Supplemental Digital Content is available in the text The angiosome concept provides practical information regarding the vascular anatomy of reconstructive and vascular surgery for the treatment of peripheral arterial occlusive disease and, particularly, critical lower limb ischemia.The aim of the study was to confirm the efficacy of direct revascularization with the angiosome concept (DR) for lower limb ischemia.Complementary manual searches were performed through the Pubmed, Cochrane Library, and EMBASE databases.We searched all randomized and nonrandomized studies (NRSs) comparing DR with indirect revascularization (IR) (without the angiosome concept) for lower limb ischemia. Only 9 nonrandomized controlled retrospective cohort studies were found and included. Trials published in any language were included.Primary endpoints were time to limb amputation and time to wound healing. Data extraction and trial quality assessment were performed by two authors independently. A third author was consulted for disagreements settlement and quality assurance.P < 0.001; I2 = 0%). In addition, DR significantly improved time to wound healing .Five NRSs involving 779 lower limbs revealed that DR significantly improved the overall survival of limbs (hazard ratio [HR] 0.61; 95% confidence interval [CI] = 0.46–0.80; All included studies were retrospective comparative studies, and no consensus was obtained in describing wound conditions in the included studies.Our results suggested that treatment of lower limb ischemia using DR is more effective in salvaging limbs and healing wounds than IR is. Additional randomized controlled studies are necessary to confirm these results. Guidelines from the Transatlantic Inter-Society Consensus II (TASC-II) and the American College of Cardiology/American Heart Association recommend multidisciplinary approaches to reduce the frequency of foot complications in CLI patients.3 Early revascularization intervention with bypass or endovascular surgery, particularly for high-risk patients, is considered to be the gold standard in reducing the possibility of hospitalization and amputation.6 Nevertheless, current revascularization intervention strategies, which restore circulation to a nontargeted artery, have a 15% failure rate in healing CLI wounds. Such a high rate suggests that increasing adequate blood supply to feeding arteries at the distal occluded lesion-site might be crucial in improving the results of intervention.Peripheral arterial occlusive disease (PAOD) is a major disease that limits active aging in elderly people. Complications of PAOD are the leading cause of hospitalization and amputation for people with lower limb ischemia, and account for billion-dollar expenditures annually in the United States.8 According to the angiosome concept, the foot is divided into 6 distinct angiosomes fed by source arteries, 3 from posterior tibial, 2 from peroneal, and 1 from anterior tibial artery, with functional vascular interconnections between muscle, fascia, and skin.9 Numerous direct arterial-to-arterial connections exist between the main arteries of the foot, and these connections provide alternative routes of blood flow when the arteries that directly supply the angiosome is either disrupted or compromised.9 Therefore, the angiosome concept suggests that recanalization of the artery that is directly supplying the ischemic and/or ulcerated angiosome, instead of revascularizing one of the other 2 major arteries hoping that existing arterial-to-arterial connections will provide blood perfusion to the ischemic and/or ulcerated angiosome, might be more successful.9The concept of angiosomes, first described by Ian Taylor, provides practical information on the application of vascular anatomy for reconstruction and vascular surgery in the treatment of PAOD, and particularly on the treatment of CLI.10 However, we performed a systematic review and meta-analysis investigating the efficacy of DR, comparing it with conventional IR for the treatment of CLI patients.It is unclear whether direct revascularization with the angiosome concept (DR) can provide superior results for CLI patients than that of conventional indirect revascularization (IR) without the angiosome concept. One recent review revealed that evidence is insufficient to recommend DR in CLI patients.11 Searches were not restricted by publication status, date, or language. The search keywords included angiosome, angioplasty, endovascular, revascularization, endoluminal, transluminal, and bypass. In addition, MeSH terms were explored. The final results were combined with the following keywords: lower limbs, extremities, and foot. The databases we used to conduct our searches were Pubmed (from 1948 to March 2013), the Cochrane Library (latest issue published March 2013), and EMBASE (January 1980–March 2013). The search strategy is provided in Appendix 1. Databases of clinical trials  reference lists of reviews had also been searched to identifying relevant trials.This protocol-driven and systematic review was conducted in accordance with PROSPERO published protocol and analysis planning .12 Reviews, case series, case reports, and trials without comparisons between DR and IR were excluded. Primary outcomes included time to limb amputation, time to wound healing, and mortality rate.In the literature search, titles, abstracts, and full texts of trials identified were independently screened by three authors . Articles with comparisons between DR and IR were included if adults (≥18 years old) with critical lower limb ischemia (as defined by TASC-II) had been treated using either endovascular surgery or conventional bypass surgery.Characteristics of studies , participants , interventions , comparisons (types of control group), and outcomes (various outcome measurements and follow-up times) were recorded. In studies with multiple arm designs, head-to-head comparison data were extracted for data synthesis. Time to amputation and time to wound healing were used as the primary outcomes.12 Because the included studies were all nonrandomized studies (NRSs), we assessed methodological quality by using the Newcastle–Ottawa Quality Assessment Scale (NOS).13 Three determinants composed the NOS system, including selection scores, outcome scores, and comparability scores.13 Studies with NOS ≥8 points were defined as high-quality studies, whereas other studies as low-quality studies.Data extraction and quality assessment were performed by two authors (T-YH and C-HY) independently. A third author (T-SH) was consulted for disagreements settlement and quality assurance. The risk of bias tool of the Systematic Reviews of Interventions from the Cochrane Handbook was utilized for determination of methodological quality.14 by using a spreadsheet developed by Tierney et al,15 as described earlier. For 12-month amputation rate and 12-month wound healing rate, we defined amputation and wound healing as events. Clinical heterogeneity were assessed by comparing the protocols and methodologies of the included studies, and assessed statistical heterogeneity with the χ2 test results , and the I2 statistic, where I2 < 25%, 25% ≤ I2 ≤ 50%, and I2 > 50% indicates mild, moderate, and substantial heterogeneity, respectively.17 Subgroup analysis based on study quality or intervention  was conducted, and data synthesis and statistical analysis were conducted using Review Manager . A funnel plot was created to evaluate publication bias, and significance level was set at 0.05.For time to event outcome, hazard ratios (HRs) with 95% confidence intervals (CIs) were extracted in primary studies as the size to estimate the overall effect of treatment. If data were not provided, the effect size was calculated in accordance with methods suggested by Parmar et al27 No randomized control trial was identified, and all studies were retrospective cohort study. Iida et al published two studies in 201020 and 2012.24 The study of 2010 analyzed patients from April 2003 to August 2008. The study of 2012 analyzed patients from April 2004 to October 2010 using matching method. Therefore, we used the study of 2012 to conduct meta-analysis. All the DR studies followed the Taylor's angiosome concept. Three studies 25, which utilized propensity score matched comparison between DR and IR groups, were analyzed. Table 28 was excluded because their results provided subgroup data only, which could not be analyzed. All included trial patients were in either Rutherford Class 5 or 6 and Fontaine Stage 4.30 The key characteristics of patients included in all studies are shown in Table 26 We did not perform further subgroup analysis because only Varela et al19 had described the subgroup of IR with collateral vessels.Overall, 10 NRSs were included, as shown in Figure The risks of bias for all NRSs are shown in Table http://links.lww.com/MD/A394, Table S2, http://links.lww.com/MD/A394, and Table S3, http://links.lww.com/MD/A394. Although 9 NRSs reported a limb salvage rate,27 and 8 NRSs reported the wound healing rate, the follow-up periods were different in individual study.27 To include more studies into our meta-analysis, we used time to amputation and time to wound healing as our primary endpoints. In addition, we could not find adequate information with regard to the data of loss-to-follow-up in primary studies. However, we analyzed 12-month amputation rate and 12-month wound healing rate using available information. A total of 719 limbs were treated with DR, and 493 limbs were treated with IR. Five studies included total endovascular treatment (EVT), another study included traditional bypass surgery, and 3 studies included both surgical and EVT interventions.Reported outcomes in primary studies were shown in Table S1, 26 recorded the limbs of the DR and IR groups (247 limbs vs 52 limbs), but only presented outcomes by counting individual patients (167 patients vs 34 patients). Therefore, we calculated the data in our meta-analysis according to the number of patients.Fossaceca et alP < 0.001; Table P = 0.69, I2 = 0%). According to study quality and intervention methods, subgroup analysis was performed, which showed little evidence of interaction .We analyzed 12-month amputation rate using 4 available studies. Our result showed the DR group significantly reduced 12-momth amputation rate compared with IR group . Subgroup analyses according to study quality and intervention methods showed little evidence of interaction .We analyzed 12-month wound healing rate using 4 available studies. Our result showed the DR group significantly improved 12-momth wound healing compared with IR group .Mortality rate was only reported in 4 studies. Neville et alPublication bias was analyzed using a funnel plot, which was symmetrical Figures  and 7.Our meta-analysis provided evidence that DR significantly improves time to amputation and time to wound healing for CLI patients. However, insufficient information was available to conduct a meta-analysis on mortality. The angiosome concept was constructed based on the anatomy of blood circulation, which demonstrates that superior blood supply can improve tissue growth and wound healing.25 The result of Iida et al showed that DR significantly decreased the amputation rate . Azuma et al and Söderström et al were also comparing the outcome of time to wound healing. Söderström et al supported that DR group significantly increased wound healing rate (P < 0.001). However, Azuma et al showed no difference for wound healing between 2 groups (P = 0.185).Three studies performed matching in their data analysis .P < 0.01) and limb salvage rate 24 months following intervention  for CLI patients. Varela et al further revealed that distal peroneal arterial connections  and the patent pedal arch played a significant role in wound healing and limb salvagability in CLI patients who were treated without using the DR model.19 This suggested that the possible cause of IR treatment failure resulted from inadequate vascular connections between the revascularized arteries and the ischemic region. Therefore, a patent pedal arch or peroneal distal branches that restore blood flow to the ischemic area through collateral vessels might show a similar result in limb salvagability and wound healing as that obtained through the specific source arteries.19Varela et al confirmed that DR model treatment improved the wound healing rate 12 months following intervention .36 Advantages of EVT intervention include less surgical trauma, a smaller wound, fewer local complications, and shorter hospital stays.32 However, subgroup analysis for comparisons between EVT intervention and surgical intervention could not be assessed in this study.Although current AHA/ACC guidelines suggest open bypass still the preferred operation for patients who would live for >2 years, traditional surgery and EVT have been compared in several studies.2 Furthermore, diabetic patients have an impaired host defense system against infections.38 Azuma et al23 stated that diabetes is one of the risk factors in prolonged tissue healing time. Iida et al showed that higher hemoglobin A1c levels were a significant predictor of major amputation in a DR group. They postulated that the increased risk is most likely attributable to poor periprocedural blood glycemic control rather than to the presence of DM during the postoperative period.24 Three studies compared DR in diabetic patients.26 However, our study compared the outcomes of these 3 studies with the others, which had a distinct percentage of diabetic patients (>64%), and minimal heterogeneity was identified . No other studies comparing diabetic and nondiabetic patients have been published. DR might have beneficial effects for wound healing in diabetic patients; however, the effects on nondiabetic patients require further investigation.DM has been recognized as a critical predicting factor for wound healing. Failure of ulcers to heal in the feet of diabetic patients might have resulted from poor vascular connections between angiosomes, which provided inadequate blood perfusion to the ischemic areas. In addition, TASC II guidelines indicate that the amputation rate was 5 to 10 times higher in diabetic patients than in nondiabetic patients because blood flow to the microvascular beds appears to be reduced in the feet of diabetic patients.23 proved that DR treatment could significantly shorten the time needed for wound healing in the entire study cohort, and that it improved limb salvage rate in end-stage renal disease (ESRD) patients (P < 0.01). However, after propensity-score matching, the differences between limb salvage rate and wound healing rate in the DR and IR groups were lost. Azuma el al concluded that the angiosome concept might be unimportant in the field of bypass surgery, unlike EVT intervention. However, our analysis revealed that the DR model concept could be consistently applied to all patients, regardless of surgical or EVT intervention.Azuma et al23 and several studies have reported that patients with ESRD have higher amputation rates.41 Johnson et al39 postulated that healing problems account for higher amputation rates rather than graft thrombosis. Thus, some studies have recommended that bypass surgery should be performed on carefully selected ESRD patients because of potential negative outcomes.43 However, no standard exists for selected ESRD patients.44 Some studies have reported that hypoalbuminemia, which might result from inflammation instead of malnutrition,46 detrimentally related to the life prognosis of ESRD patients.41 Azuma et al separated their patients into 3 groups: non-ERSD, ESRD without severely low albuminemia, and ESRD with severely low albuminemia (<3.0 g/dL). The wound healing rate of the ESRD in the low albuminemia group was significantly worse than in the other groups (P < 0.01). Their subgroup analysis demonstrated that DR significantly improved wound healing rate both in non-ESRD patients and in ESRD patients as compared with IR. However, comparing DR and IR in all patients showed no beneficial effect in their study (P = 0.185). They concluded that ESRD and the level of serum albumin were more critical than the angiosome concept.23ESRD was a crucial risk factor for wound healing and limb salvagability,47 The improvement of microvascular circulation has been reported as one of the clinical benefits of cilostazol.48 Iida et al formed 2 groups and recorded the outcome of skin perfusion pressure (in mm Hg) on the time before and after surgery.24 Skin perfusion pressure was similar to that before intervention , and it was statistically higher after EVT in the cilostazol-treated group than in the noncilostazol-treated group .24 Therefore, cilostazol might help to improve microcirculation; however, further evidence of amputation prevention or improvement in wound healing is necessary.Cilostazol was typically used in PAOD patients as an antiplatelet drug.In this study, we performed throughout literature searches. For evaluation of time-to-event outcomes, we utilized HR as the metric of the summary effect size. The calculated HR was the relative hazard of an event occurring in the DR group compared with that of the IR group. From our result, it suggested that patients who received DR had decreasing risk of limb amputation and wound unhealing by 0.61 (95% CI = 0.46–0.80) and 1.38 (95% CI = 1.13–1.69) in the PAOD patients compared with patients received IR interventions. However, our study has several limitations. First, all included studies were retrospective comparative studies. Angiosome concept was delicate. It was trivial to approach the target feeding artery for the ischemia area. Therefore, there were too many operation methods for vascular access that all the retrospective studies could not have a very specific and appropriate design. Second, another source of bias might come from the confounding bias because of patients’ condition. Only Azuma et al, Iida et al, and Söderström et al performed matching in their analyses. The bias existed among these studies resulting from the differences of the selection and grouping criteria for these patients. The mortality rate of the patients treated with IR was higher than those treated with DR might imply that the patients in the IR had more comorbidities than those in the DR group. Third, no consensus was obtained in describing wound conditions in the included studies. The Rutherford and Fontaine stage was used in only 4 studies, but they only recorded stages. The locations, numbers, infection status, and surgical debridement procedures of these wounds were not recorded in numerous studies, which were major confounders in our analysis. The wound healing rate and the treatment strategy for a gangrenous wound would be much different than that of a superficial ulcer. None of the studies reported postoperative wound care programs. Infections, antibiotic treatment, and debridement surgery are all additional concerns in the included studies. Fourth, the detail description for the vascular lesions were absent. Future randomized study should report the detail of these vascular lesions, such as length, location, stenotic status, collateral vessel of the lesions as well as the status of the pedal arch. It is possible that the patients in the DR group exhibited superior vascular quality and that the target vessel was more easily approached, whereas patients in the IR group might have had either total occlusion or vessels that were small in diameter and had degenerated. Thus, the 2 groups were at distinct stages.In conclusion, the angiosome model of revascularization was beneficial for patients with critical lower limb ischemia when considering limb salvagability and wound healing. Nonetheless, randomized controlled studies are necessary to confirm our results. Increasing the limb salvage rate is anticipated to improve daily activity and could prolong the survival of patients. Thus, a broad prospective study should be conducted to confirm the effect of the angiosome model concept. All the following characteristics should be recorded in detail, including the wound condition and location with standard recording system, the treatment of the wound , the detail description of the stenotic status, the collateral vessels, and the condition of the pedal arch. Collateral vessels should be defined more carefully because some patients, especially diabetic patients, are collateral artery-dominant blood supply. Crucial confounding factors, such as DM, ESRD, serum albumin levels, and medications, should also be reported and analyzed.


To examine the cross sectional and longitudinal relationship between cardiovascular risk factors and age-related macular degeneration (AMD) in a large British cohort study.The EPIC Norfolk Eye study is nested in a larger prospective cohort study. Data on cardiovascular risk factors were collected at baseline (1993-1997) and follow up (2006-2011) via clinical examination, validated lifestyle questionnaires and serum blood samples. AMD was ascertained using standardised grading of fundus photographs at the follow up. Logistic regression was used to examine associations between baseline and follow up risk factors with AMD.5,344 pairs  of fundus photographs were of sufficient quality for grading of AMD in participants with mean age of 67.4 years old (range 44-91) at diagnosis. There were 28 cases of late AMD =0.3-0.8%) and 645 cases of early AMD . In multivariable analysis, older people with higher levels of baseline high density lipoprotein- cholesterol (HDL-C ) and C-reactive protein (CRP) were more likely to have any signs of AMD, after adjusting for sex, education, smoking, and systolic blood pressure. In cross sectional analysis, only older age and higher HDL were significantly associated with AMD.We have found that older age and higher levels of CRP and HDL-C were associated with increased odds of AMD in this population in the longitudinal analysis, but older age and HDL-C, not CRP was significantly associated with AMD in the cross sectional analysis. The prevalence of AMD in this cohort was low compared to other cohorts in Europe, the US and Australia, and probably reflects the some selection biases in follow up participation as well as the low rate of smoking among our healthy participants. The disease can progress to either geographic atrophy (“dry” AMD) or choroidal neovascularisation (“wet” AMD), both of which can have devastating effects on central vision in late stages of the disease. AMD causes approximately 5% of global blindness.[Age-related macular degeneration (AMD) is a progressive chronic eye disease and the leading cause of low vision and blindness in developed countries.lindness., and prolindness.. Severallindness.,5 Pooledlindness.Several studies have shown a consistent effect of cardiovascular risk factors on greater prevalence and incidence of AMD.,7–11 An In this study, we examined the cross sectional and longitudinal associations of cardiovascular risk factors and AMD.36 The study was approved by the Norfolk Local Research Ethics Committee and adhered to the Declaration of Helsinki. All participants gave written informed consent.The European Prospective Investigation into Cancer (EPIC) and Nutrition study is a 10 country collaborative cohort study investigating lifestyle and nutritional risk factors for cancer. Detailed descriptions of the EPIC study methods and recruitment have been reported previously.,28,29 Th2. The measurement of habitual physical activity and visual acuity (VA) in EPIC-Norfolk has been previously described. The physical activity scale used has been validated against heart rate monitoring with individual calibration in independent studies.[The health examinations were carried out by trained nurses using standard operating protocols. Systolic and diastolic blood pressure (sBP and dBP) were recorded as the mean of two measurements separated in time, taken from the right arm with the participant seated for 5 minutes, using an Accutorr Plus blood pressure monitor . Height and weight were measured with participants dressed in light clothing and shoes removed. A stadiometer was used to measure height to the nearest 0.1metre (m), and the Tanita body composition analyser model TBF 300s  was used to measure weight to the nearest 100g. Body mass index (BMI) was calculated as weight in kg/height squared in mg for 15 minutes at 4°C, and serum samples were kept frozen at −80°C until being thawed in 2008 for CRP assaying. Non-fasting serum total and high-density lipoprotein- cholesterol (HDL-C) levels (mmol/l) were measured at both health examinations using an RA 1000 Technicon analyser . Low-density lipoprotein-cholesterol (LDL-C) was determined using the Friedewald formula. Social class was recorded according to the UK Registrar-General’s occupation-based classification system; this was based on the participant’s last occupation if they were retired. Educational level was recorded and classified into four groups according to the highest qualification achieved. The four categories are less than O levels, O levels, A levels and degree or above; O levels are qualifications gained at the end of secondary school after 11 years of schooling, and A levels after 13 years of schooling, with degrees obtained in university settings. Smoking status was recorded as current, former and never. The EPIC Norfolk database was linked to national hospital discharge data for Norfolk residents (ENCORE). Prevalent ischaemic heart disease (IHD) and stroke in this study were ascertained through self-rated disease at baseline (between 1993–1997), subsequent health examinations and any recorded episodes of IHD or cardiovascular accident (CVA) from hospital episodes statistics (HES) data accumulated from baseline until 2009.Serum high-sensitivity CRP was measured using the Olympus AU640 Chemistry Immuno Analyzer . Blood samples collected at baseline examination were centrifuged at 2,100Hard drusen size <63 μm, with more than ten hard drusen required to be present for the lesion to be classified as presentSoft drusen of size ≥125μm, with presence of one soft drusen sufficient for classification of lesion to be presentGeographic atrophyChoridal neovascularisationRetinal Pigment Epithelium (RPE) detachmentDisciform scarDigital fundus photographs of the optic disc and macula were taken using a TRC-NW6S non-mydriatic retinal camera and IMAGEnet Telemedicine System  with a 10 megapixel Nikon D80 camera  without pharmacological dilation of the pupil. AMD was categorised by independent graders using a modified Wisconsin protocol. Main feThe predominant phenotype observed or the most severe lesion for each eye was used as the final grading for that eye. Individual categorisation of AMD lesion was based on the more severely affected eye.2 test for categorical variables to compare different groups. AMD was analysed as a binary variable with any AMD (including early disease) as the main outcome due to small numbers of people with advanced AMD. Smokers were dichotomised into “never” and “ever” for current and past smokers, due to the relatively small proportion of current smokers. A right skew was evident in the CRP distribution. Therefore, CRP was categorised into clinically relevant categories, ≤1, 1.1–3, 3.1–10, ≥10mg/L for initial descriptive and univariable analysis; subsequently both categorical CRP and a log transformed continuous CRP were analysed separately to maximise power to detect any differences. Univariable associations with any AMD as the dependent variable were explored using logistic regression, tabulation and χ2 test. A stepwise multivariable logistic regression model was used to examine the effect of detected and a priori risk factors on AMD. Odds ratio for AMD were estimated per 1 standard deviation (SD) increase in log transformed CRP. Indicator variables were used with all categorical variables in the multivariable analysis, with interaction terms to test for evidence of effect modification. All statistical analyses were conducted using STATA 12 .The data were initially explored through descriptive analysis of variables using t-tests for quantitative and χOf 8,623 participants examined in the EPIC Norfolk Eye study, retinal photographs were obtained from 7,501 (87.0%) participants, of which 5,344 pairs of left and right eyes (62.0%) were of sufficient quality for grading of AMD related fundus lesions. Participants with missing or excluded photographs were older (70.8 years vs. 67.4 years p<0.01) and more likely to be male (47.5% of those missing vs 43.1% with gradable photographs p<0.01), with lower levels of education . Similar proportions of smokers did not have gradable photos compared to those with grading (8.6% vs 9.4% missing p = 0.23). The mean age of the 5,344 included participants was 67.4 years (range 48.4–91.9 years), with 56.9% women. Women were younger than men, with a mean age of 66.9 years compared to 68.1 years for men (p<0.01). Older men and women both had higher BMI, sBP, dBP, LDL-C, total cholesterol, triglycerides, and CRP. Older people were also more likely to have lower attained levels of education, to be less active, and more likely to have never smoked. Older men and women also had lower levels of HDL-C, though this was not statistically significant.The study prevalence of AMD is shown in Univariable associations between both baseline and follow up clinical variables and AMD diagnosed at the third health check are shown in Although the results indicated that people with AMD were more likely to be inactive, the association was no longer statistically significant after adjusting for age and sex. At baseline, 50 people reported strokes, with 152 recorded by third health examination, with no evidence o f an association with AMD at any time point . Similarly, 62 people had reported myocardial infarction at baseline with 161 at follow up with no associations observed with AMD  at baseline or at the third health examination .In multivariable analysis shown in In this community based study of 5344 older people, we found that higher levels of HDL and CRP at baseline, but neither smoking nor other cardiovascular risk factors, were associated with a higher occurrence of AMD. HDL was the only cardiovascular risk factor associated with AMD in the cross sectional analysis.There was an overall low study prevalence of AMD, with only 12.6% (95%CI = 11.7–13.5%) of participants with signs of AMD and 0.5% (95%CI = 0.3–0.7%) with geographic atrophy or NV-AMD. This was lower than the prevalence reported in epidemiological studies in predominantly White populations of the United States (US), Australia and the Netherlands, where late AMD (geographic atrophy or neovascular AMD) was observed in 1.6, 1.9 and 1.7% respectively.–34 The lCFH),[CFB/C2),[C3),[CFI)[The observed association with baseline CRP was consistent with findings from a review of observational studies by Hong et al., where thCFH),,36 compl[CFB/C2), complemeC2),[C3), and compC3),[CFI) are assoPrevious reports on the association between HDL and AMD have been inconsistent, with studies showing positive, inverse or no association.,19,24 WeWe did not detect an association between smoking and AMD, which contrasts strongly with several epidemiological studies and meta- analyses where smoking is associated with a two fold increase in neovascular AMD risk. EpidemioThere were limitations in our study. The present study was an average 17 year follow up of a larger longitudinal study, with healthier survivors and a lower frequency of risk factors and outcomes, which would reduce the likelihood of detecting true associations. People with AMD or cardiovascular disease were also less likely to attend due to relatively poor health and/or vision. However, both factors resulting in truncation of the distribution would be likely to attenuate any underlying associations. In addition to reduced power, there may also have been measurement error in assessment of AMD. However, the use of a standardised objective grading system would have mitigated potential systematic errors. We also used non-mydriatic photographs, and 28.8% of photos were of insufficient quality for grading. Those with ungradable photos were older and more likely to have AMD, further reducing power. The predominant phenotype approach may have reduced detection of small differences between clinical subtypes, but would have increased reliability of grading outcomes. We were only able to ascertain the outcome in the follow up study and cannot exclude prevalent cases at baseline, however, as there was a very low prevalence of late AMD In the follow up, there were unlikely to have been many cases of AMD at baseline. Furthermore, excluding late cases does not alter the interpretation of the final model, indicating that baseline risk factors were likely to have been present prior to disease, and also the associations detected relate primarily to early AMD. Additional cardiovascular risk factors known to be associated with AMD such as dietary intake of oily fish and intake of leafy green vegetables were not included in this analysis because it was beyond the scope of the current investigation, which focused on more proximal markers of cardiovascular risk. Further research into dietary factors and AMD are important because they are modifiable risk factors and could inform public health action in the prevention of AMD.In summary, we have found that older age, higher baseline and follow up levels of HDL, and baseline CRP were associated with increased odds of AMD. The prevalence of AMD in this cohort was low compared to other cohorts in Europe, the US and Australia, and possibly reflects healthier participants as well as the low rate of smoking among our participants.


Deep muscle relaxation is known to improve the surgical conditions during laparoscopic surgery. We aimed to compare the effects of deep and moderate neuromuscular block in laparoscopic colorectal surgery, including intra-abdominal pressure.Carbon dioxide  or deep  neuromuscular block by receiving a continuous infusion of rocuronium. Adjusted intra-abdominal pressure, which was titrated by a surgeon with maintaining the operative field during pneumoperitoneum, was recorded at 5-minute intervals. Perioperative hemodynamic parameters and postoperative outcomes were assessed.P < 0.001). The postoperative pain scores (P < 0.001) and incidence of postoperative shoulder tip pain were lower, whereas gas passing time (P = 0.002) and sips of water time (P = 0.005) were shorter in the deep neuromuscular block than in the moderate neuromuscular block group.Six patients from the deep and 5 from the moderate neuromuscular block group were excluded, leaving 61 for analysis. The average adjusted IAP was lower in the deep compared to the moderate neuromuscular block group (9.3 vs 12 mm Hg, Deep neuromuscular blocking showed several benefits compared to conventional moderate neuromuscular block, including a greater intra-abdominal pressure lowering effect, whereas surgical conditions are maintained, less severe postoperative pain and faster bowel function recovery. However, CO2 absorption and elevated IAP during pneumoperitoneum can cause specific pathophysiological effects such as cardiovascular, pulmonary, and splanchnic perfusion changes.2 Thus far, several studies have been conducted in an effort to reduce CO2 pressure and minimize adverse effects of pneumoperitoneum and have reported postoperative pain relief after low-pressure pneumoperitoneum.5 However, in these studies, an adequate surgical field or conditions were not considered.Compared with open surgery, laparoscopic surgery has the advantages of reduced intensity of postoperative pain and hospital stay, and improved cosmetic results and patient satisfaction.8 and the risk of a delayed respiratory function recovery due to a deep neuromuscular block (NMB) was avoided by using sugammadex.10 Sugammadex , a selective relaxant binding agent, is a modified g-cyclodextrin, especially created to bind the free plasma molecules of the neuromuscular blocking agent rocuronium to which it has high affinity.11 However, there is little evidence of a deep NMB effect that would allow the clinical use of a lower insufflation pressure in laparoscopic surgery.In previous studies, deep muscle relaxation was shown to improve surgical conditions in laparoscopic cholecystectomy, urologic, and gynecologic surgery,In the present study, we aimed to evaluate the extent of IAP reduction of a deep NMB against a conventional moderate NMB, while maintaining a satisfactory surgical exposure, and compare the perioperative physiological functions and postoperative recovery profiles of patients who received deep or moderate NMB during laparoscopic colorectal surgery.2).This was a single-center, prospective, double-blind, randomized controlled trial. The study protocol was approved by the Institutional Review Board and Hospital Research Ethics Committee of Severance Hospital at the Yonsei University Health System in Seoul, Republic of Korea, on 21 July on 2014 (# 4–2014–0559). This was registered at clinicalTrials.gov (NCT02266056) on 26 August on 2014. The informed consent was given to the all subjects, and written consent was obtained from all patients. Adult patients, who underwent elective laparoscopic colorectal resection surgery and had the American Society of Anesthesiologists (ASA) physical status classification of I–III, were assessed for eligibility. Exclusion criteria included: (1) patients aged >80 years, (2) inability to provide informed consent, (3) allergy to rocuronium or sugammadex, (4) a neuromuscular disease or personal history or family history of malignant hyperthermia, (5) a serious heart, kidney, or liver condition, (6) previous colorectal surgical history and obesity (body mass index ≥ 35 kg/mwww.randomizer.org/form.htm), and assignments were concealed in a sealed envelope. The randomization was not blocked or stratified.Patients, who were enrolled in this clinical trial, were randomly assigned to the deep NMB or moderate NMB group in a 1:1 ratio by using a computer-generated random number table . Patients were given 0.2 mg of glycopyrrolate intravenously (IV), and anesthesia was induced with a bolus administration of 1.5 to 2 mg/kg of propofol and 1 to 2 mg/kg of remifentanil. Anesthesia was maintained using 4 to 7% desflurane with an adjuvant IV infusion of 0.05 to 0.2 μg/kg/min of remifentanil. Rocuronium, which is a neuromuscular relaxing agent, 0.6 mg/kg was injected to facilitate tracheal intubation in all patients. Tracheal intubation was performed in female and male patients using a 6.5-mm and 7.5-mm  tracheal tube, respectively. The cuff pressure of the tracheal tube was maintained at 20 to 25 cm H2O throughout the procedure. Mechanical ventilation was maintained with a tidal volume of 8 mL/kg, and the ventilatory frequency was adjusted to maintain an end-tidal CO2 concentration of 35 to 45 mm Hg with an air/oxygen mixture (fraction of inspired oxygen 0.5). Body temperature was maintained at 36 °C to 37°C. BIS scores were maintained in the range of 40 and 60, and mean arterial pressure within 20% of preinduction values. After intubation, arterial cannulation was performed to monitor intraoperative continuous arterial blood pressure and perform arterial blood gas analysis during surgery.On arrival in the operating room, routine monitoring was started and it included electrocardiography, pulse oximetry, noninvasive blood pressure, and capnography. Anesthetic depth was monitored using a bispectral index (BIS) monitor  generates an electrical stimulus to the ulnar nerve and measures contractions adductor pollicis muscle13 (causing adduction of the thumb) through a sensor attached to the tip of the thumb, which was placed in a flexible adaptor that applied a constant preload to the thumb, using a current of 50 to 60 mA with a pulse width of 0.02 ms. This muscle relaxation power was maintained until completing closure of the abdominal fascia. The surgeons, patients, and those assessing outcomes were blinded to the group assignment.After induction, rocuronium was continuously infused and titrated according to the group assignment, to maintain either the post-tetanic count (PTC) at 1 to 2 in the deep NMB group, or train-of-four (TOF) response at 1 to 2 in the moderate NMB group.14At 30 minutes before the end of the surgery, ramosetron 0.3 mg IV for prophylaxis of nausea and vomiting, and paracetamol 1 g for analgesia were administered. Intravenous patient-controlled analgesia (PCA) consisting of fentanyl 15 μg/kg and nefopam 120 mg mixed with normal saline to comprise a total volume of 100 mL was administered at a basal rate of 2 mL/hour, with a bolus dose of 0.5 mL and a 15-minute lockout time, for postoperative pain control in all patients. Upon completion of the surgery, sugammadex 4 or 2 mg/kg IV was used to reverse any possible residual neuromuscular blocking effects in both groups. The tracheal tube was removed when a TOF ratio of 0.9 and spontaneous ventilation were observed and then the patients were transferred to the post-anesthetic care unit (PACU). Patients were transferred to the ward after a minimum of 30 minutes following PACU admission and when they met the modified Aldrete scoring system discharge criteria .2 pressure was initially applied for a pneumoperitoneum at 12 mm Hg, which was routine CO2 pressure in our institution for laparoscopic colorectal surgery, to expand the space within the abdominal cavity. Then, the IAP was controlled as lowly as possible by a surgeon with maintaining the same operative field compared with that of initial IAP 12 mm Hg. The surgeon continuously titrated the IAP to create a necessary operating space.Titrated average IAP during pneumoperitoneum was the primary endpoint. Setting IAP was recorded at 5-minute intervals. In both groups, the same COSecondary outcomes were (1) overall surgical conditions, including visible field and muscle relaxation, estimated by the surgeons using the 5-stage satisfaction , (2) perioperative respiratory and cardiovascular parameters measured and documented at the following time points: before the induction, before the incision, 1 hour after the pneumoperitoneum, and 10 minutes after the end of the pneumoperitoneum, (3) postoperative recovery profiles, assessed by an blinded investigator, including: postoperative abdominal pain intensity, the incidence of shoulder tip pain, analgesic consumption, antiemetic requirement, bowel function recovery parameters , and hospital stay, (4) pain and nausea intensity evaluated using a numeric rating scale (NRS) with a 0 (no pain or nausea) to 10 (worst pain imaginable or nausea) scoring system at 4 time periods .Because there were no previous studies that compared to quantify the difference of IAPs between the patients who received deep NMB and those received moderate NMB, sample size was not calculated. Instead, to analyze the data with normal distribution, 30 patients in each group would be required at least. Therefore we considered in a 20% dropout rate and enrolled 36 patients in each group.P value of <0.05 was considered statistically significant for all comparisons. Continuous variables are summarized as mean ± standard deviation (SD) or median (interquartile range [IQR]) as appropriate, whereas nominal variables are summarized as number of subjects . Normality was tested using the Kolmogorov–Smirnov test as appropriate. Between-group comparisons of continuous variables were performed with Student's 2 sample t test or the Mann–Whitney U test as appropriate. Nominal and categorical variables were compared with the Chi-square test and Fisher's exact test. All analyses were conducted using IBM SPSS 21.0 .Comparisons were made on an intention-to-treat basis, as it is evident from the results that only the patients who actually received allocated intervention were analyzed. A 2-sided Seventy-two patients, who underwent laparoscopic colorectal resection surgery between August 2014 and August 2015, were enrolled in this study. Five patients in the deep NMB group and 3 patients in the moderate NMB group dropped out after randomization due to conversions to open colorectal resection or concurrent other operations. In addition, 1 patient in the deep NMB group and 2 patients in the moderate NMB group were lost to follow-up during hospital stay because of refusal of patients. The remaining 61 patients were finally analyzed. Table P < 0.001). The surgical condition was significantly superior in the deep NMB group compared with the moderate NMB group (P < 0.001). Intraoperative estimated blood loss  and peak airway pressure after CO2 insufflation and deflation  were significantly lower in the deep than in the moderate NMB group. A significantly greater total amount of rocuronium and sugammadex was needed among patients in the deep than in the moderate NMB group . The incidence of postoperative shoulder tip pain was also significantly lower in the deep than in the moderate NMB group . Significantly lower amounts of IV morphine equivalents were administered within 6 to 24 hours after surgery in the deep than in the moderate NMB group . The number of patients who required additional rescue analgesics at 1 to 6, 6 to 24, and 24 to 48 hours after surgery was significantly lower in the deep than in the moderate NMB group . Regarding the bowel movement recovery time, gas passing time  and SOW time  were shorter in the deep than in the moderate NMB group  compared to moderate NMB (12 mm Hg). Deep NMB has benefits of a lower intensity of postoperative pain and a faster bowel function recovery.Though some studies reported of an improved quality of surgical conditions by the use of deep rather than moderate NMB during retroperitoneal laparoscopy and laparoscopic cholecystectomy,17; it still causes considerable discomfort and an increased stress response.18 As the etiology of postlaparoscopic pain can be classified into at least 3 categories: visceral, incisional, and shoulder tip pain,21 we tried to assess different types of pain including abdominal pain and shoulder tip pain. In this trial, the patients in the deep NMB group reported a significantly lower intensity of postoperative abdominal pain at all periods within postoperative 48 hours and a lower intensity of shoulder tip pain within 6 hours postoperatively. The superior postoperative pain profiles in the deep NMB group are noteworthy findings of the current trial.Surgical pain after laparoscopic surgery is less severe and has a shorter duration than after open surgery22 In the present study, gas passing time and SOW time were significantly shorter in the deep versus the moderate NMB group, although hospital stay was similar between the groups. Schwarte et al23 found that an increasing IAP decreased gastric mucosal oxygen saturation. The European Association for Endoscopic Surgery guidelines1 also suggested that an elevated IAP mechanically compresses the capillary beds, decreases splanchnic microcirculation, and thus impairs oxygen delivery to the intra-abdominal organs. During pneumoperitoneum, a 24% blood flow reduction in the superior mesenteric artery and the hepatic portal vein was reported.24Generally, pain during the postoperative period could cause an activation of the inhibitory splanchnic reflexes and result in a postoperative ileus manifested by a delay in the first bowel motion, that leads to a delayed discharge after major abdominal surgery.25 with duration longer than any other laparoscopic surgery previously researched in similar settings. The surgical duration affects the intra-abdominal organ perfusion.26 Current favorable postoperative pain and bowel function recovery results might be associated with a preserved intra-abdominal organ perfusion.Laparoscopic colorectal resection surgery is a major surgery 27 In addition, it is possible that the inducement and maintenance of a deep NMB alleviates securing a satisfactory intraperitoneal working space despite a lower insufflation pressure.28Achieving a satisfactory operative field was not an aim but rather a precondition in our study, more optimal surgical condition was reported by the surgeons regarding the overall surgical conditions in the deep NMB group. For the evaluation of overall surgical conditions, the surgeons were asked to consider their satisfaction with 5-stage, including the surgical field. The reason for this finding may be that the deep NMB seems to not only enhance the surgical view, but also prevent involuntary diaphragm movements and provide a more comfortable position for surgeons to close the abdominal muscles and fascia.29 However, the peak airway pressures 1 hour after CO2 insufflation and 10 minutes after deflation were lower in the deep NMB group compared to the moderate NMB group. These findings suggest that deep muscle relaxation may be beneficial in patients with obesity and respiratory comorbidity.In the present study, the hemodynamic parameters and arterial blood gas analysis results did not differ significantly between the 2 groups. This finding is consistent with findings from previous studies.The estimated intraoperative blood loss was lower in the deep compared to the moderate NMB group. There is no evidence that deep muscle relaxation or low IAP are associated with lesser intra-abdominal bleeding; therefore further research is needed on the association of muscle relaxation and estimated intraoperative blood loss.2 consumption during pneumoperitoneum, because of a central CO2 supply in our center. Second, the maintenance of a deep NMB has important economic repercussions. Namely, the price of sugammadex is higher than that of traditional reversing agents (pyridostigmine or neostigmine), which makes deep NMB less accessible for patients of lower socioeconomic status. Finally, the secondary endpoint analysis was underpowered, considering the low number of subjects. Further trials are needed to confirm the benefits of deep NMB over moderate NMB in laparoscopic colorectal resection.This study has several limitations. First, we could not measure the total COOur findings suggest that deep NMB has benefits over conventional moderate NMB in laparoscopic surgery, including a greater IAP lowering effect, whereas surgical conditions are maintained, less severe postoperative pain and faster bowel function recovery. Therefore, low-pressure pneumoperitoneum with deep NMB is worth considering for patients undergoing laparoscopic surgery.


EHP, investigators assess the reproductive toxicity of a mixture of chlorination DBPs in rats.Disinfection of drinking water is regarded as one of the major public health achievements of the twentieth century, resulting in drastic reductions in diseases such as cholera and typhoid fever.,,Some animal toxicity and human epidemiological studies have suggested that individual chlorination DBPs may be associated with an increased risk of birth defects, spontaneous abortion, delayed puberty, and reduced sperm quality.More than 600 unique DBPs have been identified,1 animals) continued exposure after weaning and through the births of their own litters , which were examined at birth and 6 days of age.In the current study, Simmons and colleagues assessed the reproductive health effects of a mixture of the nine regulated chlorination DBPs across three generations of rats. Throughout pregnancy and weaning, dams drank water containing 500, 1,000, or 2,000 times the maximum contaminant levels of total THMs and total HAAs allowed under current drinking water regulations. The offspring (or F1 animals, and no effects on survival or birth weight in F2 animals.The researchers reported no adverse effects on fertility, pregnancy, pup survival, or birth weight at any dose in F1 offspring showed a significant delay in the onset of puberty at the two highest exposure levels. At the 2,000× level, puberty was delayed by 5.8 days in females and 5.7 days in males. Rats generally reach puberty at about 5–7 weeks of age.Both female and male F1 male offspring, the researchers found that testosterone levels in the testis were reduced by 50% in the 2,000× exposure group, compared with controls. They also observed decreased sperm motility and nipple retention in F1 males. Nipple retention in males may signal impaired androgen production; although rats of both sexes begin developing mammary tissue in utero, androgen production causes nipple development to regress in males by birth.In F1 offspring exposed to the highest doses tended to weigh less later in life, compared with controls. This may have been due to the rats drinking less water at the 1,000× and 2,000× exposure levels—possibly because of the taste of the chemicals.Although birth weight was unaffected, F“The findings are consistent with a modest antiandrogenic effect,” says Paul Foster, chief of the Toxicology Branch at the National Institute of Environmental Health Sciences, who was not involved in the study. However, the authors suggest that reduced water intake at the highest doses may have played a role in delayed puberty as well as in body weight, and the lower body weights at these doses may, in turn, have contributed to nipple retention and compromised sperm motility.Simmons hopes the team’s research can provide a useful framework for determining health effects of complex mixtures. “Looking at a defined subset of chemicals within an environmentally realistic mixture provides a very powerful tool for determining which chemicals in a mixture may be driving risk,” she says.“Water disinfection is vitally important,” Richardson adds. “Studies like this help us to determine the safest possible processes to use.”


Objectives. The present study was aimed at investigating the wound healing effect of ethanolic extract of Cestrum nocturnum (L.) leaves (EECN) using excision and incision wound model. Methods. Wistar albino rats were divided into five groups each consisting of six animals; group I (left untreated) considered as control, group II (ointment base treated) considered as negative control, group III treated with 5% (w/w) povidone iodine ointment (Intadine USP), which served as standard, group IV treated with EECN 2% (w/w) ointment, and group V treated with EECN 5% (w/w) ointment were considered as test groups. All the treatments were given once daily. The wound healing effect was assessed by percentage wound contraction, epithelialization period, and histoarchitecture studies in excision wound model while breaking strength and hydroxyproline content in the incision wound model. Result. Different concentration of EECN (2% and 5% w/w) ointment promoted the wound healing activity significantly in both the models studied. The high rate of wound contraction (P < 0.001), decrease in the period for epithelialization (P < 0.01), high skin breaking strength (P < 0.001), and elevated hydroxyproline content were observed in animal treated with EECN ointments when compared to the control and negative control group of animals. Histopathological studies of the EECN ointments treated groups also revealed the effectiveness in improved wound healing. Conclusions. Ethanolic extract of Cestrum nocturnum (EECN) leaves possesses a concentration dependent wound healing effect.  Wound healing processes consist of integrated cellular and biochemical cascades leading to reestablishment of structural and functional integrity of the damaged tissue . Various healing .Various treatment options  are available for the wound management but majority of these therapies produce numerous unwanted side effects , 4. In rCestrum nocturnum (L.) family) is an evergreen woody shrub growing to 4 m in height. Leaves are dark green, oblong-ovate to oblong-lanceolate in shape with a pointed tip and 6–20 cm long with an entire margin. The nocturnal flowers are greenish white, having powerful sweet intoxicating fragrance [ Cestrum nocturnum like alkaloids, flavonoids, glycosides, steroids, phenols, and essential oils [Solanaceae  using excision and incision wound model in Wistar albino rats.Although need for scientific validation of herbal plants of ethnopharmacological relevance before these could be recommended for wound recovery as drugs is essential, therefore in the light of abovementioned facts about the plant, present study was designed to evaluate the wound healing potential of Cestrum nocturnum were collected in the month of February, locally from Govindpura, Bhopal, Madhya Pradesh, India. Plant material was identified and authenticated by Dr. Zia-Ul Hasan, Head of Department, Department of Botany, Safia Science College, Bhopal, and a specimen voucher (500/Bot/Safia/14), deposited in the Department of Pharmacology, Sapience Bioanalytical Research Lab, Bhopal, for future reference.Fresh leaves of Cestrum nocturnum were shade-dried for 2 weeks, then pulverized to a coarse powder, and passed through sieve number 20 to maintain uniformity. Coarsely dried powder of the leaves was first defatted with petroleum ether (60–80°C) for 72 h to remove fatty materials and then extracted with ethanol (60–70°C) using soxhlet apparatus for 36 h; the extract was collected, filtered through Whatman filter paper, and concentrated in vacuum under reduced pressure and the dried extract was stored at 4°C for further study. The percentage yield of the extract was calculated.The leaves of Cestrum nocturnum leaves (EECN) was subjected to various phytochemical screening tests for the identification of the phytoconstituents present in Cestrum nocturnum leaves using standard procedures [Ethanolic extract ofocedures .2CO3) solution was added and mixed thoroughly. The resulting mixture was incubated for 5 min at 50°C and cooled at room temperature. Absorbance of the mixture was measured at 650 nm against the reagent blank. All measurements were carried out in triplicate. Content of phenolic compounds was expressed as mg of gallic acid equivalents (GAE)/g of dry extract using the linear equation obtained from calibration curve of the standard gallic acid graph. The coefficient of determination (R2) was 0.9971.The total polyphenols content of the EECN was measured by UV spectrophotometrically according to the Folin-Ciocalteu method using gallic acid as a standard . 0.1 mL 3) ethanol solution (2%) was added to 0.5 mL of sample solution. Extract sample was evaluated at a final concentration of 0.1 mg/mL. After 1 h of incubation at room temperature, the absorbance was measured at 420 nm. All measurements were carried out in triplicate. The total flavonoid content was calculated as mg of quercetin equivalents (QE)/g of dry extract using the linear equation obtained from calibration curve of the standard quercetin graph. The coefficient of determination (R2) was 0.9964.The total flavonoid content of the EECN was determined according to aluminum chloride method using quercetin as standard . A volum ad libitum during the study. The study protocol was approved by the Institutional Animal Ethics Committee  as per Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines, India.Healthy Wistar albino rats weighing between 180 and 200 g were used for the present study. Animals were procured from the authorized animal house of Sapience Bioanalytical Research Lab, Bhopal, Madhya Pradesh. The animals were acclimatized to the standard laboratory conditions in cross ventilated animal house at 25 ± 2°C, relative humidity 44–56%, and light and dark cycle of 12 : 12 hours and fed with standard diet and waterTwo types of ointment formulations, 2% and 5% (w/w), were prepared from the extract where 5 and 10 g of the extract were incorporated into 100 g of simple ointment base British Pharmacopoeia (BP), respectively . PovidonFor excision and incision wound model, animals were divided into five groups each consisting of six animals as follows: group I, left untreated and considered as control, group II, which served as negative control (ointment base treated), group III, which served as standard and was treated with 5% (w/w) povidone iodine ointment USP (Intadine), groups IV and V which were treated with 2% and 5% (w/w) ointments of extract, respectively. All the treatments were given once daily.2 was created along the marking using toothed forceps, a surgical blade, and pointed scissors. Rats were left undressed to the open environment. The simple ointment base, formulated extract ointment, and standard drug were applied once daily from the day of the operation until the complete healing. In this model, wound contraction and epithelialization period were evaluated. Wound contraction was measured as percent contraction every 4th day after wound formation. At the end of the study, all the rats were anesthetized and from the healed wounds, specimen samples of tissue were collected from each rat, leaving a 5 mm margin of normal skin around the edges of the healed wound. Specimen tissues were stored in 10% formalin solution and used for histopathological and biochemical studies.Excision wound was created as per the method described ; five grIncision wound was created according to the method already described . The aniIn the excision wound model, wound area was measured by tracing the wound with the help of transparent sheet using millimeter based graph paper on days 0, 4, 8, 12, and 16 for all groups. Wound contraction was measured every 4th day until complete wound healing and represented as percentage of healing wound area . Percentaw wound .The tensile strength of a healing skin wound indicates the degree of wound healing. It represents how much the healed tissue resists to breaking under tension and may identify the quality of healing tissue. On the 10th day, all the animals were anesthetized by injecting ketamine hydrochloride , the sutures were removed, and the healed tissue was excised from all animals. Tensile strength of excised tissue was measured with the help of tensiometer .Excised wound tissues from all rats were analyzed for the estimation of hydroxyproline. Tissues were dried in a hot air oven at 60°C to constant weight and were hydrolyzed in 6 N HCl for 4 h at 130°C. The hydrolysates were then neutralized to pH 7.0 and were subjected to Chloramine-T oxidation for 20 min. After 5 min, the reaction was terminated by the addition of 0.4 M perchloric acid and developed color with Ehrlich reagent at 60°C. After thorough stirring the samples were analyzed at 557 nm in ultraviolet (Systronics-2203) spectrophotometer. The hydroxyproline content in the tissue samples was calculated using a standard curve of the pure L-hydroxyproline .μm thickness) were prepared by microtomy and stained with hematoxylin and eosin (H&E) dye for histological examination.At the end of the study, all the animals were anesthetized using ketamine and specimens of wound tissue were collected and preserved in glass vials containing 10% formalin solution for histological examination. Sections of wound tissue specimens (about 5 P < 0.05 levels.The results are expressed as mean ± standard error of mean (SEM). The statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer Multiple Comparisons Test employing statistical software, GraphPad, InStat 3. Differences between groups were considered significant at The percentage yield of EECN was found to be 11.78% w/w. The preliminary phytochemical investigation of the EECN revealed the presence of alkaloids, flavonoids, glycosides, tannins, triterpenoids, polyphenols, carbohydrates, and proteins .The phenolic and flavonoid contents of the EECN are represented in P < 0.001) and ointment base treated (P < 0.001) and standard groups (P < 0.01) that proportionally confer healing process. As per rate of epithelialization concern the test drug was found to show its contributory role in the accelerating epithelialization rate and required lesser time to complete epithelialization process (P < 0.01) as compared to control and the ointment base treated group (During the course of treatment the extract was found to show its preliminary effect from day 4 up to day 16 . The creed group .P < 0.01) in treated group animals in a concentration dependent manner in comparison to control and ointment base treated group (Increased hydroxyproline content ultimately responsible for increasing the collagen level confirmed the increased viability or microcirculation of collagen fibrils around the wound area. The hydroxyproline level was found to be significantly elevated (ed group . The relP < 0.01 and P < 0.001).An ideal wound healing agent must have the property of increasing the viability of collagen fibrils around the wound area that increases the tensile strength of the wound that was assessed by evaluating the tensile strength of the healed wound using tensiometer . The EECThe histopathological studies of the tissue of the excision wound were performed on the 16th day and histopathological features of the tissue of all groups of animals are shown in Figures Wound healing is an intricate process following damage to the skin and other soft tissues of the body. Wound healing involves the dynamic process of multiple biochemical consequences towards restoration of the damaged cellular structure to its regular and original state . A class Cestrum nocturnum. Recent studies suggested the valuable role of flavonoids, triterpenoids, and tannins in promoting the wound healing by multiple mechanisms, for example, wound contraction, increased rate of epithelialization, and prevention of secondary bacterial infection that would have complicated and delayed wound healing [P < 0.001) hydroxyproline levels in the granulation tissue of ointment of extract (2% and 5% w/w) treated rats indicate the elevated level of collagen content leading to swift wound healing and this venerable finding might be due to presence of flavonoid [The preliminary qualitative phytochemical screening of the EECN showed the presence of alkaloids flavonoids, terpenoids, glycosides, and tannins. Quantitative analysis of the EECN revealed rich amount of phenolic and flavonoidal content in the leaves of healing , 25. In  healing . Wound h healing . As woun healing , however healing . In the lavonoid . An incrlavonoid . HistopaIn conclusion, the results of the present study revealed that the ethanolic extract ointment of EECN contains the phytoconstituents that promote natural healing process and it could be effectively used as a wound healing agent. EECN ointment efficiently stimulates the wound strength and increases the rate of epithelialization, tensile strength, and collagen viability around the wound area. Further studies are in-process to isolate the active compound(s) responsible for wound healing and efforts shall be taken to develop the commercial preparation for wound healing.


Foodborne Hepatitis A Virus (HAV) outbreaks are being recognized as an emerging public health problem in industrialized countries. In 2013 three foodborne HAV outbreaks occurred in Europe and one in USA. During the largest of the three European outbreaks, most cases occurred in Italy . A national Task Force was established at the beginning of the outbreak by the Ministry of Health. Mixed frozen berries were early demonstrated to be the source of infection by the identity of viral sequences in patients and in food. In the present study the molecular characterization of HAV isolates from 355 Italian cases is reported.Molecular characterization was carried out by PCR/sequencing (VP1/2A region), comparison with reference strains and phylogenetic analysis.A unique strain was responsible for most characterized cases . Molecular data had a key role in tracing this outbreak, allowing 110 out of the 235 outbreak cases (46.8%) to be recognized in absence of any other link. The data also showed background circulation of further unrelated strains, both autochthonous and travel related, whose sequence comparison highlighted minor outbreaks and small clusters, most of them unrecognized on the basis of epidemiological data. Phylogenetic analysis showed most isolates from travel related cases clustering with reference strains originating from the same geographical area of travel.In conclusion, the study documents, in a real outbreak context, the crucial role of molecular analysis in investigating an old but re-emerging pathogen. Improving the molecular knowledge of HAV strains, both autochthonous and circulating in countries from which potentially contaminated foods are imported, will become increasingly important to control outbreaks by supporting trace back activities, aiming to identify the geographical source(s) of contaminated food, as well as public health interventions. Hepatitis A Virus (HAV) is a member of the Picornaviridae family, genus Hepatovirus. On the basis of genome sequence divergence, all viruses infecting humans have been classified in three genotypes , further divided into two sub-genotypes (A and B) . GenotypHAV is mainly transmitted by the fecal-oral route, e.g., through ingestion of contaminated water and food or close contacts with infected subjects .The infection is usually asymptomatic or mild in children under five years, while in adults more frequently occurs with symptoms and jaundice. Clearance of infection confers lifelong immunity. In highly endemic countries, HAV infection is acquired in early childhood and most adult population is positive for anti-HAV IgG and protected from reinfection. In contrast, in low endemicity countries, most adult population is susceptible: as a result, infections are more likely to occur in adults, in which are frequently symptomatic, and foodborne outbreaks are increasingly reported .Three different foodborne HAV outbreaks occurred in Europe and one in USA in 2013 and the vehicle of infection in all of them was a minimally processed vegetable product, in particular fresh and frozen strawberries, mixed frozen berries and pomegranate seeds ,5. TheseThe largest of the multi-country European HAV outbreaks involved 1,315 total cases as of March 31, 2014, with most cases being observed in Italy  . Local HIn the present study the molecular characterization of 355 HAV isolates from Italian cases is reported. Sequencing was crucial both to identify outbreak cases and to highlight unrelated minor outbreaks and small clusters, providing an overview of the viral strains circulating in Italy during the multi-country European epidemic.In May 2013 a specific alert was issued by the Italian Ministry of Health, recommending the LHS to enhance hepatitis A surveillance as well as to send clinical samples to the National Reference Laboratory for Hepatitis Viruses at Istituto Superiore di Sanità (NRL-ISS) or to Regional laboratories, for viral characterization. This study was done in response to a public health emergency and was thus exempt from institutional review board approval. All data were analyzed anonymously.The study population included 355 out of the 1,202 total cases notified throughout Italy to the Italian surveillance system with onset from January 1, 2013 to February 28, 2014. In particular, the study population included all cases from which a HAV sequence could be obtained from a biological sample sent by infectious disease hospitals either to the NRL-ISS or to Regional laboratories. Information about travel history, known for 287 cases, showed that the vast majority of them did not exhibit the "travel to endemic areas" risk factor: 215 cases (74.9%) reported no travel outside Italy and, among the remaining 72 cases, 14 (4.9%) travelled to an EU country, 32 (11.1%) outside EU, 26 (9.1%) did not specify the destination country in the questionnaire.The NRL-ISS received: (a) sera from nine Italian regions  and two autonomous provinces (Trento and Bolzano), leading to 108 HAV sequences; (b) sequences from five Regional laboratories corresponding to 72 cases from Lombardy, 49 from Emilia Romagna, 69 from Apulia, 35 from Veneto, 22 from Latium. Overall, sequences from 355 cases from 11 Italian regions and two autonomous provinces and with onset from January 1, 2013 to February 28, 2014 were finally included in the national sequence database at ISS for the analysis. Demographic data and risk factors were available through the National Surveillance System for Acute Hepatitis (SEIEVA), which collects notifications reporting information obtained from patients by a standard questionnaire.Viral RNA was extracted from 140 μl serum by using the QIAmp viral RNA extraction kit . One sixth extracted RNA (10 μL) was reverse transcribed by the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) with random hexamers.To obtain sequence information exactly from the same HAV genome region (VP1/2A) sequenced in other European laboratories, nested PCR and sequencing were carried out by using primers reported in a protocol kindly provided by Dr. Linda Verhoef, RIVM, Bilthoven, The Netherlands . AlthougDouble strand sequencing of purified PCR products obtained from human sera was carried out by using the GenomeLab DTCS Quick Start KiT and an automated DNA sequencer . The sequenced region encompassed the VP1/2A region of HAV genome .The NRL-ISS also received sequences, spanning the VP1/2A region, from five Regional laboratories. One laboratory (IZSLER—Emilia Romagna) carried out sequencing with the same protocol used at ISS. The four other laboratories used different protocols and, thus, sent sequences of variable size. A laboratory (UNIMI—Lombardy) sent both “long” (439 to 457 nt) and “short” sequences (270 to 349 nt), depending on whether a PCR product suitable for sequencing was obtained just at the first PCR step or required a nested PCR amplification . Another laboratory (A.O.U. Bari—Apulia) did not send sequences from individual cases, but sent four sequences representing the four strains identified in 39, 11, 1 and 18 cases; these sequences overlapped the region sequenced at ISS for 460, 338, 358 and 349 nt, respectively. A fourth laboratory (INMI—Latium) sent sequences whose size was 225 nt, and a fifth laboratory (A.O.PD—Veneto) sent sequences ranging from 226 to 247 nt.Alignment of the 225 to 358 nt sequences showed they shared a 174 nt common region,  suitable to compare them.All sequences were included in two different datasets according to their sequence length  and analyzed separately according to the rules described in the next paragraph. Accession numbers are reported in Sequence analysis was performed at the NRL-ISS. Genotype was assigned by phylogenetic analysis (Neighbour-Joining) with reference sequences see .To search for cases specifically due to the “outbreak” strain, the sequences from human sera were compared to the reference sequence Acc. No. KF182323 [In agreement with other EU member states and approval by the ECDC, different criteria were established to consider a sequence as “outbreak”, depending on its length. Sequences 419 to 460 nt were considered to be “outbreak” if 100% identity or, at most, 1 nt difference over the entire length was observed. A more restrictive rule was applied to the shorter sequences, considered to be “outbreak” exclusively if 100% identity was shown over the 174 nt common region.To investigate the phylogenetic relationships with strains from endemic and non-endemic countries available in GenBank, a third dataset (Dataset 3) was built including all collected sequences sharing at least a 330 nt region. This size was selected as a balance between the need to retain as much as possible molecular information for reliable analysis and the need to include as much as possible sequences. The dataset also included the reference strains used for genotyping  and addiPhylogenetic relationships were analyzed by constructing Maximum Parsimony trees by MEGA 6.0 . Tree reTwo different datasets were built to identify outbreak cases by comparison with the reference Italian outbreak sequence (Acc.no. KF182323). Dataset 1 included 218 sequences whose length was at least 419 nt, Dataset 2 included a 174 nt common region shared by 137 sequences whose length was 225 to 358 nt.Analysis of Dataset 1 showed that most cases were caused by the “outbreak” strain  including both sequences showing 100% identity (n = 127) and closely related sequences with no more than 1 nt difference (n = 15) see .To evaluate if the small 174 nt region in Dataset 2, coupled to the more restrictive criterium of complete identity with the reference sequence, could reliably identify outbreak cases, sequences from Dataset 1 were re-classified limiting analysis to the 174 nt portion. Only 1 out of 218 (0.6%) “long” sequences  was mis-classified: it was a sequence showing 3 nt differences over the entire length (442 nt) but no differences over the 174 nt region. This result supported the reliability of the 174 nt region for correct “outbreak” and “non-outbreak” classification of the 137 “short” sequences. Thus, 355 cases could be finally classified .From January 1, 2013 to February 28, 2014, 235 cases (66.2%) harbored an HAV isolate with the “outbreak sequence”, 92 cases (25.9%) harbored unrelated genotype IA sequences (92.3% to 97.1% identity with the reference), 27 cases (7.6%) had genotype IB and 1 case (0.3%) was infected by a genotype IIIA virus .Importantly, 110 out of the 235 “outbreak” cases (46.8%) could be recognized only by virtue of sequence identity, because consumption of frozen berries was either denied or not reported in their questionnaires.versus the reference, two clusters (4 and 5 sequences each) could be recognized, each of them containing sequences with identical nucleotide difference .One additional sequence (UNIMI-ID132/49) showed 3 nucleotide differences over 442 nt (99.3% identity). Although this isolate could not be classified as “outbreak” strain, it was obviously much more correlated to it than to the other genotype IA isolates (these latter showing 20–35 nucleotide differences The outbreak strain exceeded two-thirds of cases (range: 70.7–90%) in 6 out of 8 regions, except Piedmont and Apulia. In Piedmont a cluster of 4 cases from a Roma camp  strongly affected the distribution, because of the small sample size . In Apulia, 72.5% of cases were caused by two autochthonous genotype IA strains and only 26% by the “outbreak” strain. The tradition of eating raw mussels in Apulia represents the main risk factor associated with the two autochthonous strains, explaining the peculiar distribution.In the "mixed frozen berries" group, most cases had been infected by the outbreak strain , although only 36% of them showed exclusively this risk factor.A high frequency of the outbreak strain was also observed in the other groups  were from Moroccan patients, 10 of which reporting travel to Morocco. The median age of the Moroccan patients was 6 years (range 2–15 years). A sub-cluster of 7 identical sequences can be observed , 11 cases  and 39 cases   and with references from Russia. Two Romanian cases from Tuscany and Emilia Romagna (ISS_113_131 and IZSLER_5207_14), clustering in the same group, showed no apparent epidemiological link with the other isolates, despite 100% sequence identity.Although not all nodes were statistically supported, most remaining sequences grouped with references from the country of origin of the cases or from the same geographical area of travel .In the IB sub-tree, sequences grouped in two main clades  and in most cases their sequences were phylogenetically related to references from Africa. Two cases, occurred in Tuscany and Emilia Romagna, showed an identical IB strain and were associated with travel to Namibia. An “Egyptian” group can also be observed, although did not show bootstrap support.Finally, a statistically supported cluster unrelated to travel included 4 cases with identical sequences, 3 of them living in a Roma camp in Piedmont. Their median age was 7 years (range 5–8 years).No genotype II cases were observed. The genotype III sub-tree included the only one case observed in the studied period as well as an additional case occurred a few days later , the distribution of viral strains in the sequenced cases may roughly represent the distribution in the notified cases. Thus, the “outbreak” strain seems to be responsible for most excess notifications occurred in the studied period.The strain had been already involved in a large outbreak in the Czech Republic in 2008, with 1616 reported cases ,14. Howevice versa can be drawn, as the several nucleotide differences imply a long-lasting evolution and a possible mutual migration between continents. So, the phylogenetic and geographical origin of the outbreak strain remains an open question.The strain is phylogenetically related to Venezuelan isolates sampled in 2005–2006. Although this finding suggests a possible common evolutionary ancestor, this hypothesis cannot yet be formally proved on the basis of available data. In addition, no conclusions about the possible direct origin of the Czech strain from Venezuelan strains or The present investigation highlights, once again, the molecular characterization as an essential tool to trace outbreaks. In fact, molecular typing had a crucial role for identification of epidemic cases lacking any epidemiological link: 110 out of 235 confirmed cases (46.8%) could be correctly classified only by virtue of molecular data.Although the outbreak strain could be reliably identified also by restricting the analysis to a small region of 174 nt, closely related variants could be discerned only by analyzing longer sequences. These variants could be either the result of random variation during person-to-person transmission or the result of food contamination at production points, in which multiple related variants might circulate. The finding of two clusters of variants, each marked by a specific nucleotide variation, in cases with no apparent link supports this latter hypothesis. Recognizing variants would be valuable for tracing the origin of contaminated food by comparison with strains from endemic areas, whose better knowledge appears to be increasingly necessary.Interestingly, molecular characterization also revealed the circulation of other unrelated strains, some of them responsible for minor outbreaks and small clusters (2 to 39 cases), the smallest of which had not been recognized on the basis of epidemiological data.In Apulia, 50 hepatitis A cases linked to shellfish consumption were demonstrated to be caused by two distinct IA strains, circulating simultaneously and responsible for 11 and 39 cases each . The twoAmong the small clusters, the largest of them included 7 cases due to a IA strain likely originating from Morocco, on the basis of phylogenetic data; only 2 out of 7 cases reported “travel to Morocco”, suggesting local transmission in Italy in the remaining cases.Analysis of sporadic cases showed that in 2013 most of them were linked to travel to endemic countries, Morocco being the most frequently reported. Importantly, available data suggested that in several cases the involved strains, both IA and IB, had been imported by Moroccan children/adolescents travelling to Morocco, but were then transmitted to other children/adolescents, mainly with Moroccan nationality, in Italy. The majority of the remaining travel related cases occurred in adults and their viral sequence revealed phylogenetic relationship to strains circulating in the same country/area of travel.In conclusion, the outbreak occurred in 2013 prompted Italy to implement molecular surveillance of HAV. In addition to tracing the outbreak, the surveillance provided an overview of the strains circulating in Italy and led to establish a national database of autochthonous and imported HAV sequences that will be helpful in the next years, especially to support trace-back activities in identifying the geographical source(s) of contaminated food. However, important gaps still remain in the molecular knowledge of HAV strains circulating in those Europe and extra-Europe countries in which no molecular surveillance is established and from which potentially contaminated foods are imported. Eliminating these gaps will require improved international collaboration and data exchange, to fight emerging HAV outbreaks.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S1 FigThe tree was obtained by the Maximum Parsimony approach. The 58 sequences from the present study are reported in bold: 1 of them (reported in purple color) is representative of 235 cases of the mixed frozen berries outbreak; 2 other IA sequences represent 11 and 39 cases, respectively; the remaining 55 sequences are from individual cases. The IA and IB subtrees are shown enlarged. For each sequence from the present study the following information is reported, wherever available, in the following order: ID code, Italian region of the patient, nationality for non-Italian patients , risk factor(s) , age group , date of clinical onset (only for some relevant cases). For three IA sequences  the number of cases in which the sequence was found is also reported. For each reference sequence, information is reported in the following order: accession number, sequence name, country of isolation, risk factor(s) (if known), year of isolation, genotype/sub-genotype. Some reference sequences were from vegetable matrices . The four main clades in the genotype IA and IB sub-trees are labeled “1” to “4”. Two clusters in clade 1 are highlighted by red and blue fonts, respectively (see the main text for details). Significant bootstrap values are reported.(PDF)Click here for additional data file.