Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Somatostatin Receptor 2 Expression in Canine Meningioma.

The neuropeptide somatostatin (SST) plays an important regulatory role in the proliferation of normal and neoplastic cells. Five subtypes of somatostatin receptors (SSTRs), SSTR1-SSTR5, have been identified in human tumours. The SSTR2 subtype is identified most commonly in meningiomas. Long half-life SST analogues are now recommended for the systemic treatment of unresectable or radiation-refractory recurrent human meningiomas. In this study, SSTR2 expression was evaluated in 46 canine meningiomas; in 21 cases this was by immunohistochemistry and in 25 cases by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). In addition, SSTR2 expression was evaluated by immunocytochemistry, western blotting and RT-qPCR on primary cell cultures prepared from two canine meningiomas. SSTR2 immunohistochemical expression was observed in 17/21 cases (81%), and SSTR2 mRNA expression was detected in 14/25 cases (56%). SSTR2 protein and gene expression were not significantly correlated with the tumour histological subtype or grade. Overall, meningothelial meningiomas showed constant and diffuse SSTR2 immunohistochemical expression and the highest SSTR2 gene expression level compared with other subtypes. A tendency for loss of SSTR2 in high-grade meningiomas was observed in both immunohistochemical and RT-qPCR studies. About 90% of cultured canine meningioma cells showed SSTR2 expression. In both of the meningioma cell cultures, SSTR2 expression was also detected by western blotting and RT-qPCR. This study demonstrates for the first time that canine meningioma expresses SSTR2 and that this expression is maintained in vitro. Our results, while preliminary, provide encouragement for further studies aimed at finding novel medical treatment strategies for canine meningioma, especially for tumours that are not surgically accessible.

Sympathetic Innervation and Adrenergic Receptors in Equine Deep Digital Flexor Tendinopathy: Preliminary Results.

The aim of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses with tendinopathy by immunohistochemical labelling for tyrosine hydroxylase (TH) and α-1 adrenergic receptor (α1-AR). Twelve forelimbs were obtained from 10 horses with DDFT tendinopathy and six feet obtained from six horses were used as healthy controls. Post-mortem radiographic, ultrasonographic and gross examinations were performed on the suprasesamoidean area of the DDFT to assess the presence of tendinopathy. Longitudinal sections were collected and processed. Lesions were classified as core lesions, dorsal border lesions and parasagittal oblique splits. Immunohistochemistry was performed and the degree of immunoreaction was classified as absent, mild or marked. Seven core lesions, four dorsal border lesions and one parasagittal oblique split were identified. There was no increased expression of sympathetic innervation in samples with a dorsal border lesion of the DDFT compared with healthy samples. In contrast, core lesions showed increased expression of α1-AR and reduced expression of TH, which supports the hypothesis of a compensatory imbalance between the sympathetic mediator and the sympathetic receptors as a cause or effect of structural damage. In addition, adrenergic activation could stimulate cell proliferation and differentiation within these lesions.

Intra-articular administration of lidocaine plus adrenaline in dogs: Pharmacokinetic profile and evaluation of toxicity in vivo and in vitro.

The aim of this study was to evaluate the safety of intra-articular (IA) lidocaine plus adrenaline for improving peri-operative analgesia in anaesthetized dogs undergoing arthroscopy of the elbow. A solution of lidocaine (L) 1.98% plus adrenaline 1:100.000 was administered via the IA route and its safety evaluated in terms of cardio-, neuro-, and chondro-toxicity. No bradycardia or hypotension was recorded from induction to the last observational time point. Signs of toxicity of the nervous system could have been masked by the general anaesthesia but lidocaine concentrations detected in the blood were lower than those thought to be capable of producing toxicity. The assessment of in vitro chondrotoxicity showed a dose- and time-dependent effect of lidocaine on the viability of articular cells. Adrenaline appeared to reduce the chondrotoxicity of 1% lidocaine, following an exposure of up to 30 min.

Effect of submaximal exercise on horse homocysteinaemia: possible implications for immune cells.

Physical exercise induces a reduction of immune defences and an imbalance of red-ox status. In this study plasma levels of cysteine and homocysteine (Hcy) were determined in horses before and after submaximal treadmill exercise as well as the effect on horse lymphocyte proliferation. The exercise induced a significant increase in plasma Hcy levels, which remained high both after the 20 min recovery period and after 2 h of rest. Moreover, a reduction of lymphocyte responsiveness to the proliferative stimulus induced by Concanavalin A was observed. The effects of different Hcy concentrations on the proliferative capacity of lymphocytes in culture were also tested. The results indicated that 10 microM of this amino acid can reduce the proliferative capacity of resting lymphocytes as well as their responsiveness to mitogen. Moreover, our results suggest that homocysteinaemia could be considered one of the parameters affected by physical exercise in horses and that this amino acid could be implicated in the effects of physical exercise on the immune system.

Sympathetic innervation of the suprasesamoidean region of the deep digital flexor tendon in the forelimbs of horses.

The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue.

Effect of exercise training, selenium and vitamin E on some free radical scavengers in horses (Equus caballus).

Physical exercise increases both tissue needs for oxygen and cellular respiration and causes an overproduction of free radicals. When free radical generation exceeds the cell's antioxidant capacity tissue-damage develops due to oxidative stress. Therefore, it appears important to increase the scavenger ability of the tissues. Controlled training and dietary supplements may provide ways of doing this. As a model, we used 3-year-old racehorses (Equus caballus) which underwent a series of different physical exercise trials before and after 70 days of daily training and dietary supplements (vitamin E and selenium). The above treatments were able to increase both red blood cell resistance to the peroxidative stress induced in vitro and the glutathione peroxidase activity in lymphocytes. Moreover, they were also able to decrease malondialdehyde (MDA) concentration in the plasma as well as vitamin E consumption and the mobilisation of low molecular weight antioxidants (total peroxyl-radical trapping) following the physical exercise trials. The results obtained indicated that the training and diet supplements we used were able to significantly increase horse antioxidant defences in both the extracellular fluids and blood cells of our horses, thus decreasing peroxidative phenomena following physical exercise.

Physical exercise, oxidative stress and muscle damage in racehorses.

Since it has been suggested that lipid peroxidation following free radical overproduction may be one of the causes of physical exercise-induced myopathies and hemolysis in horses, we looked for the possible relationships between these phenomena and muscle fiber damage. We use a homogeneous group of Maremmana stallions which, after a 3-month training period, underwent a series of physical exercises of increasing intensity. We determined the contents of malondialdehyde (MDA), one of the main lipid peroxidation end-products, and glutathione the substrate of one of the most important free radical scavenger enzymes. We also measured creatine phosphokinase and serum lactate dehydrogenase isoenzyme activities whose modification may be indicative of muscle fiber damage. The results obtained indicated that the physical exercise we adopted was able to modify both MDA and glutathione contents in blood. However, its effect on some LDH isoenzyme activities suggested possible damage to tissues other than muscle.

Quantitation of phospholipids on thin layer chromatographic plates using a desk-top scanner.

A method for quantitating phospholipids separated on thin layer chromatographic plates by computer-assisted photodensitometry is described. After development, the plates are stained with molibdic reagent and the image obtained is acquired as TIFF file in the memory of a personal computer. The color intensity of the single spots of the digitalized image is analyzed using a dedicated software. Sensitivity and reproducibility are adequate for most of the needs of lipid chemist. When compared to conventional photodensitometric procedures, the present method offers the advantage of requiring a much cheaper hardware.

Platelet thromboxane A2 receptor number and function in normal and hypertensive pregnancy.

PROBLEM: Our study evaluated the number and function of platelets for thromboxane A2/prostaglandin H2 (TxA2/PGH2) and the platelet response to TxA2 receptor agonists in normal and hypertensive pregnancy. In addition, correlations between platelet membrane lipid composition and TxA2 receptor number and function were evaluated. METHODS: Ten normotensive healthy pregnant women (GC), 10 hypertensive pregnant women (PIH), and 10 normotensive nonpregnant healthy women (C) were examined. Radioligand binding and aggregation studies were performed. Lipid composition of platelet membrane was assessed as cholesterol to phospholipids ratio (CH/PL) and normalized to protein content. RESULTS: TxA2/PGH2 receptor binding sites and affinity did not differ among the groups. An enhanced response to TxA2 receptor agonist U46619 in GC and in PIH compared to C was observed, while platelet response to thrombin was not different among the groups. CH/PL was altered in PIH respect to GC and C. No significant correlation was found between CH/PL and number and function of platelet TxA2 receptors in PIH. CONCLUSIONS: In PIH there is no alteration of number or function of TxA2 receptors on platelet membrane. However, the enhanced TxA2 production associated with the increased sensitivity of platelet to TxA2 in pregnancy may contribute to the microcirculatory thrombotic changes of PIH.