Nicola Pescosolido

Biomolecular modulation of neurodegenerative events during ageing

The objective is to assess the modulation of retinal and optic nerve degenerative events induced by the combination of α-lipoic acid (ALA) and superoxide dismutase (SOD) in an animal model of ageing. For this study, 24 male Wistar-Harlan strain rats were left to age for up to 24 months. One group of rats was subjected to a diet supplemented with ALA and SOD for 8 weeks, while another group was used as a positive control and not subjected to any dietary treatment. To assess the cytoprotective effects of the antioxidants, a morphological analysis was carried out on sections of retina and optic nerve head, stained with haematoxylin-eosin, followed by an analysis of the modifications to nuclear DNA detected by the TUNEL technique. The lipid peroxidation assay was used to assess the damage induced by oxidative stress at cell membrane level. The molecules involved in apoptosis mediated by oxidative stress, such as caspase-3 and inducible nitric oxide synthase, were also assayed by immunolocalization and western blot. ALA and SOD are able to counteract senile neurodegenerative deterioration to the retina and optic nerve. Indeed, the combination of these antioxidant molecules can reduce oxidative stress levels and thus prevent both nuclear degradation and subsequent cell death.

Curcumin: Therapeutical Potential in Ophthalmology

Curcumin (diferuloylmethane) is the main curcuminoid of the popular Indian spice turmeric (Curcuma longa). In the last 50 years, in vitro and in vivo experiments supported the main role of polyphenols and curcumin for the prevention and treatment of many different inflammatory diseases and tumors.The anti-inflammatory, antioxidant, and antitumor properties of curcumin are due to different cellular mechanisms: this compound, in fact, produces different responses in different cell types. Unfortunately, because of its low solubility and oral bioavailability, the biomedical potential of curcumin is not easy to exploit; for this reason more attention has been given to nanoparticles and liposomes, which are able to improve curcumins bioavailability. Pharmacologically, curcumin does not show any dose-limiting toxicity when it is administered at doses of up to 8 g/day for three months. It has been demonstrated that curcumin has beneficial effects on several ocular diseases, such as chronic anterior uveitis, diabetic retinopathy, glaucoma, age-related macular degeneration, and dry eye syndrome. The purpose of this review is to report what has so far been elucidated about curcumin properties and its potential use in ophthalmology.

Degenerative and apoptotic events at retinal and optic nerve level after experimental induction of ocular hypertension

Ocular hypertension is a symptom of a glaucomatous condition characterized by a severe vision decrease. Blindness caused by the apoptotic death of the retinal ganglion cells and of the astrocytes of the optic nerve may eventually result. Experimental hypertension was induced by inoculation of methylcellulose in the anterior chamber. Chromatin staining, TUNEL assay, and inter-nucleosomal DNA fragmentation observed in retina and optic nerve strongly suggest that hypertension causes apoptosis. Immunolocalization of the fibrillary acidic glial protein, specific of cell stress, and caspase-3 in the same tissues, further support this mode of cell death. Activation of the ubiquitin dependant proteolytic system was also observed. Protection from apoptosis exerted by administration of the peroxide scavenger trolox, suggests that the apoptotic pathway is activated by an oxidative stress. The data presented here show that the experimental hypertensive insult induces degenerative and apoptotic events comparable to those observed in human glaucoma.

Age‐Related Changes in Rat Optic Nerve: Morphological Studies

Age‐related changes of the optic nerve were studied in 3‐month‐old (young), 12‐month‐old (adult) and 24‐month‐old (aged) male Sprague–Dawley rats. Cross sections of the intracranial portion of the optic nerves of animals of different age groups were stained with haematoxylin–eosin and examined under a light microscope at low and high magnification. Other sections were stained with crystal violet for demonstration of glial cells. A third group of sections were stained immunohistochemically to detect glial fibrillary acidic protein (GFAP) which is a marker for localizing and characterizing astrocytes. All morphological results were subjected to the quantitative analysis of images and to statistical analysis to identify significant morphometrical data. Tissue protein concentrations were determined on homogenized fragments of optic nerve. Our results demonstrate the following age‐related changes : (1) increase of the optic nerve sheaths (meningeal membranes); (2) increased number of astrocytes; (3) increase of areal density of GFAP immunoreactivity; (4) increased diameter and area of the optic nerve; (5) decreased number of nerve fibres; (6) decreased size of nerve fibres and (7) decrease of the nerve fibres/meningeal membrane ratio from 3 : 1 to 1 : 1. Moreover, the protein amount does not change with age. The rat optic nerve, therefore, appears sensitive to ageing processes and can be considered as a useful model for the studies on neuronal ageing.

Age-related changes in the human retina

BACKGROUND In a previous study, scanning electron microscopy (SEM) showed age-related changes in the rat retina. We carried out a study to evaluate age-related changes in the human retina. METHODS Samples of fresh retinal tissue obtained from younger (age 22 years or less) and older (age 66 years or more) donors were studied by means of traditional histologic methods and by SEM. Eight retinas were obtained from four donors whose corneas had been used for transplantation, and four retinas were obtained from four subjects whose eyes had been enucleated owing to injury. All morphologic results were subjected to quantitative analysis of images. The concentration of cytoplasmic (free) and structural (tissue-associated) protein in retinal tissue homogenates was determined by means of biochemical methods. RESULTS There was a decrease in all features studied with the exception of structural protein concentration. The mean retinal thickness (and standard error of the mean) was 426 (34.2) microm in the younger subjects and 261 (18.9) microm in the older subjects. The mean numbers of ganglion cells (and standard error of the mean) were 413.5/mm2 (32.3/mm2) and 256.2/mm2 (26.8/mm2) respectively, of capillaries 3.6/mm2 (1.4/mm2) and 1.8/mm2 (1.2/mm2) respectively, of synaptic bodies 122.4 (4.9) conventional units (CU)/area observed and 38.5 (1.6) CU/area observed respectively, of cellular processes 82.3 (3.1) CU/area observed and 13.1 (1.5) CU/ area observed respectively, and of intercellular connections 36.4 (2.5) CU/area observed and 14.3 (1.4) CU/area observed respectively. The mean concentration of total protein per milligram of fresh tissue (and standard error of the mean) was 92.1 (1.8) microg in the younger subjects and 78.7 (1.3) microg in the older subjects; the corresponding values for cytoplasmic protein were 27.6 (1.3) microg and 11.8 (0.8) microg, and for structural protein, 64.4 (1.6) microg and 86.9 (1.4) microg. All differences between the younger and older subjects were significant (p < 0.001) with the exception of mean concentration of cytoplasmic and of structural protein. INTERPRETATION The human retina undergoes specific changes with aging. SEM provides new morphometric information regarding age-related changes in photoreceptor cells, bipolar cells and ganglion cells that increases our understanding of this topic. Our results may be adopted as a model or as normal values when studying other changes that may occur in the human retina in pathological conditions.

Diffusive contribution to permeation of hydrogel contact lenses: theoretical model and experimental evaluation by nuclear magnetic resonance techniques

The biocompatibility of contact lenses is closely related to their oxygen permeability. In hydrogel lenses, this characteristic can be attributed to the water permeability resulting from a combination of viscous and diffusive fluxes. Hydrogel lenses were studied by means of nuclear magnetic resonance (NMR) relaxation times, resulting in a mathematical model which evaluated the water self-diffusion coefficient as a quantification of the diffusive contribution to permeation. Comparing the results obtained with the data of permeability to oxygen as measured by other techniques, a reasonable agreement was shown for lenses with a higher water content (WC) with respect to lenses with a lower WC: this difference was accounted for by considering the different contribution to permeation.

Oxidative Stress in Preretinopathic Diabetes Subjects and Antioxidants

BACKGROUND This study assessed the effect of a systemic oral treatment with antioxidants (AOs) in preretinopathic diabetes (PRD) patients, through the evaluation of oxidative stress in plasma and changes in the full-field electroretinogram (ERG). METHODS Thirty-two PRD subjects with good metabolic control were recruited. Patients were randomized in two groups, one of which received oral AO treatment with α-lipoic acid at 400 mg/day in association with genistein and vitamins, whereas the other group received a placebo. Free radicals and the AO barrier were evaluated in plasma with the Free Radical Analytical System 4 instrument (H&D srl, Parma, Italy), and the same day the electrophysiological response was measured by ERG. These analyses were performed at enrollment and after 30 days of treatment. RESULTS Statistically significant increases of plasma AO levels and ERG oscillatory potential values were observed in the group treated with AO, but not in the control group. CONCLUSIONS Results of this preliminary study suggest that an oral treatment with AOs in PRD subjects may have a protective effect on retinal cells, as detected by ERG analysis, through the strengthening of the plasma AO barrier.

L-carnitine and short chain ester in tears from patients with dry eye.

Purpose. The tear film is essential for the integrity of the ocular surface. In ocular diseases such as dry eye syndrome (DES), tear film osmolarity is increased relative to normal physiological conditions. DES can be caused by deficiency in lachrymation, hyperevaporation, or surface alterations. Carnitines, shown to have osmoregulatory properties, are thought to regulate tear film osmolarity, thus protecting the corneal surface from damage. We investigated the presence of carnitine in tears, compared tear carnitine concentrations in healthy subjects and in DES patients and speculate on carnitines potential role as a protective agent in the tear film. Methods. Tears were collected from 10 healthy subjects and 10 DES patients. Carnitine levels were assessed by high performance liquid chromatography-mass spectrometry. Results. Carnitine and its derivatives were detected in the tear samples. In DES patients, concentrations were substantially lower than in healthy subjects; the mean concentrations were l-carnitine, 3.27 ± 0.80 and 8.94 ± 0.50 &mgr;Mol/L; l-acetylcarnitine, 1.66 ± 0.50 and 3.05 ± 0.65 &mgr;Mol/L; and l-propionylcarnitine, 0.30 ± 0.11 and 0.57 ± 0.13 &mgr;Mol/L, in DES patients and healthy subjects, respectively. Conclusions. Although increased tear film osmolarity has been previously observed in DES patients, our study showed lower carnitine levels in DES patients than in healthy subjects, rather than the increased levels expected, although a causal relationship between carnitine levels and hyperosmolarity has not been established. The damage to ocular surface cells because of exposure to hypertonic tear film observed in DES may be partially because of an imbalance in the concentration of carnitine molecules in the tear film relative to the ocular surface cells. We propose, therefore, that carnitine solutions may have a role in preventing the adverse effects of observed hyperosmolarity and suggest that further studies are now warranted to investigate the clinical application of carnitine in the treatment of DES.

Lipoic acid in animal models and clinical use in diabetic retinopathy.

Introduction: Oxidative stress, a consequence of excessive production of reactive oxygen species (ROS), is a factor in the development of many diseases, including diabetes and its complications. Alpha-lipoic acid (ALA), a natural thiol antioxidant, has been shown to have beneficial effects on oxidative stress parameters in various tissues. This article is an up-to-date review of current thinking regarding ALA and its use in providing antioxidant (AO) drug therapy for ocular dysfunction due to diabetic retinopathy (DR). Areas covered: ALA prevents micro- and macro-vascular damage through normalized pathways downstream of mitochondrial overproduction of ROS, and preserves pericyte coverage of retinal capillaries. In addition, clinical studies suggest that oral administration of ALA can improve insulin sensitivity in patients with type-2 diabetes. Moreover, ALA treatment has been shown to suppress expression of vascular endothelial growth factor (VEGF), angiopoietin 2 and erythropoietin via blockade of superoxide formation. Expert opinion: The diverse beneficial effects of ALA, many of which have only recently been uncovered, suggest that it acts by multiple mechanisms on oxidative stress parameters. Consequently, ALA supplementation is an achievable adjunct therapy to help prevent vision loss in diabetic patients. Finally, further research to better understand the mechanism of ALA will be useful for the development of more effective therapies in patients affected by DR.

Age-related changes in the human optic nerve

BACKGROUND Recent morphologic research has demonstrated the presence of nerve fibres of different diameters in the human optic nerve. The purpose of this study was to investigate age-related changes in fibres of the human optic nerve. METHODS We studied the left optic nerve of 50 male cadaveric donors, 16 aged 18 to 22 years (mean 20 [standard deviation 1.2] years) and 34 aged 68 to 76 years (mean 72 [standard deviation 1.6] years). The samples were carefully harvested during autopsy from the intracranial portion of the optic nerve. Each nerve was cut into four 4-mm segments. After morphologic, histochemical and immunohistochemical staining, the optic nerve fibres were counted and measured. Each segment was evaluated under light microscopy for microanatomic details, glial cells and glial fibrillary acidic protein (GFAP) staining. The protein content was determined under biochemical analysis. We performed morphometric analysis by examining the optic nerve images quantitatively. RESULTS Compared with the younger group, in the older group there was an increase in mean diameter of the optic nerve (p < 0.001), due to an increase in the optic nerve:meningeal membrane ratio. There was also an increase in mean optic nerve area (p < 0.001) and in mean number of astrocytes and the related GFAP-immunoreactive area (p < 0.001). The mean number of nerve fibres of large diameter (greater than 4 pm) was decreased (p < 0.001). There was no difference in mean protein content of the fibres between the two groups. INTERPRETATION The human optic nerve is sensitive to the aging process and may be considered as a model for studies on neuronal aging.