Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers.

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Mineralized vectors for gene therapy.

There is an intense interest in developing materials for safe and effective delivery of polynucleotides using non-viral vectors. Mineralization of organic templates has long been used to produce complex materials with outstanding biocompatibility. However, a lack of control over mineral growth has limited the applicability of mineralized materials to a few in vitro applications. With better control over mineral growth and surface functionalization, mineralized vectors have advanced significantly in recent years. Here, we review the recent progress in chemical synthesis, physicochemical properties, and applications of mineralized materials in gene therapy, focusing on structure-function relationships. We contrast the classical understanding of the mineralization mechanism with recent ideas of mineralization. A brief introduction to gene delivery is summarized, followed by a detailed survey of current mineralized vectors. The vectors derived from calcium phosphate are articulated and compared to other minerals with unique features. Advanced mineral vectors derived from templated mineralization and specialty coatings are critically analyzed. Mineral systems beyond the co-precipitation are explored as more complex multicomponent systems. Finally, we conclude with a perspective on the future of mineralized vectors by carefully demarcating the boundaries of our knowledge and highlighting ambiguous areas in mineralized vectors. STATEMENT OF SIGNIFICANCE: Therapy by gene-based medicines is increasingly utilized to cure diseases that are not alleviated by conventional drug therapy. Gene medicines, however, rely on macromolecular nucleic acids that are too large and too hydrophilic for cellular uptake. Without tailored materials, they are not functional for therapy. One emerging class of nucleic acid delivery system is mineral-based materials. The fact that they can undergo controlled dissolution with minimal footprint in biological systems are making them attractive for clinical use, where safety is utmost importance. In this submission, we will review the emerging synthesis technology and the range of new generation minerals for use in gene medicines.

Prevalence and Predictors of Low Vitamin B6 Status in Healthy Young Adult Women in Metro Vancouver.

Low periconceptional vitamin B6 (B6) status has been associated with an increased risk of preterm birth and early pregnancy loss. Given many pregnancies are unplanned; it is important for women to maintain an adequate B6 status throughout reproductive years. There is limited data on B6 status in Canadian women. This study aimed to assess the prevalence of B6 deficiency and predictors of B6 status in young adult women in Metro Vancouver. We included a convenience sample of young adult non-pregnant women (19-35 years; n = 202). Vitamin B6 status was determined using fasting plasma concentrations of pyridoxal 5'-phosphate (PLP). Mean (95% confidence interval) plasma PLP concentration was 61.0 (55.2, 67.3) nmol/L. The prevalence of B6 deficiency (plasma PLP < 20 nmol/L) was 1.5% and that of suboptimal B6 status (plasma PLP = 20-30 nmol/L) was 10.9%. Body mass index, South Asian ethnicity, relative dietary B6 intake, and the use of supplemental B6 were significant predictors of plasma PLP. The combined 12.4% prevalence of B6 deficiency and suboptimal status was lower than data reported in US populations and might be due to the high socioeconomic status of our sample. More research is warranted to determine B6 status in the general Canadian population.

Atypical antipsychotics for insomnia: a systematic review.

BACKGROUND: Observational evidence suggests that atypical antipsychotics such as quetiapine are increasingly being used to manage insomnia. This is concerning given the uncertain efficacy and potential adverse effects associated with these medications. OBJECTIVES: The objectives of this study are to evaluate the benefits and adverse effects of atypical antipsychotics used specifically for insomnia. METHODS: The methods used in this study are systematic review and narrative synthesis. DATA SOURCES: The data were collected from PubMed; EMBASE; Cochrane Library; PsycINFO; grey literature; and the manufacturers of risperidone, quetiapine and olanzapine. PARTICIPANTS AND INTERVENTIONS: Adult patients ≥18 years of age using atypical antipsychotics specifically for primary or co-morbid insomnia for ≥ 1 week were compared to those receiving active intervention or placebo. APPRAISAL AND SYNTHESIS METHODS: Two independent reviewers screened titles, abstracts and full-text articles; extracted data; and conducted risk-of-bias analysis. Grading of Recommendations Assessment, Development and Evaluation (GRADE) assessment was completed. RESULTS: One double-blind randomized controlled trial (n = 13) met the eligibility criteria. Statistically significant differences were not observed from baseline between quetiapine and placebo after 2 weeks for primary insomnia in terms of total sleep time (mean difference (MD) 52.68 min, 95% CI -27.27 to 132.6), reduction in sleep latency (MD 72.44 min, 95% CI -2.65 to 147.5) or improved sleep satisfaction measured with a visual analogue scale out of 100 (MD 6.16, 95% CI -12.32 to 24.64), despite a trend towards improved sleep parameters. The study was rated as very low quality. CONCLUSIONS AND IMPLICATIONS: Very low quality evidence suggests that quetiapine does not significantly improve sleep parameters compared with placebo in primary insomnia, despite a trend towards clinical improvements. Atypical antipsychotics should be avoided in the first-line treatment of primary insomnia until further evidence is available.

Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Motor and somatosensory abnormalities are significant etiological factors for adolescent idiopathic scoliosis.

OBJECTIVE: In adolescent idiopathic scoliosis (AIS), we explore the role of lateralized motor and somatosensory abnormalities as a possible etiological factor. METHODS: Intraoperative transcranial electrical stimulation was performed in 15 AIS and 14 adult degenerative scoliosis (ADS) patients. Inter-side motor output balance (MOB) by comparing the ratios of right to left motor evoked potentials (MEP) amplitudes, and inter-side motor output excitability (MOE) computed with MEP amplitude, was determined separately for both patients groups. For somatosensory evoked potentials (SSEP), peak to peak P37 amplitudes from right and left lower limb SSEP and inter-side P37 amplitude ratios were obtained. RESULTS: Inter-side MOB was significantly asymmetric in AIS patients, contributed mainly by inter-side MOB changes in the upper than the lower limbs. Inter-side MOE comparisons of ipsilateral and contralateral MEP amplitudes were significantly different between AIS and ADS patients. Mean upper limb MEP amplitudes were significantly reduced in AIS patients. Amplitude of the right upper limb MEPs were positively correlated with inter-side MEP ratio. AIS patients show larger mean MEP amplitudes on the same side as the scoliotic curve. Overall, no correlation of Cobb's angle or total levels of scoliosis involvement with inter-side MOB and MOE parameters was found. Inter-side SSEP ratios were significantly higher in AIS patients. CONCLUSIONS: Primary dysfunctional and distributed motor output contributing to abnormalities of inter-side MOB and MOE changes involving the upper limbs is evident in AIS. Simultaneous but independent somatosensory and motor observations seen these patients suggest a central mechanism as an etiological factor.

High prevalence of suboptimal vitamin B12 status in young adult women of South Asian and European ethnicity.

Suboptimal vitamin B12 (B12) status has been associated with an increased risk of congenital anomalies, preterm birth, and childhood insulin resistance. South Asians - Canada's largest minority group - and women of reproductive age are vulnerable to B12 deficiency. This study aimed to assess the prevalence of and factors associated with B12 deficiency and suboptimal B12 status in a convenience sample of young adult women of South Asian and European descent in Metro Vancouver. We measured serum B12, holotranscobalamin, plasma methylmalonic acid, red blood cell and plasma folate, and hematologic parameters in 206 nonpregnant, healthy women aged 19-35 years. Categorization for B12 status adhered to serum B12 cutoffs for deficiency (<148 pmol/L) and suboptimal B12 status (148-220 pmol/L). We collected demographic, lifestyle, and dietary intake data and conducted genotyping for common genetic variants linked to B-vitamin metabolism. The prevalence of deficiency and suboptimal B12 status were 14% and 20%, respectively. Serum vitamin B12 concentrations were negatively associated with oral contraceptive use and first-generation immigrant status, and positively with dietary B12 intake and B12 supplement use. The prevalence of B12 inadequacy in this sample of highly educated women is higher than in the general Canadian population. In light of maternal and fetal health risks associated with B12 inadequacy in early-pregnancy, practitioners should consider monitoring B12 status before and during early pregnancy, especially in immigrants and women with low dietary B12 intakes including non-users of vitamin supplements.

A nitinol "U-Clip" versus sutured arteriovenous anastomosis: local tissue response and intimal hyperplasia development in a sheep model.

OBJECTIVES: This study sought to compare the local tissue response and subsequent volume of intimal hyperplasia (IH) that develops throughout the maturation of an arteriovenous fistula created using continuous/interrupted polypropylene with that of a novel, metal-alloy, penetrating anastomotic clip device. MATERIALS AND METHODS: Forty-six fistulae were created in 23 sheep under a paired design using the nitinol U-Clip (n = 23) in one hind limb and continuous (n = 20) or interrupted (n = 3) polypropylene suture for the other. Animals were killed at 4 (n = 3), 14 (n = 3), 28 (n = 10), 42 (n = 3), and 180 (n = 4) days. Histological sections were evaluated for quantitative histology and immunohistochemistry. RESULTS: Compared with continuous polypropylene, U-Clip specimens demonstrated less intima-media area per unit length (IMA/L), proliferating cells, and tissue necrosis at all time points (MANOVA, F = 9.8-24.1, all p ≤ .005; observed power >82%). Specifically, values of IMA/L were reduced by 5% (p = .97), 37% (p = .02), 33% (p < .01), 9% (p = .42), and 14% (p = .22) at the time points of 4, 14, 28, 42, and 180 days respectively. Proliferating cells were reduced by 75% (p < .01), 72% (p = .03), 76% (p = .03), 27% (p = .31), and 60% (p = .01) and tissue necrosis by 67% (p < .01), 58% (p = .02), 40% (p = .33), 21% (p = .43), 77% (p = .11). In a 28-day comparison between U-Clip and interrupted polypropylene the U-Clip group demonstrated a 4% (p = .65) reduction in IMA/L, 74% (p < .01) in proliferating cells and 49% (p < .05) in tissue necrosis. CONCLUSIONS: These results provide evidence of reduced local tissue necrosis, proliferating cells, and IH, favouring arteriovenous fistulae created using the U-Clip anastomotic device over conventional polypropylene suture techniques most evident over the first 4 weeks.

Methylmalonic acid quantified in dried blood spots provides a precise, valid, and stable measure of functional vitamin B-12 status in healthy women.

Methylmalonic acid (MMA) is a sensitive and specific functional biomarker of vitamin B-12 status, commonly assessed in plasma or serum. Dried blood spots (DBSs) allow simpler and more cost-efficient blood sampling than plasma. To facilitate convenient testing for vitamin B-12 deficiency in large-scale surveys and in population groups from remote areas, we developed a method for MMA quantification in DBSs and tested its applicability as well as the long-term stability of MMA in DBSs at various temperatures. MMA was extracted from an 8-mm DBS punch with water:methanol (95:5, v:v) and methyl-d3-malonic acid as the internal standard. After sample cleanup by ultrafiltration and hexane extraction, MMA was quantified by using reversed-phase LC-tandem mass spectrometry. Extraction conditions were optimized to maximize the detection signal and achieve DBS extract concentrations above the lowest limit of quantification (signal-to-noise ratio ≥ 10) of 10 nmol/L. Recovery was between 93% and 96%. Intra- and interassay variation (CV%) for DBS MMA was 0.49% and 2.3%, respectively. Calibrators showed linearity (R(2) = 0.998) between 10 and 10,000 nmol/L. In 94 healthy women, MMA concentrations in DBS extract (min-max: 10.2-80.5 nmol/L) and plasma (min-max: 68-950 nmol/L) were correlated (ρ = 0.90) (P < 0.001). MMA concentrations in DBSs were stable at room temperature for 1 wk, in the refrigerator for 8 wk, and at -80°C for at least 1 y. This simple and robust method allows quantification of MMA in DBSs of healthy individuals. The linear relation between plasma and DBS MMA suggests that DBS MMA could predict plasma MMA, the current reference indicator for functional vitamin B-12 deficiency. With the advantages of minimally invasive specimen collection and no need for laborious blood processing steps, this method has the potential to be a reliable, convenient, and field-applicable alternative for assessment of vitamin B-12 status.

Comparing the effects of meal replacements with reduced-fat diet on weight, sexual and endothelial function, testosterone and quality of life in obese Asian men.

Sexual dysfunction is more prevalent in obese than in normal-weight men. Meal replacements (MRs) are useful weight-loss strategies. We randomized obese (body mass index 27.5 kg m(-2), waist circumference (WC) 90 cm) Asian men (mean age 40.5 years, range 30-61) to a conventional reduced-fat diet (CD) (n=24) or MR-based plan (n=24) to reduce daily intake by 400 kcal for 12 weeks. There were significantly greater reductions in weight (4.2 ± 0.8 kg), WC (4.6 ± 0.7 cm), calorie and fat intake in the MR group, compared with the CD group (2.5 ± 0.4 kg, 2.6 ± 0.5 cm). Erectile function (International Index of Erectile Function 5-item score) improved comparably in the MR (3.4 ± 0.7 points) and CD (2.5 ± 0.5 points) groups, as did the Sexual Desire Inventory score (5.5 ± 2.3 vs 7.7 ± 2.1 points), quality of life (36-item Short Form survey score), plasma testosterone and endothelial function (Reactive Hyperemia Index). Subjects were switched to or continued CD for another 28 weeks. Weight, WC and erectile function were maintained at 40 weeks. MR induces greater reductions in weight and abdominal obesity than conventional diet, and comparable improvements in sexual and endothelial function, testosterone and quality of life.

Associations between ethnicity, body composition, and bone mineral density in a Southeast Asian population.

CONTEXT AND OBJECTIVE: Chinese men in Singapore have a higher incidence of hip fractures than Malay and Indian men. We investigated whether there were corresponding ethnic differences in peak bone mineral density (BMD) in young men and whether differences in body composition influenced peak BMD. DESIGN AND SETTING: This was a cross-sectional study of healthy volunteers in a tertiary medical center. PARTICIPANTS: A total of 100 Chinese, 82 Malay, and 80 Indian men aged 21 to 40 years, with body mass index between 18 and 30 kg/m(2) underwent dual-energy x-ray absorptiometry to assess BMD, lean mass (LM) and fat mass (FM), and magnetic resonance imaging to quantify abdominal subcutaneous and visceral adipose tissue. Multiple linear regression models, with adjustment for age and height (as a proxy for skeletal size), were used. RESULTS: Malay and Indian men had significantly higher BMD than Chinese men at the lumbar spine (Malay: B, 0.06 ± 0.02, P = .001; Indian: B, 0.03 ± 0.02, P = .049), femoral neck (Malay: B 0.04 ± 0.02, P = .034; Indian: B, 0.04 ± 0.02, P = .041), hip (Malay: B, 0.05 ± 0.02, P = .016; Indian: B, 0.06 ± 0.02, P = .001), and ultradistal radius (Malay: B, 0.03 ± 0.01, P < .001; Indian: B, 0.02 ± 0.01, P = .029), and this difference was retained after adjustment for LM and FM, except in Malay men at the femoral neck and in Indian men at the ultradistal radius. LM was an important independent determinant of BMD at all sites, whereas FM, subcutaneous adipose tissue, and visceral adipose tissue were not significantly associated with BMD at any site. CONCLUSIONS: Lower peak BMD in Chinese men may partly explain the higher fracture incidence in this ethnic group. Further studies are needed to elucidate the reasons for these ethnic differences in bone accumulation.

The epidemiology and management of adult poisonings admitted to the short-stay ward of a large Scottish emergency department.

BACKGROUND AND AIMS: The emergency department of Aberdeen Royal Infirmary receives around 68,000 new adult admissions annually. All poisoning cases are admitted to a 14-bedded short-stay ward, except those admitted to intensive care or immediately discharged. This study aimed to analyse epidemiological trends and management of short-stay ward admissions for poisonings. METHOD AND RESULTS: Adult (>13 years) poisoning presentations admitted to the emergency department short-stay ward of Aberdeen Royal Infirmary from 1 January-31 December 2009 were retrospectively reviewed using patient discharge summaries. During 2009, there were 1062 poisoning cases, of which repeat episodes were responsible for 15%. The mean age of presentation was 33.9 years (SD 14.4) and there was a female preponderance (62%). Almost half of poisonings were polypharmacy, alcohol was involved in 40% of cases and overdoses most commonly involved paracetamol (43%). Management involved basic observations only (66%), N-acetylcysteine (24%), naloxone (4%) and activated charcoal (1%). Liaison psychiatry reviewed 84% presentations and admitted 9% to the psychiatric unit. CONCLUSIONS: The short-stay ward is important for acute management of poisonings and the data gained from this study should help to direct patient services appropriately.

A comparison of the epidemiology of paediatric burns in Scotland and South Africa.

In South Africa burns affect 3.2% of the population annually and are particularly common among children. In Scotland paediatric burns are generally much less common and less severe. This study aimed to explore the epidemiological differences in the emergency presentation of paediatric burns in the Royal Aberdeen Children's Hospital (RACH) in Scotland and the Red Cross War Memorial Children's Hospital (RXH) in Cape Town. Data was retrieved retrospectively for all paediatric burns presenting in 2009 from RACH patient records and the RXH trauma database. Data was recorded in Microsoft Excel for subsequent statistical analysis. During 2009 RACH received 192 children with burns (1% total emergencies) and RXH received 994 (11% total emergencies). Children ≤ 2 years old were the most commonly injured age group in both centres. At RXH most children came from informal settlements and were of low socioeconomic status, while RACH patients were evenly distributed among all socioeconomic groups. Burn injuries were significantly more likely to present in the evening at both centres (p<0.05), and during Cape Town's winter (p<0.05), but no significant monthly variation occurred in Aberdeen. At RACH most burns involved the hands and were single site (79%) while at RXH most were multiple site (76%) and involved the face. At RACH the commonest modes of injury were scald (45%) and contact burn (43%), while at RXH scalds accounted for the majority (77%). At RACH 89% children were discharged immediately, whereas 49% of RXH patients were admitted to the burn unit. Paediatric burns are more common and generally more severe in Cape Town than in Aberdeen. All children have the right to a safe environment and protection from harm; to reduce the high burns incidence in Cape Town preventative strategies should be targeted at creating safer homes.

An arteriovenous fistula model of intimal hyperplasia for evaluation of a nitinol U-Clip anastomosis.

OBJECTIVES: The aim of this study was to create an ovine arteriovenous fistula (AVF) model which would closely replicate a human forearm fistula and use this to quantify the degree of intimal hyperplasia in those created with the U-Clip compared to a conventional sutured anastomosis. MATERIALS AND METHODS: Twenty AVFs were created in 10 Border Leicester-Merino sheep between the superficial femoral artery and vein of each hind limb. On one side the U-Clip and on the other a continuous polypropylene suture was used to perform the anastomosis. The animals were sacrificed at 2 (n = 3), 4 (n = 4), 6 (n = 3) weeks and histological slices were taken of each AVF in cross section to determine the intimal media area per unit length (IMA/L). RESULTS: Intimal hyperplasia (IH) was observed at all time points with one AVF found occluded with thrombus at the time of harvest. The IMA/L was significantly lower in the U-Clip groups by 24% at 2 weeks, 32% at 4 weeks and 23% at 6 weeks (Two-way ANOVA, p = 0.019, observed power = 0.825, time or side p ≥ 0.766, type p = 0.001; Paired t-test, p < 0.001 between matched anastomotic types). Time taken to perform the anastomosis was similar between the two anastomotic techniques (Polypropylene 14(8-18) vs. U-Clip 15.3(11-23) min; p = 0.47). CONCLUSION: This ovine AVF model results in IH similar to that seen in a human AVF. The IH that occurs with the U-Clip is less than that of continuous polypropylene suture.

Clinical and physiological effects of transcranial electrical stimulation position on motor evoked potentials in scoliosis surgery.

BACKGROUND: During intraoperative monitoring for scoliosis surgery, we have previously elicited ipsilateral and contralateral motor evoked potentials (MEP) with cross scalp stimulation. Ipsilateral MEPs, which may have comprised summation of early ipsilaterally conducted components and transcallosally or deep white matter stimulated components, can show larger amplitudes than those derived purely from contralateral motor cortex stimulation. We tested this hypothesis using two stimulating positions. We compared intraoperative MEPs in 14 neurologically normal subjects undergoing scoliosis surgery using total intravenous anesthetic regimens. METHODS: Trancranial electrical stimulation was applied with both cross scalp (C3C4 or C4C3) or midline (C3Cz or C4Cz) positions. The latter was assumed to be more focal and result in little transcallosal/deep white matter stimulation. A train of 5 square wave stimuli 0.5 ms in duration at up to 200 mA was delivered with 4 ms (250 Hz) interstimulus intervals. Averaged supramaximal MEPs were obtained from the tibialis anterior bilaterally. RESULTS: The cross scalp stimulating position resulted in supramaximal MEPs that were of significantly higher amplitude, shorter latency and required lower stimulating intensity to elicit overall (Wilcoxon Signed Rank test, p < 0.05 for all), as compared to the midline stimulating position. However, no significant differences were found for all 3 parameters comparing ipsilaterally and contralaterally recorded MEPs (p > 0.05 for all), seen for both stimulating positions individually. CONCLUSIONS: Our findings suggest that cross scalp stimulation resulted in MEPs obtained ipsilaterally and contralaterally which may be contributed to by summation of ipsilateral and simultaneous transcallosally or deep white matter conducted stimulation of the opposite motor cortex. Use of this stimulating position is advocated to elicit MEPs under operative circumstances where anesthetic agents may cause suppression of cortical and spinal excitability. Although less focal in nature, cross scalp stimulation would be most suitable for infratentorial or spinal surgery, in contrast to supratentorial neurosurgical procedures.

Combustion smoke exposure induces up-regulated expression of vascular endothelial growth factor, aquaporin 4, nitric oxide synthases and vascular permeability in the retina of adult rats.

Retinal cells respond to various experimental stimuli including hypoxia, yet it remains to be investigated whether they react to smoke inhalation. We show here that retinal cells in rats, notably the ganglion cells, Müller cells, astrocytes and blood vessels responded vigorously to a smoke challenge. The major changes included up-regulated expression of vascular endothelial growth factor (VEGF), aquaporin 4 (AQP4) and nitric oxide synthase (NOS). VEGF expression was localized in the ganglion cells, Müller cells, astrocytes and associated blood vessels. AQP4 was markedly enhanced in both astrocytes and Müller cells. Increase in vascular permeability after smoke exposure was evidenced by extravasation of serum derived rhodamine isothiocyanate which was internalized by Müller cells and ganglion cells. The tracer leakage was attenuated by aminoguanidine and N(G)-nitro-L-arginine methyl ester (L-NAME) treatment which suppressed retinal tissue NOS and nitric oxide (NO) levels concomitantly. It is suggested that VEGF, AQP4 and NO are involved in increased vascular permeability following acute smoke exposure in which hypoxia was ultimately implicated as shown by blood gases analysis. NOS inhibitors effectively reduced the vascular leakage and hence may ameliorate possible retinal edema in smoke inhalation.