Essential amino acid supplementation decreases liver damage induced by chronic ethanol consumption in rats.

The liver sustains the greatest damage from ethanol (EtOH) abuse. EtOH and its metabolites impair hepatocyte metabolism, causing intracellular accumulation of proteins and lipids and increasing radical oxygen species production. These processes are toxic to the mitochondrial respiratory chain and to mitochondrial DNA. We have recently shown that supplementating the diet of rodents with an essential amino acid-enriched mixture (EAAem) significantly increases mitochondrial mass and number in cardiac and skeletal muscles and improves mitochondrial function in aged animals. Thus, in this study we sought to test whether EAAem supplementation could reduce EtOH-induced liver damage. Groups of adult male Wistar rats were fed a standard diet and water ad libitum (the control group), drinking water with 20 percent EtOH (the EtOH group), or drinking water with 20 percent EtOH and EAAem supplementation (1.5 g/kg/day) (the EtOH+EAAem group) for 2 months. The blood EtOH concentration was measured, and markers for fat (Oil-Red-O), mitochondria (Grp75, Cyt-c-ox), endoplasmic reticulum (Grp78), and inflammation (Heme Oxigenase 1, iNOS, and peroxisomes) were analyzed in the liver of animals in the various experimental groups. EAAem supplementation in EtOH-drinking rats ameliorated EtOH-induced changes in liver structure by limiting steatosis, recruiting more mitochondria and peroxisomes mainly to perivenous hepatocytes, stimulating or restoring antioxidant markers, limiting the expression of inflammatory processes, and reducing ER stress. Taken together, these results suggest that EAAem supplementation may represent a promising strategy to prevent and treat EtOH-induced liver damage.

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.

Aluminium exposure induces Alzheimer's disease-like histopathological alterations in mouse brain.

Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer's disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing beta-amyloid (Abeta) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Abeta and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Abeta similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer's disease. So, we hypothesise that chronic Al administration is responsible for oxidative cell damage that interferes with ER functions inducing Abeta accumulation and neurodegenerative damage.

Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury.

Nitric oxide (NO), produced by nitric oxide synthase, is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. This study sought to elucidate the impact of pharmacological induction of heme oxygenase-1 (HO-1) on renal I/R injury. Rats were subjected to 45 minutes of renal ischemia followed by various times of reperfusion (30 minutes, 1 hour, or 3 hours). Plasma from sacrificed rats was obtained, and the kidneys processed for the expression of iNOS, cleaved caspase-3, p38MAPK and for immunohistochemical analysis. Furthermore, we determined renal and plasma levels of lipid hydroperoxides, total thiol groups, and plasmatic NO2-/NO3- formation. Our results showed a time-dependent increase in iNOS expression, which was also confirmed by increased plasma formation of NO2-/NO3-. Interestingly, this effect was reversed by pretreatment (12 hours) with SnCl2, a potent and specific inducer of renal HO-1 expression and activity, or by intraperitoneal injection of biliverdin (10 mg/kg). Furthermore, we observed a concomitant reduction in plasma and renal LOOH formation, a normalization of renal total thiol content, a reduction of caspase-3-mediated apoptosis, and a significant increase in p38MAPK phosphoration. Taken together, these results suggested that HO-1 and its byproduct biliverdin play major roles in the pathophysiological cascade leading to renal I/R injury.

Different experimental protocols for decontamination affect the cleaning of medical devices. A preliminary electron microscopy analysis.

The aim of the present study was to examine the efficiency of different decontamination-cleaning protocols on blood-soiled catheters used for interventional cardiology. Electrophysiology and cardiac ablation disposable devices were contaminated with bacteria-spiked human blood and underwent four different pre-sterilization protocols, including a chlorine-releasing agent, a polyphenolic emulsion, and an enzymatic detergent. Treated samples were examined by optical microscopy, scanning electron microscopy and transmission electron microscopy to identify and characterize biological and inorganic residuals. The use of chlorine as a first treatment caused denaturation of serum proteins and adherence of blood components to the surface of the device, thus hindering the cleaning efficiency of subsequent treatments with enzymatic detergents. An enzymatic/chlorine protocol was more efficient, but was considered to be a greater risk to healthcare staff. Polyphenolic-based treatments had the highest level of efficiency in bioburden removal, but interaction and adsorption of this class of chemicals onto biopolymers might lead to serious concerns about toxicity on subsequent reuse. Adequate pre-sterilization cleaning is fundamental for sterilization success and high-resolution electron microscopy can provide significant and detailed information about the efficiency of chemicals used for cleaning a blood-soiled device.

Exposure to aluminium changes the NADPH-diaphorase/NPY pattern in the rat cerebral cortex.

Aluminium (Al) impairs the glutamate-nitric oxide-cGMP pathway and reduces the number of nitroxidergic neurons in the rat somatosensory cortex. To understand better the effect of the time of exposure, we monitored the effect of aluminium administration on nitroxidergic neurons, identified by NADPH-diaphorase (NADPH-d) or by nitric oxide synthase (NOS) staining, after 0.5, 1, 2, 3, 6 and 12 months of aluminium administration. Since neuropeptide Y (NPY) is known to be colocalised with nitric oxide synthase in cortical neurons, the aim of this work was to study the effects of Al administration on the cortical expression of NADPH-d, nNOS, and NPY. NADPH-d or NOS positive neurons were found scattered in the cortex where they constituted about 1% of all neurons. Double staining using NADPH-d and NPY showed that almost all nitroxidergic neurons were co-localised with NPY neurons (NADPH-d/NPY double stained neurons) whereas some neurons were stained only with NPY (NPY single stained neurons) ; these were more numerous than NADPH-d/NPY double stained neurons. Al significantly reduced NADPH-d and nNOS positive neurons in the cerebral cortex time dependently, with the greatest effect appearing after 3 months. Also measured was the integrated optical density (IOD) of nNOS positive neurons showing a significant decrease of NOS immunostaining even in the remaining NOS positive neurons. The double staining experiment exhibited a decrease in NADPH-d/NPY double stained neurons with an apparent increase in NPY single stained neurons; these then decreased after 6-12 months. On the whole, the results confirm that Al impairs nitroxidergic pathways time dependently; moreover, the transient increase in NPY single stained neurons from 1 to 3 months suggests that there is an intraneuronal down-regulation of NOS, without affecting neuronal viability. In addition, the decrease in the NPY system found at 6 and 12 months may indicate that Al affected nitroxidergic and NPY systems at different times.

Stress proteins expression in rat kidney and liver chronically exposed to aluminium sulphate.

Aluminium (Al) is the third most widespread metal in the environment. It is toxic for the brain, bone and haematological system but unfortunately very little data exist for other organs. Stress proteins are induced or enhanced against metal toxicity with an essential role in the recovery of organules and other cellular proteins. This immunohistochemical study was performed to analyze the distribution of three stress proteins (HSP25, HSP72, GRP75) in rat kidney and liver orally exposed to Al sulphate daily for 3 and 6 months. Al-induced alterations were further studied by histopathology (H-E, PAS, Perl's, Masson) and ultrastructural morphometry. In the kidney: HSP25 was enhanced in proximal tubules after 6 months Al-exposure when abnormal brush borders were observed; HSP72 was induced in proximal tubules only after long Al-treatment; GRP75 was raised in midcortical area sometimes within nuclei. Furthermore, lysosomal and lipofuscins densities increased in the juxtamedullary tubules after 3 months Al exposure with respect to controls. In the liver: Perl's-positive deposits and fibrosis became evident after Al treatment. HSP25 was very weak; HSP72 focal in pericentral hepatocytes at 3 months and induced also in Kupffer cells at 6 months; GRP75 diffuse in periportal hepatocytes and non parenchymal cells at 6 months. Prolonged Al exposure stimulated stress proteins strictly organ-dependently in the rat. Their distribution in kidney and liver seems related to cumulative sublethal effects induced by metal and could be a sensitive index of Al susceptibility of these organs.

Mercuric chloride-induced alterations in stress protein distribution in rat kidney.

Mercuric chloride (HgCl2) induces acute renal failure associated to tubular impairment in experimental animals and humans. Stress proteins are a superfamily of proteins, comprising heat- shock proteins (HSP) and glucose-regulated proteins (GRP), enhanced or induced in the kidney in response to stress. They act as molecular chaperones that protect organelles and repair essential proteins which have been denatured during adverse conditions. The involvement of stress proteins in mercury-nephrotoxicity has not yet been well clarified. This study was undertaken to detect the tubular distribution of four stress proteins (HSP25, HSP60, GRP75, HSP72) in the rat kidney injected with HgCl2 and to quantify lysosomal and mitochondrial changes in straight proximal tubules, the main mercury target. Sprague-Dawley rats were administered i.p. with progressive sublethal doses of HgCl2 (0.25 mg/kg, 0.5 mg/kg, 1 mg/kg and 3.5 mg/kg) or saline (as controls) and sacrificed after 24 h. In dosages over 0.50 mg/kg, stress proteins increased and changed localization in a dose-dependent manner. HSP25 was focally expressed in altered proximal tubules at 1 mg/kg but in the macula densa it was at 3.5 mg/kg. HSP60 and GRP75 were intense in the nucleus and cytoplasm of proximal tubules but moderate in distal tubules. HSP72 was induced in distal tubules after low exposures but in proximal tubules it happened at the highest dose. Moreover, a significant increase in lysosomal and total mitochondria (normal and with broken cristae) area and density were progressively found after HgCl2 treatments. Stress proteins could represent sensitive biomarkers that strongly correlate with the degree of oxidative injury induced by HgCl2 in the rat proximal tubules.

Dose-dependent mercuric chloride tubular injury in rat kidney.

Mercuric chloride (HgCl(2)) produces an acute renal failure in experimental animal models. Since the mechanism of tubular injury has not completely been clarified, this morpho-quantitative study was undertaken to better understand the effects of 2 sublethal doses (T1=1 mg/kg and T3.5=3.5 mg/kg) of HgCl(2) in rat proximal tubules. Morphometrical analysis was performed to quantify both cytoplasmic and nuclear changes found in treated in respect to saline-injected proximal tubules. In the controls, single-cell damage was occasional and nucleolar changes were absent. HgCl(2) induced progressively severe proximal tubule atrophy. In the T1 group, necrosis was limited to pars recta cells and nucleolar segregation was often partial. In the T3.5 group, atrophy was extensive in both convoluted and straight tracts, the nucleolus was completely segregated and coiled body-like inclusions were detached from it. Ultrastructural analysis confirmed dose-dependent changes within straight proximal tubules, i.e., necrosis, apoptosis, nucleolar segregation, swollen mitochondria, vacuolization, and disrupted brush border. In conclusion, in the rat kidney HgCl(2) induced dose-dependent alterations not only in the cytoplasm but also in the nucleus of proximal tubule cells. These findings will be useful for better understanding of the pathogenesis of mercury nephrotoxicity and its genotoxic effect.

Cyclosporine A-induced kidney alterations are limited by melatonin in rats: an electron microscope study.

The effect of melatonin on experimental prolonged cyclosporine A (CsA) nephrotoxicity was analyzed by electron microscopy and morphometry. Twenty female Wistar rats were divided into groups that received (1) CsA at a therapeutic dose (15 mg/kg/day) sc for 40 days; (2) olive oil, CsA vehicle, as controls; (3) CsA plus melaton (1 mg/kg) ip for the same time; (4) melatonin alone, as additional controls. All controls had normal renal ultrastructure. However, in CsA group, both tubular and glomerular alterations were observed. The authors found apoptotic and necrotic proximal tubules with disrupted brush border, swollen mitochondria, abundant lysosomes; in the glomerulus, amorphous basement membrane, and abnormal mesangial matrix. CsA plus melatonin administration partly prevented these changes. Nevertheless, fibrillar deposits in podocytes and basolateral membrane dilatations in proximal tubules were observed. Histopathological analysis on semithin sections and ultrastructural morphometry were also performed. CsA induced interstitial fibrosis, atrophy, and PAS positive inclusions in the proximal tubules. Moreover, CsA reduced glomerular and mesangial volume but enhanced mesangial matrix volume and basement membrane thickness. After CsA plus melatonin, these parameters were reduced in the proximal tubules and restored in the glomerulus. In conclusion, melatonin attenuated morpho-quantitative alterations induced by CsA in the rat kidney.

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.

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.

Cell-specific expression of heat shock transcription factors 1 and 2 in unstressed rat spinal cord.

We investigated the intracellular distribution of heat shock factors 1 and 2 (HSF1, HSF2) in rat spinal cord by immunoblotting and immunohistochemistry using selective policlonal antibodies. Our results showed that both HSF1 and HSF2 were expressed in spinal cord cells (both neurons and glia) but at different intensity and cell localization. HSF1 was unusually distributed in the perinuclear compartment of selected neurons of the gray matter while astrocytes, oligodendrocytes and ependymal cells were predominantly stained in the nucleus. HSF2 was expressed at lower levels than HSF1 and was scattered in both nucleus and cytoplasm of the motoneurons of the ventral horns while glial cells again showed a nuclear positivity. This study suggested that the different ability of neurons vs. glial cells to react against adverse conditions might well be correlated with the different constitutive localization of HSFs.

Constitutive expression of heat shock proteins 70 and 90 in rat cerebellum.

Exposure to heat shock and other stressful conditions activates in cells of all organisms a specific genetic program. This enhances the synthesis of proteins with a protective role against cellular damage, called heat shock proteins (hsps). Furthermore, in the mammalian nervous system, a considerable amount of hsps is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we analysed the constitutive expression of proteins belonging to the hsp70 and hsp90 family in the rat cerebellum using immunocytochemistry with specific monoclonal antibodies. Our results showed that an intense immunostaining was evident, but was restricted in certain distinct cerebellar areas only, while no differences in the distribution of the two hsps were found. The strongest response was detected in the Purkinje neurons but deep cerebellar nuclei were also positive. In no case glial cells were found to be reactive for hsps despite their strong response for specific markers like glial fibrillary acid protein (astrocytes) and cyclic nucleotide phosphodiesterase (oligodendrocytes). These data indicate that both the hsp70 and hsp90 family have fundamental physiological functions in cerebellar neurons while they seem to play only a minor role in the metabolism of glial cells.

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.

Lysosomal changes in rat spinal ganglia neurons after prolonged treatment with cisplatin.

We performed morphometric and ultrastructural studies to determine the morphological response of rat spinal ganglion sensory neurons to prolonged administration of cisplatin up to a total dose of 18 mg/kg. We quantitated the different types of lysosomal system (LS) bodies present (primary and secondary lysosomes, lipofuscin granules) as well as multivesicular bodies in treated and control animals. Five rats were examined per group. This ultrastructural study on cisplatin-induced changes in LS of spinal ganglia neurons shows that the total area and total number of LS structures are significantly increased by cisplatin treatment. The main specific changes were increase in number of small-size lysosomes and increase in number of polymeric lipofuscin granules. Other alterations observed were presence of nucleolar segregation, patches of neurofilaments and deposits of osmiophilic material in the perikaryon and axon hillock, all indicating that sensory neurons are a major target of cisplatin.

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.

Improved immunoscintigraphy by subcutaneous injection of 99mTc or 111In labelled F(ab')2 fragments of an anti-melanoma monoclonal antibody.

Technetium-99m and/or 111In labelled F(ab')2 fragments of a melanoma associated MoAb 225.28S were injected i.v. in 80 patients affected by stage I to IV malignant melanoma. Seventy five percent of metastatic lesions already documented by other methods were detected by immunoscintigraphy, which was also capable of detecting a certain number of unknown metastases. However, we observed a lower percentage of positive scans in liver, lung and skin because of the poor tumour to background ratio. In some patients, subcutaneous (s.c.) injection allowed us to visualize documented metastases undetected by i.v. administration. An equal amount of non-specific F(ab')2 fragments (MoAb 4C4) injected s.c. as a negative control showed no positive scans. Clinical studies and chromatographic patterns of patient serum samples suggest that the s.c. route of administration offers, with respect to the i.v. route, the advantage of reducing vascular background and aspecific accumulation in liver, probably because of retention of possible contaminants by the lymphatic system.