Biomarkers of cell damage, neutrophil and macrophage activation associated with in-hospital mortality in geriatric COVID-19 patients.

BACKGROUND: The risk for symptomatic COVID-19 requiring hospitalization is higher in the older population. The course of the disease in hospitalised older patients may show significant variation, from mild to severe illness, ultimately leading to death in the most critical cases. The analysis of circulating biomolecules involved in mechanisms of inflammation, cell damage and innate immunity could lead to identify new biomarkers of COVID-19 severity, aimed to improve the clinical management of subjects at higher risk of severe outcomes. In a cohort of COVID-19 geriatric patients (n= 156) who required hospitalization we analysed, on-admission, a series of circulating biomarkers related to neutrophil activation (neutrophil elastase, LL-37), macrophage activation (sCD163) and cell damage (nuclear cfDNA, mithocondrial cfDNA and nuclear cfDNA integrity). The above reported biomarkers were tested for their association with in-hospital mortality and with clinical, inflammatory and routine hematological parameters. Aim of the study was to unravel prognostic parameters for risk stratification of COVID-19 patients. RESULTS: Lower n-cfDNA integrity, higher neutrophil elastase and higher sCD163 levels were significantly associated with an increased risk of in-hospital decease. Median (IQR) values observed in discharged vs. deceased patients were: 0.50 (0.30-0.72) vs. 0.33 (0.22-0.62) for n-cfDNA integrity; 94.0 (47.7-154.0) ng/ml vs. 115.7 (84.2-212.7) ng/ml for neutrophil elastase; 614.0 (370.0-821.0) ng/ml vs. 787.0 (560.0-1304.0) ng/ml for sCD163. The analysis of survival curves in patients stratified for tertiles of each biomarker showed that patients with n-cfDNA integrity < 0.32 or sCD163 in the range 492-811 ng/ml had higher risk of in-hospital decease than, respectively, patients with higher n-cfDNA integrity or lower sCD163. These associations were further confirmed in multivariate models adjusted for age, sex and outcome-related clinical variables. In these models also high levels of neutrophil elastase (>150 ng/ml) appeared to be independent predictor of in-hospital death. An additional analysis of neutrophil elastase in patients stratified for n-cfDNA integrity levels was conducted to better describe the association of the studied parameters with the outcome. CONCLUSIONS: On the whole, biomarkers of cell-free DNA integrity, neutrophil and macrophage activation might provide a valuable contribution to identify geriatric patients with high risk of COVID-19 in-hospital mortality.

My Mind Project: the effects of cognitive training for elderly-the study protocol of a prospective randomized intervention study.

BACKGROUND: Cognitive decline and dementia represent a key problem for public health as they heavily impair social functioning and independent living. The development of new strategies to support recommendations for patients and their caregivers may represent an outstanding step forward. AIMS: To describe the study protocol and methods of "My Mind Project: the effect of cognitive training for elderly" (Grant No. 154/GR-2009-1584108), which investigates, by the use of a multidisciplinary approach, the effects of a comprehensive cognitive training programme on performances in aged subjects with mild-moderate Alzheimer's disease, mild cognitive impairment and normal cognitive functioning. METHODS: The study is a prospective randomized intervention for the assessment of cognitive training effects in three groups of elderly subjects with different cognitive status. A total of 321 elderly people were enrolled in Marche Region, Italy. Each subject was randomly assigned to an experimental group or to a control group. Cognitive performances and biochemical blood markers have also been analysed before cognitive training (baseline), immediately after termination (follow-up 1), after 6 months (follow-up 2) and after 2 years (follow-up 3). DISCUSSION: The results will be useful to identify some efficient programmes for the enhancement of cognitive performance in elderly with and without cognitive decline. CONCLUSION: The application of a non-pharmacological approach in the treatment of elderly with cognitive disorders could have a profound impact on National Health Service.

Acetylcholinesterase inhibitors in Alzheimer's disease influence Zinc and Copper homeostasis.

BACKGROUND: Alzheimer's disease (AD) is the most common age-related neurodegenerative disease. An altered homeostasis of Zinc (Zn) and Copper (Cu), as well as a dysregulated expression of Zn-regulatory proteins have been previously described in AD. Acetylcholinesterase inhibitors (AChEI) are commonly used as AD treatment to improve cognitive function, but their effect on Zn homeostasis is still unexplored. OBJECTIVES: The aims of this study were to define the metal dyshomeostasis in AD patients, to investigate AChEI influence on Zn homeostasis and inflammation, and to analyze the relationship between cognitive impairment at two-year follow-up and metal concentrations, considering AChEI use. METHODS AND RESULTS: 84 Healthy Elderly (HE) and 95 AD patients were enrolled (62 AchEI user and 33 AchEI naïve). HE showed similar plasma Zn and Cu concentrations and Cu/Zn ratio in comparison to AChEI users, but significantly higher Zn level, as well as lower Cu amount and Cu/Zn ratio than AChEI naïve patients. Moreover, AChEI users had increased Zn plasma level, reduced Cu amount, Cu/Zn ratio, and IL1ß concentration and lower Zip2 lymphocytic expression vs. naïve patients. A multiple linear regression analysis showed that the MMSE score decline after two-year follow-up was reduced by AChEI therapy and was positively associated with plasma Zn decrease over time. CONCLUSION: Our data revealed that AChEI use may affect peripheral Zn and Cu homeostasis in AD patients, decrease Cu/Zn ratio demonstrating a general reduction of inflammatory status in patients under AChEI treatment. Finally, AChEI influence on circulating Zn could be implicated in the drug-related slowdown of cognitive decline.

Structural synaptic remodeling in the perirhinal cortex of adult and old rats following object-recognition visual training.

The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-month-old) and old (25- to 27-month-old) rats exposed to a six-session object recognition visual training were investigated by morphometric methods. The comparative analysis showed a higher synaptic numeric density, a lower synaptic average area, and a lower percentage of megasynapses (S > 0.5 microm2) in old trained rats versus controls, and a higher percentage of small (S < 0.15 microm2) junctions in adult trained rats versus controls. The more marked synaptic remodeling underlying memory consolidation in the perirhinal cortex of old rats might reflect a pre-existing lower dynamic status.

Age-related effects of moderate alcohol consumption on GAP-43 levels in rat hippocampus.

The effects of moderate intake of ethanol and ageing were investigated on the levels of the growth-associated protein GAP-43, whose expression has been used as an indicator of axonal growth during development, regeneration and remodelling of synaptic connections. Groups of female Wistar rats (12 and 24 months of age), were alcohol-fed for one month while age-matched control groups received an isocaloric diet. A quantitative evaluation of GAP-43 was performed in hippocampus and in hippocampal selected areas in view of the vulnerability of this complex to alcohol aggression by means of two different methods, namely Western blot analysis and immunohistochemistry. While the former measures total extractable GAP-43, the latter allows visualisation of in situ changes in topographical distribution of GAP-43. Western blot analysis revealed an age-dependent reduction (-47%) and an ethanol-associated increase (81%) of GAP-43 demonstrated only in the old group. Conversely, quantitative immunohistochemistry of GAP-43 in the entire hippocampus showed a non-significant ethanol-related decrement in 24-month-old rats (-30%), although the age-dependent reduction was confirmed. Ageing was associated with a decrement of GAP-43 immunostaining in CA3 stratum radiatum (CA3) and in inner molecular layer of dentate gyrus (IML). Treatment determined a decrease of GAP-43 immunostaining in adult rat CA3 and IML and no change in CA1 stratum radiatum (CA1). Our results suggest that immunohistochemistry evaluation underestimates GAP-43 levels in ethanol-treated animals possibly as a consequence of conformational changes induced by alcohol, resulting in non-targeting of the specific antibody. Western blot analysis demonstrate that although there is a reduction of GAP-43 levels in hippocampus of aged rats, this structure retain a remarkable potential to compensate for ethanol toxicity during ageing.

Mapping of mitochondrial metabolic competence by cytochrome oxidase and succinic dehydrogenase cytochemistry.

To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/microm(3) of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:1191-1192, 2001)

Cellular distribution of GAP-43 mRNA in hippocampus and cerebellum of adult rat brain by in situ RT-PCR.

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:1195-1196, 2001)

Distribution of map2 in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry.

The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging.

Quantitative immunohistochemistry of glucose transport protein (Glut3) expression in the rat hippocampus during aging.

Immunohistochemistry of Glut3 (45 kD), an integral membrane peptide mediating the transport of glucose in neurons, was carried out in the hippocampus of 3- and 28-month-old rats to assess the effect of age on energy metabolism. Free-floating sections of fixed-frozen hippocampi were processed for quantitative immunohistochemistry of Glut3. A rabbit affinity-purified antibody identified Glut3 immunoreactivity. Glut3 staining was intense in neuropil, axons, and dendrites, whereas nerve cell bodies were unstained. With aging, Glut3 reactivity was significantly decreased in the inner molecular layer of the hippocampal dentate gyrus (-46%) and the mossy fibers of the CA3 sector (-34%), whereas the stratum radiatum of CA1 did not show any difference due to age. These data document an age-dependent decrease in Glut3 expression in discrete areas of rat hippocampus. Glut3 constitutes the predominant glucose transporter in neurons and is found abundantly in regions with high synaptic density characterized by frequent bursts of function-adequate metabolic activity. Our findings therefore lend further support to the critical role of an impaired metabolism in age-related brain dysfunctions and disease.(J Histochem Cytochem 49:671-672, 2001)

Ethanol-induced decrease of the expression of glucose transport protein (Glut3) in the central nervous system as a predisposing condition to apoptosis: the effect of age.

We measured the effect of chronic ethanol administration on the expression of Glut3 in the cerebellum and hippocampus of adult and old rats. Glut3 expression significantly decreased in aging, in ethanol-treated rats vs. age-matched controls, and in adult- vs. old ethanol-treated rats. These findings lend consistent support to the hypothesis that disturbances of glucose metabolism due to ethanol may constitute an unfavorable condition predisposing to neuronal death.

Dynamic morphology of the synaptic junctional areas during aging: the effect of chronic acetyl-L-carnitine administration.

The ultrastructural features of hippocampal synaptic contact zones have been investigated by means of computer-assisted morphometry in rats of 6, 12 and 22 months of age and in age-matched animals chronically treated with ALCAR at a daily dose of 50 mg/100 g body weight from the age of 1 month up to the day of sacrifice. The number of synapses/microns 3 (Nv), the average size of the junctional areas (S) and the total area of the synaptic contact zones/microns 3 (Sv) were measured in tissue samples stained by means of the ethanol phosphotungstic acid (E-PTA) preferential technique for synaptic membranes. In control animals Nv was constant between 6 and 12 months of age, but significantly decreased in 22-month-old rats; S did not show significant differences due to age; Sv was unchanged between 6 and 12 months, but it decreased significantly in the old animals. In ALCAR treated rats Nv increased and S decreased significantly vs. Age-matched controls. Sv showed a lifespan constancy among the groups of age analysed. In ALCAR treated rats the number of contact areas smaller than 0.08 micron 2 increased by 18, 9 and 10% at 6, 12 and 22 months of age, respectively. ALCAR administration resulted in a lifespan modulation of synaptic structural dynamics. A proper metabolism at nerve terminals is accounted to play a crucial role in synaptic remodelling potential: on the basis of current research data, it is suggested that ALCAR may improve neuronal bioenergetic mechanisms.

Neuronal plasticity in aging: a quantitative immunohistochemical study of GAP-43 distribution in discrete regions of the rat brain.

Age-related changes in neuroplasticity have been investigated considering the neuronal growth-associated protein GAP-43 as a marker of nerve cell structural adaptive capabilities. We carried out a quantitative immunohistochemical study on the distribution of GAP-43 in the molecular layer of the cerebellar cortex, in the inner molecular layer of the hippocampal dentate gyrus, in the stratum radiatum of the CA1 region, in layer 1 of the cingulate cortex and in the nerve fiber layer of the main olfactory bulb of 3-, 18- and 31-month-old Wistar rats. A decrease of GAP-43 immunoreactivity was observed in the old rats in comparison with the adult animals in all the 5 areas analyzed, although these variations were only statistically significant in the dentate gyrus, cingulate cortex and olfactory bulb. In these latter zones, GAP-43 immunolabeling is reduced by 54, 42 and 38%, respectively, in the old versus the adult group. Comparing these data with the age-dependent decrease of neuron density innervating the areas investigated, we support the hypothesis that the decline of GAP-43 observed in old animals documents a consistent reduction of axon plasticity in the inner molecular layer of the dentate gyrus and in layer 1 of the cingulate cortex. These results suggest an important role of GAP-43 as a marker of age-dependent deterioration of synaptic plasticity, especially in those areas of the brain involved in memory and emotional behavior.

Morphological plasticity of synaptic mitochondria during aging.

A morphometric investigation has been carried out on the synaptic mitochondria of cerebellar glomeruli in young, adult and old rats by means of a computer-assisted image analysis technique. Mitochondrial volume density (Vv), numerical density (Nv), average volume (V) and average length (Skeleton = Sk) were investigated in tissue samples fixed, embedded and sectioned according to conventional electron microscopic methods. Vv was unchanged in the three groups of age taken into account. Nv was significantly increased in adult vs. young animals, whereas it was decreased in the old group as compared to both the other two groups investigated. V and Sk showed the same age-dependent changes: they significantly decreased in the adult vs. the young and the old groups of rats while increased significantly in the old rats vs. both the adult and young animals. A percentage distribution of Sk demonstrated that in the old group 20.6% of the population of synaptic mitochondria accounts for elongated organelles (> 5 microns) as compared to 8.6% and 5.3% in young and adult animals, respectively. The present findings match the changes previously reported by us on the ultrastructure of synaptic contact zones both in rats and human beings, and support the idea of an age-dependent dynamic adaptation in the morphology of synaptic mitochondria to cope with the metabolic needs of the pattern of synaptic connectivity they subserve.

Morphological adaptive response of the synaptic junctional zones in the human dentate gyrus during aging and Alzheimer's disease.

A computer-assisted morphometric study has been carried out on ethanol phosphotungstic acid (E-PTA) stained synaptic junctions in the human dentate gyrus supragranular layer from adult, old and Alzheimer's disease (AD)-affected patients. The number of synapses per unit volume of tissue (Nv = numerical density), the average area of the single junction (S) and the total area of the synaptic contact zones in a unit volume of tissue (Sv = surface density) were the 3 parameters taken into account. The synapse to neurone ratio was also calculated for each patient. During physiological aging, Nv and Sv significantly decreased and S increased, respectively. In the AD hippocampi, Nv and Sv underwent a further decrease which was in the range of more than 40% with reference to the adult values. S was the same as the old control group. In comparison with the adult values, the number of synapse/neurone decreased by 15.6 and 48% in old and AD patients, respectively. Nv, S and Sv, while reporting on discrete ultrastructural features of the synaptic junctional zones, are closely related to each other and, taken together per group of patients, may represent a reliable index of the morphological adaptive changes taking place at the synapses. Thus, the significant increase of S both in old and AD hippocampi may be regarded as a CNS plastic response to aging and disease, although the marked decrease of Nv and Sv supports that in AD synaptic ultrastructural alterations proceed beyond a critical threshold for functional recovery.

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella.

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

The lack of age-pigments and the alterations in intracellular monovalent electrolytes in spontaneously hypertensive, stroke-prone (SHRsp) rats as revealed by electron microscopy and X-ray microanalysis.

Male, spontaneously hypertensive, stroke-prone (SHRsp) rats established by Okamoto et al. (1974) were studied. About 80% of the males of this strain have a particularly short life span (33-41 weeks); they display a considerable hypertension (above 220 mmHg) and a tendency for plurifocal brain strokes. Hypertension and strokes can be provoked in an accelerated and synchronized fashion by supplementing 1% NaCl into their drinking water. Symptoms of the appearance of brain strokes can be judged from characteristic signs of motor disorders, and can be established also by pathohistology. Since hypertension and arteriosclerosis are frequently involved in aging, the question we intended to answer was whether these animals may represent a model of the normal aging process or not. Two approaches are described: (1) Accumulation of lipofuscin granules in their brain, liver and myocardium was followed by transmission electron microscopy before and after the appearance of strokes. It has been established that these tissues do not show any typical accumulation of lipofuscin granules, although submicroscopic signs of an enhanced damage of cell organelles (especially of mitochondria in liver and brain cells, but not in myocardium) were encountered. (2) The intracellular monovalent composition in the brain and liver was measured by using bulk-specimen X-ray microanalysis. The intracellular Na-content (mEq/kg water) was significantly higher (170-200%) in both the brain and liver cells, whereas the K-content increased only moderately (118-130%). The results suggest that although the SHRsp rats do not represent a direct model for the normal aging process from the point of view of lipofuscin accumulation, the shifts of the monovalent electrolyte contents in the brain and liver cells observed already in the youngest ages, are similar to those observed in aged normal rats. The theoretical consequences of such a conclusion are discussed.

Effects of idebenone on the intracellular water and dry mass content as well as of the intracellular monovalent ion concentrations of brain cortical cells of SHRsp rats as revealed by X-ray microanalysis.

The cerebral cortex of ten male SHRsp rats kept in conventional housing conditions were studied. Starting from the age of 3 months, five animals received placebo (5% gum arabic solution) and five other rats received 50 mg/kg idebenone suspended in the gum arabic, through a gastric tube for 4 weeks (except Sundays). During the last 2 weeks of treatment, 0.9% NaCl was added to the drinking water. Several completely untreated SHRsp rats of various ages were also involved in these studies. Blood pressure was measured weekly on the tail by means of an appropriate instrument. Serious hypertension was observed already by the end of the second week of treatment, displaying values of 250-260 mm Hg and increased further by about 20 mm Hg during the last 2 weeks of treatment. Intracellular water, dry mass and monovalent electrolyte concentrations were measured by means of a bulk specimen X-ray microanalytic method. The brain cells contained 77+/-1% water and 23% dry mass by weight in both placebo and verum-treated groups. The intracellular Na(+) content of all the male SHRsp rats was found to be significantly higher (180-200%) in the brain cells, whereas K(+) content increased only moderately, when expressed as percent of the intracellular dry mass. Idebenone treatment, however, lowered the intracellular Na(+) content of the brain cells to a significant extent (about 20%), i.e., it improved the Na(+) tolerance of the SHRsp rats, but did not alter the blood pressure.

Neurobiology of the aging brain: morphological alterations at synaptic regions.

Computer-assisted morphometric studies have been carried out on nerve cell terminal regions in rats of different ages. The numerical density (Nv), the average area (S) and the surface density (Sv) of ethanol phosphotungstic acid stained (E-PTA) synaptic junctions were evaluated in cerebellar glomeruli and dentate gyrus supragranular layers. The volume density (Vv), the average volume (V) and the numerical density (Nvm) of synaptic mitochondria were measured in the cerebellar glomeruli of young (3 months), adult (11 months) and old (28 months) animals. We found that during aging Nv and Sv undergo a significant decrease, whereas S is significantly increased. The mitochondrial Vv is unchanged in all the age groups analysed, whereas in old rats V is increased and Nvm decreased, respectively. We interpret our results as supporting that the old CNS retains part of its remodelling activity and is capable of adaptive morphological response at synaptic terminal regions.

Electron microscopic morphometric studies on the effects of idebenone on the synaptic remodelling activity in the hippocampus and cerebellum in normal old as well as in vitamin E-deficient adult rats.

Electron microscopic morphometric investigation has been carried out on the synaptic junctions of the hippocampal dentate gyri and cerebellar glomeruli of normal, old female Wistar rats (29 months of age), and vitamin E-deficient, female adult rats (11 months of age) of the same strain. The vitamin E-deficient diet was maintained from the age of 1 month for the subsequent 10-month period. Both the normal old and the vitamin E-deficient rats were treated with a daily dose of 50 mg oxidized idebenone/kg body w/day or with its solvent (5% gum arabic) through a gastric tube during the last month before killing them. The following morphometric parameters were evaluated in the hippocampal dentate gyrus and cerebellar glomerulus: the average length of the synapses (L) the surface density (S(v)) and the numerical density (N(v)) of the synaptic contact zones. Although the idebenone treatment caused a tendency to improve these parameters in both brain compartments studied, these improvements did not reach statistical significance in the cerebellum, but did so in the case of hippocampal N(v). Vitamin E deprivation caused the usual, known alterations of the synaptic parameters. Idebenone treatment during the last month of this experiment compensated the decrease of S(v) in both the hippocampus and the cerebellum; however, its protective effect was significant only in the case of hippocampus. Idebenone effect manifests itself in the increase of L, contributing mainly to the increase of S(v), since N(v) remained practically invariate. Placebo treatments did not result in any significant alterations in the vitamin E-deficient group.

Fabricated hyalS micropatterns and surface guidance of NCTC 2544 continuous cell line: an in vitro study.

Surface topography is important in establishing tissue organisation adjacent to implants, smooth surfaces generally being associated with fibrous encapsulation. By virtue of its large hydrated molecular volume and its capacity to form molecular matrix, hyaluronic acid can expand the interfibrillar collagen spaces to allow the movement of cells, although it can also hamper their locomotion. Low molecular-weight hyaluronan can also stimulate cell proliferation, especially at low concentrations. The aim of the present work was to evaluate in vitro the growth and migratory behaviour of NCTC 2544 keratinocytes cultured on different materials microstructured with hyaluronic acid or sulfated hyaluronic acid to assess the possibility of using these devices in the repair process of soft tissues. Ultrastructural morphological analyses, morphometric evaluations and detection of cytoskeletal elements were performed. Our observations provide evidence that micrometer-size parallel grooves of hyaluronic acid can influence cell growth behaviour since cells seeded onto the microstructured substrate arranged themselves according to a shape and an orientation that clearly reflected the chemotropism exerted on them by the two forms of acid. These data also highlight the importance of accurate microtexture fabrication. We intend to follow up these in vitro studies with in vivo experimental applications using PET and gelatin substrates structured with HyalS to evaluate wound healing responses, and to extend our investigations of the cytoskeletal modifications induced by different microstructures.