierarchical clustering and therefore needing to be accounted for in the model.Moo laat made up a moderate portion of the samples (148 of 597). A few mixed breed and un-labelled pigs also appeared in the sample set (31 and 19 of 597 respectively)  . Female ctively) . An addin = 587), with only ten samples of unlabelled origin. Of the submitted samples, 164 of 597 originated in a province different to their province of slaughter. Despite the question on the sample submission questionnaire, the pigs' vaccination histories were not obtained at slaughter.Most pigs were slaughtered at an appropriate BCS for marketing (2–3 out of 5) . The anin = 81 of 100) and Savannakhet (n = 58 of 100) abattoirs had the highest (81.0%) and lowest (47.7%) true seroprevalence, respectively. The CSF true seroprevalence by region of origin was 74.9%, 95% CI (68.9–80.0); 65.7%, 95% CI (57.3–73.2) and 66.1%, 95% CI (59.5–72.0) for animals of Northern (n = 173 of 231), Central (n = 88 of 134) and Southern (n = 114 of 218) origin respectively. The seroprevalence by age and breed are shown in CSF seropositive samples comprised 409 of the submitted 597 samples, with five samples returning doubtful results and a further 183 returning seronegative results. Based on these results, the true prevalence for CSF serological immunity was 68.7% (64.8–72.3) . Champasn = 60 of 98) and Luang Prabang (n = 25 of 100) abattoirs had the highest (61.3%) and lowest (25.0%) true seroprevalence, respectively. The PRRS true seroprevalence by region of origin was 39.1%, 95% CI (33.1–45.6); 58.2%, 95% CI (49.7–66.2) and 29.8%, 95% CI (24.1–36.2) for the Northern (n = 90 of 230), Central (n = 78 of 134) and Southern (n = 65 of 218) Regions respectively. The seroprevalence by age and breed are shown in The PRRS Antibody ELISA returned 236 seropositive samples of the 597 samples. For PRRS virus immunity, true seroprevalence was 39.5%, 95% CI (35.7–43.5) nationally . Luang NBrucella spp. For this reason, Brucellosis was therefore excluded from further statistical analysis in this study  of their animals. Region of origin, BCS and gender had no significant influence on CSF seropositivity . On multP < 0.001) compared with pigs >4 years of age. Animals slaughtered in the northern provinces, including Luang Namtha , Xieng Khouang  and Oudomxay  were significantly more likely to be seropositive to PRRS (P < 0.001) than animals slaughtered in Champasack. CSF seropositive animals had 2.5 times, 95% CI (1.4–4.3), the odds of being PRRS seropositive (P < 0.01)  .Table 7This study demonstrates that regularly scheduled abattoir surveillance is achievable given clear guidelines and regular funding. The pilot sites submitted 597 samples of the expected 600 samples. This sample collection schedule is impressive given the presence of ASF from June 2019 onwards in Lao PDR . The ASFet al. [This study provides a snapshot of the disease seroprevalence of Lao pigs entering the abattoir system at the same time as the ASF outbreak. Abattoir surveillance is generally biased towards populations of younger, healthier animals . In thiset al.  suggesteBrucella suis from pigs to humans, from the abattoirs and wet markets to the nationally beloved dish of raw pork mince and herbs – laarp muu [Brucella spp. seroconversion amongst Lao pigs destined for slaughter on antibody indirect ELISA, the test which is recommended by the WOAH Terrestrial Manual for population surveillance [Brucella suis in Lao pig populations, and identify this as a major area for future work. This finding suggests that Brucella suis is either not circulating or is circulating at undetectable levels in Lao PDR.Lao PDR has numerous opportunities to spread aarp muu , 28. Oureillance . The autet al. [The indirect E2 protein ELISA used in this study could not differentiate between naturally infected and CSF-vaccinated animals . The truet al.  collecteSimilar to the CSF ELISA, the PRRS ELISA was unable to differentiate between vaccinated and unvaccinated animals , and theWhilst there appears to be a relationship between CSF seropositivity and PRRS seropositivity, the differences in significant variables in the multivariate analysis are likely due to the differing proportions of animals returning positive CSF and PRRS results. In the univariate analysis both diseases showed significant results in the age group and breed categories. Interestingly, whilst likelihood of CSF seropositivity decreased with age, the opposite trend held with PRRS. This is possibly because of the older sows either being sub-productive (i.e. PRRS affected) or more likely to be vaccinated than grower populations, however further research is necessary for the Lao pork industry to manage this disease risk more effectively.Research into the pig purchasing preferences of abattoirs could shed light on the association between serological results and abattoirs. Abattoirs could preferentially purchase from producers who perform prior vaccination of their pigs, or perhaps abattoirs are choosing to buy from producers or middlemen that source consistently higher carcass yield animals which are of exotic genetics and fully vaccinated against endemic and production limiting diseases. Understanding the liveweight or dressed weight of these animals at slaughter may assist in future analyses of these animals' serological results.Whilst CSF vaccine is imported and used routinely in Lao PDR, the vaccine for PRRS is not currently routinely used in Lao pork operations . The exception being some multinational large-scale operations that privately import the PRRS vaccine for use in country . Further investigation into the presence of low-pathogenic or highly pathogenic PRRS in the Lao commercial pig industry is warranted, as PRRS vaccine may need to be imported routinely to promote future growth in this transitional economy.The presence of true infections or exposures to CSF and PRRS in the pigs is of concern due to health and welfare implications and the financial losses incurred by the diseases, as well as the food safety implications of disease-affected animals entering the food chain. Previous work investigating the knowledge, attitudes and practices of slaughterhouse workers in Lao PDR raised concerns about the conditions within abattoirs not meeting international food safety or animal welfare standards . This juA robust and representative array of national animal disease surveillance programmes are required to meet the requirements of WOAH member nation. Therefore the motivation exists to pursue the implementation of programme such as this. Based on the results achieved, the sample sizes are adequate to contribute to estimates of the seroprevalence of a disease. However, further research and governmental organisation are required to provide more confidence about the spatial distribution of disease. Furthermore, lifetime traceability documents would assist in accurately tracing pork products, allowing the separation of vaccinated from naturally infected animals in future serosurveys. This study demonstrated the feasibility and initial outputs of a pilot abattoir surveillance system in Lao PDR, and based on the results of this study the work was extended to all provinces at the end of 2019.


Acacetin is reported as a potential drug candidate for the treatment of atrial fibrillation. However, clinical applications are limited by poor water solubility, limited ethanol solubility, and extremely low oral bioavailability.The present study prepared and evaluated acacetin-loaded microemulsion (ME) to achieve efficient pharmacokinetics together with no or minimal invasiveness for transdermal delivery.ex vivo permeation with Franz diffusion cell and excised mice skin. In vivo pharmacokinetic studies were also performed at 8 mg/kg in rats within 6 h by transdermal administration.The formulation of ME was determined by the water titration method based on solubility results. The optimized formulation was achieved by the simplex lattice experiment design. The optimized ME formulations FA, FB and FC  were evaluated by 0-∞  than that FA alone (121.4 ± 33.1 min × μg/mL).The optimal ME (FA) was comprised of 12.2% caprylic acid decanoate monoditriglyceride (MCF-NF), 39.8% Smix (RH40: Trans = 2:1 w/w) and 48% water, respectively. Acacetin-loaded FA with particle size 36.0 ± 3.6 nm and drug solubility 803.7 ± 32.1 mg/g was prepared. FB had significantly higher cumulative amounts and higher AUCThe formulation of ME combined with the penetration enhancer can effectively improve the solubility and percutaneous absorption efficiency of acacetin, providing a new option for the non-invasive delivery of acacetin. Saussurea involucrata (Kar. et Kir.) Sch.-Bip (Asteraceae), was reported to possess the beneficial effects of anticancer  calculated by the Bliss method . In recent studies, we have prepared a highly water-soluble phosphate prodrug of acacetin for first aid, which exert an effective termination of experimental persistent AF induced by atrial triggering with vagal nerve stimulation in beagle dogs by infusion. The therapeutic window was determined as 61.1 ∼ 650 ng/mL by the correlation analysis of pharmacodynamics and pharmacokinetics. However, in general terms, drug delivery aims to achieve efficient pharmacokinetics together with no or minimal invasiveness. Thus, it’s still necessary to develop a novel formulation with good clinical compliance and longer therapeutic plasma concentration for daily use.Acacetin, a natural flavone first found in Transdermal drug delivery system (TDDS) is preferred for drugs with significant first-pass effect from the standpoint of translation medicine research and clinical application. TDDS could provide a prolonged period of administration, during which plasma concentrations could be maintained within the therapeutic window, and avoid the first-pass effect for compounds with short biological half-life. However, skin is only permeable to a small number of molecules of a few hundred Daltons and an appropriate octanol-water partition coefficient  preparation for the transdermal delivery of acacetin. ME formulations were designed, optimized and evaluated comprehensively. Pseudo-ternary phase diagrams and the simplex lattice experiment design were employed in the process of formulation design and optimization. The effects of enhancers on the permeability of ME were evaluated. The Acacetin was synthesized by Yaomingkangde Pharmaceutical Company Limited. (2-Hydroxypropyl)-®-cyclodextrin, PEG300, PEG400, RH40 Cremophor® (RH40), Cremophor®EL (CrEL), oleic acid, isopropyl myristate (IMP) and Tween 80 were purchased from Sigma; Labrafac Lipophile WL 1349(1349), Labrasol® (Labra), Transcutol® (Trans), lipoid MCT were generous gifts from Gattefosse ; mineral oil, ethyloleate were purchased from Advance Company . Methanol and acetonitrile were of chromatographic grade. All of the other chemicals and reagents used were of analytic grade.Solubility studies were conducted to select the appropriate components of ME formulation with high drug loading capacity. In the process, an excess amount of acacetin was separately added into 0.1 g of solvents such as ethanol, PEG300, PEG400; oils such as Lipoid MCT, mineral oil, IMP; surfactants like CrEL, Tween 80, Labra; co-surfactants like Trans. Then samples were vortexed for 5 min thoroughly mixed and followed by constantly shaken at 37 °C for 24 h to achieve the dissolution equilibrium. After that, the samples were centrifuged at 14 000 rpm for 30 min to precipitate the un-dissolved particles. The concentration of acacetin in the supernatants was qualified by HPLC analysis. All experiments were performed in triplicate.The HPLC system was equipped with a C-18 column  and mobile phase was composed of a mixture of water and methanol  using an isocratic elution . The flow rate was 1.0 mL/min. The UV absorbance detector was set at 330 nm.T) was marked and calculated by Vimage 2014 . After the formulation of ME was determined, the weight ratio of km was varied from 1:3 to 3:1. At each specific km, the pseudo-ternary phase diagrams were constructed by water titration method as mentioned above, and also the areas of AT were compared.On the basis of solubility study and HLB value, RH40 was selected as surfactant, 1349 and MCF-NF were selected as oil phase, PEG300 and Trans as co-surfactants. Pseudo-ternary phase diagrams were constructed by water titration method , the corresponding amount of each component, e.g., oil, surfactant and co-surfactant were weighed out exactly and blended together adequately. Then measured amount of acacetin was dissolved into very small amount of dimethyl sulfoxide (DMSO) and added into the oily mixtures. Ultimately, a weighed quantity of distilled water was added into the system drop by drop under mild magnetic stirring for 5 min. The whole process was performed at R.T. The particle size of acacetin loaded ME formulation were measured by Dynamic Light Scattering at 25 °C using Malvern Zetasizer Nano-ZS . All experiments were performed in triplicate.Preparation of skin: Skin permeation study was carried out according to the National Institute of Health Guide for Care and Use of Laboratory Animals (1996) and was approved by the Institutional Animal Care and Use Committee of Changzhou University (NO. Y20210019). Male Sprague-Dawley (SD) rats weighing about 250 g were obtained from Laboratory Animal Unit of University of Hong Kong. The rats were housed in cages and supported with standard laboratory diet and water. Before the experiment, the rats were fasted overnight. The abdominal skin was excised surgically after rats were anesthetized by pentobarbital and the hair on the skin was shaved with electrical shaver. Then the subcutaneous tissues of skin were removed. The obtained skin was washed with normal saline and stored at −80 °C. The skins were inspected for integrity before further use to ensure that the samples were free from surface defects  containing 1% sodium dodecyl sulfate (SDS) to maintain sink condition. The whole system was kept at 37 °C and under magnetic stirring at 600 rpm throughout the entire process. Caution was taken to remove all air bubbles between the underside of the skin and the receptor solution. Then, 0.5 mL of acacetin-loaded Mes to be tested or other samples, all containing an equal amount of drug, e.g., 5 mg, was given to the stratum corneum (SC) side of the skin, respectively. Afterwards, the donor compartment was sealed with paraffin film to prevent water evaporation from the system. For each experiment, 0.3 mL of the sample was taken out from the receptor compartment at pre-determined time point and replenished instantly with an equal volume of fresh receiver medium. All samples were treated with 10 times volume of methanol, vortexed for 2 min and then filtered through 0.22 μm membrane filters before HPLC analysis. The cumulative amount of acacetin that permeated through the excised skin  was calculated based on the reported methods  was calculated from the slope of the Qt-time graphs. Apparent permeability coefficient  was calculated by the following equation:Permeation experiment: In order to evaluate the permeability of the prepared formulation or the drug itself, the Where Cd is acacetin concentration in the donor compartment .The maximum flux of acacetin and acacetin-prodrug were also evaluated by dissolved in 50% Tween 80.n = 3) for transdermal administration. The rats were anesthetized by ip 50 mg/mL pentobarbital and the upper back region was very carefully shaved with an electric hair clipper to keep the integrity of the shaved skin. 200 μL of the transdermal formulations  was applied every 1 h within 6 h, and the reservoir was covered with Tegaderm film , in order to prevent the possible solvent evaporation. Blood samples 0.5 mL were collected at 0, 60, 120, 180, 240, 360, 480 and 1440 min from the orbital venous plexus at predetermined-time point over 24 h after formulation application. Plasma was separated by centrifuged  and frozen at −80 °C until analysis.Pharmacokinetic: Rats were divided into 3 groups  at 1, 25, and 400 ng/mL were prepared in blank plasma. Secondly, frozen serum samples were thawed at room temperature (R.T.). The post-treatment of plasma samples was the same as plasma standards as described above.HPLC-MS analysis: Firstly, a set of plasma standards of acacetin were prepared by mixing of different amount of acacetin with 100 μL of blank rat plasma to give final acacetin concentrations of 0.5 ∼ 1000 ng/mL in plasma, respectively. Then an aliquot (1 mL) of the internal standard (IS) solution (30 ng/mL pentamethylquercetin in methanol) was added to each plasma standards. After vortexed mixing for 5 min to precipitate plasma protein, followed by centrifugation at 12,000 m/z 285.0→m/z 241.8 for acacetin and m/z 373.0→m/z 312.0 for the IS. The retention time of IS and acacetin was 1.97 and 2.02 min, respectively.An UFLC system  coupled with a tandem mass spectrophotometer system  was used for the quantitative analysis of acacetin. Chromatographic separations were achieved on an RP C18 column  at 40 °C. The gradient mobile phase consisted of 5 mM ammonium formate containing 0.1% formic acid (A) and acetonitrile (B) was pumped at a flow rate of 0.4 mL/min. The initial mixture (90 A:10B) was maintained for 0.4 min; mobile phase B was increased to 90% at 1.9 min and held at 2.5 min. The mixture returned to initial conditions at 2.6 min, followed by 1.9 min of equilibration. The total run time was 4.5 min. The sample injection volume was 1 μL. The analytical method was validated according to the FDA guidelines for bioanalytical method validation (Tiwari and Tiwari n = 5). Samples were stable after 4 h on the autosampler.Linearity range with 1/x/x weighting was from 1 ∼ 500 ng/mL. The lower limit of quantification was 0.5 ng/mL, and the limit of detection was 0.1 ng/mL for acacetin. Assay accuracy at low, medium, and high QCs was 100 ± 3.18%, 104 ± 2.89% and 97.5 ± 5.12%, respectively (n = 3). The statistical analysis of the data was carried out using a one-way analysis of variance (ANOVA) with p < 0.05 as the minimal level of significance unless otherwise indicated.Statistical data analysis: Data reported were expressed as the mean ± SEM (The equilibrium solubility of acacetin in various vehicles is shown in T were compared (the solid and dashed line represent the starting and ending points of the titration respectively). As seen in T. Thus, we fixed Km as 2:1, 1349 and MCF-NF as oil phase, PEG300 and Trans as co-surfactant, respectively. As compared in T were compared and listed in On the basis of preliminary screening results of the solubility study, 1349, PEG300 and RH40 were firstly employed as oil phase, co-surfactant and surfactant. The Km was set as 1:1, 2:1 and 3:1. The pseudo-ternary phase diagrams were drawn and AA simplex lattice experiment design was utilized to screen and optimize the ME formulation subsequently. As a higher solubility warrants the drug loading capacity in the optimized formulation and a smaller particle size is supposed to promote drug release and transport through membranes. Based on the results from pseudo phase graphs, the range of independent variables was selected as follows: for Oil , Smix  and water . A simplex lattice experiment design containing 16 runs generated by Design-Expert 8.0 software and corresponding response were presented in P-values, lack of fit and PRESS values for comparing the models so as to select statistically significant models for the desired response, such as the smaller size and PI, higher solubility. After ANOVA analysis of the suggested models obtained from the aforesaid process, the models that possessed the smallest P-values and PRESS values were built for PI and solubility, as shown in Hereafter, analysis and optimization of the obtained data were conducted. In the process of fit summary, the regression calculations were carried out to fit all of the polynomial models such as linear, special cubic and full cubic polynomials to the selected response. It produced statistics like The optimal ME formulation comprised of 12.2% MCF-NF, 39.8% Smix and 48% water was recommended and selected. Acacetin loaded ME with particle size 36.0 ± 3.6 nm, PI 0.337 ± 0.023 and drug solubility 803.7 ± 32.1 mg/g was prepared thereafter.The skin is impermeable to most molecules and the SC is generally considered to be the main barrier, due to its unique composition of laterally overlapping, quasi-columnar stacks of corneocytes embedded in intercellular lipid multi-layers. Chemical penetration enhancer (CPE) in the formulation is generally employed to overcome SC barrier and achieve therapeutically plasma concentration by transdermal delivery , FA with 10% DMSO (FB) and solubilizing formulation with 50% DMSO (FC). The formulation FC was composed of 50% DMSO, 30% CrEL, 10% ethanol and 10% PEG300. And the drug concentration was determined as 10 mg/mL in all formulations to be tested.In addition, the compatibility between CPE and drug should be considered, such as if the skin residence time of CPE is very different from the drug  percutaneously were depicted in p < 0.01) at T = 30, 90, 120, 240 min, and extremely significant differences (p < 0.001) at T = 180 min. These results demonstrated that enhanced skin permeability of ME could be achieved by 10% DMSO significantly. High percent of DMSO (50%) acting as CPE in FC was verified at every time point, especially before 120 min, compared with FA and FB.0-t) was calculated by the linear up-log-down rule. As shown in 0-t of FC is slightly higher than FB, but it’s not significant due to big variation. FC presented a faster permeation rate than FA and FB especially at the early post-administration time points. These results are in good agreement with the ex vivo permeability data.The mean acacetin plasma concentration-time profiles after transdermal administration are shown in 0-t and AUC0-∞ between FB and FC groups certified the promising feasibility of a combination of ME and DMSO (FB) to be employed for the transdermal delivery of lipophilic drugs  is the default starting position. The permeability of acacetin prodrug was compared with the parent drug. As listed in ex vivo permeability studies verified that parent water-insoluble acacetin is a good candidate compared with water soluble acacetin prodrug. Thus, the parent acacetin was selected and studied thereafter.Owing to the extremely low oral bioavailability of acacetin and short biological half-life, the development of TDDS is considered. Given that the skin’s principal function is to act as a protective barrier, and that the rate of molecular transport through (in particular) the outermost layer, the SC, is highly constrained and very slow. Therefore, the evaluation of the molecule’s skin penetrability by maximum flux across the skin  the homogeneous state of ME size, (2) the certainty of the bicontinuous phase and discontinuous phase.w/o to o/w. The effect of formulation on the size is not a linear process or any process could be described by some known mathematical models. As shown in However, we didn’t obtain a significant model for size. It is presumed that with the water adding during the titration the type of emulsion will gradually change from via carrier mechanism  altering physicochemical properties of SC; (2) by changing a hydrating property of SC; (3) by altering structure of lipids and protein in intercellular channel Ex vivo permeation studies were conducted to compare the skin permeation ability among formulations of FA, FB and FC. Enhanced skin permeability of ME could be achieved by 10% DMSO (FB) and 50% DMSO(FC) at every time point. The enhanced permeability mechanisms of DMSO might due to (1) DMSO is a polar aprotic solvent able to dissolve polar and nonpolar small molecules, fluidize skin lipid molecules for vesicles permeation  and reduce the occurrence of skin irritation and systemic adverse effects involved (Marren The results from d Marren . So, theex vivo permeation study, FA with 10% DMSO showed significant higher cumulative amounts of acacetin permeated after 4 h application than FA itself. It can be concluded that ME is a potential carrier for the transdermal delivery of acacetin. Further work is required to determine whether ME may ultimately be incorporated into a transdermal patch and take advantage of the favorable delivery rate as well as the lower skin irritation potation.In the present study, acacetin-loaded ME formulations were successfully developed and evaluated for transdermal delivery. Pseudo-ternary phase diagrams were employed to depicted the ME regions for the screening of the ME ingredients. The formulations were further employed with a simple lattice experiment design. The optimized formulation FA showed an average particle size of 36 nm. In


Venous drainage distribution is significantly inversely correlated with oscillatory CSF volume at the level of the foramen magnum plane [−0.37 (0.04)] and not significantly correlated at the C2C3 level [−0.37 (0.05)] over our entire population. This correlation maintained the same trend in patients with syrinxes [−0.80 (<0.01)] and disappeared in patients without a syrinx [−0.05 (0.81)]. Conclusion: The distribution of venous drainage is an important factor in intracranial homeostasis. Impaired venous drainage would lead to greater involvement of the CSF in compensating for arterial blood influx, thus contributing to syrinx genesis.Background: The pathophysiology of this association of type 1 Chiari malformation (CM1) and syrinxes is still unknown. There is an alteration in the dynamics of neurofluids  during the cardiac cycle in CM1. Our objective is to quantify CSF or arterial blood or venous blood flow in patients with Chiari syndrome (CS) with and without syrinxes using phase-contrast MRI (PCMRI). Methods: We included 28 patients with CM1 . Morphological MRI with complementary PCMRI sequences was performed. We analyzed intraventricular CSF, subarachnoid spaces CSF, blood, and tonsillar pulsatility. Results: There is a highly significant correlation ( Chiari type I malformations have a radiological definition based on ptosis of the cerebellar tonsils beyond the foramen magnum ,2,3,4. CUnder physiological conditions and during each cardiac cycle, the interactions between cerebrospinal fluid (CSF) and arterial and venous blood are well known ,11,12,13Intracranial venous drainage increases in the diastolic phase to compensate for the systolic arterial blood brain volume expansion. Thus, intracranial pressure decreases and leads to reversed CSF flow from the spinal to the intra cranial compartment. These rapid and physiological back and forth movements of the CSF are continuously active during each cardiac cycle, even if instantaneously intracranial neurofluid volume is not constant. It is important to note that over the mean time of the cardiac cycle the intracranial volume remains unchanged. The time scale of cardiac cycles is shorter than the time scale of the Monro–Kellie doctrine, which describes preservation of mean intracranial volume ,13. ThisPhase-contrast MRI (PCRMI) is a unique non-invasive tool to quantify CSF and venous and arterial blood flow dynamics in the craniospinal system during one cardiac cycle ,14,15. BIn the context of CS, cervical CSF flushing may be impaired. This CSF compensatory mechanism in response to intracranial systolic vascular volume expansion is therefore altered, leading to probable alteration of intracranial pressure . In CS, The association of CS with a syrinx has been the subject of many pathophysiological proposals . The firThese mechanisms are present in many cases of CS with an associated or unassociated syrinx. Associated factors must exist. A few studies have highlighted the possibility of a hemodynamic origin to syrinxes. These hemodynamic disorders would potentially be related to venous drainage ,28.Our objective is to show and quantify by PCMRI the existence of CSF or arterial blood or venous blood flow in patients with CS with and without syrinxes.This is a retrospective observational study in two populations of patients with Chiari malformation with and without syrinxes.Twenty-eight patients older than 18 years of age (37 ± 13 (19–74) years) with CS were retrospectively included from 2017 to 2022. The symptomatology was secondary to CS and confirmed after examination by a neurosurgeon. Patients with associated spina bifida  and those younger than 18 years of age were excluded.The diagnostic workup required brain and spinal cord MRI, which allowed the patients to be divided into two groups: the syrinx group and the non-syrinx group.A total of 9 patients had an associated syrinx (CS/syrinx group) and 19 patients were included in the group of patients without a syrinx (CS group).All patients underwent a 3T MRI investigation of the head and spine based on the morphological sequences of the conventional diagnostic workup. Complementary phase-contrast sequences were added to investigate CSF and blood flows.The planes were positioned perpendicular to the axis of the studied flow regions. Vascular investigations were performed at the intracranial level for the internal carotid and the arteries and through the straight and superior sagittal sinuses . CSF invThe PCMRI sequence parameters are presented in https://www.flow-software.com. Semi-automatic segmentation was used for delineation of the regions of interest: the blood flows, the CSF oscillations and the tissues oscillations. By multiplying the area of the regions of interest by the velocities, flow dynamic curves were calculated over the 32 points of the cardiac cycle.Data were analyzed using our in-house software (public), For the oscillating flows of CSF and the movement of tissues, their stroke volume (SV) was calculated. SV represents the volume displaced through the region of interest during the cardiac cycle  both in a) was defined as the sum of the flow through the internal carotid and basilar artery. Cerebral venous blood flow (CBFv) was measured as the sum of the flow of the straight sinus and superior sagittal sinus. As CBFv does not drain all the CBF itself , a venous correction factor (α factor) was calculated equal to the mean(CBFa)/mean(CBFv). It was thus possible to provide a theorical correctedCBFv flow curve = α factor × CBFv flow. When this venous factor is equal to 1, correctedCBFv is equal to CBFv. This corresponds to exclusive jugular venous drainage. When the correction factor is >1, it shows the magnitude of accessory drainage paths.Cerebral arterial blood flow  during one cardiac cycle.SV-CSF-cerv, which corresponds to the stroke volume of CSF oscillating through the upper cervical SAS at the level of the C2C3 intervertebral disc during one cardiac cycle.SV-syrinx, which corresponds to the stroke volume of CSF oscillating into the syrinx during one cardiac cycle.SV-CBFa, which represents mean cerebral arterial blood flow during one cardiac cycle.-blood, which represents the volume of blood oscillating into the cranial compartment during one cardiac cycle.SV-Venous correction factor α, which is the ratio between CBFa and mean venous flow. It corresponds to the cerebral venous drainage repartition. When the α factor is 1, there is exclusive sinus drainage. When the α factor is greater than one, it means that the accessory venous drainage pathway contributes to the sinuses to drain all cerebral blood flow. According to the example, if the α factor equals 2, that means that the sinuses participate in only 1/2 (or 50%) of total cerebral drainage.The study parameters were:We used Xstat for statistical analysis.p-value.Mann–Whitney tests were used to compare hemodynamic parameters between patients with a syrinx and those without. The threshold of significance was set to a 0.05 p-value.The correlation study was performed using a Pearson correlation and linear regression. The threshold of significance was set to a 0.05 All results are presented in There were no differences found between the group of CS patients without a syrinx and the group of patients with syrinxes. All results are shown in 2 = 0.16; p < 0.001); SVblood and CBFa were slightly correlated .Across the overall population, the α factor and the CBF parameters were slightly correlated . This was less correlated across the overall CS population  and no significant correlation was found in the CS with a syrinx population . Results are presented in Only one hydrodynamic parameter correlates with SV2 = −0.80; p < 0.01). This correlation is not present in the CS group without syrinxes . All results are shown in A significant negative correlation was found between the α factor in the CS group with syrinxes  is related to an increase in the recruitment of accessory drainage pathways in the CS patient population associated with syrinxes. In this population, we also demonstrated that cervical CSF stroke volume was not correlated with vascular stroke volume. There is an alteration to “CSF mobile compliance”. The existence of a negative correlation between the stroke volume of the cervical CSF and the α venous correction factor may be a result of the implementation of new parameters of compensation for the incompetence of the CSF in its compliance role. The increase in the α venous correction factor would allow for the capacities of the system to be increased to compensate for the intracranial vascular volume changes during systole .In 2000, a study by Pinna  performeOur study shows a more important hydrodynamic and craniospinal hemodynamic alteration in CS associated with a syrinx. These alterations may be secondary to the arteriovenous delay, which would be lengthened and thus act on the temporal distribution between systolic and diastolic durations. If there is an arteriovenous alteration related to an alteration to venous vascular compliance, the CSF should play a role as a compensatory mechanism. In CS, the CSF has, by definition, a disturbance of free flow. This can lead to the formation of a syrinx. The genesis of this syrinx would be potentially secondary to an alteration to CSF flow but also to an alteration to venous blood flow.In many CSF pathologies, potential alterations in venous drainage have been demonstrated ,32,33,34Our groups were heterogeneous in size. This is due to the recruitment methods, which were retrospective. This is a rare orphan disease. The presence of a syrinx is also rare. This study shows the interest of a prospective analysis of this major aspect of this still unexplored pathology.In addition to a prospective analysis, we propose a temporal analysis of hemodynamics and hydrodynamics. We have observed profiles that may be linked to an increase in cardiac systole duration. We propose an analysis of arteriovenous delay in populations with and without a syrinx.We have shown that there are differences in hemodynamic and hydrodynamic interactions depending on the presence or absence of an associated syrinx in patients with CS. PCMRI allows for the quantification of variations in intracranial vascular volume and the response of the CSF. CSF is a passive liquid in terms of its flow and responds to physical laws such as pressure gradients and resistance to flow, thus also responding to the flow surface. Disturbances related to venous drainage can have several origins and can be both an element of the genesis of syrinxes in CS and a consequence. These disturbances of hemohydrodynamic interactions could open a new field of pathophysiological but also clinical exploration.


During her first year of junior high school, a 12-year-old Japanese girl with Down syndrome experienced dizziness, gait disruption, paroxysmal weakness in her hands, and sluggish speaking. Regular blood tests and a brain MRI revealed no abnormalities, and she was tentatively diagnosed with adjustment disorder. Nine months later, the patient experienced a subacute sickness of chest pain, nausea, sleep problem with night terrors, and delusion of observation. Rapid deterioration then developed with simultaneous fever, akinetic mutism, loss of facial expression, and urine incontinence. These catatonic symptoms improved after a few weeks after admission and treatment with lorazepam, escitalopram, and aripiprazole. After discharge, nonetheless, daytime slumber, empty eyes, paradoxical laughter, and declined verbal communication persisted. Upon confirmation of the cerebrospinal N-methyl-D-aspartate (NMDA) receptor autoantibody, methylprednisolone pulse therapy was tried, but it had little effect. Visual hallucinations and cenesthopathy, as well as suicidal thoughts and delusions of death, have predominated in the following years. Cerebrospinal IL-1ra, IL-5, IL-15, CCL5, G-CSF, PDGFbb, and VFGF were raised in the early stage of initial medical attention with nonspecific complaints, but were less prominent in the later stages of catatonic mutism and psychotic symptoms. We suggest a disease concept of progression from Down syndrome disintegrative disorder to NMDA receptor encephalitis, based on this experience. Acute or subacute regression in adolescents with Down syndrome (DS) has been increasingly recognized in the last decade and has been designated variously as Down syndrome disintegrative disorder (DSDD), Down syndrome regression disorder, or unexplained regression in Down syndrome –5. AlthoA multifactorial etiology has been proposed for DSDD, including prior psychosocial stressors, neuroinflammation, and incipient AD pathology and/or developmental neurotransmitter dysregulation –4. ThereHerein, we present a case of DSDD, with three separate phases: the first, nonspecific complaint and declining speech, the second, autistic regression with acute catatonia syndrome, and the third, psychosis with profuse hallucinations. CSF anti-N-methyl-D-aspartate (NMDA) receptor autoantibody was negative in the first and second phases of the cell-based assay but became positive in the third phase. Unexpectedly, some CSF inflammatory cytokines were elevated in the first phase but less prominent in the second and third phases. They included certain molecules whose involvement in the etiology of AD have been assumed. The relevance of CSF results, as well as the diagnostic boundary between DSDD and NMDA receptor encephalitis (NMDARE) in this instance, are discussed.The girl with mosaic DS had a mild intellectual handicap and had been attending a special educational class since the age of 9, but she had no medical complications as a result of trisomy 21. Her premorbid demeanor was upbeat, and she enjoyed dancing and singing. She was somewhat obstinately diligent in academic works, and would cry with stress when she was called on in class but could not answer well to the questions. No autistic traits had been reported. Her intellectual quotient was assessed as 58 on the Wechsler Intelligence Scale for Children, 4th edition, at the age of 11 years.Shortly after starting junior high school at the age of 12 years, the patient complained of fatiguability and paroxysmal weakening of fingers and acquired an unsteady gait over 3 months period. Slowness in the speech was also observed . Blood tNine months later, a few weeks after the COVID-19 pandemic was settled down and the school activity restarted, the girl's linguistic communication and eye contact continued to deteriorate, as did her sleep maintenance insomnia, and night terrors. Within a week, she complained of chest pain, nausea, the delusion of observation, and the perception of how each piece of furniture was feeling. The next morning, she was discovered incontinent and unresponsive to her surroundings, her facial appearance being completely lost. When she arrived at our hospital, her body temperature was 38.1°C, which subsided soon after admission. Her awareness level fluctuated substantially, between E4V1M6 and E1V1M4 on the Glasgow Coma Scale. She demonstrated her intent by shaking or nodding her head. No focal neurological signs were noted on examination. However, she was akinetic and completely mute, stared into space, and held a fixed posture or showed waxy flexibility in her extremities. Drooling and retching, were common, as was oral dyskinesia. Blood and CSF tests, including herpes simplex virus DNA, came back within normal ranges. Brain MRI indicated several high-intensity spots in the frontal white matter, but no abnormalities in the hippocampus sections; sleep EEG revealed a widespread high voltage slow-wave activity, that progressed to irregular activity with a frequency range of 3–10 Hz upon awakening , C, F. LSoon thereafter, the patient came to manifest with a silly smile, paradoxical laughter, and monology. The patient claimed that there are many, invisible people or goods in her house, that dwarf and fairly are sitting on the air cleaner or her head/arms/clothing, that she was talking with someone in her ear and/or neck, that there are tubes in her ear/mouth/body, and that bad ideas come into her head prompting an urge to stab someone with a pair of scissors. A SPECT examination found hypoperfusion in the bilateral parietal and left occipital lobes , contradShe then claimed to have delusions that her voices were recorded, that another person arose while she was fake, and that the present day was the dinosaur age. These psychotic symptoms were relieved by increasing the dosage of aripiprazole. During the period of preparation for high school admission, however, complaints of somatic symptoms or cenesthopathy predominated including throat discomfort, feeling like her face was slipping off, stubbed or dirty abdomen, dysesthesia in the distal extremities, and difficulty in chewing. Coupled with an auditory hallucination that someone was requesting and/or ordering her to die, the patient was influenced by thoughts over suicide and/or life expectancy, as well as the belief that she was already dead. She is currently a high school student, with persistent psychiatric issues. Such an extended course of hallucinations and delusions prompted us to examine the CSF specimen with cell based assay (CBA), which qualified a positive result of anti-NMDA receptor antibodies for the specimen taken just before the intravenous methylprednisolone  9). The. The9). CSF levels of IL-5, IL-15, CCL-5, granulocyte colony-stimulating factor, platelet-derived growth factor (PDGF)-bb, and vascular endothelial growth factor (VEGF) were raised throughout the course, highest at the first examination at the age of 12 years  10, 11), 1110, 1Aside from autoantibody testing, cytokine study on CSF samples from DSDD patients, or even DS people without DSDD, has never been done. This is the first case of DS in which the CSF levels of cytokines and anti-NMDA receptor autoantibodies were measured before, during, and after the subacute catatonic regression. These three times corresponded clinically to the first phase with nonspecific complaints and declining speech, the second period of acute regression and catatonia, and the third phase of psychotic features.The scores of catatonia symptoms peaked at the second admission , 13 Fig, which fTherefore, clinical features of the present patient until the third admission were typical for DSDD, rather not NMDARE. In the paraclinical data, normal CSF results and SPECT hypoperfusion in posterior areas were unusual for NMDARE. Through these multimodal diagnostics, we assumed that the diagnosis of DSDD was most appropriate for the patient. However, the patient began to experience profuse and extended hallucinations during and after the recovery from the catatonia and autistic regression. This evolution coincided with the positive CBA results of the cell-surface immunolabelling of cultured neurons expressing GluN1/GluN2B, when the criteria for “definite” NMDARE were fulfilled , 8. In oAIRE) gene product alters the phenotypic of lymphocytes in the thymus, perhaps leading to the development of anti-TPO autoantibody. The positive results of the anti-TPO antibody in the current case, as well as the higher prevalence of this antibody in DSDD, support the hypothesis of autoimmunity in the pathogenesis of DSDD (APP) gene, which is thought to develop the amyloid plaques with the resultant provocation of inflammation in DS brains. Amyloid plaques in DS can be detected on neuropathology as early as 8–9 years of age when there are no signs of dementia. From this perspective, it is intriguing that IL-5, IL-15, PDGFbb, and VEGF were found to be raised in the CSF of the current patient, all of which are upregulated in AD brains, but not in NMDARE in general  for the publication of this case report.YM, MS, FT, HM, MO, YO, AY, and YW were in charge of the clinical management of the subject and decisions concerning the treatment. YT examined the anti-NMDA receptor antibody assays and measured the cerebrospinal cytokines, chemokines, and other molecules. YM and YS wrote the draft. MS, YW, ST, and YT critically reviewed the manuscript. All authors have read and approved the final manuscript.