Objective: The protective effect of silymarin (SMN) on mycophenolate mofetil (MMF)–induced duodenal disorders was investigated. Materials and Methods: Forty-two Wistar rats were assigned to seven groups including control and test groups. The control animals received saline and the test animals were treated with MMF  and saline, MMF and SMN , MMF and Celecoxib , and MMF and SMN plus CLX for 14 consecutive days. The antioxidant status and myeloperoxidase activity were determined and the histopathological examinations on duodenal section also were performed.Results: Biochemical analyses revealed that SMN and CLX individually and in combination therapy could reduce the MMF-increased nitric oxide (NO) content, myeloperoxidase (MPA) activity, and malondialdehyde (MDA) level, while the MMF-reduced level of total thiol molecules (TTM) was increased significantly (p<0.05) by given compounds. Concurrent administration of SMN and CLX resulted in a synergistic effect on the reduction of MDA level and MPO activity. SMN and CLX were able to improve the MMF-induced histopathological damages including the villus atrophy and inflammatory cells infiltration.Conclusion: Our data suggest that the MMF-induced duodenal disorders may attribute to the elevated NO and MDA levels and myeloperoxidase activity that resulted in pathological injuries. Moreover, the biochemical alterations and histopathological injuries due to MMF administration were reduced by SMN alone or in combination with CLX indicating its protective effect.  Mycophenolate mofetil (MMF) as an immunosuppressive pro-drug has been used to prevent graft rejection over the last decade. MMF is rapidly metabolized into its active metabolite - mycophenolic acid (MPA) - which inhibits non-competitively the rate-limiting enzyme of inosine 5-monophosphate dehydrogenase . Previously, we showed that the MMF-induced GI disorders are largely related to local inflammatory reactions, which were highlighted with increased nitric oxide (NO) and myeloperoxidase activity in the gastrointestinal tract , is used globally as a hepatoprotective remedy in human medicine , 5.5’-dithiobis-2-nitrobenzoic acid (DTNB), N-(1-naphtyl) ethylendiamine.2HCl (NED), hexadecyl trimethyl ammonium bromide, and tetramethyl benzidine were purchased from Sigma Chemical Co. . N-(1-naphthyl) ethylenediamine.2HCl was obtained from Sigma-Aldrich (Germany). Thiobarbituric acid, phosphoric acid (85%), dimethyl sulfoxide (DMSO), sodium nitrite, and ethanol were purchased from Merck (Germany). N-Butanol was obtained from Carl Roth, GmbH Co. (Germany). Phosphoric acid, dimethyl sulfoxide (DMSO) and ethanol were obtained from Merck (Germany). Sulfanilamide was purchased from ACROS, New Jersey (USA). All other chemicals were commercial products of analytical grade. Commercially available standard kit was used for determination of alkaline phosphatase .Iran) was obtained from a local drug store  and was suspended in saline. MMF was provided by Hoffman La Roche,  and was suspended in saline containing 0.4% Tween 80, 0.9% benzylalcohol  were obtained from the animal resource of the Faculty of Veterinary Medicine, Urmia University. The rats were acclimatized for one week and had free access to food and water during adaptation and experimental periods. The experimental protocols were approved by the ethical committee of Urmia University in accordance with principles of laboratory animal care . Animals were assigned into control and test groups (n=6). Animals in the test group were subdivided to following groups: MMF group: animals in this group received MMF . SMN 25 group: animals in this group received MMF  and SMN .  SMN 50 group: animals in this group received MMF  and SMN . SMN 100 group: animals in this group received MMF  and SMN .  CLX group: rats in this group received MMF  and . SMN+CLX group: animals in this group received MMF , SMN  and CLX . The control group received only saline  containing the same amount of the test compound solvent during the 14-day experiment period. Animals received saline and/or test compounds via gastric gavage. The selected dose levels for MMF and SMN were based on our previous reports  sections of the duodenum were removed and rinsed with chilled saline. The samples were then divided into two parts which the first part was fixed in 10% formalin in phosphate buffer saline for pathological examinations and the second part was snap frozen in liquid nitrogen and kept in -70 °C till further biochemical analyses. The anesthetized animals were euthanized using COMeasurement of serum level of alkaline phosphatase (ALP) Serum level of ALP was measured using commercially available standard kit  and according to manufacturer's instructions. NO measurement2. In reaction with sulphanilamide, HNO2 forms a diazonium salt, which reacts with N-(1-naphthyl) ethylenediamine. 2HCL to form an azo dye that can be detected by absorbing at 540 nm wavelength. The NO content of the examined organs was expressed as nmol per mg of protein in samples. Total nitrate/nitrite content of duodenal tissue was measured according to the Griess reaction  determination g for 10 min; 0.5 ml of the supernatant was mixed with 3 ml phosphoric acid (1% V/V) and then after vortex mixing, 1 ml of 6.7 g L−1 TBA was added to the samples. The samples were heated at 100 °C for 45 min, and then chilled in ice. After addition of 3 ml N-butanol, the samples were centrifuged at 3000 × g for 10 min. The absorbance of supernatant was measured spectrophotometerically , at 532 nm and the amount of lipid peroxidation end product was calculated according to the simultaneously prepared calibration curve using MDA standards. The amount of MDA was expressed as nmol per mg of protein. The protein content of the samples was assessed based on Lowery method .To determine the lipid peroxidation rate in the control and test groups, MDA content of the duodenal samples was measured using the thiobarbituric acid (TBA) reaction as described previously   g for 10 min. To 0.2 ml of the supernatant of tissue homogenate, 0.6 ml Tris-EDTA buffer  and thereafter 40l 5.5’-dithiobis-2-nitrobenzoic acid (10 mM in pure methanol) were added. The final volume of this mixture was made up to 4.0 ml by an extra addition of pure methanol. After 15 min incubation at room temperature, the samples were centrifuged at 3000 × g for 10 min and ultimately the absorbance of the supernatant was measured at 412 nm. The TTM capacity was expressed as nmol per mg of protein in samples. The protein content of the samples was measured according to the Lowry method .Total sulfhydryl level in the duodenal section was measured as described previously .Myeloperoxidase (MPO) activity, as a biochemical indicator of neutrophil infiltration into the gastrointestinal tissue, was measured in collected duodenal region of the GUT samples as previously described . For each animal in the test and control groups, at least three slides from the duodenum were prepared and the averages of scored marks were analyzed. To count the mono- and poly-morphonuclear cells, 45 microscopic fields were randomly chosen in the lamina properia part of duodenal region and at the 400 × magnification the cells were counted. The histopathological studies were conducted by a pathologist who was unaware of the study purposes. Statistical analysesThe mean and standard deviation of the measured parameters were calculated. The results were analyzed using Graph Pad Prism software . The comparisons between groups were made by analysis of variance (ANOVA) followed by Bonferroni post-hoc test. p<0.05 was considered statistically different.SMN and CLX recovered the MMF-reduced body weight gain and lowered the MMF-elevated ALP levelThe body weight gain (BWG) in the animals that received MMF for 14 days reduced significantly (p<0.01) in comparison with the control group, while SMN- and CLX-treated groups showed remarkable improvement in the MMF-reduced BWG. Co-administration of CLX and SMN improved the MMF-reduced BWG, nevertheless no synergism between two compounds on BWG could be achieved . SMN at SMN reduced the MMF-enhanced Myeloperoxidase activity and NO level The NO content of duodenal region in the MMF-received animals was elevated. On the contrary, SMN and CLX reduced the NO level in the duodenal tissue, with maximum reduction at 50 mg/kg dose level of SMN. Although maximum reduction in the NO-content of duodenal tissue was obtained in animals which received both test compounds simultaneously, no synergistic effect was obtained between SMN and CLX statistically . The MMFSMN attenuated the MMF-elevated MDA content and protected from the MMF-induced TTM depletionTo analyze the antioxidant status following 14-day MMF administration and providing various treatment approaches for protection from the MMF-induced duodenal disorders, the rate of lipid peroxidation and total thiol molecules (TTM) of duodenal region were measured. Results showed that the MDA content of duodenal region in the MMF-received group was significantly (p<0.05) elevated, while SMN and CLX both lowered the lipid peroxidation rate. SMN was able to reduce the MDA level remarkably at 50 mg/kg dose level when it was given individually. Co-administration of CLX and SMN showed a synergistic effect on the MDA reduction . TTM conSMN prevented from the MMF-induced villus atrophy and attenuated the mono- and poly-morphonuclear inflammatory cells infiltration in the duodenal region The histopathological examinations of the duodenal samples in the control group showed no remarkable pathological changes . The irrThe current study showed that the MMF-induced duodenal disorders are accompanied with severe inflammatory reactions characterized by mono- and poly- nuclear inflammatory cells infiltration in the lamina propria of the duodenum. Moreover, the high level of produced MDA and remarkable elevation of MPO activity confirmed the lipid peroxidation and neutrophil infiltration as two inflammatory biomarkers. NO content increase and TTM depletion in the duodenal region of the MMF-received animals also indicated that both nitrosative and oxidative stress are involved in the MMF-induced duodenal disorders. Additionally, this study showed that SMN and CLX in individual and combination therapies are able to protect from the MMF-induced GI damages. In some biochemical and histopathological examinations, a synergistic effect between SMN and CLX was obtained. Undoubtedly, GI disorders which are clinically characterized by reversible abdominal pain and diarrhea, are the dominant side effects of the MMF administration as an immunosuppressive agent  loss compared with the control animals, which may be related to anorexia, low feed intake, and more importantly to the MMF-induced GI damages. Those animals which concurrently with MMF received SMN and/or CLX individually or in combination, showed a remarkable improvement in body weight gain, suggest that these compounds either increased the feed intake or lowered the MMF-related GI damages and consequently improved the nutrient absorption through the GI tract.The effect of MMF via CNS on the feed intake has not been reported, yet. Therefore, the observed BW loss due to the MMF-administration may be attributed to the local injuries, which likely reduced the intestinal nutrient absorption. Similar clinical symptoms including body weight loss have been reported in human patients who after organ transplantation received MMF for a long time (Herrero et al., 2007The significant elevation of ALP in the MMF-received animals indicates an inflammation, which indirectly also affects the feed intake in involved animals. Previous studies in rodents have demonstrated that the ALP elevation could be a biomarker of intestinal inflammation. Moreover, the increase of ALP activity in enterocytes has been microscopically demonstrated in the dextran sulfate sodium-induced model of inflammation (Renes et al., 2002In this study, two histopathological points of villus height to crypt depth ratio and mono- and poly-nuclear inflammatory cells infiltration in the MMF-received animals and concurrently in the protective compounds-received rats were highlighted. Our computer-assisted program to measure the villus height and crypt depth ratio showed that MMF significantly declined the ratio. This finding was strongly supported by light microscopic examinations, which demonstrated remarkable villus atrophy in the MMF-received animals suggesting a crucial role of villus atrophy in the MMF-induced GI disorders. Another supportive finding of the current study is that in the MMF-received animals, recruitment of leukocytes including lymphocytes, neutrophils, and plasma cells in the duodenal region were significantly increased. The later issue may reflect an inflammatory reaction in the region. de novo pathway of purine synthesis (Behrend, 2001The villus atrophy as a long recognized histopathological manifestation of the MMF-induced GI disorders has been reported. The reason for villus atrophy may be related to the enterocytes dependency on the Next to the histopathological examinations, biochemical analyses such as assessment of oxidative/nitrosative stress biomarkers which were determined by measuring the TTM and NO levels and also the produced MDA level in the duodenal region support the MMF-induced damages. The elevated level of NO and MDA and simultaneously reduced level of TTM indicate locally occurred oxidative/nitrosative stress in the MMF-received animals. It seems that MMF by reducing the TTM level as an important source of antioxidant capacity and at the same time by enhancing the NO content of the duodenal region facilitates events which end up with remarkable lipid peroxidation. In the current study, to clarify the pathogenesis of MMF-induced damages, the MPO activity was measured as a biomarker of neutrophil infiltration. Indeed, our results showed a significant elevation of MPO activity in the MMF-treated animals. The increased MPO activity may be accounted as a strong source of elevated NO content in the damaged tissues. The contribution of neutrophils in the production of NO in the initial phase of murine asthma and in the polyethylene oxide-modified polyurethane-induced neutrophil activation which resulted in a great NO production, have been previously reported (Fernandes et al., 2007The second part of the current study was devoted to show the protective effect of SMN and CLX individually and in combination on the MMF-induced duodenal disorders including histopathological damages and biochemical changes. In the previous study, we showed that the MMF-induced damages in the duodenal region are similar to those of aspirin-created injuries and a local inflammation alongside with oxidative stress were the dominant feature of occurred injuries (Malekinejad et al., 2011CLX also lowered the MMF-induced inflammatory reactions particularly the neutrophil infiltration and consequently the NO production and MPO activity, confirming the nature of the MMF-induced pathological impact as an inflammation (Parfitt et al., 2006Another interesting finding of this study was that we demonstrated a synergistic effect between SMN and CLX, when they were co-administered. Although statistically, in all performed experiments, we could not prove this. However, in the biochemical analyses which were in the same direction including the neutrophil infiltration, MPO activity, and MDA production, the synergism between two compounds was confirmed. The histopathological findings did not show any synergistic effect between two given compounds. Additionally, in the animals that received both compounds, the MMF-induced damages including the atrophy of villus were not completely diminished. The reason for this discrepancy could be a possible interaction between two tested chemicals and short study period for histopathological damages to be cured. Undoubtedly, to clarify the effectiveness of such a combination therapy, further pharmacokinetics studies during longer experimental period are warranted. We showed that in the MMF-induced duodenal disorders, in addition to histopathological damages which are characterized by the villus atrophy and mono- and poly-nuclear cell infiltration, the biochemical alterations including a significant elevation of MPO activity, NO and MDA content along with a remarkable reduction of TTM level in the duodenal region were involved. Our findings indicate that the MMF-induced duodenal damages were reduced by SMN alone or in combination with CLX. This protective effect of SMN may be due to its anti-inflammatory and antioxidant properties.


Adolescents are characterized by impulsive risky behavior, particularly in the presence of peers. We discriminated high and low risk-taking male adolescents aged 18–19 years by assessing their propensity for risky behavior and vulnerability to peer influence with personality tests, and compared structural differences in gray and white matter of the brain with voxel-based morphometry (VBM) and diffusion tensor imaging (DTI), respectively. We also compared the brain structures according to the participants' actual risk-taking behavior in a simulated driving task with two different social conditions making up a peer competition situation. There was a discrepancy between the self-reported personality test results and risky driving behavior . Comparison between high and low risk-taking adolescents according to personality test results revealed no significant difference in gray matter volume and white matter integrity. However, comparison according to actual risk-taking behavior during task performance revealed significantly higher white matter integrity in the high risk-taking group, suggesting that increased risky behavior during adolescence is not necessarily attributed to the immature brain as conventional wisdom says. Adolescence is characterized by novelty-seeking and impulsive risky behavior The human brain undergoes dramatic structural changes during childhood and adolescence, and adolescents' heightened propensity for risky behavior is thought to reflect maturational imbalance between affective reward processing and cognitive control systems In support of this ‘dual systems’ theory, a recent functional magnetic resonance imaging (MRI) study According to MRI-based morphometry studies, gray matter (GM) and white matter (WM) also show different developmental trajectories. Specifically, GM volume rapidly increases early in development, peaking at around age 4, and decreases thereafter To date, brain imaging research on adolescent risk-taking (and peer influence) has been focused on functional brain activation associated with reward processing and cognitive control, particularly in different age groups e.g., , while rIn the present study, we compared both GM volume and WM integrity of high and low risk-taking groups of adolescents assessed by personality tests on propensity for risky behavior and vulnerability to peer influence. We also evaluated differences in the brain structure according to the participants' actual risk-taking behavior verified in a simulated driving task with two different social conditions involving peer influence. The social context was manipulated by informing the participants that the performance would be compared among their peers.Adolescent participants were recruited from a local vocational school after assessing their propensity for risky behavior and vulnerability to peer influence with personality tests. A total of 215 students completed the tests administered online in a computer class. Excluding females and left-handers, 187 were further examined for extreme cases on the scales for impulsivity (sensation-seeking) and resistance to peer influence, and 43 high risk-taking (high impulsivity and low resistance to peer influence) and 46 low risk-taking (low impulsivity and high resistance to peer influence) respondents were selected, and 17 from each group agreed to participate in the experiment. We confirmed with histograms and a scatter plot that the scores (n = 187) were normally distributed, and the selected respondents and participants for each group were scattered randomly within its quadrant divided by the average scores. Thus, the participants were 34 right-handed male adolescents aged 18–19 years with no history of neurological or psychiatric problems.The study was approved by the Ethics Committee of the Hospital District of Southwest Finland, and conducted at the Department of Radiology of the Turku University Hospital (TYKS), Turku, Finland, according to the Declaration of Helsinki. The participants gave written informed consent and received monetary compensation (150 euro) for their participation.After the experiment, MRI data of some participants were excluded due to failure to follow instructions or problems in data registration. Thus, between-group MRI analyses included 32 participants (16 per group) and correlation analyses between MRI measures and the social factor included 29 participants (16 high and 13 low risk-taking groups).We used Zuckerman-Kuhlman-Aluja Personality Questionnaire (ZKA-PQ) We used a simple computerized driving task modified from the Stoplight Game The traffic light at the first intersection always remained green, and thus only the other 19 trials were included in the data analysis. Successfully traveling through an intersection without braking (Go) resulted in no delay, whereas braking (Stop) and waiting for the red light to turn green resulted in a short 3-s delay. Unsuccessful traveling with a crash at an intersection results in a longer 6-s delay. Risk-taking  was encouraged by instructing to complete the track as quickly as possible.The game script included varying distance between intersections, or inter-trial interval (11–13 s), and timing of the traffic light turning yellow before entering an intersection (1.5–3.0 s). The probability of the crash at the intersection also varied as another car, invisible in advance, crossed the track either immediately  or 2.0 s  after the participant's car arrived at the intersection. Details of the task parameters will be reported separately. Participants were given 5 min to complete the track and the worst case with 12 stops and 7 crashes took 5 min 12 s to reach the end of the track. There were six variants of the game script, two for practice sessions and the other four for the actual experiment, presented in a counterbalanced order across participants.The participant was first given instructions and performed a short guided game of 10 trials, sitting in a chair outside the MRI room. The participant then performed two full-length practice runs with instructions to reach the end of the track as quickly as possible. The participant was then placed in an MRI scanner and completed four runs of the task under two different social conditions, the first two runs in the non-competition condition and the other two runs in the competition condition. In the non-competition condition, the participant was simply instructed to reach the end of the track as quickly as possible, in the same way as in the practice runs, whereas in the competition condition, they were told that the results of their performance would be presented at school and compared among their peers to reveal the fastest record. The change of social context was a surprise manipulation. Instructions on the social condition were given before starting every run of the task.It took less than 1 h to complete six runs of the task (including two practice runs), and the whole experimental session including instructions, positioning in the scanner, and structural MRI scans before and after task performance took about 2 h.The number of Go responses (or risk-taking), and Go and Stop response (or decision-making) time were compared between two groups (high and low risk-taking) and two social conditions . The behavioral data showed that the personality tests could not predict actual risk-taking behavior in the driving task. The questionnaire and behavioral data will be reported separately. In short, participants took more risks and spent more time in making either Go or Stop decision during the competition condition, without any difference between the questionnaire-based groups. Thus, we also considered performance-based group comparisons for the MRI data using the number of Go responses.An index of social influence was calculated as a ratio between the number of Go responses registered in the conditions with and without a peer competition situation. This index was used in the GM volume and fractional anisotropy (FA) analyses. Only 29 subjects  had valid social indices: Two subjects reported after experiment that they did not hear or understood the instruction about a shift to competition, and one subject made no risky decisions during non-competition sessions.2) applied in 64 isotropically distributed encoding directions. The diffusion imaging was performed using 7300 ms TR, 92 ms TE, 90° flip angle, 56 axial slices with 2 mm cubic voxel size, and 112×112 mm acquisition matrix. Image processing and statistical analysis was performed with Statistical Parametric Mapping  A set of structural images including T1-weghted MRI and diffusion data set was obtained with the Verio 3T MRI scanner  at the Medical Imaging Centre of Southwest Finland, Turku, Finland. A high-resolution T1-weighed 3D magnetization-prepared rapid gradient-echo (MP-RAGE) T1-weighted images were segmented into tissue classes of GM, WM, and cerebrospinal fluid (CSF) using the New Segment toolbox in SPM8. Then, the GM and WM images were entered into the diffeomorphic anatomical registration through exponentiated Lie algebra (DARTEL) procedure http://www.mccauslandcenter.sc.edu/mricro/mricron/index.html). The diffusion tensor for each voxel was estimated by the ordinary method of least squares implemented in the artefact correction in diffusion MRI (ACID) toolbox for SPM Diffusion-weighed images were corrected for head motion and eddy currents Each type of data (GM volume and FA) was analyzed in three ways, all based on the general linear model as implemented in SPM8. First, between-group differences in high and low risk-taking groups based on either questionnaire or performance data were explored by two-sample t-tests. Second, in order to check if using a continuous regressor, instead of group comparison, could be more sensitive for some activations, an additional model (multiple regression) was used including both total number of Go responses and questionnaire score of risk-taking propensity as covariates. Finally, because only 29 subjects had a valid social influence index , a separate model including the index as a single regressor of interest was used to estimate the influence of peer competition.In the GM volume analysis, each model also included a regressor of individual differences in total intracranial volume (GM + WM + CSF) to account for the global effect and the analysis was restricted by a binary mask that was created by using an optimal threshold A cluster-level criterion for significance was used for all obtained local effects in both GM volume and FA. A cluster-defining uncorrected voxel threshold was set at p<0.001 We found no significant effects in the GM volume analyses with group comparisons (High vs. Low risk-taking group) or correlation using the total number of Go responses.Regarding DTI data, between-group comparisons did not reveal any differences between questionnaire-based groups, whereas for performance-based groups, the High > Low risk-taking group contrast revealed significant FA differences. Similarly, only positive correlation between the total number of Go responses  and FA were found. Because both between-group and covariate analyses were based on the same measure (number of Go responses), their results expectedly demonstrated partial overlap , Table 1No significant correlation between the index of social influence and either GM volume or FA was found.We discriminated high and low risk-taking adolescents by assessing their propensity for risky behavior and vulnerability to peer influence with personality tests, and compared structural differences in gray and WM of the brain with VBM and DTI, respectively. We also verified their actual risk-taking behavior using a simulated driving task including a peer competition condition, but there was a discrepancy between the self-reported personality test results and task performance. Thus, we also evaluated differences in the brain structure according to task performance using Go response rates (risky decisions).Comparison between high and low risk-taking adolescents according to personality test results revealed no significant difference in GM volume and WM integrity. However, comparison according to actual risk-taking behavior during task performance revealed significantly higher WM integrity in the high risk-taking group. These results are consistent with previous findings that engaging in risky behavior was associated with more mature WM These findings suggest that adolescents who engage in more dangerous  behavior may have structurally more mature brain than relatively conservative peers. The maturation of the adolescent brain can be influenced by both environmental experience and genetic factors. Thus, adolescents who engage in exploratory and risky behavior may gain more experience in various domains, promoting maturation of the brain, while their conservative peers may not have much experience in life and thus have less mature brains. Biologically, precocious development may predispose some adolescents to risky behavior. It has been posited that puberty leads individuals to biological maturity sooner than society permits Higher WM integrity has been observed particularly in the frontal cortex in the present and a previous study Although previous studies Increased risky behavior during adolescence, especially in the presence of peers, is thought to imply weak prefrontal cognitive control over behavior as compared with a more rapidly developing subcortical motivation system e.g., . HoweverAs a limitation of the study, the sample was limited to 18–19 years old due to their relevance to driving behavior , which can only capture a very limited range of the dynamic changes that occur across adolescence. The findings were also based on individual behavioral differences rather than developmental differences in age or puberty stages. The lack of developmental analyses can limit the interpretation of the data in the context of adolescent development, for instance, without comparing developmental differences between groups, the differences in WM integrity cannot be solely attributed to brain maturation but also to individual differences of the same age group or developmental stage. Finally, given the small sample size (n = 13–16 in each group), the lack of differences in GM volume or between questionnaire-based groups might be due to a lack of statistical power. Thus, further investigation is needed with a larger sample covering a wide age range and direct developmental comparisons.


Octodon degus) is a diurnal rodent with dichromatic color vision. Its retinal structure is similar to that in humans in many respects, therefore, it is well suited to study retinal aging. Histological, cell type-specific and ultrastructural alterations were examined in 6-, 12- and 36-months old degus. The characteristic layers of the retina were present at all ages, but slightly loosened tissue structure could be observed in 36-month-old animals both at light and electron microscopic levels. Elevated Glial fibrillary acidic protein (GFAP) expression was observed in Müller glial cells in aging retinas. The number of rod bipolar cells and the ganglion cells was reduced in the aging specimens, while that of cone bipolar cells remained unchanged. Other age-related differences were detected at ultrastructural level: alteration of the retinal pigment epithelium and degenerated photoreceptor cells were evident. Ribbon synapses were sparse and often differed in morphology from those in the young animals. These results support our hypothesis that (i) the rod pathway seems to be more sensitive than the cone pathway to age-related cell loss; (ii) structural changes in the basement membrane of pigment epithelial cells can be one of the early signs of degenerative processes; (iii) the loss of synaptic proteins especially from those of the ribbon synapses are characteristic; and (iv) the degu retina may be a suitable model for studying retinal aging.The retina is sensitive to age-dependent degeneration. To find suitable animal models to understand and map this process has particular importance. The degu ( The vertebrate retina, like other parts of the central nervous system, is subjected to degenerative changes caused by aging. The retina is also the site of diseases for which age is a major risk factor, including macular degeneration and glaucoma . This latter species presents several advantages for studying different pathological conditions. The degu is a diurnal, highly visual South American hystricomorph rodent native to Chile, which in old age expresses cognitive deficits, anxiety . Since degu retina is more similar to the human retina than to the retina of other rodents, this description will provide a strong foundation for future studies where experimental manipulations and/or neuroprotective agents can be studied.n = 8), 12 (n = 8), and 36-months of age (n = 12) were used. This latter group is considered as aging (but not old) group. Degus were housed individually in opaque glass cages (40 × 25 × 25 cm) at the animal facilities of the University of Murcia. Throughout the study, the experimental room was maintained under controlled temperature (21 ± 1°C) and 12 h light/dark cycle (lights on at 7:00 a.m. and off at 19:00 p.m.). The floors of the cages were covered with wood shavings that were changed once a week. Food and water were provided ad libitum by placing 120 g food pellets  per day and water bottles on a grid located on the top of the tank. The water in the tank was changed daily. All experiments were performed in accordance with relevant regulatory standards, experimental guidelines and procedures complied with the European Community Council Directive (2010/63/UE) and the ethical committee of the University of Murcia.A total of 28 female degus (body weight 180–270 g) of 6 , administered with a continuous flow vaporizer , and then sacrificed by decapitation. Both eyes were immediately removed and distinctly disposed according to histological or electron microscopic procedure to be performed.n = 2–5 measurements from one tissue block). The following parameters were measured: (i) cross-section of the retina from the outer limiting membrane (OLM) to the inner limiting membrane (ILM); (ii) the width of individual retinal layers. Statistical comparisons were made using one-way ANOVA test followed by Tukey-B posthoc analysis. Data were presented as mean ± SEM (GraphPadPrism5.0).For histology eyes were fixed in 4% paraformaldehyde  dissolved in 0.1M phosphate buffer . The eyecups were dissected and embedded in epoxy resin  as we previously described . Sections were cut at 70 nm in Reichert Ultracut S and counterstained with Reynold’s lead citrate. Samples were examined and photographed in a JEOL 1200EX electron microscope.Electron microscopy was performed on eyes fixed with 4% PFA supplemented with 1% glutaraldehyde dissolved in 0.1M PB. After washing in PB, tissue samples were treated with 1% OsOEyes were dissected immediately after sacrifice in ice-cold phosphate buffer with saline (PBS) and fixed in 4% PFA at room temperature. Tissues were then washed in PBS and cryoprotected in 20% sucrose at 4°C. For cryostat sectioning, retinas were embedded in tissue-freezing medium , cut in a cryostat  at 10 μm radially. Sections were mounted on subbed slides. Primary antibodies and peanut agglutinin-conjugated with FITC (PNA) were used overnight at room temperature .The number of RGCs , outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and GCL . The number of RGCs (Brn3a-positive cells)/100 μm retina length was significantly decreased in the 36-month-old degu retina -positivity was selectively localized to endfeet of Müller cells Figures . Müller Basement membrane proliferation of the RPE in the areas of age-related retinal peripheral degeneration was observed in the 36-month-old degus retinas. The thickening of Bruch’s membrane and fibrosis of the choriocapillary were evident. The somas of the RPE cells were pressed toward this thinner basement membrane in the 36-month-old retinas, suggesting an initial age-related alteration of the RPE. Early peripheral changes of the RPE included increased basal infoldings, phagolysosomes and lipofuscin deposits, as well as atrophy and whirling extensions of the basement membrane into the cytoplasm Figure . The relWe observed that the cones composed a nest between the rods in all age groups, in accordance with the cone-dominant retinal structure of the degu  were more electron dense and cup-shaped spherules, while cone terminals were bowl-shaped, less dense pedicles Figures . The onlPhotoreceptors transmit their signals at ribbon synapses in the OPL, the first synaptic region in the retina, whereas bipolar cells make their ribbon synaptic contacts in the IPL. A large number of regularly aligned synaptic vesicles were tethered to the ribbon. The rod photoreceptor ribbon synapses had horseshoe-shaped structure in the OPL, however, the cone photoreceptor (OPL) and bipolar cell ribbons (IPL) had a dot-like appearance at light microscopic level. We analyzed the structure of the OPL with retina-specific ribbon synapse markers presynaptic CtBP2 and Bassoon labeling. The rod ribbon synaptic profiles were marked with horseshoe-shaped ribbons by CtBP2 and continuous distribution of punctate staining by Bassoon protein Figures . Near thThe pan-bipolar cell marker Chx10 reveals the organization of bipolar cells in the INL of the retina . The most prominent difference was at the age of 36 months with a significant reduction in these parameters  rod/cone ratio is similar to the human ratio; (ii) the diurnal behavior is charactheristic to this species and its activity pattern resembles that of the human; (iii) its lifespan is considerably longer than those of the other experimental models, therefore the age-related changes can be better monitored and compared to those described in the human retina; and (iv) other, age-related diseases  often appear spontaneously in this species  depends on the underlying structure of neural networks. Any loss of the pre- and/or postsynaptic profiles of the retinal neurons causes changes in their morphology and function. As a consequence, these retained neurons may be capable to establish new synaptic contacts , so the retina may undergo marked remodeling. In the aging degu retina, the synaptic rearrangements/alterations in the OPL and potentially concomitant synaptic alterations in the IPL efficacy would reduce transmission, while the loss of function of the RPE cells would alter the homeostasis of the retinas. The neuronal elements of the vertical pathway such as rod bipolar and ganglion cells were seen affected already at 36-month of age. We think that these structural changes become more obvious, will reach other cells and will more seriously affect the synaptic complexes with advancing age.The proven similarities between the degus and the human retina in their structure and aging processes offer a possibility to develop potential treatments and therapies for retinal age-related alterations and diseases.KS researched data and wrote, edited and reviewed the manuscript; CE, EF-V, ET, VI and GS Jr. researched data; RG and MTH researched data, reviewed and edited the manuscript; DR and AT reviewed the manuscript.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.


PET-CT is becoming more and more important in various aspects of oncology. Until recently it was used mainly as part of diagnostic procedures and for evaluation of treatment results. With development of personalized radiotherapy, volumetric and radiobiological characteristics of individual tumour have become integrated in the multistep radiotherapy (RT) planning process. Standard anatomical imaging used to select and delineate RT target volumes can be enriched by the information on tumour biology gained by PET-CT. In this review we explore the current and possible future role of PET-CT in radiotherapy treatment planning. After general explanation, we assess its role in radiotherapy of those solid tumours for which PET-CT is being used most.In the nearby future PET-CT will be an integral part of the most radiotherapy treatment planning procedures in an every-day clinical practice. Apart from a clear role in radiation planning of lung cancer, with forthcoming clinical trials, we will get more evidence of the optimal use of PET-CT in radiotherapy planning of other solid tumours. Cancer treatment has undergone major progress in the past decades. Many previously untreatable malignancies are now-days being successfully cured mostly by the combination of various treatment modalities. Radiotherapy (RT) is almost always part of them. When prescribing radical radiotherapy to the patients we need to achieve two goals; the target volume of the tissue irradiated to high-dose must encompass the entire tumour and any microscopic extensions of disease and the dose to the normal tissues should be kept as low as possible to avoid major acute and late complications. To arrive at these goals we have to combine technical improvements and clinical experiences.1The most important technical improvements consist of integration of computed tomography (CT) imaging into treatment planning and introduction of computer controlled multileaf collimator system. They enable development of more efficient techniques for dose delivery, such as 3-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT) and volumetric arc radiotherapy (VMAT).3For target delineation we need accurate tumour assessment. Until recently, morphologic  imaging with CT and/or MRI scans, was the only information available in the treatment planning process.2Namely, alongside with the innovations in medical physics and nuclear medicine imaging, there has been a major leap in the understanding of tumour biology. It is now recognized that cancer is not a homogeneous ensemble of cancer cells with similar attributes, but that consists of subvolumes with very different radiobiological properties such as hypoxic areas that are known to be highly radio-resistant, regions with uncontrolled cellular proliferation.3The integration of [18F]-fluorodeoxyglucose-positron emission tomography with computed tomography imaging (18FDG-PET-CT) has become an essential part in the evaluation of various types of malignancies.13Positron Emission Tomography (PET) scanning has become a paradigm for molecular imaging.15e.g. brain tumour or lung cancer), or when the intent of RT is to deliver higher than standard doses of radiotherapy  to the tumour in order to kill as many tumour cells as possible and damage as few as possible normal tissue cells .In radiotherapy planning, biologic imaging is particularly useful when the patient has poorly defined target volumes,  need to be defined in order to deliver RT: gross tumour volume (GTV) includes macroscopically visible tumour and involved lymph nodes; clinical target volume (CTV) is derived from GTV by adding margins to it and includes all subclinical disease; and planning target organ (PTV) which is derived from variations in the size/position of CTV  and the patient .21e.g. 40%, 50%), threshold depending on the background signal, to defined absolute standardized uptake value (SUV) (e.g. 2 ± 0.4), and, the most commonly used, the visual interpretation of the PET scan.et al. compared different techniques of tumour contour definition by FDG-PET that lead to substantially different volumes, especially in patients with inhomogeneous tumors.et al. tried to determine the cut-off value of SUV by matching the pathologic gross tumour volume from whole-mount serial section of stage I non-small cell lung cancer (NSCLC) to PET-based GTV.et al. questioned use of various automatic delineation methods for several reasons: poor resolution of functional imaging, tumour motion, and distinct pathologic sub-types.When PET or PET-CT is used for RT planning, precise protocols should be followed and consistently applied.For more accurate results, innovations in image quality and reconstruction are required in order to determine the most proficient delineation technique.The following sections provide a review of the clinical application of PET-CT in radiation treatment planning of some common cancers.et al. reported in a prospective study that for patients with NSCLC treated with radical intent, PET-CT-based staging was significantly more powerfully correlated with overall survival than conventional imaging-based staging, across all staging and within cohorts of patients given any particular form of therapy.30When available PET-CT should be used to select patients with NSCLC for treatment with radical RT.In comparison to conventional imaging with CT or PET alone, integrated PET-CT can distinguish malignant lesions from benign lesions with an accuracy of 82% with varying sensitivity and specificity values .33et al. revealed that the degree of early metabolic change already after the second week of chemo-radiotherapy can predict the response to treatment.38Evaluation of the neoadjuvant chemo-radiotherapy response before potential surgery is another possible implication of functional imaging.Main limitation of FDG-PET as re-assessment tool is that it is not as good as in primary staging of mediastinal lymph nodes . Currently it is not recommended as the only diagnostic tool in therapeutic decisions when restaging patients after induction therapy in stage III NSCLC.34et al. disclosed that areas with residual FDG uptake after high dose (chemo)-radiotherapy largely overlapped with the areas of high FDG uptake locations before treatment.40Aerts PET-CT significantly changes lymph node staging in the thorax, usually by showing more positive lymph nodes than CTIn cases with atelectasis, PET-CT helps to define the border between tumour and atelectasis, allowing a smaller volume of lung to be treated.Of all the common cancers, lung cancer has been most intensively studied by integrating dual imaging into the radiation treatment management of patients.Differences between tumour and atelectasis on PET-CT image are shown in et al. have reviewed the results of 18 trials involving 661 patients with lung cancer that compared delineated target volumes, GTV, CTV and PTV using CT alone with the target volumes delineated using additional FDG-PET.44Nestle vs. 53% and 37%).50Use of PET-CT can influence the treatment strategy of head and neck cancer patients in various ways. The greatest impact usually results from changes of nodal status before therapy, detection of distant metastasis and/or treatment evaluation.et al.51PET may also be of help in searching for the index tumour in patients presented with lymph node metastases of squamous cell carcinoma from unknown primary to the neck.et al.)54The assessment of residual disease in the neck by PET-CT after chemoradiotherapy has become a standard for recognizing patients, who may avoid unnecessary neck dissection.e.g. brainstem or optic chiasm).et al. examined 20 head and neck cancer patients and studied the potential impact of PET-CT imaging on more tailored IMRT plans, with the exclusion of prophylactic irradiation of PET-negative regions.Recent studies have mainly focused on the feasibility of integrating FDG-PET with radiotherapy planning CT with the goal of enhancing tumour localization for IMRT, so the tumour coverage and normal tissue sparing can be optimized.et al. compared GTVs delineated at CT, MRI and PET-CT images with resected tumour specimen.et al. tried to correlate the SUV to pathologic specimen size and found that fixed threshold of 40% of maximum SUV would offer the best compromise between accuracy and the risk of underestimating tumour extent.47Several published studies on head and neck cancer patients that compared RT volumes on PET images with standard CT-based planning volumes, observed significant differences in target delineation depending on the imaging technique.et al.et al. reported that the changes in primary tumour SUV during and soon after completed treatment were also highly predictive of tumour recurrence compared to CT imaging alone.Several investigators analyzed the correlation between pre-therapeutic SUV and disease outcome and were reviewed by Inokuchi i.e. prescribing dose to points in the target depending of biologic signal intensity.et al., the median dose was escalated either to 80.9 Gy to the high-dose CTV or to 85.9 Gy to the GTV.As already said, FDG uptake correlates with outcome in head and neck cancer patients and most of loco-regional recurrences occur within FDG-avid areaset al. performed pretreatment FMISO-PET on 73 head and neck cancer patients and found that both the degree of hypoxia and the size of hypoxic volume measured by FMISO-PET were independent predictive factors of survival.et al. found similar prognostic value of FAZA PET-CT imaging, demonstrating a significant difference in disease free survival rate in patients with non hypoxic tumours and patients with hypoxic tumours .64Two other promising PET tracers are 18F-fluoromisonidazole (FMISO) and 18F-fluoroazomycin arabinoside (FAZA), which provide quantitative measurements of tissue hypoxia, one of the main factors affecting treatment resistance in head and neck cancer.Despite the progress in anatomical and functional imaging in defining tumour borders, meticulous, peer reviewed clinical examination cannot be replaced by any of the imaging techniques when determining mucosal extent of primary tumour.et al., authors found a significant improvement of the sensitivity (93%), accuracy (92%) and negative predictive value (98%) in the assessment of locoregional lymph nodes by integrated imaging, compared to the use of CT or PET alone.67FDG-PET-CT can provide incremental staging information compared to combined CT and endoscopic ultrasonography in up to 40% of patients and can change management in one third of patients with oesophageal carcinoma.et al.in vivo, since the FDG uptake in pathologic areas on one side can be compensated on the other side by inflammation.67There is no doubt that addition of FDG- PET in radiotherapy planning has a significant impact on target volume delineation (in 20–94% of patients), resulting in either reduction or increase in target volumes based on CT images as it is reviewed by Muijs Some prospective studies70et al. showed significantly increased concordance rate in GTV definition (i.e. ratio of the intersection of the GTVs to their union) in inter- and intraobserver comparison, when incorporating FDG-PET-CT images in comparison to CT images of cervical oesophageal carcinoma.et al. found no significant effect of the additional use of FDG-PET on the interob-server variability.67Another method to utilize FDG-PET in RT treatment planning is to reduce inter-and intra-observer variability. Toya et al. evaluated the potential prognostic value of FDG uptake in oesophageal cancer patients.Omloo i.e. FIGO stage is ≥IB2), with significantly better sensitivity and specificity in comparison to other morphological imaging (PET-CT sensitivity ranging from 77% to 96% for pelvic lymph nodes (PELN) and 50% to 100% for para-aortic lymph nodes (PALN), specificity from 55% to 75% for PELN and 83% to 95% for PALN, as reviewed by Magne et al.74T2 weighted MRI is currently the gold standard for primary tumour staging, especially in determining tumour extension in the parametria  of PET positive nodal metastases correlate well with recurrence-free and disease-free survival.77Although the presence of metastatic lymph nodes in the PELN and PALN regions does not alter the clinical stage of disease, it alters radiation treatment strategy .et al. attempted to develop a treatment plan to deliver an escalated irradiation dose to involved nodal regions without harming adjacent organ at risk.et al. prospectively compared toxicity and clinical outcomes of cervical cancer patients treated with PET-CT-guided IMRT and conventional radiation therapy, treated to the same prescription dose. The late bowel and bladder toxicity (of Grade 3 or more) were present in only 6% in IMRT group compared to 17% in non-IMRT group. The IMRT group also showed (unexpectedly) better overall and disease specific survival and a trend towards better recurrence free survival.80Esthappan et al. proposed the implementation of routine post-treatment PET-CT, which could affect the approach and timing of salvage therapy of patients with cervical cancer recurrence and would also provide long-term prognostic information only 3 months after completion of therapy.et al. found that even earlier assessment (after receiving 54–60 Gy) of metabolic response of PELN in patients with initial PELN metastases correlates to disease free survival rate and could be a useful milestone for selecting patients who need more intense treatment.81Several studies demonstrated that post-treatment metabolic response immediately after RT and 3 months after RT are predictive of patient outcome.Recent advances in PET-CT can broaden the radiation oncologist’s knowledge on the extent of the disease, in order to avoid missing the tumour, with consequent reduction of local control probability, and, on the other hand, avoiding the unjustified irradiation of healthy tissue. Furthermore, the correct definition of the disease stage is mandatory for selection of the most appropriate therapeutic strategy. Another important information for the radiation oncologist relates to biological characteristics of treated tumour, which could affect the response to radiotherapy and are of paramount importance for personalized and biologically oriented radiotherapy. The consensus of using PET-CT information for automated target volume delineation has not been reached yet and is awaiting further validation. PETCT can answer many questions regarding correct disease staging and its biologic characteristics, but its exact role in every-day RT treatment planning is expected to be defined in future clinical trials.


Smoke-free policies can effectively protect nonsmokers from secondhand smoke (SHS) exposure in multiunit housing. We surveyed all affordable multiunit housing properties in North Carolina to determine the statewide prevalence of smoke-free policies and to identify predictors of smoke-free policies.2 analysis and multivariate logistic regression to identify correlates of smoke-free policies.Representatives of affordable housing properties in North Carolina completed a mailed or online survey during June through October 2013. The primary outcome measure was presence of a smoke-free policy, defined as prohibiting smoking in all residential units. We used χOf 1,865 eligible properties, responses were received for 1,063 (57%). A total of 16.5% of properties had policies that prohibited smoking in all residential units, while 69.6% prohibited smoking in indoor common areas. In multivariate analysis, an increase in the number of children per unit was associated with a decrease in the odds of having a smoke-free policy at most properties. Newer properties across all company sizes were more likely to have smoke-free policies. Accessing units from interior hallways predicted smoke-free policies among medium-sized companies.More smoke-free policies in affordable multiunit housing are needed to protect vulnerable populations, particularly children, from SHS exposure. Public health professionals should continue to educate housing operators about SHS and the benefits of smoke-free policies at all properties, including older ones and ones where units are accessed from outside rather than from an interior hallway. The home is a primary source of secondhand smoke (SHS) exposure . Home SHSmoke-free policies can protect nonsmokers from SHS exposure in MUH ,7, but sThis study was a cross-sectional, observational study. Our sample of affordable housing properties included all multiunit properties in North Carolina that receive site-based federal subsidies or state low-income housing tax credits. This sample comprised properties owned by for-profit and nonprofit private owners and did not include public housing properties. We defined MUH as housing with more than 1 living unit and excluded properties classified as group homes.This study was an establishment survey, with the unit of analysis being the property itself and not the individual answering the survey. In some cases, a single respondent completed multiple surveys because he or she managed more than 1 property; however, all survey questions were objective questions about policies and events occurring at the property and none assessed respondent attitudes.We created a sampling frame of affordable housing properties in North Carolina by obtaining lists from the US Department of Agriculture’s (USDA’s) Rural Development program, the US Department of Housing and Urban Development (HUD), and the North Carolina Housing Finance Agency (NCHFA), the state’s purveyor of low-income housing tax credits. We cross-checked this frame with lists of properties from 8 large affordable housing management companies and added missing properties. After removing duplicates, we identified 1,865 properties, all of which were eligible to participate in this census survey.We developed the survey instrument by reviewing previous MUH operator surveys and obtaining input from smoke-free MUH researchers. We also consulted affordable housing property managers, who provided feedback regarding use of industry-specific terms and appropriateness of survey response categories. We pretested the survey with 18 property managers and made revisions to eliminate confusing wording and skip patterns and reduce respondents’ difficulty with finding information needed to answer the questions.The Office of Human Research Ethics at the University of North Carolina at Chapel Hill (UNC-CH) ruled that this study did not constitute human subjects research under federal regulations and did not require its approval. We called properties to confirm the appropriate respondents’ names and contact information and their preference for an online or paper survey. For large companies, we contacted corporate headquarters to determine the most appropriate survey respondent for their properties.The Carolina Survey Research Laboratory (CSRL) at UNC-CH collected data from June to October 2013. Respondents who chose to complete the survey online received a mailed letter containing the URL and password for the survey. Respondents who preferred paper surveys were mailed a questionnaire in booklet form with a prepaid return envelope. The CSRL contacted nonrespondents by telephone during July and August 2013 and sent them either online survey information or mailed questionnaires by August 30, 2013. Several medium and large companies (defined below) instructed their property managers to participate in the survey and contacted nonrespondents twice during the data collection period to encourage participation. Respondents who chose to be compensated received a check for $25.To ensure representation among small companies and single owners, we stratified and weighted properties by management company size. Using tertiary cut points, we defined as small those companies managing 1 to 27 properties in North Carolina, as medium those managing 28 to 79, and as large those managing 80 to 171. We produced weights as the inverse stratum-specific sampling rates and then adjusted for differential response based on stratum-specific response rates. SAS software (version 9.2)  was used in all statistical analyses and reported significance at the .05 level.2 goodness-of-fit tabulations to compare characteristics such as year built and location, subsidy type and source, and type of access to units in smoke-free and smoking-allowed properties. We used logistic regression to identify relationships between a smoke-free policy and possible predictor variables. Variables included in the logistic regression model were those that were significant in bivariate analysis or identified by North Carolina housing operators as being important in determining whether to adopt smoke-free policies. The model included the following predictor variables: year built ; company size; type of access to residential units ; number of stories in residential buildings (1 or 2 stories or ≥3 stories); and number of children per unit . The quadratic term for number of children per unit was also included because it indicated curvilinearity in the relationship between the number of children per unit and the log odds of a property’s being smoke-free. Family subsidy status was included in an early version of the model but was not significant and not included in the final model.We used χA total of 1,063 completed surveys were received for the 1,865 eligible properties, for a response rate of 57%. Almost two-thirds (62.3%) of the respondents completed the survey online. Most surveys (87%) were completed by on-site managers.The responding properties had an average of 55.2 units and 8.0 buildings per property (weighted numbers) . Three-qP < .001), with 48.5%  of properties built before 2002 receiving HUD subsidies and 34.2%  receiving NCHFA tax credits.Bivariate analysis indicated a relationship between subsidy source and year built. HUD properties were older than NCHFA properties  allowed smoking in all residential units, while 16.5%  did not allow smoking in any residential units, and 2.4%  allowed smoking in some residential units. Of the very small percentage of properties that allowed smoking in some residential units, 73.6%  allowed current residents to smoke under a “grandfather” policy, with the intention of being completely smoke-free once all grandfathered smokers had moved out. Among properties prohibiting smoking in residential units, 58.3%  had converted to smoke-free and 41.7%  had opened as smoke-free. Most properties banned smoking in indoor common areas , while few properties prohibited smoking in outdoor common areas  or outdoor private areas .For purposes of comparing smoke-free and smoking-allowed properties, we categorized properties as having a smoke-free policy if smoking was not allowed in any residential units, and smoking-allowed if smoking was allowed in some or all residential units. Children were less likely than adults to live in properties with smoke-free policies, with 10.5%  of children living in smoke-free properties and 15.1%  of adults doing so.P = .01). Properties without family subsidies were more likely to be smoke-free than those with family subsidies (P = .01). There was no association between smoke-free policies and receiving USDA subsidies, while HUD subsidies were negatively associated with smoke-free policies (P < .001) and NCHFA tax credits were positively associated with smoke-free policies (P < .001). More medium-sized companies had smoke-free policies than large or small companies (P = .01). Type of access to residential units was also associated with having a smoke-free policy (P < .001); a larger proportion of properties with units accessed from the interior had smoke-free policies (23%) than properties with units accessed from outdoors (13%). Number of stories in residential buildings, metropolitan statistical area status of the county in which the property is located  between company size and other variables  indicated evidence of effect moderation. Because of this interaction effect, we have presented the effect of the predictor variables on smoke-free policies by company size .This study was the first statewide survey at the property level to assess the availability of smoke-free policies in affordable MUH. While most properties banned smoking in indoor common areas, few prohibited smoking in residential units, suggesting significant potential for exposure to SHS for residents. Although North Carolina is a tobacco-growing state, this prevalence of smoke-free policies is similar to findings from operator surveys in other areas of the country –10. Few We found that children in North Carolina affordable housing are at increased risk of SHS exposure. An increasing concentration of children was associated with decreasing odds of having a smoke-free policy at most properties. Also, children were more likely to live in smoking-allowed properties than adults; however, the strength of this latter finding is in question because of overlapping confidence intervals. This study adds to the small body of literature regarding children’s SHS exposure in MUH. Children living in MUH have significantly higher levels of exposure to SHS than children living in detached houses . SupportOlder properties across all management company sizes were less likely to have smoke-free policies. This finding, also observed in a New York study of MUH , suggestOur study also found an association between the existence of a smoke-free policy at a property and its source of subsidy: HUD-subsidized properties were less likely to be smoke-free, while properties subsidized by NCHFA tax credits were more likely to be smoke-free. This difference may be attributed to the age of the property: HUD properties were older than properties that received tax credits, which are used to finance new construction or renovations . SimilarWe examined variables not tested in other studies, including management company size and type of access to residential units. Regarding management company size, small and large companies were less likely to have smoke-free policies than medium companies. Small companies may lack knowledge about trends in smoke-free policies, and large companies may be reluctant to convert a large portfolio of properties to smoke-free. More research is needed to determine reasons for this finding and to guide targeted outreach to promote smoke-free policies among small and large management companies. Regarding type of access to residential units, buildings with units accessed from the outside were less likely to have smoke-free policies than units accessed from an interior hallway; interior hallway access predicted smoke-free policies among medium companies. Housing operators may believe there is less smoke transfer between units that do not share a common indoor hallway, so that properties of this style are a lower priority for smoke-free conversion. North Carolina housing operators suggested adding this question to the survey, demonstrating the importance of engaging stakeholders to identify relevant fields of inquiry. Public health professionals should emphasize in their communications to housing operators that residents of apartments accessed from outdoors would benefit from smoke-free policies as well, because smoke can travel between these units through shared walls and ceilings and inside from outdoor balconies and patios.Strengths of this study include incorporating input from housing industry professionals into the survey design, sampling at the property level, using government lists to create the sampling frame, and having a large sample size and a high response rate in comparison to previous operator surveys. However, this study has several limitations. First, the study examined only affordable housing properties and the results may not be generalizable to market-rate housing. It is possible that market-rate housing has a higher prevalence of smoke-free policies because of lower smoking rates among higher income individuals . This quMore smoke-free policies in affordable MUH are needed to protect vulnerable populations, particularly children, from SHS exposure. The availability of smoke-free policies in affordable housing properties in North Carolina is low, though increasing. After data collection was completed, 2 large management companies converted their properties to smoke-free in 2014, increasing the percentage of smoke-free properties in North Carolina from 16.5% to 27.9% in the period of a year. To ensure this trend continues in North Carolina and is replicated throughout the country, public health professionals should continue to educate housing operators about the benefits of smoke-free policies at all multiunit properties.


Why is another measure of disturbed eating necessary? While there is a growing literature identifying links between certain emotion regulation styles and eating disturbances, much contention and contradictory evidence exists regarding the distinction between different specific eating disorder (ED) diagnoses in such relationships. This is possibly due to the common and overlapping features of different ED diagnoses and rather what is needed is exploration of the link between certain emotional styles and specific ED characteristics. However, in order to explore such relationships, a freely available self-report measure that reliably and validly identifies specific characteristics of eating disturbances is required. The current study aimed to develop such a measure. To this end an initial 66-item pool was devised and tested in Study 1 with a sample of 403 women. An exploratory factor analysis revealed a four-factor structure retaining 59 items. A confirmatory factory analysis (CFA) supported this factor structure and lead to the identification of four sub-scales 'Drive for thinness' (31-items), 'Binge' (10-items), 'Purge' (9-itmes), and 'Extreme-restraint' (9-items). These sub-scales were found to have high internal consistency and good reliability and validity. In Study 2 this factor structure was again found in a CFA with a sample of 749 women.


A new scan that matches an aided Inertial Navigation System (INS) with a low-cost LiDAR is proposed as an alternative to GNSS-based navigation systems in GNSS-degraded or -denied environments such as indoor areas, dense forests, or urban canyons. In these areas, INS-based Dead Reckoning (DR) and Simultaneous Localization and Mapping (SLAM) technologies are normally used to estimate positions as separate tools. However, there are critical implementation problems with each standalone system. The drift errors of velocity, position, and heading angles in an INS will accumulate over time, and on-line calibration is a must for sustaining positioning accuracy. SLAM performance is poor in featureless environments where the matching errors can significantly increase. Each standalone positioning method cannot offer a sustainable navigation solution with acceptable accuracy. This paper integrates two complementary technologies—INS and LiDAR SLAM—into one navigation frame with a loosely coupled Extended Kalman Filter (EKF) to use the advantages and overcome the drawbacks of each system to establish a stable long-term navigation process. Static and dynamic field tests were carried out with a self-developed Unmanned Ground Vehicle (UGV) platform—NAVIS. The results prove that the proposed approach can provide positioning accuracy at the centimetre level for long-term operations, even in a featureless indoor environment. High-precision dynamic positioning is in great demand in Unmanned Aerial Vehicle (UAV) and Unmanned Ground Vehicle (UGV) applications. The most popular technology is the integration of carrier-phase-based differential global navigation satellite systems (GNSS) and commercial-grade or tactical-grade Inertial Navigation Systems (INS) to provide centimetre-level high-accuracy navigation with good GNSS availability . HoweverVarious methods to reduce or bound INS drift have been addressed in the past. A feature-aided INS with external sensors can be placed in odometers, magnetometers, or cameras for Light Detection and Ranging (LiDAR) as an alternative to satellite-based navigation technology in a GNSS-denied environment. The most popular feature-aided INS solutions are visually aided and LiDAR-aided systems ; they ofetc. carried out pilot research by proposing a LiDAR scan matching method with a Gauss-Newton algorithm; the matching results were fused with the measurements of an IMU to estimate a full 3D-motion of a moving platform. It is now available as an open-source package for Robot Operating Systems (ROS) [Most existing LiDAR-aided INSs currently use similar positioning methods with a visually aided system; they extract geometric features  from laser scans ,15,16,17ms (ROS) ; the accms (ROS) . As alrems (ROS) , the posFeature-aided inertial navigation can be divided into two categories: relative-based systems and absolute-based systems . Features in consecutive images or scans are detected and relatively matched to determine the rigid-body transformation (translation and rotation) of the sensing platform ,15,20. APrevious research has shown that an Extended Kalman Filter (EKF) is a relatively robust and efficient error estimating framework suitable for dynamic motion systems ; in partA global scan matching aided INS method is thus proposed to establish an efficient navigation system for GNSS denied environment; the entire system consists of 2D LiDAR and a commercial-grade IMU sensor. The low-cost IMU provides a short-term coarse transformation of position and attitude. A 2D laser scanner with a self-developed improved probabilistically motivated Maximum Likelihood Estimation (IMLE) algorithm  then useThe rest of this paper is organized as follows: The INS navigation frame (n-frame) is a local geodetic frame with the x-axis pointing towards geodetic north, the z-axis orthogonal to the reference ellipsoid pointing down, and the y-axis completing a right-handed orthogonal frame. It is also called a north-east-down (NED) system. The body frame (b-frame) is defined at the IMU centre. The dynamic equations in the n-frame are given by ,24:(1)rShin, 2001 and 2005 [i.e., If there were no additional errors, the above mechanization equations could estimate the position and velocity of the system from the raw data of the IMU. However, the IMU outputs contain errors and cause the navigation results to rapidly drift; compensating for this drift is difficult. Thus, an error propagation model must work alongside the system motion model to further correct and obtain a better navigation solution; this is also the theoretic basis of the INS and LiDAR EKF fusion model of this paper. A classic perturbation analysis via a first-order Taylor series expansion is applied, and the error state vector and state-space model are defined as follows:and 2005 ,26; ∅k iLiDAR is an active range measuring sensor with a laser source that can detect the geometric information of the environment. However, most existing LiDAR-aided inertial navigation systems use feature  extraction and matching methods for assisted navigation. The workflow is more complicated and the extraction process may eliminate effective matching features. Moreover, this method is unstable and unreliable, particularly in featureless environments. Thus, a full scan-matching aided INS is proposed in this paper.The proposed LiDAR scan-matching algorithm-IMLE is a probabilistic scan matching method based on the feature uncertainty model of a LiDAR sensor ,28. A liAs shown in Based on the above model, a brute search algorithm can be deployed to estimate the best body transformation An Extended Kalman Filter is selected to fuse the measurements of the INS and LiDAR scan matching; an overview of the system architecture is shown in i.e., The EKF observation functions are as follows:The estimates of the EKF prediction functions are:The Kalman gain isThe state vector is updated asFinally, the estimated error 2) [A series of field tests were performed to evaluate the proposed LiDAR-aided inertial navigation system based on . As show1 cm/s2) ,31; The Two groups of experiments were carried out on the second floor of the Finnish Geospatial Research Institute (FGI) main building: the first group were stationary tests and the second group were dynamic tests along the corridor to evaluate the precision and effectiveness of the position and attitude estimates.The error distribution of the LiDAR scan-matching method is stepwise and the error distribution of the IMU + LiDAR resembles white noise. As previously mentioned, the LiDAR scan-matching algorithm is an IMLE, which is a likelihood grid map-based searching method that determines the optimum position from candidates. The likelihood map is divided into small cells as candidate positions according to the map resolution and the angle searching intervals, which are set to 1 cm and 0.25° in this test. We believe this is the primary reason for stepwise error distribution. However, In the IMU + LiDAR combined method, Gaussian error predominates the EKF model, resulting in a white-noise distribution positioning error.The estimated positioning and heading results of LiDAR scan matching are better than the IMU + LiDAR fused method. In the static condition, the incoming laser scan has no feature changes. The LiDAR range measure noise is the only stochastic noise source, and with this optimized condition the IMLE easily detects the platform as stationary. However, when a commercial grade IMU is integrated with LiDAR, the accumulated drift of the gyroscope and accelerometer undermines the accuracy of the final positioning result, although it is verified by LiDAR in an EKF. However, the heading errors are minor and can be neglected for positioning processing. It is anticipated that when a higher-grade IMU  is integrated, the positioning error can be mitigated. The positioning results of the y-axis are better than the results of the x-axis, regardless of whether the IMU is integrated. The stationary positioning experiment was performed at the beginning of the corridor for approximately 3 min. The NAVIS was installed on a cart at an installation height of approximately 1.3 m. As seen in In stationary positioning, the overall estimated accuracy of position and attitude is higher in the LiDAR scan-matching solution than in the combined IMU + LiDAR solution. The accumulated gyroscope and accelerator drift, as measured by a commercial-grade IMU, deteriorates the final position result, even though the positioning accuracy is still at the centimetre level and the heading estimate RMS error is under 0.2 degrees. The EKF model can be used for LiDAR-aided inertial navigation in certain GNSS-denied environments. This result shows that when an IMU detects a platform as stationary, a LiDAR standalone solution should be deployed rather than the combined solution. To prove the effectiveness of the LiDAR-aided inertial navigation system, further dynamic field tests were performed. The NAVIS was installed on a cart and the cart was driven along the corridor of the third floor of the FGI main building several times by an operator. The results of the IMU + LiDAR combined solution and the LiDAR standalone solution were analysed and compared. It is well known that an inertial navigation system includes both attitude and range estimation. In an IMLE, a normalized A series of Moreover, 36 key points of environmental features are selected as a reference network to evaluate the mapping and positioning accuracy of the proposed method, as shown in In summary, this paper proposes a LiDAR scan-matching aided inertial navigation system based on a commercial-grade IMU and LiDAR combined into one system, with raw IMU outputs used to refine the search scope of SLAM to optimize brute search efficiency. The positioning results calculated by an IMU mechanization algorithm are fused with the SLAM results on a navigation frame with a loosely coupled EKF. This combination mitigates the corresponding inherent drawbacks of a standalone solution to establish stable long-term navigation in a GNSS-denied environment. The results of stationary and dynamic field tests confirm that (1) in a feature poor environment, LiDAR scan matching provides more accurate navigation state estimation than a low-cost IMU + LiDAR system can offer under stationary conditions. In addition, (2) IMU mechanization provides better attitude estimation in dynamic tests, regardless of whether environmental features are rich; it can significantly mitigate the inherent heading estimation errors introduced by scanning matching methods in feature-poor environments. Moreover, (3) LiDAR scan matching contributes to range estimation along the moving direction, which is the basis for accurate heading estimation. Finally, (4) the dynamic positioning accuracy remains at the centimetre level with the proposed combined solution, even in a featureless environment. The proposed LiDAR-aided INS system can overcome the drawbacks of each standalone system and achieve centimetre-level positioning accuracy. It can also be successfully applied in a GNSS-denied environment.Only 2D navigation was investigated in the current platform configuration. In future work, two LiDARs will be installed vertically and horizontally on the mobile platform for 3D position estimation to verify the performance of 3D navigation in more complicated situations. In this paper, we also found that the outputs of a commercial-grade IMU degraded the accuracy of the combined solution in a stationary survey. Several tactical IMUs will be integrated into the current NAVIS setup to evaluate how these higher-accuracy IMUs could benefit indoor mapping accuracy.


Glaucoma is not only an eye disease but is also associated with degeneration of brain structures. We now investigated the pattern of visual and non-visual brain structural changes in 25 primary open angle glaucoma (POAG) patients and 25 age-gender-matched normal controls using T1-weighted imaging. MRI images were subjected to volume-based analysis (VBA) and surface-based analysis (SBA) in the whole brain as well as ROI-based analysis of the lateral geniculate nucleus (LGN), visual cortex (V1/2), amygdala and hippocampus. While VBA showed no significant differences in the gray matter volumes of patients, SBA revealed significantly reduced cortical thickness in the right frontal pole and ROI-based analysis volume shrinkage in LGN bilaterally, right V1 and left amygdala. Structural abnormalities were correlated with clinical parameters in a subset of the patients revealing that the left LGN volume was negatively correlated with bilateral cup-to-disk ratio (CDR), the right LGN volume was positively correlated with the mean deviation of the right visual hemifield, and the right V1 cortical thickness was negatively correlated with the right CDR in glaucoma. These results demonstrate that POAG affects both vision-related structures and non-visual cortical regions. Moreover, alterations of the brain visual structures reflect the clinical severity of glaucoma. By comparing retinal nerve fiber layer (RNFL)1214In fact, glaucoma has been thought of as a neurodegenerative disease12et al. measured the volumes of 93 structures in the brain and observed widespread structural brain changes even in non-visual structures such as the bilateral inferior occipital gyri, right inferior temporal gyrus and other structures as wellet al.Morphological brain changes were observed even outside the visual pathway throughout the whole brain as shown by different methods31516However, the aforementioned studies of the whole brain structures just focused on structural changes using a single morphological marker. Moreover, some of these studies suffered from the methodological flaw of using relatively liberal corrections for multiple comparisons during statistical analyses or even no correction at all. This raises the question how widespread brain morphological alterations really are.To overcome these limitations, we now investigated brain structural changes in glaucoma patients using three independent analysis tools: volume-based analysis (VBA), surface-based analysis (SBA) and ROI-based analysis. In the statistical process, the most conservative family-wise error (FWE) correction was applied to reduce the likelihood of type 1 errors using multiple comparison statistics. Finally, we correlated the morphological markers with the clinical measures.The study was conducted according to the Declaration of Helsinki and was approved by the Committee at Beijing Tongren Hospital. Written informed consent was obtained from all the subjects which comprised 25 primary open angle glaucoma (POAG) patients  and 25 age-sex-matched normal controls .All the normal controls underwent a routine clinical examination, which included optometric examination, intraocular pressure examination, inquiry of personal disease history and family disease history. This was done to assure that they had no glaucoma or other visual diseases. The diagnoses of POAG in patients were based on a thorough history and ophthalmologic examinations, including optic intraocular pressure, gonioscopy, fundoscopy and standard automated perimetry. The diagnosis criteria included high intraocular pressure, open anterior chamber angle, typical vision loss, glaucomatous abnormalities of the optic nerve head and optic disk. The glaucoma diagnosis was made by three independent glaucoma clinicians in a masked (blinded) manner.The patients underwent 30-2 Swedish Interactive Testing Algorithm (SITA) fast visual field tests with standard automated perimetry  to measure visual field loss (mean deviation: MD). The iVue 100  was used to measure CDR and RNFL thickness. Because all patients were recruited from the outpatient service between 2009 and 2012, which was a long time before the current retrospective research was carried out, some clinical data of ophthalmologic examinations were missing when assembling the medical records for our study so that about half of our patients had incomplete data sets and could not be included in the correlation analysis. All the available clinical data of POAG patients (n = 14) are listed in Because RNFL thickness, CDR and the visual field MD are the three most commonly used clinical parameters to diagnose glaucoma, these parameters were correlated with the abnormal brain morphological measures.o, matrix = 256 × 256, FOV = 24 × 24 cm2.Brain MRI scans were made in a GE 3T scanner to acquire T1-weighted structural MRI images with the following scanner parameters: TR/TE = 8.9/3.5ms, slice thickness = 1 mm, flip angle = 13We investigated structural abnormalities in glaucoma with three independent measures: VBA, SBA and ROI-based analysis. For VBA, gray matter volume was defined by voxel-based morphometry (VBM). SBA assessed the vertex-based cortical thickness and for the ROI-based analysis, we studied the volumes of five selected brain structures in each hemisphere . Finally, in glaucoma patients we correlated brain morphological measures with retinal measures and vision loss as quantified by mean deviation obtained by standard automated perimetry .http://www.fil.ion.ucl.ac.uk/spm/).The image processing included GM/WM/CSF segmentation of all T1 weighted imagest-test (age and gender as covariates) and corrected by FWE of All T1-weighted images were analyzed by VBM with DARTEL in Statistical Parametric Mapping software . The image process included non-uniform intensity correction, skull-strip of the braint-tests to assess the thickness differences between two groups with age and gender as covariates t- tests were conducted to compare the volume of each ROI between the two groups controlling for age and gender  by taking into account the effect of age, gender and intracerebral volume (ICV).Glaucoma patients showed no significant GM volume alterations in any region compared to normal controls. However, surface-based analysis revealed significantly reduced thickness of the right frontal pole cortex (FPC) in glaucoma patients .When compared to normal controls, glaucoma patients had significant volume shrinkages in the LGN bilaterally (left: ce in V2 . In orde right: ).Correlation coefficients were calculated between brain morphological markers of glaucoma patients  and retinal morphological marker (RNFL thickness and CDR) or the visual field MD see . The lef right:) , i.e. a ght:)  . Furthert CDR  . Thus, sBy applying VBA, SBA and ROI-based analysis, we were able to confirm brain structural changes in the primary visual system in glaucoma patients6789In central visual structures of glaucoma patients we noted abnormalities in the primary visual relay center (LGN) and the primary visual cortex (V1). The LGN is an important visual relay center transferring visual information from retina to primary visual cortex306789Surface-based analysis of whole brain morphology revealed that only the right FPC was found to be thinner in glaucoma patients compared to normal controls. This is compatible with other structural imaging studies showing protracted change in the FPC gray matter volume32FPC is also altered in other diseases, such as schizophrenia3841Another main finding of our study is that glaucoma patients suffer from abnormalities in the amygdala as well, which adds an emotional component and suggests that glaucoma is not just a pure “vision disorder” but a more complex brain syndrome. The amygdala plays a role in the control of emotion43442323There is a close connection of the amygdala with the hippocampus, both of which are part of the Papez circuit that regulates emotion. The hippocampus is a structure controlling memory formation and its main function is to consolidate information from short-term memory to long-term memory. Indeed, memory impairments were reported in some glaucoma patientsVisual field MD and CDR are important clinical parameters in glaucoma diagnosis. The visual field MD is a functional sign of pathology; the smaller the visual field MD, the greater the extent of damage in glaucoma and the greater is the CDR. Both the right FPC thickness and the left amygdala did not correlate with standard clinical measures of disease severity. This indicates that the non-visual structural changes may occur independently of the atrophy of the optic nerves and other brain structures in glaucoma. The positive correlation between the right visual field MD and the right LGN volume is consistent with our expectations that they are related with each other in glaucoma. Furthermore, the negative correlation between bilateral CDR and left LGN volume as well as between right CDR and right V1 thickness is also a sign that a higher CDR is associated with degeneration in LGN and V1. The correlation results suggest that abnormalities in central visual structures reflect the glaucoma severity and demonstrate that the abnormal indices found by us may be used as the biomarker of the glaucoma.It is unclear why in our study significant structural alterations were found only in one brain hemisphere: a thinner right FPC, right V1 and left amygdala. This is rather surprising since the damage to the optic nerve should affect both hemispheres equally. Because an asymmetric loss of the visual field can not explain the laterality , we rather suspect that the lateralization of brain structural changes shows that the peripheral and central visual degeneration may not be controlled by the exact same factors; in other words, central degeneration does not simply mirror the peripheral damage in a one-to-one (retinotopic) manner. One possible independent component might be brain lateralizationOur study confirms that glaucoma patients suffer from brain structural abnormalities as demonstrated by volume-based and surface-based analyses. When a FWE correction was applied during statistical analysis, significant structural brain alterations were only seen when the surface-based analysis was used. This demonstrates that surface-based morphometry is the more sensitive method to reveal brain alterations than volume-based analysis, which is consistent with previous studies344748et al.et al.15A volume-based analysis did not reveal any significant alterations between glaucoma patients and normal controls. This result differs from studies by Li There are some limitations of our study. Firstly, the sample size of the cross-sectional design is relatively small, because only a smaller subsample of 14 out of 25 POAG patients had complete clinical records. Secondly, although the ages of two groups match statistically .