Search

Background Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. Methods Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Key Results Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Conclusions & Inferences Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity. [ABSTRACT FROM AUTHOR]

Background The optimal duration of clopidogrel treatment following percutaneous coronary intervention (PCI) and the patient population that would benefit most are still unknown. In a porcine coronary injury model, we tested two different durations of clopidogrel treatment on severely or moderately injured arteries and examined the arterial response to injury. To understand the molecular mechanism, we also investigated the effects on transcription factors nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1). Materials and methods In 24 cross-bred pigs, one coronary artery was only moderately injured by percutaneous transluminal coronary angioplasty (PTCA) and one coronary artery was severely injured by PTCA and subsequent beta-irradiation (Brachy group). Animals received 325 mg aspirin daily for 3 months and 75 mg clopidogrel daily for either 28 days [short-term (ST) clopidogrel group] or 3 months [long-term (LT) clopidogrel group]. Results After 3 months, the number of proliferating cells per cross-section differed significantly between ST and LT in both injury groups (PTCAST 90·2 ± 10·3 vs. PTCALT 19·2 ± 4·7, P < 0·05; BrachyST 35·8 ± 8·4 vs. BrachyLT 7·5 ± 2·0, P < 0·05). Similar results were seen for inflammatory cells (CD3+ cells): PTCAST 23·5 ± 3·55 vs. PTCALT 4·67 ± 0·92, P < 0·05; BrachyST 83·17 ± 11·17 vs. BrachyLT 20 ± 4·82, P < 0·05). Long-term administration also reduced the activity of NF-κB and AP-1 by 62–64% and 42–58%, respectively. However, the effects of different durations of clopidogrel administration on artery dimensions were not statistically significant. Conclusions Regarding inflammation and transcription factor activity at the PCI site, long-term clopidogrel administration is superior to short-term administration, especially in severely injured arteries. Transferring our results to the human situation, patients with more severely diseased arteries may benefit from a prolonged clopidogrel medication after PCI. [ABSTRACT FROM AUTHOR]

Background: Tissue factor (TF), the initiator of coagulation, circulates in blood and contributes to thrombosis in patients with coronary artery disease (CAD). TF is present in the α-granules of platelets. Therapy with clopidogrel results in inhibition of platelet degranulation. Whether clopidogrel affects circulating TF is unknown. This study examined the effect of clopidogrel on TF level in the blood of patients with stable CAD and ST-elevation myocardial infarction (STEMI) as well as healthy controls. Methods: Thirty-three patients with CAD and twenty with STEMI were studied pre and post clopidogrel therapy (loading dose 300 mg, then 75 mg daily). All were treated with aspirin 100 mg/d. The control groups consisted of thirty healthy male volunteers also treated with clopidogrel and ten patients with CAD treated with aspirin only. TF concentration in blood drawn pre and 96 h post clopidogrel administration was measured by enzyme-linked immunosorbent assay. Results: Patients with CAD and STEMI had significantly more TF in blood than healthy controls. Clopidogrel reduced TF in stable CAD patients to levels seen in healthy controls. No alterations in TF were found in controls and patients with STEMI post clopidogrel therapy. Clopidogrel reduced sCD40L level in stable CAD patients, but not in STEMI patients. A correlation between TF and sCD40L was found for the combined CAD and control, but not STEMI group. Conclusion: Clopidogrel leads to a reduction of not only sCD40L but also TF in stable CAD. The reduction of TF may lead to a reduced thrombogenicity, contributing to the benefits of clopidogrel therapy. [ABSTRACT FROM AUTHOR]

SummaryAims Platelet activation is known to be associated with arrhythmic effects in myocardial ischaemia. The present study attempts to clarify whether diabetic cardiovascular autonomic neuropathy (CAN) is associated with intravascular platelet activation. Methods Platelet activation was assessed by flow cytometry analysis in 30 patients with Type 1 diabetes mellitus screened for diabetic complications. Fifteen patients showed evidence of CAN as assessed by a battery of standard cardiovascular autonomic reflex tests. Fifteen patients without CAN were then selected as a matched control group. Platelet activation was assessed by flow cytometric detection of activation-dependent platelet membrane antigens (P-selectin (CD62), thrombospondin, lysosomal GP53 (CD63) and ligand-induced binding site-1 of GPIIb/IIIa (LIBS-1)). Results Significantly more activated platelets were detected in the patients with CAN showing 20.9% (coefficient of variation (CV) 44%) CD63+ (vs. 17.2% (CV 19%) in controls, P ≤ 0.05), 6.4% (CV 87%) CD62+ (vs. 4.1% (CV 37%), P ≤ 0.05), and 6.7% (CV 55%) thrombospondin+ (vs. 4.6% (CV 39%), P ≤ 0.01) platelets, respectively. LIBS-1 on platelets was not significantly different between patients with and without CAN. No correlation was found between glucose metabolism and platelet activation. Conclusions Cardiovascular autonomic neuropathy is associated with platelet activation in Type 1 diabetes mellitus. The high platelet activation may reflect an increased prothrombotic state in diabetic cardiovascular autonomic dysfunction. [ABSTRACT FROM AUTHOR]

The gastrointestinal tract of pig is a good and available (slaughterhouse material) model to study the enteric nervous system in relation to nutrition, inflammation, development and disease in general. In order to investigate the responses of the enteric nervous network to a variety of stimuli, e.g. growth factors, hormones, extracellular matrix components, but also noxious compounds in a controlled manner, an in vitro experimental set-up is most appropriate. Methods to obtain in vitro cultures of enteric neurons of rodents, chicken and human are well described. This study attempted to use these methods on pig material. The muscle layer containing the myenteric plexus was dissected from pieces of fetal, neonatal and adult pig jejunum. They were rinsed in Hanks basal salt solution (BSS) in which antibiotics were added. Pieces of ±25 mm2 were transferred to BSS with 1 mg/ml collagenase and 1 mg/ml trypsin inhibitor and incubated for 60 min (5% CO2, 90% RH). Subsequently, they were vortexed for 20 s and ganglia were selected. The procedure was repeated ±4 times. Myenteric ganglia could then be plated or further dissociated in 1 mg/ml trypsin in BSS for 30 min in the incubator. Afterwards, the dissociated ganglia were centrifuged (5 min 1500 rpm) and the trypsin-solution was replaced with Dulbecco's minimal essential medium (DMEM). The explants or dissociated myenteric ganglia were transferred on non-coated or coated (poly-L-lysine, laminin, fibronectin, collagen or extracellular matrix gel) cover slips. After incubating for 45 min they were topped with 1 ml of DMEM with antibiotics and with or without fetal calf serum (5%). Medium was replaced twice a week. Using immunohistochemistry, both neurons (PGP9.5) and glial cells (S100) could be identified in both culture types. When cultivated under harsh conditions, the dissociated cultures gave rise to neurosphere-like bodies, containing neurons and glial cells. Thus, the digestion and dissociation technique is applicable to pig material. [ABSTRACT FROM AUTHOR]

The chondroitin sulphate proteoglycan brevican is one of the most abundant extracellular matrix molecules in the adult rat brain. It is primarily synthesized by astrocytes and is believed to influence astroglial motility during development and under certain pathological conditions. In order to study a potential role of brevican in the glial reaction after brain injury, its expression was analysed following entorhinal cortex lesion in rats (12 h, 1, 2, 4, 10, 14 and 28 days and 6 months post lesion). In situ hybridization and immunohistochemistry were employed to study brevican mRNA and protein, respectively, in the denervated outer molecular layer of the fascia dentata and at the lesion site. In both regions brevican mRNA was upregulated between 1 and 4 days post lesion. The combination of in situ hybridization with immunohistochemistry for glial fibrillary acidic protein demonstrated that many brevican mRNA-expressing cells are astrocytes. In the denervated zone of the fascia dentata, immunostaining for brevican was increased by 4 days, reached a maximum by 4 weeks and remained detectable up to 6 months post lesion. Electron microscopic immunocytochemistry showed that brevican is a component of the extracellular matrix compartment. At the lesion site a similar time course of brevican upregulation was observed. These data demonstrate that brevican is upregulated in areas of brain damage as well as in areas denervated by a lesion. They suggest a role of brevican in reactive gliosis and are compatible with the hypothesis that brevican is involved in the synaptic reorganization of denervated brain areas.