Evaluation of the effect of eltrombopag therapy on the platelet collagen receptor glycoprotein VI (GPVI) expression and soluble GPVI levels in young patients with immune thrombocytopenia.

BACKGROUND: Platelet glycoprotein VI (GPVI) receptor is essential for platelet adhesion and aggregation. Eltrombopag is as an effective treatment for chronic immune thrombocytopenia (ITP); yet, its effect on platelet function is not fully characterized. AIM: This prospective study investigated the effect of eltrombopag therapy on platelet function through assessment of GPVI receptor expression and soluble GPVI levels among pediatric patients with persistent or chronic ITP. METHODS: Thirty-six children and adolescents with persistent or chronic ITP were divided equally into two groups either to receive eltrombopag therapy or the standard of care. All patients were followed-up for 12 months with assessment of bleeding score and complete blood count (CBC). Evaluation of GPVI expression using flow cytometry and measurement of its soluble form by ELISA was done at baseline and at 6 months. RESULTS: ITP patients on eltrombopag had significantly lower bleeding score after 6 months of therapy while the quality of life has significantly improved. Platelet count was significantly increased throughout the study. GPVI expression by flow cytometry and soluble GPVI levels were significantly increased after eltrombopag therapy. After 12 months, ITP patients on eltrombopag were able to maintain a good quality of life and low bleeding score. CONCLUSION: Our data suggest that eltrombopag, through its effect on the GPVI receptor expression and its soluble form, might reduce bleeding manifestations and improve the quality of life of chronic and persistent ITP children independent of its effect on the platelet count.

Cardiac tamponade in venoarterial extracorporeal membrane oxygenation.

Cardiac tamponade is a challenging diagnosis in the unstable patient requiring extracorporeal membrane oxygenation. We present a case of cardiac tamponade secondary to hemorrhagic pericardial effusion that developed in the setting of cardiopulmonary resuscitation and venoarterial extracorporeal membrane oxygenation. Specifically, we aim to discuss the pertinent diagnostic challenges in confirming this diagnosis.

Myocardial Irritation from a Left Ventricular Assist Device Resulting in Refractory Ventricular Tachycardia.

BACKGROUND: Due to an increasing prevalence of heart failure but a steady rate of heart transplantation, the number of left ventricular assist devices (LVADs) implanted is growing. These patients present to emergency departments (EDs) with a variety of complications from their implanted device as well as their baseline cardiomyopathy. One-third of patients will present with a dysrhythmia, the most common of which is ventricular tachycardia. CASE REPORT: A 77-year-old man with nonischemic cardiomyopathy and HeartMate II LVAD presented with sustained ventricular tachycardia and 43 automatic implantable cardioverter-defibrillator (AICD) discharges. Due to left ventricular remodeling, ongoing diuresis, and positioning of his LVAD inflow cannula against his interventricular septum, a likely dysrhythmogenic foci, he quickly decompensated with sedation while in the ED. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Refractory ventricular tachycardia is a common dysrhythmia for LVAD patients and may lead to full cardiopulmonary arrest. Common strategies such as chest compressions are used only in limited scenarios, but medical management is possible. This should focus on resolution of the dysrhythmia and identification of the etiology, including possible mechanical compromise.

daf-16/FOXO and glod-4/glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans.

AIMS/HYPOTHESIS: The aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms. METHODS: Nematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment. Additionally, the activity of glyoxalase-1 and superoxide dismutase (SOD) and the formation of reactive oxygen species (ROS) and AGEs were determined. RESULTS: Insulin and its analogues reversed the life-shortening effect of HG conditions and prevented the glucose-induced neuronal impairment. Insulin treatment under HG conditions was associated with reduced concentration of glucose, as well as a reduced formation of ROS and AGEs, and increased SOD activity. These effects were dependent on the Forkhead box O (FOXO) homologue abnormal dauer formation (DAF)-16. Furthermore, glyoxalase-1 activity, which was impaired under HG conditions, was restored by human insulin. This was essential for the insulin-induced lifespan extension under HG conditions, as no change in lifespan was observed following either suppression or overexpression of glyoxalase-1. CONCLUSIONS/INTERPRETATION: Human insulin and its analogues prevent the reduction in lifespan and neuronal damage caused by HG conditions. The effect of human insulin is mediated by a daf-2/insulin receptor and daf-16/FOXO-dependent pathway and is mediated by upregulation of detoxifying mechanisms.

Influence of insulin and glargine on outgrowth and number of circulating endothelial progenitor cells in type 2 diabetes patients: a partially double-blind, randomized, three-arm unicenter study.

BACKGROUND: Endothelial progenitor cells (EPC) are bone marrow-derived cells which can undergo differentiation into endothelial cells and participate in endothelial repair and angiogenesis. Insulin facilitates this in vitro mediated by the IGF-1 receptor. Clinical trials showed that the number of circulating EPCs is influenced by glucose control and EPC are a predictor of cardiovascular death. To study direct effects of insulin treatment on EPCs in type 2 diabetes patients, add-on basal insulin treatment was compared to an escalation of oral medication aiming at similar glucose control between the groups. METHODS: 55 patients with type 2 diabetes (61.6±5.9 years) on oral diabetes medication were randomized in a 2:2:1 ratio in 3 groups. Patients were treated additionally with insulin glargine (n=20), NPH insulin (n=22) or escalated with oral medication (n=13). Number of circulating EPC, EPC-outgrowth, intima media thickness, skin microvascular function and HbA1c were documented at baseline and/or after 4 weeks and 4 months. RESULTS: HbA1c at baseline was, 7.3+/-0.7% in the oral group, 7.3+/-0.9% and 7.5+/-0.7% in the glargine and NPH insulin respectively (p=0.713). HbA1c after 4 months decreased to 6.8+/-0.8%, 6.6+/-0.7% and 6.7+/-0.6%, in the oral, glargine and NPH insulin group respectively (p=0.61). FACS analysis showed no difference in number of circulating EPC between the groups after 4 weeks and 4 months. However, the outgrowth of EPCs as detected by colony forming assay was increased in the NPH insulin and glargine groups (29.2+/-6.4 and 29.4+/- 6.7 units respectively) compared to the group on oral medication (23.2+/-6.3, p=0.013) after 4 months of treatment. A significant decrease of IMT from 0.80mm (+/-0.14) at baseline to 0.76mm (+/-0.12) after 4 months could be observed in all patients only (p=0.03) with a trend towards a reduction of IMT after 4 months when all patients on insulin treatment were compared to the oral treatment group (p=0.06). Skin microvascular function revealed no differences between the groups (p=0.74). CONCLUSION: The study shows that a 4-month treatment with add-on insulin significantly increases the outgrowth of EPC in patients with type 2 diabetes mellitus. TRIAL REGISTRATION: (Clinical Trials Identifier: NCT00523393).

Impact of Covid-19 lockdowns on the anthropometric development in primary school children in the Rhein-Neckar Region, Germany.

BACKGROUND: Published data suggests that lockdowns during the COVID-19 pandemic may have negatively affected children's weight development. This study aims to assess the prevalence of overweight and obesity after the COVID-19 lockdowns as well as anthropometric development among primary school children in the Rhein-Neckar Region, Germany. METHODS: In this cross-sectional study, schools were selected in cooperation with the local health authority to include different socioeconomic backgrounds. Participation was voluntary at school and individual level, requiring written informed consent from legal guardians. Study visits in schools were conducted between October 2021 and July 2022. Anthropometric data from nationally recommended medical examinations at 4 years (U8) and 5 years (U9), data on nutrition, physical activity, and socioeconomic data was collected using questionnaires. zBMI and weight category were calculated based on German reference data. RESULTS: 256 children with a mean age of 8.0 years (7.1-9.3 years) were included in the study. Most participants were from households with an above average income. 5.1% of the children were overweight, 4.7% were obese, 15.6% were underweight, and 74.6% were normal weight with a mean zBMI of -0.25 (SD 1.10), which is significantly lower than the mean zBMI of the German reference population (p < 0.001). No significant changes in zBMI were observed between U8 and U9 (p = 0.16). The mean zBMI decreased by 0.17 (SD 0.72) between U9 and the study visit (p = 0.02). A zBMI decrease of 0.5 was documented for the subgroup of overweight and obese children (p = 0.028) as well as a decrease of 0.23 (SD 0.63) for the normal-weight children subgroup (p < 0.001). CONCLUSIONS: Contrary to previous reports mean zBMI decreased significantly in the children studied. No significant changes in zBMI were observed between U8 and U9 examinations, which supports the hypothesis that the decrease in zBMI could be attributed to lockdown measures. The study was registered at clinicaltrials.gov on September 21st 2021 under the registration number NCT05077059.

Mortality Associated With Proportionality of Secondary Mitral Regurgitation After Transcatheter Mitral Valve Repair: North American Mitraclip for Functional Mitral Regurgitation Registry.

The association, if any, between the effective regurgitant orifice area (EROA) to left ventricular end-diastolic volume (LVEDV) ratio and 1-year mortality is controversial in patients who undergo mitral transcatheter edge-to-edge repair (m-TEER) with the MitraClip system (Abbott Vascular, Santa Clara, CA). This study's objective was to determine the association between EROA/LVEDV and 1-year mortality in patients who undergo m-TEER with MitraClip. In patients with severe secondary (functional) mitral regurgitation (MR), we analyzed registry data from 11 centers using generalized linear models with the generalized estimating equations approach. We studied 525 patients with secondary MR who underwent m-TEER. Most patients were male (63%) and were New York Heart Association class III (61%) or IV (21%). Mitral regurgitation was caused by ischemic cardiomyopathy in 51% of patients. EROA/LVEDV values varied widely, with median = 0.19 mm2/ml, interquartile range [0.12,0.28] mm2/ml, and 187 patients (36%) had values <0.15 mm2/ml. Postprocedural mitral regurgitation severity was substantially alleviated, being 1+ or less in 74%, 2+ in 20%, 3+ in 4%, and 4+ in 2%; 1-year mortality was 22%. After adjustment for confounders, the logarithmic transformation (Ln) of EROA/LVEDV was associated with 1-year mortality (odds ratio 0.600, 95% confidence interval 0.386 to 0.933, p = 0.023). A higher Society of Thoracic Surgeons risk score was also associated with increased mortality. In conclusion, lower values of Ln(EROA/LVEDV) were associated with increased 1-year mortality in this multicenter registry. The slope of the association is steep at low values but gradually flattens as Ln(EROA/LVEDV) increases.

The effect of GLP-1 receptor agonist lixisenatide on experimental diabetic retinopathy.

AIMS: Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC-MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. RESULTS: Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. CONCLUSIONS: Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.

Protective Effects of Transient Glucose Exposure in Adult C. elegans.

C. elegans are used to study molecular pathways, linking high glucose levels (HG) to diabetic complications. Persistent exposure of C. elegans to a HG environment induces the mitochondrial formation of reactive oxygen species (ROS) and advanced glycation endproducts (AGEs), leading to neuronal damage and decreased lifespan. Studies suggest that transient high glucose exposure (TGE) exerts different effects than persistent exposure. Thus, the effects of TGE on ROS, AGE-formation and life span were studied in C. elegans. Four-day TGE (400 mM) as compared to controls (0mM) showed a persistent increase of ROS (4-days 286 ± 40 RLUs vs. control 187 ± 23 RLUs) without increased formation of AGEs. TGE increased body motility (1-day 0.14 ± 0.02; 4-days 0.15 ± 0.01; 6-days 0.16 ± 0.02 vs. control 0.10 ± 0.02 in mm/s), and bending angle (1-day 17.7 ± 1.55; 3-days 18.7 ± 1.39; 6-days 20.3 ± 0.61 vs. control 15.3 ± 1.63 in degree/s) as signs of neuronal damage. Lifespan was increased by 27% (21 ± 2.4 days) after one-day TGE, 34% (22 ± 1.2 days) after four-days TGE, and 26% (21 ± 1.4 days) after six-days TGE vs. control (16 ± 1.3 days). These experiments suggest that TGE in C. elegans has positive effects on life span and neuronal function, associated with mildly increased ROS-formation. From the perspective of metabolic memory, hormetic effects outweighed the detrimental effects of a HG environment.

Sulforaphane and Vitamin E Protect From Glucotoxic Neurodegeneration and Lifespan Reduction In C. Elegans.

Caenorhabditis elegans is an established model organism in neurodegeneration and aging research. Oxidative stress and formation of advanced glycation endproducts (AGEs), as they occur under hyperglycemic conditions in diabetes mellitus, contribute to neuronal damage and lifespan reduction. Sulforaphane (SFN) is an indirect antioxidant, alpha-tocopherol (vitamin E) is a direct antioxidant that acts as a free radical scavenger. Aim of this study is to investigate the protective effects of SFN and vitamin E against glucotoxic damages to the neuronal system and lifespan in C. elegans. Culture conditions that mimic clinical hyperglycemia increased the formation of reactive oxygen species (ROS) (p<0.001) and the accumulation of methylglyoxal-derived advanced glycation endproducts (MG-derived AGEs) (p<0.01) with subsequent neuronal damage and neuronal dysfunction, ultimately leading to a significant shortening of lifespan (p<0.01). Treatment with both, 20 µmol/l SFN and 200 µg/ml vitamin E, completely prevented the increase in ROS and MG-derived AGEs, abolished the glucotoxic effects on neuronal structure and function, and preserved lifespan, resulting in a life expectancy similar to untreated controls. These data emphasize the relevance of indirect and direct antioxidants as potential therapeutic options for the prevention of glucotoxic pathologies.

Kidney injury as post-interventional complication of TAVI.

Transcatheter aortic valve implantation (TAVI) is an accepted treatment approach of aortic stenosis. In the beginning, this technique was executed in high-risk patients only. Today, intermediate-risk patients are also amenable for TAVI, as long as the transfemoral approach is chosen. Numerous predictors have been identified that could lead to periprocedural complications and are defined by patient co-morbidities as well as being inherent to the technical approach. Although vascular complications and postinterventional paravalvular regurgitation have been minimized over the past years by revised technologies and techniques, there is a prevailing individual risk brought about by the specific pathophysiology of the cardiorenal syndrome.

Pharmacist-Initiated Management of a Suspected Case of Risperidone-Induced Neuroleptic Malignant Syndrome in an Aged-Care Resident. The Role of Residential Medication Management Reviews in Medication Safety.

A 70-year-old female aged-care resident was referred by her general practitioner for a residential medication management review after nurses reported difficulties with swallowing, episodes of hyperthermia, elevated blood pressure, and tachycardia. These symptoms were accompanied by increasing confusion and drowsiness. Risperidone had recently been prescribed to treat behavioral and psychological symptoms of dementia. This case study describes the pharmacist-initiated management of the symptoms through a national medication review program. It demonstrates the valuable role collaborative medication reviews play in managing adverse drug reactions in aged-care.

Impaired dexamethasone resorption in two patients with pseudo-Cushing after bariatric surgery: Implications for immunosuppressive treatment.

Two cases of middle-aged female patients treated by gastric bypass surgery for weight loss presented to our clinic for a follow-up examination 3-6 months after the surgical procedure (a mini gastric bypass and a modified single anastomosis sleeve-ileostomy). In both patients increased ACTH levels and either high serum cortisol or an increased urinary cortisol excretion was apparent and triggered further endocrine testing. Serum cortisol could not be suppressed adequately by 2 and 4 mg dexamethasone in the standardized oral overnight suppression test while midnight salivary cortisol dropped well below the desired cut-off. This led to the hypothesis of an impaired dexamethasone resorption and could be further substantiated by suppression of serum cortisol below the cut-off by an intravenous dexamethasone application. The data presented point to an impairment of enteral synthetic corticosteroid resorption in patients after gastric bypass surgery and could be of importance for individuals in need for immunosuppressive treatment. In view of the growing number of bariatric procedures, pharmacokinetics of corticosteroids and other drugs should be tested in clinical trials.

The model Caenorhabditis elegans in diabetes mellitus and Alzheimer's disease.

Diabetes mellitus, with its complications, and Alzheimer's disease (AD) share many similarities. Both are age-related and associated with enhanced formation of advanced glycation endproducts (AGEs) and oxidative stress, factors that can be observed during the normal aging process as well. AGE deposits can be found in areas of atherosclerotic lesions in diabetes and in senile plaques and neurofibrillary tangles in AD. A classical model organism in aging research is the nematode Caenorhabditis elegans (C. elegans). Though C. elegans lacks a vascular system, it has been introduced in diabetes and AD research since it shares many similarities at the molecular level to pathological processes found in humans. AGEs accumulate in C. elegans, and increased AGE-formation and mitochondrial AGE-modification are responsible for increased oxidative stress and limiting life span. Moreover, C. elegans has an accessible and well characterized nervous system and features several genes homologous to human genes implicated in AD like amyloid-beta protein precursor, presenilins and tau. In addition, human genes linked to AD, such as amyloid-beta or tau, can be expressed and studied in C. elegans. So far, C. elegans research has contributed to a better understanding of the function of AD-related genes and the development of this disease.

External electric muscle stimulation improves burning sensations and sleeping disturbances in patients with type 2 diabetes and symptomatic neuropathy.

OBJECTIVE: External muscle stimulation (EMS) of the thighs was previously shown to have beneficial effects in a pilot study on painful diabetic neuropathy. However, differential effects on specific symptoms of neuropathy as well as determinants of treatment response have not been described. DESIGN: Ninety-two type 2 diabetes patients with different neuropathic symptoms were included in a prospective uncontrolled trial. Patients were treated twice a week for 4 weeks. Symptoms were graded on numeric scales at baseline, before the second and the eighth visit. RESULTS: Seventy-three percent of the participants reported marked improvement of symptoms. Subjective treatment response was positively and independently associated with symptom intensity but independent of disease extent, metabolic factors, age, or gender. Total symptoms graded by patients on numerical scales decreased significantly after 4 weeks of treatment. Patients in the upper tertile of symptom intensity showed significant improvement of paresthesia, pain, numbness and most pronounced for burning sensations and sleeping disturbances. CONCLUSIONS: In an uncontrolled setting, EMS seems to be an effective treatment for symptomatic neuropathy in patients with type 2 diabetes, especially in patients with strong symptoms.

Dietary Intervention with Oatmeal in Patients with uncontrolled Type 2 Diabetes Mellitus - A Crossover Study.

BACKGROUND: In a pilot study, we evaluated the efficacy of two days of oatmeal on insulin resistance and glucose metabolism and found a marked decrease of insulin requirements. The most important shortcoming of that study was that the interventions were not isocaloric (diabetes adapted diet: 1500 kcal/d vs. oatmeal 1100 kcal/d). To address these drawbacks we designed the OatMeal And Insulin Resistance (OMA-IR) study. METHODS: The study was a randomized, open label crossover dietary intervention study with consecutive inclusion of 15 patients with uncontrolled type 2 diabetes. The intervention comprised two days of oatmeal on days 3 and 4 of a 5 days hospital stay. During the control period, patients received a diabetes mellitus adapted diet only. The primary endpoint was the daily insulin requirement and glycemic control. RESULTS: Upon oatmeal treatment, the required insulin dose could be significantly reduced on the third and fourth day as compared to the second day of inpatient stay (82.0±30.3 and 69.9±29.9IU versus 112±36.2IU;P<0.001). During control treatment, insulin requirement did not change. There were no significant differences in the changes of mean blood glucose or fasting glucose between both treatments. HbA1c was lower four weeks after the oatmeal intervention. CONCLUSION: In this crossover study, two days of oatmeal intervention allowed a highly significant reduction of required daily insulin doses while maintaining adequate metabolic control as compared to a diabetes adapted diet only. The beneficial effects of the intervention might last for several weeks as shown by the lower HbA1c four weeks after the intervention.

RAGE expression in the human peritoneal membrane.

BACKGROUND: Experimental animal models have demonstrated that the interaction of advanced glycation end-products (AGE) with their receptor RAGE is, at least in part, responsible for peritoneal damage. This study investigates the in vivo expression of RAGE in the peritoneal membrane of uraemic human patients. METHODS: Peritoneal biopsies of 89 subjects (48 uraemic and 41 healthy age-matched patients) were examined. The expression of CD3, IL-6, activated NFkappaBp65, VEGF, transforming growth factor (TGF)-beta1, smooth-muscle actin (SMA), methylglyoxal (MGO) and RAGE was analysed immunohistochemically. Additionally, in 4 of the 48 uraemic patients, peritoneal biopsies were repeated after 15 months at the time of catheter removal to analyse the above parameters and the extent of NFkappaB-binding activity determined by electrophoretic mobility shift assay (EMSA) in the long-term follow-up. RESULTS: In comparison to the healthy controls, uraemic patients showed a significant increase in fibrosis, angiogenesis, submesothelial thickness, MGO-derived protein adducts, RAGE, IL-6, VEGF, TGF-beta1, SMA and NFkappaBp65 in their peritonea. Four patients, followed up longitudinally from peritoneal dialysis (PD) catheter insertion to removal, demonstrated further significant increase in the above parameters, particularly in RAGE expression and NFkappaB activation. CONCLUSIONS: Along with a higher expression of several indicators for inflammation, angiogenesis, fibrosis and AGE accumulation, the peritoneal membrane of the uraemic patients showed an increased submesothelial thickness and a marked induction of RAGE expression and NFkappaB-binding activity, which both further increased after PD treatment. These findings in human peritoneum support the concept of the AGE-RAGE interaction being crucial in peritoneal damage due to uraemia and PD.

Metabolic syndrome in a large chemical company: prevalence in a screened worksite sample.

The metabolic syndrome (MS) leads to serious health problems like diabetes and has serious economic consequences for multinational companies. Thus, the workplace is an important setting for primary prevention. Aim of this study was to evaluate the prevalence of MS in a mixed working population to provide a basis for interventional strategies. In 2006, 1,594 employees attended a screening program at BASF Ludwigshafen, the number of employees with MS was determined and the distribution of impaired glucose tolerance (IGT), type 2 diabetes and cardiovascular disease (CVD) analyzed. The study-population consisted of 1,075 men and 519 women, aged 17-64. 374 individuals (23.5%) were classified to be affected by MS, of which 86.9% were male (prevalence MS in men 30%, in women 9.7%). Subjects with MS had higher BMI (P < 0.01), blood pressure (P < 0.01), heart rate (P < 0.01), liver enzymes (P < 0.01), uric acid (P < 0.01) and LDL (P < 0.01), while HDL was significantly lower (P < 0.01). (Pre)-Diabetes and CVD were found more frequently in subjects with MS. There were no significant differences between individuals with different types of employment ("white collar vs. blue collar" workers) or smoking status. We found a high prevalence of MS in our working population, thus interventional programmes should be implemented. The workplace-setting can be used to promote long-term prevention strategies in this adult working population.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Neuronal damage and shortening of lifespan in C. elegans by peritoneal dialysis fluid: Protection by glyoxalase-1.

Glucose and glucose degradation products (GDPs), contained in peritoneal dialysis (PD) fluids, contribute to the formation of advanced glycation end-products (AGEs). Local damaging effects, resulting in functional impairment of the peritoneal membrane, are well studied. It is also supposed that detoxification of AGE precursors by glyoxalase-1 (GLO1) has beneficial effects on GDP-mediated toxicity. The aim of the current study was to analyze systemic detrimental effects of PD fluids and their prevention by glyoxlase-1. Wild-type and GLO1-overexpressing Caenorhabditis elegans (C. elegans) were cultivated in the presence of low- and high-GDP PD fluids containing 1.5 or 4% glucose. Lifespan, neuronal integrity and neuronal functions were subsequently studied. The higher concentrations of glucose and GDP content resulted in a decrease of maximum lifespan by 2 (P<0.01) and 9 days (P<0.001), respectively. Exposure to low- and high-GDP fluids caused reduction of neuronal integrity by 34 (P<0.05) and 41% (P<0.05). Cultivation of animals in the presence of low-GDP fluid containing 4% glucose caused significant impairment of neuronal function, reducing relative and absolute head motility by 58.5 (P<0.01) and 56.7% (P<0.01), respectively; and relative and absolute tail motility by 55.1 (P<0.05) and 55.0% (P<0.05), respectively. Taken together, GLO1 overexpression protected from glucose-induced lifespan reduction, neurostructural damage and neurofunctional damage under low-GDP-conditions. In conclusion, both glucose and GDP content in PD fluids have systemic impact on the lifespan and neuronal integrity of C. elegans. Detoxification of reactive metabolites by GLO1 overexpression was sufficient to protect lifespan, neuronal integrity and neuronal function in a low-GDP environment. These data emphasize the relevance of the GLO1 detoxifying pathway as a potential therapeutic target in the treatment of reactive metabolite-mediated pathologies.