Metabolic syndrome and chronic simvastatin therapy enhanced human cardiomyocyte stress before and after ischemia-reperfusion in cardio-pulmonary bypass patients.

Metabolic syndrome (MetS) is a set of metabolic alterations including high levels of low-density lipoprotein (LDL), which increase the risk of cardiomyopathy often leading to surgery. Despite inducing myopathy, statins are widely used to lower LDL. Cardiopulmonary bypass (Cpb) causes oxidative stress and metabolic injury, altering mitochondrial expression (Grp75) and endoplasmic reticulum (Grp78) chaperones, apoptotic enzymes (Bcl2 family) and increasing cardiomyocyte lipid/lipofuscin storage. We believe that cardiomyocytes from patients with MetS may be more sensitive to surgical stress, in particular after simvastatin therapy (MetS+Stat). The study group included ten patients with MetS, ten patients with Mets+Stat and ten healthy subjects. Myocardial biopsies were obtained both before and after-Cpb. Grp75, Grp78, Bax, Bcl2, lipids, lipofuscin and fibrosis were evaluated by immuno/histochemistry. MetS cardiomyocytes had higher Grp75, Bax, fibrosis and lipofuscin. MetS+Stat had lower Grp75 and higher Grp78 expressions, high Bax, fewer fibrosis and higher lipofuscin content. Cpb did not vary the fibrosis and lipids/lipofuscin content, although it influenced the chaperones and Bax expression in all groups. These changes were more profound in patients with MetS and even more so in patients with MetS+Stat. The results suggest that MetS and MetS+Stat cardiomyocytes were more highly stressed after-Cpb. Interestingly, simvastatin caused high stress even before-Cpb.

Supplementation with essential amino acids in middle age maintains the health of rat kidney.

Chronic kidney diseases are a social and economic problem, and diet has long been recognized as a fundamental modulator of kidney health in human and experimental models. Age-dependent alterations in mitochondrial function play a crucial role in the development of diseases of aging, and mitochondrial disorders have been observed in experimental models of kidney failure. Recently, the beneficial dietary effect of a specific mixture of essential amino acids (EAA) has been studied in elderly subjects, but no data were collected from the kidney. The aim of this study was to assess whether daily supplementation of the diet with EAA at the beginning of senescence could preserve renal health. We used middle-aged (18-month-old) male Wistar rats fed a standard diet and water ad libitum (M-aged group) or a diet with added EAA (1.5 g/kg per day) dissolved in drinking water for 3 months (M-aged+EAA group). Young (2-month-old) rats fed a standard diet for 3 months were used as controls. Mitochondrial morphology and markers for collagen, cyt-c-oxidase, HSP60, GRP75, eNOS, iNOS, Bax, Bcl2 and VEGF were analyzed in glomeruli and tubules. EAA supplementation limited fibrosis and increased the capillary tuft area in the glomeruli of M-aged rats. VEGF and eNOS were enhanced in glomeruli and the peritubular space with the EAA-supplemented diet. Mitochondrial cyt-c oxidase, Bcl2, and chaperones increased in the distal tubules of the EAA group to levels similar to those observed in the young group. Mitochondrial area and density after EAA intake did not differ from young groups. The results suggest that prolonged EAA intake could represent a strategy for maintaining the healthy status of the kidney in M-aged animals.

Essential amino acids improve insulin activation of AKT/MTOR signaling in soleus muscle of aged rats.

Essential amino acids (EAA) improve basal muscle protein synthesis in the elderly. Nevertheless, in settings of prolonged supplementation, putative signal pathways of EAA are currently unknown. The purpose of this study was to test the effects of prolonged supplementation of EAA enriched mixture (12-L-Amin) on Insulin/Insulin-like Growth Factor-1 (IGF1) pathway by measuring total and phosphorylated Akt (Ser473) and its upstream (IRS1 at Ser636) and downstream (mTOR at Ser2448, p70S6K at Thr389) targets in basal conditions and following acute insulin (0.1 U/L) incubation in vitro. To this aim, soleus muscles were dissected from male Wistar rats divided in three groups of 7 each: adults (AD, 10 mo of age), elderly (EL, 22 mo of age) and elderly supplemented (EL-AA, 12-L-Amin 1.5gr/Kg die in drinking water for 3 mo). EL showed reduced basal and post-insulin mTOR and p70S6K activation and reduced post-insulin IRS1 degradation relative to AD. EL-AA showed an increase of post-insulin Akt activation, no change in basal and post-insulin phospho-mTOR, lower reduction of phospho-p70S6K and increased post-insulin IRS1 degradation relative to AD. These results demonstrate that chronic 12-LAmin administration exerts anti-ageing effects on the activation/inactivation of the Insulin/IGF1/mTOR pathway which is identified as putative target of EAA in the elderly.

Nitric oxide involvement in the trigeminal hyperalgesia in diabetic rats.

Trigeminal hyperalgesia frequently appears in diabetic neuralgia altering the transmission of orofacial sensory information. This study was designed to explore the effects of trigeminal hyperalgesia in streptozotocin-induced diabetes monitoring the expression of nitric oxide synthase in the trigeminal ganglion cells. The threshold to heat noxious stimuli decreased in diabetic animals. The number of NADPH-diaphorase (NADPH-d)-positive neurons significantly decreased in the diabetic rats compared with controls. Insulin treatment prevented the decreased nociceptive threshold and reduction of the number of NADPH-d-positive neurons. These findings point out that there is a relationship between the trigeminal nociceptive perception and NADPH-d neuronal expression suggesting that NO may play a role in the pathogenesis of trigeminal sensory neuropathy.

Distribution of heat shock proteins in kidneys of rats after immunosuppressive treatment with cyclosporine A.

Cyclosporine A (CsA), a fungal undecapeptide, is the most common immunosuppressive drug used in organ transplantation and auto-immune diseases. However, it has severe side effects mainly on renal structures and functions. Therefore, nephrotoxicity is the major limiting side effect. Heat shock proteins (HSPs) are molecular chaperones, that are induced or expressed at high levels in mammalian cells due to a variety of adverse effects. HSPs have beneficial roles in protein processing and protection against cell injury. In the present study, we examined immunohistochemically levels of expression and localization patterns of various HSPs in rat kidneys after administration of a therapeutic CsA dose during 30 days. After CsA treatment, both constitutive HSP 25 and alpha B-crystallin immunoreactivity became stronger in glomeruli, proximal tubules and collecting ducts. Nuclear translocation of these proteins was detected in renal tubules. HSP 47 was detected in the interstitial space between tubules, vascular smooth muscle and medullary rays. Finally, HSP 72 was induced in the cytoplasm of epithelial cells of proximal and distal tubules, and in the cytoplasm of epithelial cells of Henle limbs and collecting ducts. These data demonstrate that CsA clearly induces increased immunoreactivity of HSPs in defined structures of rat kidneys. These findings suggest that these proteins are functionally involved in the defence against renal cellular damage caused by prolonged drug treatment in rat.

[Spontaneous rupture of the excretory tract following renal colic in kidney malformation]. / Rottura spontanea della via escretrice a seguito di colica renale in malformazione renale.

This paper reports a group's experience in treating a case of excretory tract rupture caused by a renal colic. After careful analysis and description of this case as well as related literature analysis, they have hypothesised the dynamic physiopathological events involved in this case. In addition, they have offered clinical considerations in preventing and detecting the development of such a lesion.

Does methylene blue protect the kidney tissues from damage induced by ciclosporin A treatment?

Ciclosporin A (CsA) is the first-choice immunosuppressant universally used in allotransplantation and autoimmune diseases. However, it has been demonstrated that this drug produces negative side effects in several organs and in particular in the lymphoid organs and in the kidney. It has been suggested that the CsA causes deleterious effects because it increases the oxygen free radical production. Here we wanted to test whether antioxidants protect the kidney parenchyma from the toxicity induced by CsA. We used methylene blue (MB), because it inhibits the formation of oxygen free radicals. The study was carried out in four groups of Wistar rats. Group I animals were intraperitoneally injected with MB (1 mg/kg/day) for 21 days; group II animals were subcutaneously injected with CsA (15 mg/kg/day) for 21 days; group III animals were treated with CsA combined with MB at the same doses and for the same periods as groups I and II, and group IV animals were injected subcutaneously with olive oil for 21 days as controls. The kidneys and the thymuses were subsequently removed and examined by conventional morphological staining (hematoxylin-eosin and Masson's trichrome) and enzymatic (NADPH-diaphorase, cytochrome, c oxidase, and superoxide anion production) and immunoenzymatic (inducible nitric oxide synthase--iNOS, endothelial nitric oxide synthase--eNOS) techniques. The thymuses were used to check the persistence of CsA-immunosuppressive effects during MB administration. Group I, III, and IV animals showed a normal kidney architecture and low levels of NADPH-diaphorase and of superoxide anion in all structures studied (proximal and distal tubules, glomeruli and the Henle loops). The cytochrome c oxidase showed a strong activity in proximal tubules, a moderate activity in distal tubules, and a weak activity in glomeruli and in the Henle loops. The expression of iNOS was weak in the proximal tubular epithelial cells and negative in the glomeruli, while eNOS was found to be moderately positive in the glomeruli and in the interstitial arteries, but not in the tubules and in the Henle loops. Degenerative changes with tubulointerstitial injury in the cortex of CsA-treated kidneys (group II) and increases of NADPH-diaphorase levels, iNOS activity, and superoxide staining were found in all structures. The expression of eNOS did not change in group I, III and IV animals. MB combined with CsA prevented the degenerative changes caused by CsA, preserving the structural, enzymatic, and immunoenzymatic integrity of the renal parenchyma. The mechanism by which MB exerts its protective action is not yet clear, but it seems to be due to its ability to inhibit xanthine oxidase and to quench nitric oxide production. Moreover, these data have been also supported by the following: (1) the superoxide anion levels were very high after CsA treatment and reduced after CsA-MB treatment, and (2) the iNOS levels increased in CsA-treated rats and showed normal levels after CsA-MB treatment. Moreover we demonstrated that MB administration did no compromise the CsA immunosuppressive effects, since the thymus showed a cytoarchitecture like that observed in CsA-treated rats.

Immunohistochemical study of cyclosporin-A on rat thymus after various treatment times.

The effects of the immunosuppressive agent cyclosporin A (CsA) after 4, 8, 15, and 21 days of treatment were investigated in the thymus of 12 week-old rats by an immunochemical method using the TRPM1 and TRPM2 monoclonal antibodies. Major changes were not observed after 4, 8 and 15 days' treatment. But thymus histology completely changed after 21 days: Interdigitating (TRPM1-positive) cells completely disappeared and only a small number of (TRPM1-positive) macrophages were observed in the thymus cortex. TRPM2-positive cells also decreased, most markedly in the outer cortex and cortex. These findings indicate that inhibition of T-lymphocyte maturation is linked to disappearance of interdigitating cells which produce cytokines with proliferative and differentiation properties.

Ultrastructural study of the alterations in spinal ganglion cells of rats chronically fed on ethanol.

A study was conducted to find the effects of chronic alcohol (EtOh) administration on the rat dorsal root ganglion (DRG) cells in vivo. Morphoquantitative changes of the cytoplasmic organelles in neurons and satellite cells (SC) of lumbar DRG of animals fed with 20 and 40% of EtOH for 6 months were determined at the electron microscopic level. Stereological methods were used to quantitatively evaluate the changes in the neuronal Golgi fields, in the lysosomal system components called dense bodies (DB), in the mitochondria, and in the cytoplasmic perikaryal projections (PP) characteristic of DRG neurons. Prolonged consumption of 20% EtOh was well tolerated by neurons. There were, however, some structural modifications in the studied organelles, and there was a significant increase in the neuronal surface. In SC the number of mitochondria and DB increased significantly. Treatment with 40% EtOH produced massive organelle alterations in both neurons and SC, including disruption of the PP, markedly reducing the neuronal surface area. The architecture of the SC sheath appeared disorganized. The alterations resembled those of senescence, and indicated that a high dose of EtOH (or its metabolites) had a profound disruptive effect on the organelles and on the membrane systems of the DRG cells. The SC of the DRG units from the animals fed with EtOH were the first to show significant morphological alterations. When the architecture of the SC sheath already showed evident signs of disorganization, the neuronal body was just beginning to show morphological damage. These results suggest that the progressive disorganization of the SC sheath is a probable source of complication in peripheral neuropathy.

Effects of cyclosporin A on some accessory cells of rat thymus.

We used immunohistochemistry with monoclonal antibodies (TRPM1, TRPM2) and histochemistry (acid phosphatase (AcP)) to investigate the effects of cyclosporin A (CsA) on macrophages and interdigitating cells (IDCs) in adult rat thymus after 21 days of treatment, and 21 days after stopping treatment. We also studied the development of IDCs and macrophages in 2, 6, 12, 20 and 30-day-old rats after 21 days of CsA administration to the pregnant mothers. In adult rats after 21 days of CsA treatment, IDCs were absent and only a small number of macrophages were present in the cortex; 21 days after stopping treatment the distribution of IDCs and macrophages had become similar to that in normal adults. The AcP+ macrophages in treated adult rats disappeared, as shown by immunohistochemistry, 21 days after CsA treatment and were again present, similarly to control animals, 21 days after stopping treatment. Therefore CsA causes the thymus medulla of adult rats to disappear and also a significant decrease in the macrophage population. We also found that while in normal rat neonates the thymus has the features of the adult thymus by the 12th day, in neonates from CsA treated mothers this did not appear until the 30th day. CsA treatment to pregnant rats delays thymus development in the young animals but does not cause persisting morphological alterations. This last finding was similar to that observed in adult rats 21 days after the end of CsA treatment.

A light and electron microscope study of rat abducens nucleus neurons projecting to the cerebellar flocculus.

Injection of horseradish peroxidase (HRP) into the cerebellar flocculus of the rat was employed to identify neurons in the abducens nucleus that project to the flocculus. The number, ultrastructural features and precise localisation of these neurons in the nucleus were examined. They were present bilaterally and represented about 7% of the total neuronal population of each nucleus. They were localised principally in the dorsomedial area of the cranial half of each nucleus and did not display the typical ultrastructural features of motoneurons. It is concluded that the localisation and ultrastructural characteristics of these HRP-positive neurons are useful for distinguishing them from other neuronal populations within the nucleus.

Bilateral tonsillar metastasis of gastric adenocarcinoma.

The occurrence of metastatic tumors in the tonsils, although rare, must be considered in the differential diagnosis of tonsillar lesions. In particular, the review of the literature reports only 7 cases of tonsillar metastasis of gastric carcinoma. The authors describe a case of adenocarcinoma involving bilaterally the palatine tonsils and the lingual tonsil, in a patient previously operated due to gastric adenocarcinoma. The clinical appearance of the lesion could be also consistent with lymphoproliferative or infectious diseases. The clinical and pathologic differential diagnosis of this peculiar case is discussed, together with a brief review of the literature.

Supraspinal connections and termination patterns of the parabrachial complex determined by the biocytin anterograde tract-tracing technique in the rat.

We have re-evaluated, using the anterograde tracer biocytin, supraspinal efferent projections from the parabrachial complex (PBN) to gain new information about the nature of its connections and nerve terminal patterns. We selectively injected biocytin into the 3 main regions of the nucleus (lateral PBN, medial PBN and Kölliker-Fuse nucleus). We observed distinct groups of ascending and descending fibres of different calibre from the PBN running throughout the brain and reaching many brain areas involved in the regulation of autonomic function. Here we detected labelled bouton-like terminals and fibres with en-passage varicosities. The ascending efferents from the lateral PBN mainly reached the reticular, raphe and thalamic nuclei, the zona incerta (ZI), central nucleus of the amygdala (CeA) and lateral area of the periaqueductal grey (PAG). Thin descending efferents reached the ventral region of the solitary tract nucleus (STN). The ascending efferents from the medial PBN were seen in the raphe nuclei, reticular nuclei, ventral and lateral areas of the PAG, thalamic nuclei, and in the medial and lateral nuclei of the amygdala. Descending efferents were seen in the STN and in some reticular nuclei. The ascending projections from the Kölliker-Fuse targeted the ventral area of PAG, CeA, ZI, lateral hypothalamic area, ventromedial thalamic nucleus and, with only a few terminals, the ipsi and contralateral reticular area. A large number of descending efferents reached STN, caudal and paragigantocellular reticular nuclei. The higher sensitivity of biocytin compared with other types of markers allowed us to determine more effectively the distribution, nature and extent of the supraspinal PBN connections. This suggested that in several nerve circuits the PBN probably plays a more important role than previously thought.

The rat abducens nucleus: a histo- and immunohistochemical study.

The number and proportion of motoneurons and interneurons present in the rat abducens nucleus was determined by the use of ChAT immunostaining and of HRP staining after retrograde transport from the injected right lateral rectus muscle. After HRP injection 67% of abducens neurons took up HRP and were hence motoneurons to the muscle. The cell bodies were mainly located in the middle third of the nucleus and were either spindle-shaped or pyriform. By ChAT-immunohistochemistry, 77% of the rat abducens neurons were ChAT-positive. After considering and discarding the hypotheses that the lateral rectus muscle could be incompletely filled by HRP, and that other muscles may be innervated by abducens motoneurons, it is concluded that some interneurons of the abducens nucleus of the rat are probably cholinergic.

Cytoplasmic changes in satellite cells of spinal ganglia induced by cisplatin treatment in rats.

The effects of cisplatin (cis-DDP) therapeutic treatment on the cytoplasmic compartment of satellite cells (SC) of rat dorsal root ganglia (DRG) were evaluated. Female Wistar rats were treated once a week with i.p. injection of cis-DDP (2 mg/kg) for 9 weeks. Morpho-quantitative changes of the cytoplasmatic organelles in SC cytoplasm from L4-L6 DRG were determined at the electron microscopic level. The quantitative changes in the lysosomal system components called dense bodies (DB) and in the mithocondria were stereologically evaluated. The data from SC were compared to those from the neurons. The cis-DDP treatment induced a great increase in DB and mithocondria volume of SC. Furthermore, the SC sheath showed an increase of the cytoplasmic lamellar expansions responsible of the physical dissociation of SC sheath from the nerve cell body surface. The comparative analysis from SC and neurons showed that the drug affected primarily the SC, supporting the idea that SC could be the initial target of cis-DDP molecule. The alterations of the anatomical relationships between SC and neurons could modify the cell control on extracellular solutions, altering the functional role barrier attributed to SC. It appears that not only the DRG neurons but also and principally the SC were involved in the peripheral neuropathy mechanisms caused typically from therapeutic cis-DDP administration.