Predicting the Radiation Sensitivity of Male and Female Rhesus Macaques Using Gene Expression.

Radiosensitivity differs in humans and likely among closely-related primates. Reasons for variation in radiosensitivity are not well known. We examined preirradiation gene expression in peripheral blood among male and female rhesus macaques which did or did not survive (up to 60 days) after whole-body irradiation with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study in 142 irradiated rhesus macaques. Animals were untreated (placebo), or treated using recombinant human IL-12, G-CSF or combination of the two. We evaluated gene expression in a two-phase study design where phase I was a whole genome screen [next generation sequencing (NGS)] for mRNAs (RNA-seq) using five RNA samples from untreated male and female animals per group of survivor and non-survivor (total n = 20). Differential gene expression (DGE) was defined as a statistically significant and ≥2-fold up- or downregulation of mRNA species and was calculated between groups of survivors and non-survivors (reference) and by gender. Altogether 659 genes were identified, but the overlapping number of differentially expressed genes (DGE) observed in both genders was small (n = 36). Fifty-eight candidate mRNAs were chosen for independent validation in phase II using the remaining samples (n = 122) evaluated with qRT-PCR. Among the 58 candidates, 16 were of significance or borderline significance (t test) by DGE. Univariate and multivariate logistic regression analysis and receiver operating characteristic (ROC) curve analysis further refined and identified the most outstanding validated genes and gene combinations. For untreated male macaques, we identified EPX (P = 0.005, ROC=1.0), IGF2BP1 (P = 0.05, ROC=0.74) and the combination of EPX with SLC22A4 (P = 0.03, ROC=0.85) which appeared most predictive for the clinical outcome for treated and combined (untreated and treated) male macaque groups, respectively. For untreated, treated and both combined female macaque groups the same gene (MBOAT4, P = 0.0004, ROC = 0.81) was most predictive. Based on the probability function of the ROC curves, up to 74% of preirradiation RNA measurements predicted survival with a positive and negative predictive value ranging between 85-100% and associated odds ratios reflecting a 2-3-fold elevated risk for surviving per unit change (cycle threshold value) in gene expression. In conclusion, we identified gender-dependent genes and gene combinations in preirradiation blood samples for survival prediction after irradiation in rhesus macaques.

Differentiating Total- or Partial-Body Irradiation in Baboons Using mRNA Expression Patterns: A Proof of Concept.

To better predict clinical outcome after radiation exposure, it is very important to know the absorbed dose and body areas exposed. Previously we found that 22 miRNAs appeared to predict total- and partial-body irradiation (TBI and PBI, respectively) patterns and were suggestive of the percentage of the body exposed in a baboon model. Motivated by these results, we performed a similar analysis on the transcriptional level (mRNAs) using whole genome microarrays. From 17 irradiated baboons, blood samples were taken before, and at 1, 2, 7, 28 and 75-106 days postirradiation to an equivalent TBI dose of 2.5 or 5 Gy applied either to the total body or to different parts of the body such as the upper body (UBE) or left hemibody (LHB). We compared quantile normalized log2-transformed gene expression values with three exposure pattern comparisons, namely TBI vs. PBI, TBI vs. LHB and UBE vs. LHB using Kruskal-Wallis and logistic regression analysis for receiver-operator characteristic (ROC) calculation. We found several hundred significantly (P < 0.05) and ≥2-fold deregulated mRNAs per exposure pattern comparison with a peak of 163-860 mRNAs at day 28. Lower numbers on day 2 (60 mRNAs) and day 7 (91-162 mRNAs) were observed, with the lowest number of deregulated mRNAs at day 75-106 (22-58 mRNAs). The 14 most promising mRNAs (e.g., LTF, DEFA3, OLFM4) appeared 10.1-46.2-fold upregulated and the exposure groups were completely or almost completely discriminated (ROC between 0.8-1.0). Several of the mRNA gene expression changes were significantly associated with the percentage of the body exposed. The numbers of overlapping genes used for diagnosis on consecutive days postirradiation were mostly 0 or less than 10. Bioinformatic analysis confirmed that at each time point different biological processes predominated. Our results suggest mRNA changes over time may be used to retrospectively determine radiation exposure patterns as partial or total body. mRNA gene expression changes likely could be applied over a longer time frame (2-75 days postirradiation) than miRNA, but due to the transient gene expression changes a different set of candidate mRNAs appears to be required at each day after irradiation.

miRNA Expression Patterns Differ by Total- or Partial-Body Radiation Exposure in Baboons.

In a radiation exposure event, a likely scenario may include either total-body irradiation (TBI) or different partial-body irradiation (PBI) patterns. Knowledge of the exposure pattern is expected to improve prediction of clinical outcome. We examined miRNA species in 17 irradiated baboons receiving an upper-body, left hemibody or total-body irradiation of 2.5 or 5 Gy. Blood samples were taken before irradiation and at 1, 2, 7, 28 and 75-106 days after irradiation. Using a qRT-PCR platform for simultaneous detection of 667 miRNAs, we identified 55 miRNAs over all time points. Candidate miRNAs, such as miR-17, miR-128 or miR-15b, significantly discriminated TBI from different PBI exposure patterns, and 5-to-10-fold changes in gene expression were observed among the groups. A total of 22 miRNAs (including miR-17) revealed significant linear associations of gene expression changes with the percentage of the exposed body area (P < 0.0001). All these changes were primarily observed at day 7 postirradiation and almost no miRNAs were detected either before or after 7 days. A significant association in the reduction of lymphocyte counts in TBI compared to PBI animals corresponded with the number of miRNA candidates. This finding suggests that our target miRNAs predominantly originated from irradiated lymphocytes. In summary, gene expression changes in the peripheral blood provided indications of the exposure pattern and a suggestion of the percentage of the exposed body area.

Impact of Inter-Individual Variance in the Expression of a Radiation-Responsive Gene Panel Used for Triage.

In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.

Improved tendon healing using bFGF, BMP-12 and TGFß1 in a rat model.

Several growth factors (GFs) are expressed as tendons heal, but it remains unknown whether their combined application enhances the healing process. This matter was addressed by applying a combination of basic fibroblast growth factor (bFGF), bone morphogenetic protein 12 (BMP-12) and transforming growth factor beta 1 (TGFß1) in a rat Achilles tendon transection model. GFs were applied in one of the three following ways: i) direct application of all three factors at the time of surgery; ii) sequential, tiered percutaneous injection of individual factors immediately after surgery, 48 h and 96 h later; iii) load of all three factors onto a collagen sponge implanted at the time of surgery. After 1, 2, 4 and 8 weeks, healing was assessed based on tendon length and thickness, mechanical strength, stiffness and histology. Best results were achieved when GFs were loaded onto a collagen sponge - with a rapid increase in mechanical strength (load to failure, 71.2 N vs. 7.7 N in controls), consistent tendon length over time (9.9 mm vs. 16.2 mm in controls) and faster tendon remodelling, as measured by histology - followed by tiered injection therapy over 96 h. In conclusion, implantation of a GF-loaded collagen sponge could provide a promising treatment, especially in high-performance athletes and revision cases prone to re-rupture. For conservative treatment, tiered percutaneous GF application could be an option for improving clinical outcome.

Correlation of Radiation Dose Estimates by DIC with the METREPOL Hematological Classes of Disease Severity.

The degree of severity of hematologic acute radiation syndrome (HARS) may vary across the range of radiation doses, such that dose alone may be a less reliable predictor of clinical course. We sought to elucidate the relationship between absorbed dose and risk of clinically relevant HARS in humans. We used the database SEARCH (System for Evaluation and Archiving of Radiation Accidents based on Case Histories), which contains the histories of radiation accident victims. From 153 cases we extracted data on dose estimates using the dicentric assay to measure individual biological dosimetry. The data were analyzed according to the corresponding hematological response categories of clinical significance (H1-4). These categories are derived from the medical treatment protocols for radiation accident victims (METREPOL) and represent the clinical outcome of HARS based on severity categories ranging from 1-4. In addition, the category H0 represents a post-exposure hematological response that is within the normal range for nonexposed individuals. Age at exposure, gender and ethnicity were considered as potential confounders in unconditional cumulative logistic regression analysis. In most cases, victims were Caucasian (82.4%) and male (92.8%), who originated from either the Chernobyl (69.3%) or Goiânia (10.5%) accident, and nearly 60% were aged 20-40 years at time of exposure. All individuals were whole-body exposed (mean 3.8 Gy, stdev ±3.1), and single exposures were predominantly reported (79%). Seventy percent of victims in category H0 were exposed to ≤1 Gy, with rapidly decreasing proportions of H0 seen at doses up to 5 Gy. There were few HARS H4 cases reported at exposed dose of 1-2 Gy, while 82% of H4 cases received doses of >5 Gy. HARS H1-3 cases varied among dose ranges from 1-5 Gy. In summary, single whole-body radiation doses <1 Gy and >5 Gy corresponded in general with H0 and H3-4, respectively, and this was consistent with medical expectations. This underlines the usefulness of dose estimates for HARS prediction. However, whole-body doses between 1-5 Gy poorly corresponded to HARS H1-3. The dose range of 1-5 Gy was of limited value for medical decision-making regarding, e.g., hospitalization for H2-3, but not H1 and treatment decisions that differ between H1-3. Also, there were some H0 cases at high doses and H2-4 cases at low doses, thereby challenging an individual recommendation based solely on dose.

Validating Baboon Ex Vivo and In Vivo Radiation-Related Gene Expression with Corresponding Human Data.

The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004-0.018 Sv); radiotherapy for prostate cancer (0.25-0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0-4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model ( WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments ( FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001-5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.

The influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis.

The present in vitro study investigated the influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis. Fifteen juvenile female piglets (18-20 kg body weight) were randomly assigned into three groups (n=5 animals each): i) control (clinically healthy animals, without doxazosin treatment), ii) animals with induced inflammation of the urinary bladder, but without doxazosin treatment (experimental group I) and iii) animals with inflamed bladder, treated orally with doxazosin (0.1 mg/kg body weight for 30 days; experimental group II). Thereafter, the pigs were sacrificed and strips of the bladder trigone were suspended in organ baths. The tension and amplitude of the smooth muscles was measured before and after exposition to 5-hydroxytryptamine (5-HT; 10-6-10-4 M), acetylocholine (ACh; 10-5-10-3 M) and norepinephrine (NE; 10-9-10-7 M). 5-HT caused an increase in the tension of contractions in all the groups and the amplitude in the experimental groups, however, the effect was higher in the experimental group I than in group II as compared to that found in the pre-treatment period. ACh caused an increase in the tension in the control group and a decrease in the amplitude in both experimental groups; these changes significantly differed between the control and doxazosin-treated group. NE caused a decrease in the tension in both experimental groups and amplitude in all the groups, however, the effect was most strongly expressed in doxazosine-treated group. The present study has revealed that long-term administration of doxazosin causes a desensitization of the detrusor smooth muscle to in vitro applied mediators in the autonomic nervous system.

Using Clinical Signs and Symptoms for Medical Management of Radiation Casualties - 2015 NATO Exercise.

The utility of early-phase (≤5 days) radiation-induced clinical signs and symptoms (e.g., vomiting, diarrhea, erythema and changes in blood cell counts) was examined for the prediction of later occurring acute radiation syndrome (ARS) severity and the development of medical management strategies. Medical treatment protocols for radiation accident victims (METREPOL) was used to grade ARS severities, which were assigned response categories (RCs). Data on individuals (n = 191) with mild (RC1, n = 45), moderate (RC2, n = 19), severe (RC3, n = 20) and fatal (RC4, n = 18) ARS, as well as nonexposed individuals (RC0, n = 89) were generated using either METREPOL (n = 167) or the system for evaluation and archiving of radiation accidents based on case histories (SEARCH) database (n = 24), the latter comprised of real-case descriptions. These data were converted into tables reflecting clinical signs and symptoms, and submitted to eight teams representing five participating countries. The teams were comprised of medical doctors, biologists and pharmacists with subject matter expertise. The tables comprised cumulated clinical data from day 1-3 and day 1-5 postirradiation. While it would have reflected a more realistic scenario to provide the data to the teams over the course of a 3- or 5-day period, the logistics of doing so proved too challenging. In addition, the team members participating in this exercise chose to receive the cumulated reports of day 1-3 and 1-5. The teams were tasked with predicting ARS incidence, ARS severity and the requirement for hospitalization for multiple cases, as well as providing the certainty of their diagnosis. Five of the teams also performed dose estimates. The teams did not employ harmonized methodologies, and the expertise among the members varied, as did the tools used and the means of analyzing the clinical data. The earliest report time was 3 h after the tables were sent to the team members. The majority of cases developing ARS (89.6% ± 3.3 SD) and requiring hospitalization (88.8% ± 4.6 SD) were correctly identified by all teams. Determination of ARS severity was particularly challenging for RC2-3, which was systematically overestimated. However, RC4 was correctly predicted at 94-100% by all teams. RC0 and RC1 ARS severities were more difficult to discriminate. When reported RCs (0-1 and 3-4) were merged, on average 89.6% (±3.3 SD) of all cases could be correctly classified. Comparisons on frequency distributions revealed no statistically significant differences among the following: 1. reported ARS from different teams (P > 0.2); 2. cases generated based on METREPOL or SEARCH (P > 0.5); or 3. results reported at day 3 and 5 postirradiation (P > 0.1). Dose estimates of all teams increased significantly along with ARS severity (P < 0.0001) as well as with dose estimates generated from dicentric chromosomal-aberration measurements available for SEARCH cases (P < 0.0001). In summary, early-phase radiation-induced clinical signs and symptoms proved to be useful for rapid and accurate assessment, with minor limitations, toward predicting life-threatening ARS severity and developing treatment management strategies.

The influence of resiniferatoxin on the chemical coding of caudal mesenteric ganglion neurons supplying the urinary bladder in the pig.

Resiniferatoxin (RTX) is used as experimental drug therapy for a range of neurogenic urinary bladder disorders. The present study investigated the chemical coding of caudal mesenteric ganglion (CaMG) neurons supplying the porcine urinary bladder after intravesical RTX instillation. The CaMG neurons were visualized with retrograde tracer Fast Blue (FB) and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), calbindin (CB), galanin (GAL) and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 6) and RTX-treated pigs (n = 6), the vast majority (92.3 ± 2.7% and 93.1 ± 1.3%, respectively) of FB-positive (FB+) nerve cells were TH+. Intravesical instillation of RTX caused a decrease in the number of FB+ / TH + neurons immunopositive to NPY (91.0 ± 2.2% in control animals vs. 58.8 ± 5.0% in RTX-treated pigs) or VIP (1.7 ± 0.5% vs. 0%) and an increase in the number of FB+ / TH+ neurons immunoreactive to SOM (3.4 ± 1.5% vs. 20.6 ± 4.3%), CB (1.8 ±0.7% vs. 13.4 ± 2.3%), GAL (1.5 ± 0.6% vs. 7.5 ± 1.0%) or nNOS (0% vs. 10.9 ± 3.4%). The present results suggest that therapeutic effects of RTX on the mammalian urinary bladder can be partly mediated by CaMG neurons.

First Generation Gene Expression Signature for Early Prediction of Late Occurring Hematological Acute Radiation Syndrome in Baboons.

We implemented a two-stage study to predict late occurring hematologic acute radiation syndrome (HARS) in a baboon model based on gene expression changes measured in peripheral blood within the first two days after irradiation. Eighteen baboons were irradiated to simulate different patterns of partial-body and total-body exposure, which corresponded to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. Blood samples taken before irradiation served as unexposed control (H0, n = 17). For stage I of this study, a whole genome screen (mRNA microarrays) was performed using a portion of the samples (H0, n = 5; H1-2, n = 4; H2-3, n = 5). For stage II, using the remaining samples and the more sensitive methodology, qRT-PCR, validation was performed on candidate genes that were differentially up- or down-regulated during the first two days after irradiation. Differential gene expression was defined as significant (P < 0.05) and greater than or equal to a twofold difference above a H0 classification. From approximately 20,000 genes, on average 46% appeared to be expressed. On day 1 postirradiation for H2-3, approximately 2-3 times more genes appeared up-regulated (1,418 vs. 550) or down-regulated (1,603 vs. 735) compared to H1-2. This pattern became more pronounced at day 2 while the number of differentially expressed genes decreased. The specific genes showed an enrichment of biological processes coding for immune system processes, natural killer cell activation and immune response (P = 1 × E-06 up to 9 × E-14). Based on the P values, magnitude and sustained differential gene expression over time, we selected 89 candidate genes for validation using qRT-PCR. Ultimately, 22 genes were confirmed for identification of H1-3 classifications and seven genes for identification of H2-3 classifications using qRT-PCR. For H1-3 classifications, most genes were constantly three to fivefold down-regulated relative to H0 over both days, but some genes appeared 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) over H0. For H2-3, some genes appeared four to sevenfold up-regulated relative to H0 (RNASE3, DAGLA, ARG2), but other genes showed a strong 14- to 33-fold down-regulation relative to H0 (WNT3, POU2AF1, CCR7). All of these genes allowed an almost completely identifiable separation among each of the HARS categories. In summary, clinically relevant HARS can be independently predicted with all 29 irradiated genes examined in the peripheral blood of baboons within the first two days postirradiation. While further studies are needed to confirm these findings, this model shows potential relevance in the prediction of clinical outcomes in exposed humans and as an aid in the prioritizing of medical treatment.

Expression of aquaporin 1 and 5 and their regulation by ovarian hormones, arachidonic acid, forskolin and cAMP during implantation in pigs.

Aquaporin proteins (AQPs) are a family of channels expressed in numerous mammalian tissues, where they play a fundamental role in regulating water transport across cell membranes. Based on reports that AQPs are present in the reproductive system and participate in reproductive processes, our aim was to investigate the effect of progesterone (P(4)), estradiol (E(2)), oxytocin (OT), arachidonic acid (AA), forskolin (FSK) and cyclic adenosine monophosphate (cAMP) on AQP1 and AQP5 expression at mRNA and protein levels in porcine uterine explants from Days 14-16 of gestation in order to determine if they play a role in implantation period in pigs. Quantitative real time PCR and Western-blot analysis revealed that the uterine explants treated with FSK and cAMP produce delayed, but long-term effects on AQP1 abundance (24 h) while AQP5 had a rapid and sustained response to FSK and cAMP in protein content (3 and 24 h). AA increases gene and protein content of AQP1 after longer exposition whereas AQP5 increases after 3 h only at the protein level. Both AQPs potentially remains under control of steroid hormones. OT has been shown to increase AQP1, and decrease AQP5 mRNA, without visible changes in protein content. P(4), E(2), AA, FSK and cAMP caused the appearance of AQP5 expression in the basolateral plasma membrane of the epithelial cells. The staining represents most likely AQP5 functioning mechanism for both absorption and reabsorption across the glandular epithelium.

Early Results of Physician Modified Fenestrated Stent Grafts for the Treatment of Thoraco-abdominal Aortic Aneurysms.

OBJECTIVES: The aim was to determine whether physician modified stent grafts (PMSGs) are safe and effective for the treatment of high risk patients with thoraco-abdominal aortic aneurysms (TAAAs). DESIGN: This was a retrospective single institution study. MATERIAL: Consecutive patients with TAAA undergoing endovascular repair using a PMSG between January 2012 and June 2014 were evaluated. METHODS: Fenestrations to preserve branch vessels were created in TX2 thoracic (Cook Medical) stent grafts. Pre- intra- and post-operative data were recorded by means of a prospectively maintained database. RESULTS: Eleven high risk patients with TAAA (type I, n = 4; type III, n = 3; type IV, n = 3; type V, n = 1) underwent fenestrated endovascular repair using PMSGs. Indications were painful aneurysm (n = 5), >70 mm rapidly enlarging aneurysm (n = 4), saccular aneurysm (n = 1), and visceral patch false aneurysm after open repair of a type IV TAAA (n = 1). In four asymptomatic patients, an additional fenestration was created for temporary selective sac perfusion and occluded 2-4 weeks later. Median duration for stent graft modifications was 2 hours (range 1-3 hours). The median number of fenestrations was three (range 2-4). One patient died during the post-operative period from colonic ischemia, giving a 9% in hospital mortality rate. Four (36%) patients presented with moderate to severe complications. One (9%) patient presented with a paraparesis that resolved completely after spinal fluid drainage. Among surviving patients, four required early endovascular re-intervention for type III endoleak (n = 2), type Ia endoleak (n = 1), or target vessel cannulation failure (n = 1). The median follow up time was 6 months (range 3-20 months). During follow up, no other complications occurred and all target vessels remained patent. One patient presented with a persistent type II endoleak. CONCLUSION: PMSGs provided acceptable short-term results and may be a management option for the treatment of TAAA in selected high risk patients. Durability concerns need to be assessed in additional studies with long-term follow up.

Editor's Choice - ECAR (Endovasculaire ou Chirurgie dans les Anévrysmes aorto-iliaques Rompus): A French Randomized Controlled Trial of Endovascular Versus Open Surgical Repair of Ruptured Aorto-iliac Aneurysms.

OBJECTIVES/BACKGROUND: ECAR (Endovasculaire ou Chirurgie dans les Anévrysmes aorto-iliaques Rompus) is a prospective multicentre randomized controlled trial including consecutive patients with ruptured aorto-iliac aneurysms (rAIA) eligible for treatment by either endovascular (EVAR) or open surgical repair (OSR). Inclusion criteria were hemodynamic stability and computed tomography scan demonstrating aorto-iliac rupture. METHODS: Randomization was done by week, synchronously in all centers. The primary end point was 30 day mortality. Secondary end points were post-operative morbidity, length of stay in the intensive care unit (ICU), amount of blood transfused (units) and 6 month mortality. RESULTS: From January 2008 to January 2013, 107 patients (97 men, 10 women; median age 74.4 years) were enrolled in 14 centers: 56 (52.3%) in the EVAR group and 51 (47.7%) in the OSR group. The groups were similar in terms of age, sex, consciousness, systolic blood pressure, Hardman index, IGSII score, type of rupture, use of endoclamping balloon, and levels of troponin, creatinine, and hemoglobin. Delay to treatment was higher in the EVAR group (2.9 vs. 1.3 hours; p < .005). Mortality at 30 days and 1 year were not different between the groups (18% in the EVAR group vs. 24% in the OSR group at 30 days, and 30% vs. 35%, respectively, at 1 year). Total respiratory support time was lower in the EVAR group than in the OSR group (59.3 hours vs. 180.3 hours; p = .007), as were pulmonary complications (15.4% vs. 41.5%, respectively; p = .050), total blood transfusion (6.8 vs. 10.9, respectively; p = .020), and duration of ICU stay (7 days vs. 11.9 days, respectively; p = .010). CONCLUSION: In this study, EVAR was found to be equal to OSR in terms of 30 day and 1 year mortality. However, EVAR was associated with less severe complications and less consumption of hospital resources than OSR.

Changes in chemical coding of sympathetic chain ganglia (SChG) neurons supplying porcine urinary bladder after botulinum toxin (BTX) treatment.

Botulinum toxin (BTX) is a neurotoxin used in medicine as an effective drug in experimental therapy of neurogenic urinary bladder disorders. We have investigated the influence of BTX on the chemical coding of sympathetic chain ganglia (SChG) neurons supplying the porcine urinary bladder. The toxin was injected into the wall of the bladder. SChG neurons were visualized by a retrograde tracing method with fluorescent tracer fast blue (FB) and their chemical coding was investigated by double-labelling immunohistochemistry with antibodies against dopamine ß-hydroxylase (DßH; a marker of noradrenergic neurons), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), Leu(5)-enkephalin (L-ENK) and neuronal nitric oxide synthase (nNOS). In both the control (n = 5) and BTX-treated pigs (n = 5), the vast majority (91 ± 2.3 % and 89.8 ± 2.5 %, respectively) of FB-positive (FB+) nerve cells were DßH+. BTX injections caused a decrease in the number of FB+/DßH+ neurons that were immunopositive to NPY (39.5 ± 4.5 % vs 74.5 ± 11.9 %), VIP (8.9 ± 5.3 % vs 22.3 ± 8.8 %), SOM (5.8 ± 2.3 % vs 17.4 ± 3.7 %) or GAL (0.9 ± 1.2 % vs 5.4 ± 4.4 %) and a distinct increase in the number of FB+/DßH+ neurons that were immunoreactive to L-ENK (3.7 ± 2.9 % vs 1.1 % ± 0.8 %) or nNOS (7.7 ± 3.5 % vs 0.8 ± 0.6 %). Our study provides novel evidence that the therapeutic effects of BTX on the mammalian urinary bladder are partly mediated by SChG neurons.

Inhalable desert dust, urban emissions, and potentially biotoxic metals in urban Saharan-Sahelian air.

Saharan dust incursions and particulates emitted from human activities degrade air quality throughout West Africa, especially in the rapidly expanding urban centers in the region. Particulate matter (PM) that can be inhaled is strongly associated with increased incidence of and mortality from cardiovascular and respiratory diseases and cancer. Air samples collected in the capital of a Saharan-Sahelian country (Bamako, Mali) between September 2012 and July 2013 were found to contain inhalable PM concentrations that exceeded World Health Organization (WHO) and US Environmental Protection Agency (USEPA) PM2.5 and PM10 24-h limits 58 - 98% of days and European Union (EU) PM10 24-h limit 98% of days. Mean concentrations were 1.2-to-4.5 fold greater than existing limits. Inhalable PM was enriched in transition metals, known to produce reactive oxygen species and initiate the inflammatory response, and other potentially bioactive and biotoxic metals/metalloids. Eroded mineral dust composed the bulk of inhalable PM, whereas most enriched metals/metalloids were likely emitted from oil combustion, biomass burning, refuse incineration, vehicle traffic, and mining activities. Human exposure to inhalable PM and associated metals/metalloids over 24-h was estimated. The findings indicate that inhalable PM in the Sahara-Sahel region may present a threat to human health, especially in urban areas with greater inhalable PM and transition metal exposure.

Long-term prognosis of nerve palsy after total hip arthroplasty: results of two-year-follow-ups and long-term results after a mean time of 8 years.

INTRODUCTION: Nerve damage is a rare but serious complication after THA. There exist only little data about the outcome of these patients particularly regarding the long-term results later than 2 years postoperatively. Aim of this study is to answer the following questions: Is the recovery to be expected for light nerve lesions different from the severe ones? Is there a possibility of nerve recovery more than 2 years after THA? Is the potential of nerve recovery depending on the affected nerve? MATERIALS AND METHODS: This study investigates 2,255 primary THA as well as revision surgeries performed from 1988 to 2003 relating to iatrogenic nerve lesion. We classified the nerve lesion according to the core muscle strength in severe (M0-M2) and light (M3-M4) nerve damage and differentiated between femoral, sciatic and superior gluteal nerve, according to the electromyography. RESULTS: We found 34 cases of iatrogenic nerve damage representing an incidence of 1.5 %. 17 of 34 (50 %) patients showed a complete recovery after 2 years. Out of the remaining 17 patients, six out of seven patients with a final examination after a median time of 93 months achieved further improvement. The different nerves showed no significant different potential of recovery. CONCLUSIONS: In contrast to the literature, an improvement beyond the limit of 2 years is probable and independent of the nerve affected.

Changes in the cholinergic innervation pattern of porcine ovaries with cysts induced by dexamethasone administration.

We revealed earlier that induction of ovarian cysts in gilts by dexamethasone phosphate disodium salt (DXM) administration from the follicular phase of the estrous cycle (EC) changed the cholinergic innervation of the gonad. In the present study, the innervation of porcine ovaries by vesicular acetylcholine transporter (VAChT)-, neuronal nitric oxide synthase (nNOS)-, vasoactive intestinal peptide (VIP)- and somatostatin (SOM)-immunoreactive (IR) fibres, after induction of cystic changes from the middle luteal phase of the EC, was determined. The cystic changes were induced by DXM injections from days 7 to 21 of the EC, and 11 days later, the ovaries were collected. In the cystic ovaries, VAChT-, nNOS- and SOM-IR fibres were found around cysts and small tertiary follicles; nNOS-IR and also VAChT-IR fibres were observed near secondary follicles and veins; and VAChT- and nNOS-IR fibres were not found around cortical arteries. The number of VIP-IR fibres increased near the cysts and within the ground plexus, while the number of VAChT-IR fibres decreased within the medullar part of this structure. Thus, our study showed changes in the cholinergic innervation pattern of the porcine cystic ovaries induced from the middle phase of the cycle and confirmed that cystic ovary innervation depends partly on the phase of the EC in which the induction of cysts was started.

The noradrenergic innervation and steroidogenic activity of porcine cystic ovaries.

The aim of the present study was to investigate the distribution and density of noradrenergic nerve fibres (NNFs), content of catecholamines (CATs) and steroids in the cystic ovaries of gilts receiving DXM from middle luteal phase. Cystic status of ovaries was induced by i.m. DXM injections on days 7-21 of the estrous cycle. During the same time, gilts in the control group received saline. The ovaries were collected on predicted day 11 of the second studied estrous cycle. The cystic ovaries were supplied by more numerous NNFs than the control gonads. Moreover after DXM injections, the content of CATs and progesterone and androstendione (A(4)) in the cystic wall were elevated, while the levels of A(4), testosterone and estradiol-17beta in the cystic fluid were lowered. Our results show that in the porcine cystic ovaries, induced by DXM injections from middle phase of estrous cycle, increased the density of NNFs and level of CATs, and that it was accompanied by changes in the content of steroids. Moreover, this study is a further confirmation that the morphological and functional changes of cystic ovaries are partly dependent on phase of the estrous cycle in which the induction of the ovarian cysts was initiated.

Tetrodotoxin induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder.

Tetrodotoxin (TTX) mode of action is based on a blocking of fast sodium channels in nerve cell membrane what, in turn, abolishes the propagation of the action potential along the nerve fibers. TTX is currently used in experimental therapies focused on neoplastic or neurogenic pain, however, as for now there is no data concerning the influence of TTX on dorsal root ganglion (DRG) sensory neurons function. Thus, the present study was aimed at characterization of neurochemical coding of porcine sensory bladder-projecting cells after bladder instillation with TTX. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall of six juvenile female pigs and three weeks later bladder instillation with TTX (12 microg per animal) was carried out in all animals. A week later, DRGs of interest were harvested from all animals and the neurochemical characterization of FB+ neurons was performed using routine double-immunofluorescence labeling technique on 10-microm-thick cryostat sections. In TTX-treated animals the number of FB+ cells containing galanin (GAL), nitric oxide synthase (NOS), somatostatin (SOM) and calbindin (CB) was 2.5%, 2%, 0.25% and 0.2%, respectively and that of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive (IR) cells was 43%. These data when compared with previous reports, demonstrated that TTX profoundly changed the chemical coding of porcine bladder-projecting sensory neurons thus implicating that it may be used in case of hypoactivity of afferent part of reflex arc responsible for transmission of sensory information from the urinary bladder.