Internalized chitosan nanoparticles persist for long time in cultured cells.

Chitosan-based nanoparticles (chiNPs) are considered to be potentially good carriers for the sustained intracellular delivery of specific molecules. However, scarce attention has been paid to the long-lasting permanence of these NPs in the intracellular milieu, as well as to their intracellular fate (i.e., distribution, interaction with cell organelles, and degradation) in the long term. In the present study, the presence and subcellular location of FITC-labelled chiNPs were monitored in HeLa cells up to 14 days post-administration using multicolor-fluorescence confocal microscopy and diaminobenzidine photo-oxidation at transmission electron microscopy. The main result of the present study is the demonstration that internalized chiNPs persist inside the cell up to two weeks, occurring in both the cytoplasm and nucleus; accordingly, chiNPs are able to pass from mother to daughter cells through several mitotic cycles. The cells did not show increased mortality or structural damage up to 14 days after chiNP exposure.

Kinematic analysis of the wheelchair tennis serve: Implications for classification.

The aim of the present study was to assess the validity of the classification system used in Open-class wheelchair tennis by investigating the relationship between post-impact ball velocity in the serve (measured using a sports radar gun) and the severity of impairment. Shoulder and wrist angles at the instant of ball impact were also estimated from 2D motion analysis. Forty-three nationally ranked Italian Open-class wheelchair tennis players were assigned to four groups (A­D) according to descending level of activity limitation. Ten successful flat serves (WFSs) and 10 successful kick serves (WKSs) for each player were recorded. One-way ANOVA showed that the severity of impairment significantly (P < 0.05) affected post-impact ball velocity and shoulder angle at the instant of ball impact. Furthermore, the mean value of post-impact ball velocity in WFS increased from group A to group D, i.e., with descending level of activity limitation. The results of this cross-sectional study indicate that the severity of impairment per se is associated with velocity of the wheelchair tennis serve, suggesting that the current classification is flawed in that it overlooks the impact of severity of impairment on players' performance.

The effects of three different rear knee angles on kinematics in the sprint start.

The purpose of this study was to investigate the rear knee angle range in the set position that allows sprinters to reach greater propulsion on the rear block during the sprint start. Eleven university-track team sprinters performed the sprint start using three rear knee angle conditions: 90°, 115° and 135°. A motion capture system consisting of 8 digital cameras (250 Hz) was used to record kinematic parameters at the starting block phase and the acceleration phase. The following variables were considered: horizontal velocity of the centre of mass (COM), COM height, block time, pushing time on the rear block, percentage of pushing time on the rear block, force impulse, push-off angle and length of the first two strides. The main results show that first, horizontal block velocity is significantly greater at 90° vs 115° and 135° rear knee angle (p<0.05 and p<0.001 respectively) at block clearance and the first two strides; second, during the pushing phase, the percentage of pushing time of the rear leg is significantly greater at 90° vs 135° rear knee angle (p<0.01). No significant difference was found for block time among the conditions. These results indicate that block velocity is the main kinematic parameter affected by rear knee angle during the starting block phase and acceleration phase. Furthermore, the 90° rear knee angle allows for a better push-off of the rear leg than larger angles at the set position. The findings of this study provide some direction and useful practical advice in defining an efficient rear leg biomechanical configuration at the set position.

Forced mild physical training improves blood volume in the motor and hippocampal cortex of old mice.

OBJECTIVES: To assess the effect of mild forced physical training on cerebral blood volume (CBV) and other brain parameters in old mice. SETTING: Treadmill in the animal house. PARTICIPANTS: Thirty old (>25 mo) male mice were randomly assigned to one of three groups, exercise (E), exercise plus testosterone (T) (ET), and rest (C). INTERVENTION: Mild physical training on treadmill (30 min a day at belt speed = 8 m/min, five days a week) with or without one weekly injection of testosterone. MEASUREMENTS: CBV, quantitative transverse relaxation time (T2) maps, and cortical thickness were measured by magnetic resonance imaging. RESULTS: A significant increase of CBV was found in the motor and hippocampal cortex of E and ET mice; cortical thickness was not affected. T2 maps analysis suggested that water distribution did not change. T administration did not add to the effect of physical training. CONCLUSION: This work provides first quantitative evidence that exercise initiated at old age is able to improve the hemodynamic status of the brain cortex in key regions for movement and cognition without inducing edema.

Tracing nanoparticles and photosensitizing molecules at transmission electron microscopy by diaminobenzidine photo-oxidation.

During the last three decades, diaminobenzidine photo-oxidation has been applied in a variety of studies to correlate light and electron microscopy. Actually, when a fluorophore is excited by light, it can induce the oxidation of diaminobenzidine into an electron-dense osmiophilic product, which precipitates in close proximity to the fluorophore, thereby allowing its ultrastructural detection. This method has very recently been developed for two innovative applications: tracking the fate of fluorescently labeled nanoparticles in single cells, and detecting the subcellular location of photo-active molecules suitable for photodynamic therapy. These studies established that the cytochemical procedures exploiting diaminobenzidine photo-oxidation represent a reliable tool for detecting, inside the cells, with high sensitivity fluorescing molecules. These procedures are trustworthy even if the fluorescing molecules are present in very low amounts, either inside membrane-bounded organelles, or at the surface of the plasma membrane, or free in the cytosol. In particular, diaminobenzidine photo-oxidation allowed elucidating the mechanisms responsible for nanoparticles internalization in neuronal cells and for their escape from lysosomal degradation. As for the photo-active molecules, their subcellular distribution at the ultrastructural level provided direct evidence for the lethal multiorganelle photo-damage occurring after cell photo-sensitization. In addition, DAB photo-oxidized samples are suitable for the ultrastructural detection of organelle-specific molecules by post-embedding gold immunolabeling.

Chitosan nanoparticles are efficient carriers for delivering biodegradable drugs to neuronal cells.

Chitosan nanoparticles (NPs) are biocompatible drug carriers able to cross the blood-brain barrier and represent a promising drug delivery system to the central nervous system. We used chitosan NPs to deliver the D-Ala2-D-Leu5-enkephalin (DADLE) to neuronal cells in vitro. DADLE is a hypometabolising synthetic opioid potentially useful for biomedical applications, but its short plasmatic half-life makes its in vivo administration ineffective. Here, we demonstrate by immunoelectron microscopy that (1) chitosan NPs are capable to deliver the opioid to neuronal cells; (2) DADLE is released from the internalised, opioid-loaded NPs up to 48 h; (3) in the nucleus, DADLE binds the transcription/splicing sites; (4) cells treated with DADLE-loaded NPs undergo a decrease in transcription factor amounts and proliferation rate without damage to cell organelles. In this model, chitosan NPs protected the loaded opioid from degradation, thereby prolonging its intracellular effects. These findings suggest that these NPs are efficient for the systemic and tissue administration of opioids in vivo.

Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabelling in cultured cells.

Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.

Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy.

Chitosan-based nanoparticles (NPs) deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier) and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB) photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing.

Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of premRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing.

Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of pre-mRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

Forced mild physical training-induced effects on cognitive and locomotory behavior in old mice.

OBJECTIVES: To assess the effect of mild forced physical training on cognitive and locomotory behavior in old (26 mo.) mice. DESIGN: Randomized, controlled study. SETTING: Open-field in the behavioral laboratory. PARTICIPANTS: Sixteen old sedentary male mice randomly assigned to one of two groups, exercise (E) or rest (R). INTERVENTION: group E underwent treadmill running for one month at moderate intensity (belt speed=8 m/min, 45 min, five days a week), group R was only allowed spontaneous locomotor activity. MEASUREMENTS: exploratory and locomotor behavior were evaluated in an enriched environment (Ethovision recording). RESULTS: motor patterns were significantly reduced (chi2 test, p<0.05) in the E vs R group after one month of training; exploratory patterns were not different, both groups showing modest exploratory activity. CONCLUSIONS: mild forced physical training initiated at old age may have detrimental effect on motor behavior in male mice without improving cognitive parameters.

The solitary chemosensory cells and the diffuse chemosensory system of the airway.

Solitary chemosensory cells (SCCs), which resemble taste bud cells, are present in the epidermis and oropharynx of most primary aquatic vertebrates. Recent studies have led to the description of SCCs also in mammals too. In the airway and digestive apparatus, these elements form a diffuse chemosensory system. SCCs do not aggregate into groups and in SCCs, as in taste bud cells, immunoreactivity forthe G-protein subunit alpha-gustducin and for other molecules of the chemoreceptive cascade was found. Questions remain about the role of the diffuse chemosensory system in control of complex functions (e.g. airway surface liquid secretion) and about the involvement of chemoreceptors in respiratory diseases. Therapeutic actions targeting chemoreceptors could be tested in the treatment of respiratory diseases.

Leptin in adipose tissue - Morphological aspects

Leptin is a peptide hormone encoded by the obgene and released by adipocytes. Over the past few years, the synthesis, regulation and effects of leptin have been extensively investigated in view of its pleiotropic role in human (patho)physiology. This work aimed at reviewing current morphological knowledge on leptin synthesis and secretion in adipocytes. It is concluded that the intracellular trafficking of leptin and the structural basis of leptin secretion remain to be defined. Further morphological work, especially at the ultrastructural level, is needed to firmly establish the intracellular distribution and the release pathways of the hormone (AU)

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Neuronal intermediate filaments in the developing tongue of the frog Rana esculenta.

The expression of several neuronal intermediate filament (NIF) proteins was investigated in the tongue of metamorphosing tadpoles (stage 38-45 of Gosner) and in adult individuals of the frog, Rana esculenta by means of immunohistochemistry. Results showed that nerve fibres at early stages of tongue development expressed peripherin (a NIF protein usually found in differentiating neurones) as well as the light- and medium molecular weight NIF polypeptide subunits (NF-L and NF-M, respectively); in the adult frog, peripherin was still found in nerve fibres reaching the fungiform papilla together with NF-M, but NF-L immunoreactivity was absent therein. Clusters of epithelial cells expressing peripherin were found in the early developing tongue before differentiation of taste organs, and NF-L and NF-H immunoreactivities were present in basal (Merkel) cells of the adult frog taste disc. Results indicate that neurones innervating the adult frog's taste disc maintain a certain plasticity in their cytoskeleton and that neuronal-like cells are present in the undifferentiated and differentiated tongue epithelium possibly playing a role in the developing and mature taste organ.

Quantitative ultrastructural changes of hepatocyte constituents in euthermic, hibernating and arousing dormice (Muscardinus avellanarius).

Hibernating animals represent a suitable model for investigating the structural effects of drastic changes in cell activity under physiological conditions. In this study we investigated by means of electron microscopy and morphometrical analysis the fine structural counterpart of functional rest in hepatocytes of the hibernating dormouse, Muscardinus avellanarius, in comparison with arousing and euthermic dormice. Our observations demonstrate that during hibernation several structural constituents of the hepatocyte undergo modifications. In particular, during deep hibernation, the total cell and cytoplasm area significantly reduced, as well as the total and percent glycogen and residual body area, and the Golgi apparatus almost disappeared. Upon arousal, the amount of glycogen was minimal, whereas total cell and cytoplasm area significantly increased towards the euthermic value as well as total and percent residual body area. In comparison with the euthermic condition, the total and percent cell lipid area significantly increased in early hibernation, reduced in deep hibernation and almost disappeared during arousal. Taken together, our findings give quantitative ultrastructural support to the marked reduction found in hepatocyte functional activities during hibernation. Such a reduced activity involves profound rearrangement of the euthermic cell structure, which is rapidly resumed upon arousal.

Proton and carbon NMR spectroscopic analysis of rat liver postnatal development.

BACKGROUND: In the present paper compositional changes of rat liver from birth to adult age were investigated by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. METHODS: The water-soluble and organic (lipid) fraction of pooled liver tissue from rats aged 1,7, 21 days, and 5 months were extracted according to Folch's method and analysed by proton and carbon nuclear magnetic resonance spectroscopy at 500 MHz. Phospholipids separated by chromatography from the organic fraction were also analysed. RESULTS: In proton spectra of the water-soluble fraction several metabolites were identified. Developmental changes in the ratio between betaine and choline and between 3-hydroxybutyrate and several other organic acids were shown. In proton spectra of the total organic fraction, signals from fatty acids chain, phophocholine and glycerol were assigned unambiguously. Phospholipids fatty acid acyl chain length, mean unsaturation and mean polyunsaturation increased from birth to weaning. The relative amounts of unsaturated fatty acids did not show obvious changes during the investigated period. CONCLUSIONS: This study shows that NMR spectroscopy is a useful tool to investigate postnatal developmental changes in the liver chemical constituents with a minimum of preparative procedures.

NMR spectroscopic analysis of rat brain development: in vitro proton and carbon studies of whole tissue and its phospholipid fraction.

The developmental patterns of the rat brain at several postnatal time points were investigated in Folch (chloroform-methanol) extracts. The chloroform- (lipid-containing) and water-soluble (cytosolic) fractions of whole-tissue extracts and the phospholipid fraction separated from the organic fraction by chromatography were analysed by means of high-resolution (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. Analysis of the cytosolic fraction showed the changing patterns of several brain metabolites during postnatal maturation, in full agreement with data obtained from perchloric acid extracts. (1)H NMR spectroscopy of the phospholipid fraction allowed for quantitative evaluation of fatty acid acyl chain length, mean unsaturation and mean polyunsaturation. It was found that both mean unsaturation and polyunsaturation are lower in adult brain phospholipids than during the first 3 postnatal weeks. (13)C NMR spectroscopy of the same fraction showed that the molar percentage of C(18) fatty acids (oleic, linoleic, linolenic) in brain phospholipids is similar at all the investigated time points. These results indicate that the combination of Folch extraction with simple chromatographic procedures and NMR analysis yields useful data to define the chemical maturation of the brain.

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice.

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.

In vivo octreotide administration acutely reduces exocrine granule size in the human pancreas.

BACKGROUND: Octreotide has been found to be effective in the prevention of postoperative complications of pancreatic surgery, but the benefit of preoperative octreotide administration has not been assessed. AIMS: To evaluate the ability of octreotide in reducing the amount of digestive enzymes in the pancreas before surgery, a morphometric ultrastructural study of the gland was undertaken in patients undergoing demolitive pancreatic surgery. METHODS: Twenty-three inpatients received saline (n = 8) or octreotide (100 micrograms s.c.) before surgery either once (n = 5), or three (n = 5) or six (n = 5) times at 8-hour intervals. At surgery, biopsies of the pancreas were taken and processed for electron microscopy. Several parameters were assessed in exocrine cells by means of ultrastructural morphometry. RESULTS: A single administration of octreotide significantly reduced the exocrine granule number and the mean and total granular surface sectional area, and the ratio between granule area and cytoplasmic area. Repeated octreotide administrations were associated with partial (3 administrations) and complete (6 administrations) recovery of all parameters to control values. CONCLUSION: Preoperative administration of octreotide, the synthetic analogue of somatostatin, acutely reduces exocrine granule number and size in the pancreatic cell. This finding can partially explain the prophylactic effect of the drug on early complications of pancreatic surgery. Such an effect is not maintained over multiple administrations of the somatostatin analogue. Possible explanations for this latter finding are discussed.