In vitro study, tongue cancer cells treated with everolimus, the specific mTOR inhibitor, or transfected with mTOR-mediated siRNAs dramatically attenuated the abilities of cell proliferation by MTT and BrdU assays. In 4-NQO-induced tongue cancer murine model, mTOR inhibitors significantly decreased the incidence of tongue SCC. In conclusion, p-mTOR overexpression was independently associated with poor prognosis of patients with tongue SCC. In vitro and vivo, mTOR inhibition showed the promising activity in tongue SCC. Our results suggest that inhibition of mTOR signaling pathway may be a novel therapeutic target for tongue SCC.Despite improvement in preoperative imaging, surgical technique, and adjuvant therapy, the prognosis of patients with tongue squamous cell carcinoma (SCC) is still unsatisfactory. The mammalian target of rapamycin (mTOR) play a key role in the regulation of tumor cell proliferation and survival. However, the significance of mTOR on the prognosis of tongue SCC remains largely undefined. In the present study, immunohistochemistry was performed to evaluate the expression of phosphorylated mTOR (p-mTOR) in 160 surgically resected tongue SCC, and correlated with survival. Univariate analysis revealed that p-mTOR overexpression (P = 0.006) was associated with inferior overall survival. In multivariate comparison, p-mTOR overexpression  remained independently associated with worse overall survival.  More than 90% of head and neck cancers are squamous cell carcinoma (SCC)2. In Taiwan, the incidence of head and neck cancer increased rapidly, and was ranked as the fourth leading cancer death in males in 20113. Tongue cancer is a common form of head and neck cancer and accounted for 28.11% of patients in 2011 in Taiwan3. Tongue has a rich supply of lymphatic drainage and neurovascular bundle which increase the incidence of cervical lymph node metastasis. The presence of cervical lymph node metastasis is a particularly reliable predictor of regional and distant failure4. Therefore, the prognosis of tongue SCC is worse than that in the other sites of oral cavity. The treatment for early-stage tongue SCC is usually single modality, either surgery or radiotherapy. The treatment for locally advanced tongue SCC is multimodal, with either surgery followed by adjuvant radiation or chemoradiotherapy5. Despite advances in preoperative imaging, surgical technique, and adjuvant therapy, the survival of patients with tongue SCC is still unsatisfactory over the past several decades6. Therefore, discovery of prognostic biomarkers for tongue SCC may provide an useful insight that help the development of potential novel target.Head and neck cancer is one of the ten most frequent cancers worldwide, with an estimated over 500 000 new cases being diagnosed annually7. In response to a variety of biochemical pathways such as hypoxia, intracellular energy change, growth factor signaling molecules, hormones, and cytokines, mTOR can be activated by phosphorylation of Ser2448 through the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway7. Previous studies have reported that dysregulation of mTOR signaling pathway is associated with tumorigenesis, tumor angiogenesis, metastasis, and anticancer drug resistance9. In particular, recent development of the mTOR inhibitors, rapamycin analogs such as Everolimus  and Temsirolimus , have generated considerable excitement in both the clinical and basic cancer research communities, as it exhibits potent activity against a wide panel of cancers11. Therefore, further evaluation of the mTOR signaling in human cancers is important for development of anti-neoplastic therapy targeting the mTOR signaling pathway. For patients with head and neck SCC, recent studies14 showed the inconsistent findings of prognostic significance of mTOR signaling activation, indicating that the effect of mTOR signaling activation may be diverse in different head and neck site. Therefore, we conducted the present study to evaluate the significance of mTOR activation on the prognosis of tongue SCC. Besides, we also investigate the functional relevance of mTOR signaling pathway in vitro and in vivo.The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that plays an important role in regulating ribosomal protein translation, cap-dependent translation, and protein synthesisThe clinicopathologic parameters of the 160 patients with tongue SCC are described in Table The correlations between clinicopathological parameters and immunohistochemical expression of p-mTOR are summarized in Table th AJCC Stage III/IV (P < 0.001), depth of invasion ≥4 mm (P = 0.046), vascular invasion (P = 0.007), perineural invasion (P = 0.025), extracapsular spread (P < 0.001), positive/close surgical margin (P = 0.003), and smoking history (P = 0.027) were significantly associated with inferior overall survival. Additionally, p-mTOR overexpression , histologic grade 2/3 (P = 0.031), vascular invasion (P = 0.015), perineural invasion (P = 0.004), extracapsular spread (P < 0.001), positive/close surgical margin (P = 0.015), and smoking history (P = 0.042) were significantly associated with inferior disease-free survival.Correlations of clinicopathological parameters and p-mTOR expression with overall survival and disease-free survival are shown in Table th AJCC Stage III/IV . For disease-free survival, p-mTOR overexpression , extracapsular spread , pathological 7th AJCC Stage III/IV , and histologic grade 2/3  represented an independent adverse prognosticator. The 5-year overall and disease-free survival rates were 41% and 39% in patients with overexpression of p-mTOR, and 64% and 57% in patients with low expression of p-mTOR, respectively.In multivariate comparison, p-mTOR overexpression  remained independently associated with worse overall survival, together with extracapsular spread , and pathological 7First, we used siRNA to silence endogenous mTOR expression/or activity for functional studies in tongue cancer cells. Two specific mTOR siRNAs were transfected into SAS and HSC-3 cells; both effectively suppressed the expression of p-mTOR/mTOR Fig. . Using tWe next determined whether the inhibitory effect of mTOR inhibitor, everolimus, exists in 4-NQO-induced tongue cancer murine model. Among the 75 mice exposed to 4-NQO, 21 of the 25 mice in placebo group, 18 of the 25 mice in low-dose everolimus group, and 20 of the 25 mice in high dose everolimus group survived at the end of 28 weeks. Histopathologically, all of the surviving mice in three groups had hyperplasia and dysplasia. Papilloma was found in 16 of the 21 surviving mice in placebo group, 11 of the 18 surviving mice in low-dose everolimus group, and 11 of the 20 surviving mice in high-dose everolimus group. There were no significant differences in papilloma between three groups Fig. . Invasiv7. Numerous drugs have been developed to target different components of this pathway11. However the prognostic value of mTOR signaling activation remained largely undefined in cancer patients. For head and neck cancer, Garcia -Carracedo et al.12 reported a positive correlation between the mTOR activation and a less aggressive phenotype, resulting in a significantly improved survival in patients with laryngeal cancer. Naruse et al.13 and Monteiro et al.14 showed that p-mTOR overexpression was correlated with poor survival rate in patients with oral SCC. These incongruous findings suggest that mTOR signaling may be various in different tumor site of head and neck cancer. Further studies on different tumor site of head and neck cancer are thus necessary to fully evaluate the effect of mTOR signaling activation. To the best of our knowledge, large series study focusing on tongue SCC only to assess the prognostic role of mTOR activation is lacking. Therefore, we conducted the present study. We found that p-mTOR overexpression was associated with inferior OS and DFS in 160 patients with tongue SCC, which further support previous findings from Naruse et al.5 and Monteiro et al.14 and highlight the important role of mTOR activation if tongue SCC.The activation of mTOR signaling pathway is common in solid tumorsDespite advance in imaging tool and surgical technique, there is still a proportion of patients with tongue SCC who develop recurrences after surgery. Thus, identification of patients at high risk for recurrence who may benefit from post-operative adjuvant therapy is worthwhile. In the present study, p-mTOR overexpression represented biological aggressive phenotype. The 5-year OS and DFS rates were 41% and 39%, respectively, in patients with p-mTOR overexpression, and 64% and 57%, respectively, in patients with low expression of p-mTOR, suggesting that p-mTOR status might be used to select some patients for adjuvant therapy after surgery. However, our clinical observation has an important limitation. It was a retrospective analysis.10. The present study explored the effect of everolimus on tongue cancer cell lines. The results showed that everolimus significantly reduced the tumor growth and BrdU incorporation of SAS and HSC-3 cells. Furthermore, by using siRNA approach, the abilities of cell proliferation were attenuated in loss-of function of mTOR in both SAS and HSC. Altogether, these results suggest that silencing mTOR expression/activity may provide a potential strategy for the treatment of tongue squamous cell carcinoma.Blockage of mTOR activity by an mTOR inhibitor, everolimus, exhibits an effect of anti-tumor development and is approved by the FDA for the treatment of several types of cancerset al.15 described that rapamycin prevents Early Onset of tongue SCC in an oral-Specific K-ras and p53 two-hit carcinogenesis mouse model. Pten ablation led to of PI3K/AKT/mTOR activation. Compared to control mice, Squarize et al.16 reported that intraoral administration of 4-NQO resulted in the faster development of oral-specific carcinomas in genetically defined mice displaying reduced PTEN expression achieved by the conditional deletion of PTEN using the keratin promoter 14 CRE-lox system. These studies from mice models further support the importance of the mTOR signaling pathway in the progression of tongue SCC.In 4-NQO-induced tongue cancer murine model, we found that chronic administration of everolimus halts the malignant progression of carcinogen-induced tongue SCC. Raimondi 17. 4-NQO treatment of mice induced carcinoma in the tongue simulates many aspects of human tongue carcinogenesis, such as hyperplasia, dysplasia, severe dysplasia, in situ carcinoma and squamous cell carcinoma. 4-NQO-induced cancer model may have increased heterogeneity and genetic complexity of tumor cells, but it still has some limitations. For example, 4-NQO-induced tumor is not from human origin may not reflect relevant carcinogenic exposures and limit the utility as a preclinical platform for therapeutic research. In recent, patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. PDX is based on transfer of human tumor tissue from a patient into immunosuppressed mice, with no interfering ex vivo cell culture18. PDX models retain the architecture and stromal components of the original tumor and preserve tumor heterogeneity. In addition, PDX model also provides the potential tool for the personalized drug therapy19. Despite the advantages of PDX, it still have some limitations, particularly, that an immunodeficient host is still required20. Our current study is limited that the therapeutic efficacy and response of everolimus by using PDX tongue cancer model should be further investigated in the future.Preclinical models of mouse with cancers are crucial tools for the study of testing novel therapeutic efficacy and treatment responses of drugs. The ideal model is cost effective to use, easily manipulable, and recapitulates the biology of human disease. To date, a seldom preclinical model of tongue cancer entirely executes all these criteria. 4-NQO, a synthetic water soluble carcinogen, has been used extensively to study oral carcinogenesisIn conclusion, p-mTOR overexpression was independently associated with poor prognosis in patients with tongue SCC. Inhibition of mTOR activity showed the promising activity in tongue SCC cell lines and mouse model. Our results suggest that inhibition of mTOR signaling pathway may be a novel therapeutic target for the treatment of tongue SCC.th American Joint Committee on Cancer (AJCC) staging system21. Adjuvant therapy, such as radiation alone or concurrent chemoradiation with platinum-based chemotherapy, was used in patients with adverse pathologic features, which was mostly based on the National Comprehensive Cancer Network (NCCN) guidelines. The radiation technique was intensity-modulated radiation therapy. Overall survival (OS) was calculated from the time of surgery to death as a result of all causes. Disease-free survival (DFS) was computed from the time of surgery to the recurrence or death from any cause without evidence of recurrence.This study included 160 patients with available paraffin block who underwent primary surgical resection at Kaohsiung Chang Gung Memorial Hospital for treatment of tongue SCC. This retrospective study was approved by Chang Gung Medical Foundation Institutional Review Board. All procedures were conducted in accordance with guidelines provided by the Ethics Committees and Institutional Review Boards at Chang Gung Memorial Hospital. The informed consent was signed by all study participants. Patients with synchronous cancers in the other organ and patients receiving preoperative chemoradiotherapy, preoperative chemotherapy, or preoperative radiotherapy were excluded. Clinicopathologic information was obtained retrospectively from clinical records and pathology reports. The pathological TNM stage was determined according to the 723.Immunohistochemistry staining was performed using an immunoperoxidase technique. Staining was performed on slides (4 mm) of formalin-fixed, paraffin-embedded tissue sections with primary antibodies against p-mTOR . Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (95 °C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for at least 18 hours at 4 °C overnight. Immunodetection was performed using the LSAB2 kit  followed by 3–3′-diaminobenzidine for color development and hematoxylin for counterstaining. For p-mTOR, incubation without the primary antibody and normal esophageal squamous epithelium were used as a negative control, and normal gastric gland were used as a positive control23. The fraction of tumor cells with significant staining (0–100%) was recorded, and the average value of 2 pathologists was calculated in each patient. The p-mTOR overexpression was defined as the presence of at least staining in ≧ 10% of tumor cells22.The staining assessment was independently carried out by 2 pathologists (S.L.W. and W.T.H.) without any information about clinicopathologic features or prognosis. We followed the previously published method to score the expression of p-mTOR3 cells/well in 96-well plates for culturing 48 h; for everolimus experiments, 5 × 103 cells in 96-well plates treated with or without everolimus as indicated concentration for 48 h. After that the culture medium was removed and the cells were washed with PBS. Cells were then incubated with 0.5 mg/ml MTT, in culture medium without FBS, for 4 h at 37 °C in a 5% CO2 atmosphere. The medium was removed and 100 μL DMSO buffer was added and incubated in the dark for 10 min. Absorbance was measured on a microplate reader at 540 nm. The OD values were normalized with the value for the control group. For bromodeoxyuridine (BrdU) incorporation assay, cells were seeded at a density of 5 × 103 cells/well in 96-well culture plates following treated with or without everolimus for 48 h. BrdU incorporation analysis was performed using the cell proliferation ELISA kit  according to the manufacturer’s instructions.The tongue cancer cell lines SAS and HSC-3 were obtained from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The viability of sub-confluent cells was analyzed by 3--2,5-diphenyltetrazolium bromide (MTT) reduction assay. For siRNA experiments, cell were seeded at 5 × 1025 or placebo control was started 16 weeks after initiation of 4-NOQ treatment, and each group had 25 mice. Mice dying prior to the end of the experiment were excluded from the analysis. At the end of the experiment, mice were sacrificed, and tongues were excised, fixed in 10% buffered formalin, embedded in paraffin blocks, and sectioned for hematoxylin and eosin staining. The histological determination were performed by two pathologists (S.L.W. and W.T.H.) according to the criteria described previously26. The lesions observed were classified into four types: epithelial hyperplasia; dysplasia; papilloma; and invasive squamous cell carcinoma. Hyperplasia was defined as thickened epithelium with prominent surface keratinization and with or without elongated rete ridges. Dysplasia was defined as loss of polarity in the epithelial cells, nuclear pleomorphism and hyperchromasia, abnormal single cell keratinization (dyskeratosis), and increased or abnormal mitoses. Papilloma was defined as noninvasive exophytic growth of neoplastic cells, and invasive squamous cell carcinoma was defined as a lesion with invasion into the subepithelial tissues.The carcinogen, 4-NQO  stock, was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at −20 °C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol  and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of 100 μg/mL. All animal experiments were carried out in accordance with protocols approved by the Animal Use and Management Committee of Kaohsiung Chang-Gung Memorial Hospital. Fifty six-week-old C57Bl/6 mice were treated with 100 μg/ml 4-NQO in drinking water for 16 weeks and then resumed with normal autoclaved drinking water for another 12 weeks . Everolimus is the mTOR signaling pathway inhibitor. The treatment of the everolimus t test. For all analyses, a P value < 0.05 was considered statistically significant.For patient data, statistical analysis was performed using the SPSS 17 software package. The Chi-square test or Fisher’s exact test were employed to compare data between the two groups. For survival analysis, the Kaplan–Meier method was used for univariate analysis, and the difference between survival curves was tested by a log-rank test. In a stepwise forward fashion, parameters with P value < 0.1 at univariate level were entered into Cox regression model to analyze their relative prognostic importance. For 4-NQO-induced tongue tumor mice experiments, statistical analyses of the incidence of tongue tumors were performed using This retrospective study was approved by Chang Gung Medical Foundation Institutional Review Board. All procedures were conducted in accordance with guidelines provided by the Ethics Committees and Institutional Review Boards at Chang Gung Memorial Hospital. The written informed consent was obtained from patients.The datasets supporting the conclusions of this article are included within the article.Supplementary information


Purpose. This study aims to evaluate the process of brain development in neonates using combined amide proton transfer (APT) imaging and conventional magnetization transfer (MT) imaging. Materials and Methods. Case data were reviewed for all patients hospitalized in our institution's neonatal ward. Patients underwent APT and MT imaging (a single protocol) immediately following the routine MR examination. Single-slice APT/MT axial imaging was performed at the level of the basal ganglia. APT and MT ratio (MTR) measurements were performed in multiple brain regions of interest (ROIs). Data was statistically analyzed in order to assess for significant differences between the different regions of the brain or correlation with patient gestational age. Results. A total of 38 neonates were included in the study, with ages ranging from 27 to 41 weeks' corrected gestational age. There were statistically significant differences in both APT and MTR measurements between the frontal lobes, basal ganglia, and occipital lobes . Furthermore, APT and MTR in above brain regions exhibited positive linear correlations with patient gestational age. Conclusions. APT/MT imaging can provide valuable information about the process of the neonatal brain development at the molecular level.  Brain development is a gradual process of continuous maturation, with varying degrees of brain maturity associated with each different developmental period. Maturation of the neonatal and infant brain is a rapid process, occurring predominantly from five months gestational age to approximately one year after birth. During this period, maturation of the developing brain primarily involves neuronal myelination, accomplished via oligodendroglial cell proliferation. This neuroglial cell proliferation is characterized by the synthesis of various proteins, leading to increased total protein content of developing brain tissue.However, the process of myelination is not homogeneous, generally progressing from caudal to rostral, central to peripheral, and dorsal to ventral –5. Conse1H-MRS, the changes of N-acetylaspartate (NAA), Creatine (Cr), Choline (Cho), myo-Inositol (mI), glutamate (Glu), glutamine (Gln), Glu + Gln (Glx), and so forth in the neonatal period with gestational age/region can be observed [31P-MRS can evaluate brain development from the perspective of energy metabolism with changes of phosphomonoesters (PME), phosphodiesters (PDE), phosphocreatine (PCr), and so forth [With the development and wide availability of MR, the diffusion tensor imaging (DTI) is used to evaluate the neonatal brain development more frequently, especially in showing the structure of white matter fiber with obvious advantages. DTI can quantify and display the diffusion properties of water molecule in brain tissues, and it is usually used to display white matter microstructure , 8. Magnobserved , 10. 31Pso forth . DTI andMT is an MRI technique that offers the ability to quantify structural differences in the central nervous system (CNS). MT imaging contrast is achieved through interactions between protons bound to semisolid macromolecules and the free water protons of biological tissue . MT-MRI APT imaging, a newer technique derived from MT imaging, accomplishes molecular MR imaging based on the principles of CEST , 15. APTThus, the purpose of this study was to investigate the relationship between APT and MT signal and gestational age during neonatal brain development in order to further elucidate the complex mechanisms of maturation during this period of brain development.Case data were reviewed for all patients hospitalized in our institution's neonatal ward between December 2013 and June 2014. The patient population of this study were hospitalized for the reasons of respiratory tract infections, fever, skin infections, and diarrhea. Clinically, these reasons may result in brain lesions. When clinicians suspect that there may be brain lesion, a head MR examination should be ordered. We screened the neonates without nervous system disease for further study. Exclusion criteria included history of brain abnormality established prenatally, birth asphyxia, congenital malformations of the brain, mental retardation, and other diseases of the central nervous system. No intravenous contrast was administered for any portion of the MRI examinations. This study was approved by the local Ethical Committee . We obtained informed consent from the patients' guardians and permission from their primary clinicians based on clinical status before additional APT/MT imaging. Conventional MRI examination is followed by APT/MT imaging where sedation has to be used only once for the same patient. The sedative is chloral hydrate with high security for newborns.0 shimming method through a pencil beam volume shimming algorithm) and second-order shimming , using a body coil for transmission and an eight-channel sensitivity-encoding (SENSE) receiver coil. Each examination was interpreted separately by two experienced radiologists.Patients underwent sedation prior to MRI using a 5% chloral hydrate (50 mg/kg) enema administered by an anesthesiologist 30 minutes prior to the study and were monitored by the clinically responsible physician throughout the examination. All examinations were performed on a Philips 3.0 Tesla (3 T) MRI system with pencil beam  sequence used for T2WI were as follows: TR of 4.6 ms; TE of 200 ms; FOV of 180 × 155 mm2; matrix of 224 × 162; slice thickness of 5 mm. Parameters for the spin-echo (SE) sequence used for DWI were as follows: TR of 2500 ms; TE of shortest time; FOV of 200 × 200 mm2; matrix of 124 × 124; slice thickness of 5 mm.The conventional brain MRI examination included T1WI, T2WI, and DWI sequences. A fast-field echo (FFE) sequence was performed for T1WI using the following parameters: TR of 200 ms; TE of 2.3 ms; FOV of 180 × 161 mmWe used a single protocol that could be processed to generate both APT and MT images simultaneously. We used the raw data to calculate both APT and MTR values. The listed parameters apply to both imaging techniques.0) inhomogeneity and optimize signal noise ratio (SNR) while maintaining an acceptable duration of scanning time for clinical applications. The protocol selected for APT/MT imaging employed an RF saturation time of 500 ms [Axial T1WI was used for positioning of the neonates at the level of the basal ganglia prior to image acquisition. The APT/MT imaging protocol was tailored to minimize local magnetic field (Bf 500 ms  (the maxTSE with a turbo factor (TF) of 38 was used for single-slice acquisition. A multiacquisition method with multiple RF pulses was performed to enhance SNR for MT and APT and included eight acquisitions at ±3.5 ppm offset from water frequency . Image aAs mentioned above, APT/MT imaging included multiple acquisitions with multiple RF pulses. Over the course of the acquisition process, the images were obtained using different frequency offsets from water. The specific selected frequency offsets are as follows (parentheses indicate multiple acquisitions and the number performed): 0, ±0.25, ±0.5, ±0.75, ±1, ±1.5, ±2, ±2.5, ±3 (2), ±3.25 (4), ±3.5 (8), ±3.75 (4), ±4 (2), ±4.5, ±5, ±6 ppm, and 15.6 ppm. An unsaturated image was used to normalize the signal.0 field and subsequently to obtain field correction of the Z spectrum. The corrected Z spectrum data were applied to the MTR asymmetry (MTRasym) analysis using symmetrical ±3.5 ppm offset data points. Finally, APT images were generated from the MTRasym values that were calculated at the selected offsets using the following equation: MTRasym (3.5 ppm) = Ssat/S0 (−3.5 ppm) − Ssat/S0 (3.5 ppm), where Ssat/S0 represents the ratio of signals obtained with (Ssat) and without (S0) saturation. The measured APT values were used to reflect the relative magnitude of APT-weighted effects on generated images.For the ATP analysis, raw data from the image acquisition was imported to an interactive data language program  used for data analyses and reconstruction of pseudo-color images. This software was used to calculate a voxel-based Z spectrum. A 12th-order polynomial was then employed to fit the entire Z spectrum and identify the point of lowest intensity on the Z spectrum. This information was used to characterize the inhomogeneity of the BSsat/S0. The measured MT spectra  were corrected for B0 field heterogeneity effects on a pixel-by-pixel basis. Conventional MTR images were calculated from the saturated images at a selected offset of 15.6 ppm [With the same technique and similar method, the MTR was defined according to the equation: MTR = 1 − 15.6 ppm .Following automated analysis of the raw acquisition data, APT and MT images generated by the software were comparatively analyzed by both senior diagnostic radiologists. With T1WI and T2WI images used as references, the process of image analysis began with the selection of ROIs. For all neonates in the study population, the ROIs included deep white matter in both frontal lobes, bilateral basal ganglia, and deep white matter in both occipital lobes . For ROIEach ROI was carefully defined using the drawing function on the clinical workstation; the APT and MT values measured within the ROIs were recorded. The mapping process was performed three times for each ROI to yield the average APT/MT values. The magnitude of APT/MT values measured in the regions of acquisition was used to reflect the relative signal intensity of the ROIs on APT and MT images, with higher values manifesting increased signal intensity.P < 0.05 was interpreted as statistically significant.Statistical analyses were performed using SPSS for Windows . Quantitative data were reported as mean ± standard deviation  were analyzed for correlation with gestational age (in days) via Pearson's correlation analysis. ANOVA was applied to assess for statistically significant differences between APT and MTR values individually measured in frontal lobe deep white matter, basal ganglia, and occipital lobe deep white matter.Independent two-sample A total of 38 neonates without brain abnormalities were included in the study. The patient population included both preterm and full-term infants, with corrected gestational age ranging from 27 to 41 weeks and median gestational age of 36 weeks ± 4 days.P = 0.031, frontal lobe versus basal ganglia P = 0.00, and basal ganglia versus occipital lobe P = 0.00. MTR represents frontal lobe white matter mean ± SD = 12.09 ± 1.28, basal ganglia mean ± SD = 18.16 ± 2.34, and occipital lobe white matter mean ± SD = 12.90 ± 1.09 and frontal lobe versus occipital lobe P = 0.034, frontal lobe versus basal ganglia P = 0.00, and basal ganglia versus occipital lobe P = 0.00.For all the neonates, both APT and MTR showed significant differences between the regions. The measured APT and MTR values were highest in the basal ganglia, followed by the occipital lobe and lowest in the frontal lobe Tables  and 2: Ar = 0.87), frontal lobe white matter (r = 0.85), and basal ganglia (r = 0.80).APT values measured at all three regions exhibited a linear, positive correlation with gestational age . The strWe observed the MTR measurements  in all tInitially proposed by Wolff and Balaban in 1989 , MT-MRI Normally the components of the brain's internal environment and its physical and chemical properties are vital to the survival of brain cells and brain development , 34. HowThe progressive and vigorous increase in cell density and protein content during neonatal brain maturation produces highly variable water content and complex biochemical changes in the developing brain tissue, in stark contrast to the comparative stability of adult brain tissue . This dyR2 values were relative small compared to the APT findings. This is due to the fact that brain development is a gradual process of myelination and glial cells proliferation. In the brain, MT effect primarily originates from semisolid macromolecular including cholesterol and other lipids [Our results demonstrated linear, positive correlation between APT and MT values measured in different regions of the brain and patient gestational age, consistent with known physiological changes during CNS development, namely, neuroglial cell proliferation and myelination . The obsr lipids , 32, 38,r lipids , which cAPT and MT measurements both showed significant differences between the three brain regions evaluated in this study. These regional disparities, however, can be explained by the asymmetric regional progression of neonatal brain maturation with associated underlying differences in tissue composition and inhomogeneity of local internal environments. The basal ganglia, which are rich in perikaryons and dendrites, develop earlier than the frontal and occipital lobes. Moreover, the deep white matter of the occipital and frontal lobes is composed of nerve fibers that are unmyelinated at birth. Furthermore, myelination of white matter occurs later in the frontal lobe than in the occipital lobe. Mean APT and MT values in the current study measured highest in the basal ganglia, followed by the occipital lobe deep white matter, and lowest in the frontal lobe deep white matter. This observed pattern is concordant with the demonstrated directional pattern of myelination –43, showZhang et al. have also recently performed an imaging study of brain development . They evDespite the promising results of our study, several technical and performance issues must be addressed prior to routine clinical application of the APT imaging. For example, issues that limit APT/MT in its current state include eliminating direct water saturation effect, increasing the SNR, and improving image contrast , 45. SafThere were several limitations in our study regarding the use of APT/MT imaging for evaluation of neonatal brain development. Firstly, our investigation is limited by the technical limitations of our available APT/MT imaging sequence, which permits only single-slice imaging. Thus, for the axial basal ganglia level selected in this study, other areas and structures were neither shown nor evaluated. Also, our study was limited to some degree by the somewhat small size of the patient population. Theoretically, the small number of patients within each gestational age group could have fostered slight bias in measured correlations between APT/MT signals and different gestational ages. However, we do not expect that the size of the patient population group should have affected the trend of APT/MT measurements across the different gestational ages.Neonatal brain development is associated with cell proliferation and increased protein content. APT and MT offer noninvasive means of characterizing this process through the quantification of protein content and myelination. Applied to the neonatal brain, APT and MT imaging offer effective new tools for the characterization of brain maturation that could enhance our understanding of normal and pathologic brain development.


Previous studies have shown that the increased coverage of antiretroviral therapy (ART) could reduce the community viral load (CVL) and reduce the occurrence of new HIV infections. However, the impact on the reduction of HIV transmission among men who have sex with men (MSM) is much less certain. The frequency of HIV infections in MSM have been rapidly increasing in recent years in Hangzhou, China. The “Treatment as Prevention” strategy was implemented at a population-level for HIV-infected MSM from January 2014 to June 2016 in Hangzhou; it aimed to increase the ART coverage, reduce the CVL, and reduce HIV transmission.We investigated a subset of MSM diagnosed with HIV pre- and post-implementation of the strategy, using random sampling methods. Viral load (VL) testing was performed for all enrolled individuals; the lower limits of detection were 20 and 50 copies/mL. The data on infections were collected from the national epidemiology database of Hangzhou. Logistic regression analyses were conducted to identify factors associated with the differences in social demographic characteristics and available VL data.P < 0.05). The undetectable VL (≤400 copies/mL) rate pre-implementation period was 50.0% which increased to 84.7% post-implementation (P < 0.001). The mean CVL at the county level significantly decreased in each county post-implementation .The ART coverage increased from 60.7% (839/1383) during the pre-implementation period to 92.3% (2183/2365) during the post-implementation period in Hangzhou. A total of 940 HIV-infected MSM were selected for inclusion in this study: 490 (52.1%) and 450 (47.9%) MSM in the pre- and post-implementation periods, respectively. In total, 89.5% (841/940) of patients had data available on VL rates. The mean CVL was 579 copies/mL pre-implementation and this decreased to 33 copies/mL post-implementation . The mean CVL decreased for all variables investigated post-implementation of the treatment strategy (Our study confirmed a population-level association between increased ART coverage and decreased mean CVL; overall 84.7% of HIV infected MSM had an undetectable VL and were no longer infectious. The Joint United Nations Programme on HIV/AIDS implemented the “90–90-90 strategy” in 2014 with the aim of ending the HIV epidemic; the strategy aims to achieve 90% diagnosis, 90% treated, and 90% virally suppressed for those living with HIV by 2020 . MSM usuART has been proven to reduce the VLs of infected individuals, and might reduce the risk of sexual transmission to uninfected persons . CommuniIn China, the frequency of MSM is exponentially growing; the sentinel surveillance of MSM conducted from 2010 to 2016 showed the HIV prevalence increased from 5.7 to 7.8% . In HangIndividuals were considered for inclusion in this study if they were older than 18 years of age, residents in Hangzhou for over 3 months, HIV-infected MSM, and if they belonged to the follow-up management group of Hangzhou epidemiologic database, regardless of whether they were in care or lost to follow-up.The data on infectious diseases were collected from the national epidemiologic database of Hangzhou, which tracks everyone who is diagnosed with HIV in China. The following data were collected: date of HIV diagnosis, education and other social demographic characteristics, CD4 counts, transmission route, and routine follow-up and treatment information. The above databases have been described in detail in previous publications , 25. ProFrom the Hangzhou epidemiological data base, a random sampling method was used in order to select HIV-infected MSM to be included in the study; patients’ ID numbers in the epidemic database were sorted, and research subjects were randomly selected based on the sample size calculations. The pre-implementation period data were collected from the national epidemiologic database (data until the end of December 2013), while the post-implementation data were collected from the database (data until June 2016). This was a prospective intervention study. From January 2014 to June 2016, the “Treatment as Prevention” strategy was implemented in Hangzhou; the goal of this strategy was to increase ART coverage to 90% among HIV-infected MSM and decrease the population-level CVLs by 50% by June 2016. Random sampling methods were used to select MSM individuals with HIV infection in Hangzhou from before January 2014 (pre-implementation period) and after June 2016 (post-implementation period). The sample size was calculated using WINPEPI (PEPI- for-windows) version 9.5 software. The sample size calculations were performed based on the increased ART coverage rate and decreased mCVL, which would achieve 90% power to detect significant mean differences in empowerment scores at a level of 0.05. The minimum sample size required was 400. Data were prospectively collected from other surveillance databases in order to ascertain trends in HIV incidence and frequency of new infections.VLs were not tested for enrolled patients on ART who had a VL test conducted within the past 6 months. For such patients the previous VL results were used for this study. Patients not on ART or those on ART whom did not have VL tests performed in the past 6 months, had VL testing completed by study investigators. Two sensitive polymerase chain reaction (PCR) techniques for measuring VLs were used in this study. The lower limits of detection were 20 and 50 copies/mL for these techniques, which were the same as those for patients with VLs completed in the past 6 months. Blood sampling was completed within a month after the completion of the selection of the HIV-infected MSM subjects for enrollment in the pre- (before January 2014) and post-implementation (after July 2016) periods.The mCVL was defined as the mean VL of HIV-infected MSM in Hangzhou. The Log10-transformed value was calculated according to the individual VL value, and then the arithmetic mean was calculated. Arithmetic mean log10-transformed individual VLs are approximately normally distribution. When calculating the mean value, we used half of the detection limit to calculate the VL below the detectable limit. Undetectable VLs were defined as an individual mean of ≤400 copies/mL due to the variation in the lower limits of detection using different testing platforms.The investigations of HIV incidence and new infections were conducted in the first halves of 2014 and 2016 for the pre- and post-implementation periods, respectively. The HIV incidence was calculated using the sentinel surveillance data for MSM in Hangzhou; all of the positive infections were detected using the BED capture enzyme immunoassay (BED-CEIA), and were used to calculate HIV incidence. New HIV infections included newly diagnosed HIV cases in 2014 and 2016 using BED detection with CD4 tests conducted within 3 months of diagnosis, showing CD4 counts ≥600 cells/μL . BED-CEIP < 0.10 and/or those previously shown to be associated with the differences in social demographic characteristics and VLs were included in the multivariate regression models, adjusted odds ratios (AOR) were calculated along with coinciding 95% confidence intervals (CI). In order to mitigate the effects of missing VL data, SPSS MCMC was used to conduct multiple imputations for VLs on the log-transformed level based on the factors associated with the available VL data [P≤0.05 level and β = 0.1. Data were analyzed using SPSS version 19.0.Categorical variables are presented as frequencies and proportions while continuous variables are presented as medians and interquartile ranges (IQR). Differences in the general demographic characteristics were calculated using the Student’s T test or chi-squared (χ2) and Kruskal-Wallis tests. We determined the mean (standard deviation [SD]) and median (IQR) for mCVL. Logistics regression analyses were used to identify the factors associated with the differences in social demographic characteristics and VLs in the pre- and post-implementation periods. Factors from univariate analysis with  VL data , 27. We P < 0.001). A total of 940 HIV infected MSM were included in the study: 490 (52.1%) and 450 (47.9%) individuals in the pre- and post-implementation periods, respectively. The ART coverage was 51.0% (250/490) and 83.8% (377/450) in the pre- and post-implementation periods (P < 0.001). There were significant differences in marital status (P = 0.017), education levels (P < 0.001), and CD4 counts (P = 0.010) between individuals in the pre- and post-implementation periods. There were no significant differences in the other demographic characteristics investigated (P > 0.05). The multivariate logistic regression analysis showed that level of education  and CD4 counts  were significantly higher in the pre- rather than post-implementation period  in January 2014 to 92.3% (2183/2365) in June 2016 (383 in JaP = 0.233). HIV-infected MSM who did not receive ART (P < 0.001) and residents registered as nonlocals (P < 0.001) had more missing VL data than those who received ART and local residence. Multivariate logistic regression analysis demonstrated the following: those on ART had 4.81  times the odds of having available VL data than those not on ART; nonlocal residents had 0.46  times the odds of having available VL data than locals; those in the post-implementation period had 0.41  times the odds of having available VL data than those treated in the post-implementation period  of patients had data available on VL rates; there were no differences in available VL rates for those in the pre- (90.5%) and post-implementation 88.2%) periods , . The median VL was 18,451 copies/mL  and 10 copies/mL (IQR 10–10) in the pre- and post-implementation periods, respectively. The undetectable VL rate was 50.0% in the pre-implementation period; however, this increased to 84.7% in the post-implementation period (P < 0.001), education (P = 0.016), age (P < 0.001) and treatment with ART (P < 0.001). There were also significant differences in the mCVL in the post-implementation period based on marital status (P = 0.007), education (P = 0.012), CD4 count (P = 0.034), treatment with ART (P < 0.001) and place of registered residence (P = 0.022). The mCVL decreased for all variables investigated post implementation of the treatment strategy (P < 0.05),  and CD4 count (P = 0.215); however, significant differences existed based on marital status (P = 0.007), age (P = 0.031), and treatment with ART (P < 0.001) in the post-implementation period. The undetectable VL rate increased for all variables investigated post implementation of the treatment strategy with the exception of CD4 count <200 cells/μL and those who never received ART. Those HIV-infected individuals who never received ART had an undetectable VL rate of 10.8% before implementation and this increased to 15.1% after implementation (P = 0.326),  in the post-implementation period. Fig. .Fig. 1MeP > 0.05). Using BED-CEIA methods it was determined that there were 77 newly infected individuals from January to June in 2014 and 73 newly infected individuals from January to June in 2016. Meanwhile, the frequency of patients with CD4 counts ≥600 cells/μL was 27 and 21 in the pre- and post-implementation periods, respectively.The sentinel surveillance of MSM in Hangzhou showed the HIV incidence decreased from 5.71% (95% CI 2.36–9.07) to 3.58% (95% CI 0.38–6.78) in the pre- and post-implementation periods (Following the implementation of the “Treatment as Prevention” strategy in Hangzhou among HIV-infected MSM, the ART coverage increased from 60.7 to 92.3%, the mCVL decreased from 579 to 33 copies/mL, and the undetectable VL increased from 50.0 to 84.7%. Concurrently, the HIV incidence decreased from 5.71 to 3.58%. Despite this finding, the frequency of new HIV infections did not increase. There were differences in education levels and CD4 counts pre- and post- implementation of this strategy. The difference in CD4 counts in the pre- and post-implementation periods was mainly due to lack of ART that led to increased CD4 counts in the pre-implementation period; the intervention strategy was to encourage patients to receive ART. The rapid increase in the frequency of MSM in recent years in Hangzhou has occurred mainly among young, highly educated individuals. Therefore, the social demographic characteristics were balanced between the study periods.The findings of this study are consistent with various other studies conducted in different countries. In a study conducted in New York , which i3 to indicate new versus old HIV infection. Moreover, several previous studies used new HIV diagnoses as a direct proxy for new infections [Similar to the studies in San Francisco , Canada fections , 18, 31,fections , 18. Allfections . Recentlfections ; thus, uThe information on available VLs was distinctively different in the previous research studies; the proportion of patients with available VL data in San Francisco, New York, and Washington was 75% , and 48%Our study was consistent with the findings from previous ecological studies that focused on associations between ART coverage and aggregate outcomes associated with CVL trends and the frequency of new HIV infections in a given geographical area , 18. We Another study conducted in South Africa  showed tThere were several limitations in our study. The strategy was only implemented in Hangzhou, according to the migratory patterns of MSM, because the migratory infections were introduced into Hangzhou over a short period of time. This led to the new infections occurring in the region while local infections were transmitted outside of the region. It would be not be valid to assess the HIV transmission based on the migratory patterns of MSM. A study in Canada  showed tIn conclusion, after the implementation of this “Treatment as Prevention” strategy among HIV-infected MSM in Hangzhou, our study confirmed a population-level association between increased ART coverage and decreased mCVL. Overall, 84.7% of HIV-infected MSM had undetectable VLs and were therefore no longer infectious. Thus this strategy was effective in curbing the incidence and frequency of new HIV infections among MSM. The “Treatment as Prevention” strategy can be used to decrease HIV incidence and frequency of new infections, particularly among MSM.


To evaluate the effect of applied suction during microkeratome-assisted laser in situ keratomileusis (LASIK) procedure on peripapillary retinal nerve fiber layer (RNFL) thickness as well as macular ganglion cell-inner plexiform layer (GC-IPL) thickness. 89 patients (124 eyes) with established myopia range from −3.0 to −8.0 diopters and no associated ocular diseases were included in this study. RNFL and GC-IPL thickness measurements were performed by spectral domain optical coherence tomography (SD OCT) one day before LASIK and at 1 and 6 months postoperatively.μm while the first month and the sixth month postoperatively were 94.01 ± 12.04 μm and 94.46 ± 12.27 μm, respectively. Comparing results, there is no significant difference between baseline, one month, and six months postoperatively for mean RNFL (p > 0.05). Mean GC-IPL thickness was 81.70 ± 7.47 μm preoperatively with no significant difference during the follow-up period . Mean RNFL thickness prior to LASIK was 93.86 ± 12.17  RNFL and GC-IPL complex thickness remained unaffected following LASIK intervention. Laser in situ keratomileusis (LASIK) is currently the most common refractive surgery procedure. However, LASIK application among glaucoma patients or glaucoma suspects still remains controversial, mainly because of the short-term increase in intraocular pressure (IOP) during the creation of the corneal flap, that is, during the application of the suction ring. At that point, IOP values may reach up to 65 mmHg . It has Spectral domain OCT (SD OCT) represents a prevalent imaging technique that among others enables a valid quantitative and qualitative analysis of the peripapillary RNFL –9. MoreoPurpose of the present study was to assess the effect of microkeratome-assisted LASIK procedure on the peripapillary RNFL and the GC-IPL using spectral domain OCT (SD OCT).This is a prospective clinic-based observational study that was conducted at the “Maja” Clinic in Nis, Serbia, and was approved by the hospital's ethics committee. All participants were enrolled in the study from the refractive surgery service in a consecutive-if-eligible basis and obtained written informed consent according to the tenets of the Helsinki Declaration.Inclusion criteria were that participants should have stable refraction for over a year and a spherical equivalent in the range between −3.00 and −8.00 diopters (D) and to be over 18 years old. Exclusion criteria were the presence of any other associated ocular disease, ocular surface disorder, glaucoma, corneal thickness below 500 microns, and irregular corneal topography, as well as any history of systemic disease. Patients who had previous ocular or refractive surgeries were excluded from the study.All participants received a complete ophthalmological examination, including best-corrected visual acuity, IOP measurement by Goldmann applanation tonometry, gonioscopy, slit lamp and fundus examinations, Schirmer test, corneal pachymetry, and tomography to rule out any LASIK contraindications.Peripapillary RNFL was measured using the glaucoma analysis mode of Cirrus® SD OCT device . The optic nerve head (ONH) was automatically scanned over an area of 6 × 6 mm by 200 × 200-pixel resolution axial scan. The RNFL thickness within the whole circle circumference, the linear maps in 12 hour positions, and the circular maps in each quadrant was recorded for each patient. The ganglion cell analysis algorithm of the Cirrus SD OCT was used to process and measure the thickness of macular GC-IPL. The average, minimum, and six sectoral  GC-IPL thicknesses were measured from the elliptical annulus centered on the fovea. All measurements were performed by an experienced surgeon. Medicament mydriasis was achieved by tropicamide 1% drops before recording. Images with a signal power more than seven were used for analysis. Measurements were performed one day prior to and 1 and 6 months after LASIK.All LASIK procedures were performed by the same experienced surgeon. Proxymetacaine hydrochloride 0.5% drops were used for local anesthesia, while lids and lashes were sterilized with povidone-iodine (10%) scrub solution. The Moria One Use-Plus SBK microkeratome was used for the creation of the flap. The negative pressure of the suction ring was set at 600–620 mmHg, and the velocity of the head movement was constant (3 mm/seconds). The hinge was created at the 12 o'clock position. The Alcon WaveLight Allegretto Wave Eye-Q® excimer laser 400 Hz was used for all ablations. After ablation, the flap was repositioned with an irrigation cannula and the interface was thoroughly irrigated. In postoperative period, all patients were administered fixed combination of tobramycin and dexamethasone q.i.d. for a 10 days and preservative free artificial tears for 2 months.p < 0.05. All statistical analyses were performed with the Statistical Program for Social Sciences (SPSS) version 20.0 for Windows software package .Kolmogorov-Smirnov testing was applied to the assessment of the normality of the measured data. All parameters were expressed as mean ± standard deviation (SD). Differences between pre- and postoperative measurements were evaluated by means of Mann–Whitney test. The level of statistical significance was set at μm. Comparing results, there is no significant difference between baseline, one month and six months postoperatively for mean RNFL (p > 0.05) as well as regarding the clock hours and quadrants RNFL thickness.In total, 89 patients (124 eyes) were included in the study; 56 were female (62.92%), mean age 32.08 ± 7.7 years (range 18–51 years). The mean preoperative spherical equivalent was −4.81 ± 2.48 D. Preoperative and postoperative RNFL values at all quadrants and in each of the 12 clock-hour sectors  are shown in μm while the minimum value of GC-IPL thickness was 75.21 ± 12.57 μm. The GC-IPL complex thickness did not change significantly from preoperative to any postoperative visit (p > 0.05). In addition, the mean central subfield thickness was 263.47 ± 21.46, preoperatively, with any significant differences being detected at follow-up visits , superior (S), superonasal (SN), inferonasal (IN), inferior (I), and inferotemporal (IT) layer have been reported in p visits .A series of previous studies regarding the influence of transient intraoperative IOP elevation during LASIK on RNFL thickness, implementing conventional SLP with fixed compensator, reported a postoperative thinning of RNFL, raising considerations concerning the safety of LASIK in patients with glaucoma or glaucoma suspects. Nevertheless, this finding was attributed to alterations in corneal birefringence following LASIK that affected the accuracy of the instrument's readings , 14–17. A great importance in establishing glaucoma diagnosis and in monitoring structural changes in glaucomatous patients has lately been attributed to GCC –12. GCC In the current study, we attempted to evaluate the effect of applied suction during microkeratome-assisted LASIK on OCT-derived RNFL and macular GC-IPL thickness, in a cohort of myopic patients without any other ocular pathology. According to our findings, suction-induced IOP elevation during LASIK produces nonsignificant alterations on the OCT-derived peripapillary RNFL, which is in agreement with previous literature reports , 19, 20.In conclusion, to the best of our knowledge, this is one of the first studies to report on the impact of microkeratome-assisted LASIK on peripapillary RNFL, as well as the macular GC-IPL thickness, excluding influence of macular NFL. Our results suggest that suction-induced IOP elevation has no clinically significant impact on RNFL and macular GC-IPL thickness, namely, the retinal structures that are most susceptible to elevated IOP.


P = 0.005). CBV ASPECTS 0–7 remained independent prognostic factors for HT after adjustment for clinical baseline variables. CBV ASPECTS could be of value to predict HT risk after acute cardioembolic stroke and may be a quick risk assessment approach before reperfusion therapy.Alberta Stroke Program Early Computed Tomography (CT) score (ASPECTS) has been applied to CT perfusion (CTP) with good interrater agreement to predict early ischemic stroke, and it can be useful in decision making in acute ischemic stroke. The aim of the present study was to assess the predictive value of CTP ASPECTS of hemorrhagic transformation (HT) in acute cardioembolic stroke. This is a single-enter, retrospective study. All patients hospitalized with acute cardioembolic stroke from January 2008 to September 2013 were included. ASPECTS of baseline non-contrast CT, CTP maps of cerebral blood volume (CBV), cerebral blood flow, and mean transit time were collected from 52 consecutive patients with less than 12-h anterior circulation ischemic stroke. MRI scan was performed within 72 h of symptom onset after index stroke including T2*-weighted gradient echo to identify HT. For bleeding risk assessment, CTP and diffusion-weighted imaging ASPECTS were categorized into 0–7 or 8–10. Baseline characteristics, ASPCETS scores and HT were compared. Eighteen (34.6%) patients had HT and four (7.7%) developed symptomatic HT. On univariate analysis, the proportion of patients with CBV-ASPECTS 0–7 was significantly higher in HT patients as compared to patients without HT (44 versus 9%,  Hemorrhagic transformation (HT) can cause devastating consequence of ischemic stroke, especially for cardioembolic stroke, occurring in up to 90% of patients within the first week after symptom onset –4. In reAlberta Stroke Program Early CT score (ASPECTS) has been applied to CTP with good interobserver variability, and CTP ASPECTS is more accurate at predicting the extent of reversible and irreversible ischemia than non-contrast CT (NCCT) , 10. Difhttps://ccs.mgh.harvard.edu/main.php)  .Patients were included in the present analysis if 1) they had acute cardioembolic stroke in the anterior circulation and undergone NCCT and CTP within 12 h of symptom onset and (2) an MRI scan was performed within 72 h of symptom onset after index CTP including DWI, T2*-weighted gradient echo (GRE), and MR angiography (MRA). Patients with poor image quality, no GRE sequence, no MRA or CT angiography (CTA), posterior circulation infarction, and anterior cerebral artery infarction were excluded for further analysis. HT was defined as GRE documented hemorrhage in the ischemia occurred within 3 days after treatment onset , providing 16, 5-mm thick slices through the anterior circulation.All patients underwent NCCT, CTP, and CTA according to a standardized protocol as part of routine clinical assessment. Whole-brain NCCT was performed using 120 kV, 250 mA, and slice thickness 5 mm. The following CTP used 40 ml non-ionic iodinated contrast at a rate of 4 ml/s b-values , apparent diffusion coefficient, time of flight MR angiography, and T2*-weighted GRE sequence. The MRI parameters for DWI and GRE were a slice thickness of 5 mm (no gap between slices) with a matrix size of 128 × 128, 24–30 axial slices, and a field-of-view of 240 mm. DWI (EPI spin echo) parameters were a repetition time (TR) of 4,528 ms, an echo time (TE) of 103 ms. Other imaging parameters were as follows: GRE , FLAIR , T1 , and T2 .MRI scans were performed on 1.5 T scanner. MRI protocol included T1- and T2-weighted, fluid-attenuated inversion recovery (FLAIR), axial trace DWI with 2 All ASPECTS were assessed independently by two experienced investigators, a neuroradiologist (ZJ) and a neurologist (CY) both blinded to patient identity and clinical information. A template of two axial NCCT/CTP/DWI slices with markers for the 10 regions being scores by the NCCT/CTP/DWI ASPECTS was offered. Scoring of NCCT, CTP and DWI was done separately.Non-contrast CT ASPECTS were accessed for focal parenchymal low attenuation or swelling Figure  20). Al. Al20). Leukoaraiosis was defined on MR FLAIR image as hyperintense supratentorial white matter lesions. Fazekas scale was used both in periventricular  and subcortical areas  , 26. LA To evaluate of the present of vessel occlusion, two observers (Jinglong Zhao and Yanyan Cao) independently reviewed the circle of Willis maximum intensity projection reconstructions of acute CTA and graded middle cerebral artery as proximal and distal occlusion. Follow-up MRA was also graded in the same manner.t tests for normal distribution (Kolmogorov–Smirnov test) and the Mann–Whitney U for non-normality. Second, ASPECTS were dichotomized (scores 0–7 versus scores 8–10) according to previous CT-based study . The patients were divided into two groups by the presence of HT at follow up GRE image. First, Mean ASPECTS on NCCT, CTP, and DWI modalities were compared with each other between groups using ed study , 28. Theed study .Of 61 patients suspected of having an acute cardioembolic stroke in the anterior circulation territory, 9 patients were excluded due to no GRE imaging (1 patient), poor imaging of CTP (3 patients), posterior circulation or anterior cerebral artery infarction (5 patients). Therefore, a total of 52 cardioembolic ischemic stroke patients were included in the analysis . The NIHSS score on admission was 10.5 ± 7.0 . Median time from symptom onset to NCCT and CTP imaging was 5.2 ± 4.8 h  and from symptom onset to MRI imaging was 30.7 ± 30.0 h . Of 24 patients who received reperfusion therapy, 15 patients were treated with standard dose intravenous recombinant tissue plasminogen activator (rt-PA). Seven patients received intravenous rt-PA and mechanical thrombectomy, and two patients were given intravenous and intra-arterial rt-PA. The interval time from onset to reperfusion therapy is 2.6 ± 1.0 h . Table The mean ASPECTS showed significant difference between modalities in the following order Table : NCCT anP = 0.005; Table P = 0.008; Table P = 0.005; Table Eighteen 34.6%) patients had HT. Six patients (11.5%) had parenchymal hemorrhage, and four (7.7%) developed sICH. All the sICH patients were given reperfusion therapy. On univariate analysis, the proportion of patients with CBV-ASPECTS 0–7 was significantly higher in HT patients as compared to patients without HT  and negative predictive value (NPV) for HT prediction are provided in Table P = 0.008)]. Likewise, DWI-ASPECTS 0–7 was an independent risk factor in this binary logistic regression model [OR 11.61 ].In a binary logistic regression model, CBV-ASPECTS 0–7 was independently associated with HT when controlling for reperfusion therapy [odds ratio (OR) 10.26 (95% CI 1.83–57.64, Our results indicate that CBV and DWI ASPECTS 0–7 are associated with HT after cardioembolic ischemic stroke.The ASPECTS scoring system was initial designed to improve EIC detection on CT scans , 29. AltIn most institutions, CTP is still the preferred choice of rapid diagnosis of hyperacute ischemic stroke by generating maps of CBV, CBF, and MTT. Low CBV the same as DWI, is an indicator of infarction. Several studies explored the predictive value of CBV and showed that lower relative cerebral blood volume and very low cerebral blood volume were more powerful predictor of HT than other parameters of CTP in acute ischemic stroke , 31. ButPrevious study has showed that CBV ASPECTS closely predicted final infarct in patients with major reperfusion, and CBF and MTT ASPECTS predicted final infarct in patients without major reperfusion . In our In our analysis, the similar ASPECTS of MTT and DWI indicate that most reperfusion therapy is ineffective, which is in accordance with previous study that delayed recanalization after acute cardioembolic stroke is an independent predictor of HT . FurtherSimilar to prior studies, we have shown that CBF ASPECTS are approximate to DWI ASPECTS and CBV ASPECTS underestimate DWI volumes –36. SincThe main limitation of our study is the retrospective nature of the design. DWI ASPECTS were not performed in the hyperacute stage of stroke, which does not support the feasibility of using DWI ASPECTS in real time. In most institutions CT and CTP are still the first choice of evaluation the ischemia of stroke. Due to the higher sensitivity of CTP for detection of early ischemia as compared to CT, it may therefore be possible that CTP ASPECTS were more reliable than CT ASPECTS. Therefore, the relationship of CBV ASPECTS and HT risk may have been established in the present analysis. Other limitations are the relatively small number of stroke patients, which was a consequence of our strict inclusion criteria.In conclusion, our study indicates that as a widely applicable and semiquantitative methodology that does not require advanced automated software analysis, CBV ASPECTS could be of value in predicting HT risk after acute cardioembolic ischemic stroke and may be a quick risk assessment approach before reperfusion therapy. Obviously, our findings should be prospectively assessed in a large study.This is a chart review study approved by The Institutional Review Board of the Beth Israel Deaconess Medical Center of the Harvard Medical School.QW and LC had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: QW and LC. Acquisition of data: LL, QW, YC, JZ, and ND. Analysis and interpretation of data: LL, QW, and BW. Drafting of the manuscript: LL, QW, and LC. Critical revision for important intellectual content: LL, QW, BW, and LC. Study supervision: QW and LC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer NY and handling editor declared their shared affiliation.


Athletes generally met able-bodied recommendations for macronutrients. Male and female athletes often failed to meet the Recommended Dietary Allowance (RDA) or Adequate Intake (AI) for vitamin D, vitamin E, pantothenic acid, magnesium, and potassium. On average, females did not meet the RDA for iron and calcium, whereas males did not meet the RDA for vitamin A and folate. Commonly consumed supplements were vitamin D, protein powder, sport bars, and sport drinks. Analysis of diet and supplement use within this population shows several micronutrient deficiencies and irregular use of specific supplements. Athlete support and education is required to optimize nutrition in Paralympic athletes.Dietary intakes and supplement use in Paralympic athletes remains largely unexplored, and specialized recommendations are lacking. The aim of this study was to evaluate nutrient intakes and supplement use in high-performance athletes with physical disabilities using three-day food records and a validated dietary supplement use questionnaire. A secondary aim examined gender differences in nutrient and supplement intakes. Male ( The Paralympic games are highly competitive, and athletes need to maximize performance—one crucial aspect of which is nutrition. Proper sports nutrition should be incorporated into an athlete’s lifestyle, beginning at the training phase. Specifically, athletes should focus on optimal amounts and timing of macronutrient consumption while ensuring daily micronutrient recommendations are met . IncorpoSport nutritional needs are non-transferable between athletes and are dependent upon the type, duration, and intensity of the activity performed . The addThis lack of nutritional guidelines, combined with the fact that athletes with impairments generally have lower energy intakes, may lead to low levels of specific micronutrients; therefore, dietary supplementation (DS) may be necessary . AlthougWith little research surrounding nutrition in athletes with physical disabilities, the primary objective of this study was to assess macro- and micronutrient intakes and types of dietary supplements being consumed. The secondary objective was to report preferred sources of nutritional information for athletes with impairments. Awareness of dietary concerns, types of dietary supplements, and preferred sources of information can be used to develop educational programs and guide future research.Male and female athletes 18 years and older who met the criteria for an athlete with a physical or visual disability, as set out by the International Paralympic Committee (IPC) [The study was approved by the Mount Royal University Human Research Ethics Board (Ethics ID 2015-35). Athlete written consent was provided prior to completing the Dietary Supplement Questionnaire ,10 Supp and Threhttps://www.youtube.com/watch?v=Hpra2GoyWRA&feature=youtu.be) [Dietary intakes were collected via Three-Day Food Records—a protocol tested for validity and reliability and previously used in athletes with spinal cord injuries . Athleteoutu.be)  was provhttp://www.nutritionco.com/FWnutrients.htm [The Three-Day Food Records were analyzed using FoodWorks version 17 . FoodWorks derives the nutritional information for its food sources from over 40,000 food references, including USDA Standard Reference 27, the Food and Nutrition Database for Dietary Studies, and the Canadian Nutrient File 2010. Additionally, direct data from seven fast food restaurants was used. A complete nutrient list can be found at ents.htm . When a A dietary supplement questionnaire ,10 was uParticipants were mailed or emailed a copy of the consent form, along with the Dietary Supplement Questionnaire and Three-Day Food Records, and a postage paid return envelope.t-test for normally distributed data or Mann–Whitney U test for data that was not normally distributed. Differences between genders in supplement use were determined by a chi square test and sources of information using a Fisher’s exact test. All analyses were performed using SPSS Statistics version 24 .Athletes were categorized into groups based on gender: males 18–50 years and females 18–50 years. Intakes of macronutrients are presented as absolute values, percent of total calories, and grams per kilogram of body weight. Percent of total calories for carbohydrates, sugar, and protein was determined by multiplying the grams by 4 kcal/g, dividing by the total calories, and multiplying by 100. Calculations that adjust for dietary fiber were not conducted, as there is no clear consensus on the energy value per gram of dietary fiber. Percent of total calories from fats was calculated by multiplying the grams by 9 kcal/g, dividing by the total calories and multiplying by 100. All micronutrient intakes are presented as the percent of the Recommended Dietary Allowance (RDA) or Adequate Intake (AI). Percent of the Tolerable Upper Intake Levels (UL) was included for sodium. Percent RDA, AI, and UL were calculated by taking each athlete’s intake and dividing it by the established RDA, AI, or UL for their age and gender  and multA total of 42 athletes were sent questionnaires, and 40 athletes responded. Descriptive characteristics of the participants are outlined in p = 0.013). There were no differences in carbohydrate and fat intakes based on body weight (BW) between genders. Males had greater protein intakes based on body weight at 1.6 g/kg BW compared to females at 1.4 g/kg BW (p = 0.017). Females consumed a significantly greater percentage of their total calories from sugar as compared to males (p = 0.049).Macronutrient intakes are presented in p = 0.031), selenium (p = 0.016), and iron (p < 0.001) as compared to males.Micronutrient intakes are presented in p = 0.648).When athletes were asked to rank their diet as a whole, 11% of males and 5% of females ranked their diet as “not healthy”, 61% of males and 73% of females ranked their diet as “average”, and 28% of males and 23% of females thought their diet was “very healthy”. There was no difference between genders (p = 0.492).Athlete use of dietary supplements is presented as those who consume the supplement regularly, occasionally, or never over the past three months . The majMales most regularly used sport bars (38.9%), protein powder (38.9%), and sport drinks (33.3%), whereas females most regularly used vitamin D (40.9%), protein powder (22.7%), and fatty acids (18.2%).p = 0.008). There were no other significant differences between genders in supplement use. Complete results for dietary supplement use are provided in When analyzed according to gender, males were more likely to use branched chain amino acids (BCAAs) (When asked “What are your reason(s) for taking dietary supplements?” athletes reported their top five reasons as “stay healthy” (50.0%), “increase energy” (42.5%), “medical” (40.0%), “enhance overall athletic performance” (37.5%), and “improve exercise recovery” (37.5%). Males reported their top three reasons as “improve exercise recovery” (50.0%), “increase energy” (44.4%), and “enhance overall athletic performance” (44.4%). Females’ top reasons for taking dietary supplements were “stay healthy” (59.1%) and “medical (your doctor told you to)” (50.0%). There were no significant differences between males and females in reasons for supplementation.p = 0.006). The least common sources for both genders included pharmacist (2.5%), television (5%), naturopath/chiropractor (7.5%), physio/massage therapist (7.5%), health store (7.5%), and print media (7.5%).When asked the question “Where do you get information about dietary supplements?” athletes reported their top three sources as dietician/nutritionist (62.5%), medical physician (doctor) (40%), and athletic trainer (27.5%). Both genders reported dietician/nutritionists as their number one source. Females reported medical physician (doctor) (45.5%) and product labels (18.2%) as their second and third choices. Males reported athletic trainer (50.0%) as their second choice and medical physician (doctor) (33.3%), internet (33.3%), and teammates/friends (33.3%) tied for third. Males were significantly more likely to get information from athletic trainers than females (p = 0.033). Though not significant, “coach or athletic trainer” should be noted, as 61.1% of males reported preferring this source, whereas only 31.8% of females preferred this source for nutritional information (p = 0.110).When the question was changed to “Which way do you prefer to receive information about dietary supplements?” athletes preferred “individual nutrition consultation” (80%), “coach or athletic trainer” (45%), and “doctor/chiropractor/physiotherapist, etc.” (45%). Thirty-three percent of males preferred to receive information from “family/friends” as compared to 4.5% of females (This research analyzes dietary intakes and supplement use in elite Canadian athletes with physical disabilities, an understudied population, and highlights areas of potential dietary improvements.Our findings show that elite Canadian athletes with physical disabilities tend to consume a diet that is adequate in most macro- and micronutrients. Female energy intakes were significantly lower when compared to males—a trend that has been cited in previous literature . Total eAthletes’ energy needs will fluctuate during seasonal training phases, and are often higher than energy intakes in able-bodied endurance athletes . When coThe standardization of macronutrient recommendations for athletes with physical disabilities will be complicated since nutritional needs vary from athlete to athlete. Despite the challenges, some guidelines should be developed, similar to those available for AB athletes . In the Protein recommendations for AB athletes range from 1.2 to 2.0 g/kg BW depending on type, duration, and intensity of physical activity . Our stuDietary fat is important in health and athletics, as it provides the body with energy, aids in the absorption of fat-soluble vitamins, and is a component of cell membranes . DietaryAlthough no standards are available, it is suggested that athletes may require higher levels of select micronutrients , and a dCalcium and vitamin D have roles in bone-building and muscle strength and function ,20,21,22Iron intakes did not meet the RDA, on average, in female athletes. Iron deficiency can promote anemia, which can cause weakness, fatigue, poor concentration, and limit physical performance ,27. FemaMales did not meet the RDA for folate and vitamin A, which have roles in cellular functioning  and immuThis study demonstrates that athletes with physical disabilities have a high usage of a wide variety of dietary supplements, as 95% of the participants reported usage over the past three months. To our knowledge, there are few studies investigating supplement use in this population. One study reported 58% of athletes with an impairment consumed dietary supplements within the previous six months . Lower rAmong the most commonly used supplements were multi-vitamin/multi-minerals, essential fatty acids, and protein powders, which are similar to those reported in other studies ,6. Vitampreferred source of nutrition information in the current study. This may be concerning, as athletes may receive false or inaccurate information that is unsupported by research [It is pivotal to know where athletes seek information on DS in order to develop and implement educational strategies for those involved in the training and education of athletes . Althougresearch . To counThe information gained from this study is highly relevant to athletes with physical disabilities and their support personnel. High-performance Canadian athletes with physical disabilities could optimize their dietary intakes and supplement use to achieve world-class performance. Future studies should evaluate the impact of the level of impairment on energy and macronutrient intakes to work towards recommendations. In order to adequately support these athletes, educational programs and counseling from sport nutrition professionals are urgently needed.