The Expression of Major Facilitator Superfamily Domain-Containing Protein2a (Mfsd2a) and Aquaporin 4 Is Altered in the Retinas of a 5xFAD Mouse Model of Alzheimer’s Disease

Cerebral amyloid angiopathy (CAA) is characterized by amyloid β (Aβ) accumulation in the blood vessels and is associated with cognitive impairment in Alzheimer’s disease (AD). The increased accumulation of Aβ is also present in the retinal blood vessels and a significant correlation between retinal and brain amyloid deposition was demonstrated in living patients and animal AD models. The Aβ accumulation in the retinal blood vessels can be the result of impaired transcytosis and/or the dysfunctional ocular glymphatic system in AD and during aging. We analyzed the changes in the mRNA and protein expression of major facilitator superfamily domain-containing protein2a (Mfsd2a), the major regulator of transcytosis, and of Aquaporin4 (Aqp4), the key player implicated in the functioning of the glymphatic system, in the retinas of 4- and 12-month-old WT and 5xFAD female mice. A strong decrease in the Mfsd2a mRNA and protein expression was observed in the 4 M and 12 M 5xFAD and 12 M WT retinas. The increase in the expression of srebp1-c could be at least partially responsible for the Mfsd2a decrease in the 4 M 5xFAD retinas. The decrease in the pericyte (CD13+) coverage of retinal blood vessels in the 4 M and 12 M 5xFAD retinas and in the 12 M WT retinas suggests that pericyte loss could be associated with the Mfsd2a downregulation in these experimental groups. The observed increase in Aqp4 expression in 4 M and 12 M 5xFAD and 12 M WT retinas accompanied by the decreased perivascular Aqp4 expression is indicative of the impaired glymphatic system. The findings in this study reveal the impaired Mfsd2a and Aqp4 expression and Aqp4 perivascular mislocalization in retinal blood vessels during physiological (WT) and pathological (5xFAD) aging, indicating their importance as putative targets for the development of new treatments that can improve the regulation of transcytosis or the function of the glymphatic system

The supplementation of a high dose of fish oil during pregnancy and lactation led to an elevation in Mfsd2a expression without any changes in docosahexaenoic acid levels in the retina of healthy 2-month-old mouse offspring

IntroductionDuring fetal development, the proper development of neural and visual systems relies on the maternal supplementation of omega-3 fatty acids through placental transfer. Pregnant women are strongly advised to augment their diet with additional sources of omega-3, such as fish oil (FO). This supplementation has been linked to a reduced risk of preterm birth, pre-eclampsia, and perinatal depression. Recently, higher doses of omega-3 supplementation have been recommended for pregnant women. Considering that omega-3 fatty acids, particularly docosahexaenoic acid (DHA), play a crucial role in maintaining the delicate homeostasis required for the proper functioning of the retina and photoreceptors the effects of high-dose fish oil (FO) supplementation during pregnancy and lactation on the retina and retinal pigmented epithelium (RPE) in healthy offspring warrant better understanding.MethodsThe fatty acid content and the changes in the expression of the genes regulating cholesterol homeostasis and DHA transport in the retina and RPE were evaluated following the high-dose FO supplementation.ResultsOur study demonstrated that despite the high-dose FO treatment during pregnancy and lactation, the rigorous DHA homeostasis in the retina and RPE of the two-month-old offspring remained balanced. Another significant finding of this study is the increase in the expression levels of major facilitator superfamily domain-containing protein (Mfsd2a), a primary DHA transporter. Mfsd2a also serves as a major regulator of transcytosis during development, and a reduction in Mfsd2a levels poses a major risk for the development of leaky blood vessels.ConclusionImpairment of the blood-retinal barrier (BRB) is associated with the development of numerous ocular diseases, and a better understanding of how to manipulate transcytosis in the BRB during development can enhance drug delivery through the BRB or contribute to the repair of central nervous system (CNS) barriers

The high-dose fish oil (FO) supplementation increased Mfsd2a expression in the retina of healthy mice

Mfsd2a is expressed mainly in the endothelial cells and is an essential regulator of blood vessel transcytosis. Therefore, decrease in Mfsd2a expression can be a risk factor for developing leaky blood vessels. Mfsd2a is also the main docosahexaenoic acid (DHA, C22:6n3) transporter. DHA, an omega-3 fatty acid, is one of the main structural lipids of the neuronal and vascular retina, crucial for the normal functioning of photoreceptors (PRs). However, the capacity of the retina to synthesize DHA is limited, and the maintenance of retinal DHA content relies on the uptake from bloodborne lipids. The currently recommended FO doses yielded low PUFAs tissue bioavailability, and supplementation with higher doses has been increasingly recommended. Nevertheless, the effects of higher FO doses on retinal Mfsd2a expression and blood vessels coverage are unknown. Western blot and qPCR analyses showed that high dose FO supplementation increased Mfsd2a expression in the retina. Immunohistochemical analyses of Mfsd2a expression on retinal blood vessels (labeled with 488-conjugated Lycopersicon esculentum, lectin) and subsequent ImageJ analyses revealed 1.32-fold increase in the Mfsd2a retinal blood vessel coverage. In the same time the pericyte blood vessel coverage (CD13+ cells) was not affected with FO supplementation, and the increase in Mfsd2a blood vessel expression is not the result of the increased pericyte coverage. Therefore, the high-dose FO supplementation emerges as the prophylactic fortifier of the retinal blood vessels that can serve either as prophylaxis in the healthy eye or as an adjuvant in developing targeted manipulations of the barrier during diseases.Poster Session: Brain Metabolism & Dietary Intervention

Prolonged Zaleplon Treatment Increases the Expression of Proteins Involved in GABAergic and Glutamatergic Signaling in the Rat Hippocampus

Zaleplon is a positive allosteric modulator of the γ-aminobutyric acid (GABA)A receptor approved for the short-term treatment of insomnia. Previous publications on zaleplon have not addressed the proteins involved in its mechanism of action but have mostly referred to behavioral or pharmacological studies. Since both GABAergic and glutamatergic signaling have been shown to regulate wakefulness and sleep, we examined the effects of prolonged zaleplon treatment (0.625 mg/kg for 5 days) on these systems in the hippocampus of male Wistar rats. Western blot and immunohistochemical analyses showed that the upregulated components of GABAergic signaling (glutamate decarboxylase, vesicular GABA transporter, GABA, and α1 subunit of the GABAA receptor) were accompanied by increased protein levels in the glutamatergic system (vesicular glutamate transporter 1 and NR1, NR2A, and NR2B subunits of N-methyl-d-aspartate receptor). Our results, showing that zaleplon enhances GABA neurotransmission in the hippocampus, were not surprising. However, we found that treatment also increased glutamatergic signaling. This could be the result of the downregulation of adenosine A1 receptors, important modulators of the glutamatergic system. Further studies are needed to investigate the effects of the zaleplon-induced increase in hippocampal glutamatergic neurotransmission and the possible involvement of the adenosine system in zaleplon’s mechanism of action

Deciphering ciprofloxacin-induced neurotoxicity: behavioral and molecular profiling of ciprofloxacin treatment in rats

Introduction: Ciprofloxacin is a fluoroquinolone antibiotic commonly used to treat various bacterial infections, with a potential to induce adverse mood effects in patients. Since the molecular mechanism of ciprofloxacin-induced neurotoxicity is poorly understood, we aimed to identify behavioral changes and corresponding neurotransmitter pattern after its prolonged administration in rats. We screened for untoward effects of ciprofloxacin on locomotor activity, despair, anhedonia, object recognition memory, and anxiety, as behavioral domains affected in various psychiatric diseases. Methodology: Three-month old male Sprague-Dawley rats were orally gavaged with ciprofloxacin (20 or 100 mg/kg) or solvent (0.5% methyl cellulose solution) each day for 4 weeks (n=80). One group of animals (n=40) passed the open field (OF), novel object recognition test (NORT), and forced swimming test (FST). Another group (n=40) underwent elevated plus maze (EPM) and sucrose preference test (SPT). After the completion of behavioral battery, the prefrontal cortex and cerebrospinal fluid (CSF) were collected. The neurotransmitters and metabolites of the kynurenine pathway were determined in the prefrontal cortex (PFC) through HPLC-MS/MS. Additionally, levels of interleukin-2 (IL-2) in CSF were quantified with Luminex. Behavioral and molecular parameters were analyzed by one-way ANOVA followed by Dunnett post hoc test in GraphPad Prism 9. Results: In FST, the treatment with high dose of ciprofloxacin decreased the swim time compared to control, which could be related to induction of despair-like behavior (p<0.05). The ciprofloxacin treatment did not affect object memory in NORT. In OF, the distance travelled and the number of rotations were not changed after treatment with ciprofloxacin compared to the control group. Further, animals treated with ciprofloxacin did not show changes in parameters in EPM and SPT. The acetylcholine levels in PFC were increased after ciprofloxacin treatment (p<0.05) in comparison with controls, which could be associated with depressed mood states. In line with that, high dose of ciprofloxacin treatment showed the tendency to decrease and increase levels of GABA and dopamine, respectively, but without reaching the statistical significance (p=0.07 and p=0.06). No changes in kynurenine pathway were observed after the treatment. The IL-2 concentration in CSF was increased after prolonged administration of low dose of ciprofloxacin treatment compared to the control levels (p<0.05), which could imply immunological stimulation of T lymphocytes and potential neuroinflammation. Conclusion: The despair behavior after treatment with high dose of ciprofloxacin was accompanied by increased levels of acetylcholine in PFC. Furthermore, the high dose of ciprofloxacin treatment showed tendency to decrease GABA levels, and increase dopamine levels in PFC, which could be connected to psychiatric adverse effects. Nonetheless, further studies are essential to confirm these neurotransmitter changes. On the other hand, the low dose of ciprofloxacin treatment elicited the increase of IL-2, which could be a marker of neuroinflammation-related neurotoxicity. In the future, efforts should be made to examine the role of IL-2 in the interaction of the immune system and the central nervous system, as its potential significance as a biomarker

The effect of light/dark cycle changes on vascular permeability, inflammation and visual cycle in streptoyotocin-induced diabezic retinophaty in rats

Introduction: Diabetic retinopathy (DR) is not cured efficiently and changes of lifestyle measures may alleviate its course. The aim of our study was to investigate the effects of shortened daily photoperiod on inflammation and visual cycle in rat retina and retinal pigment epithelium (RPE) in DR. Methodology: Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with a single intraperitoneal injection of streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18h for 3 months (6/18); 4. diabetic group exposed to light/dark cycle 6/18h (DM+6/18). Retinal blood vessel permeability was estimated immunohistochemically based on lectin staining, while the expression of genes involved in the visual cycle (SOX9, LRAT, RPE65, OTX2), and inflammation (IL-1, TNF-α) was determined by qRT-PCR in the retina and RPE. Results: Shortened photoperiod reduced neovascularisation and the expression of IL-1 and TNF-α in both retina and RPE. The expression of IL-1 and TNF-α genes in the retina was significantly higher in DM vs. control group (P=0.001). In contrast, retinal IL-1 and TNF-α expressions were significantly lower in DM+6/18 vs. DM group (P=0.001). The expression of IL-1 and TNF-α in RPE was significantly higher in DM vs. control group, however the expression of these genes was significantly lower in DM+6/18 vs. DM group (PIL-1=0.008 and PTNF-α=0.002). The expression of visual cycle genes was significantly up-regulated in RPE in DM+6/18 vs. DM group (P=0.001). Conclusion: Shortened daily photoperiod reduces blood vessel permeability in DR via its anti-inflammatory effect associated with accelerated visual cycle in the retina.Poster Session: Neuroimmunoendocrine Interaction

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors

The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.United States. National Institutes of HealthNew York State Stem Cell ScienceNational Institute of Neurological Disorders and Stroke (U.S.)Starr Foundation (Tri-Institutional Starr Stem Cell Scholars Fellowship

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication