Morphological and functional changes in experimental ocular hypertension and role of neuroprotective drugs.

Glaucoma is a neurodegenerative disease characterized by progressive loss of retinal ganglion cell axons and their cell bodies in the retina. Elevated intraocular pressure (IOP) is considered to be the major risk factor associated with the development of this neuropathy. Randomized controlled clinical trials have demonstrated that in some patients the disease progresses, even after lowering the IOP. Several researchers have devised ways to induce elevated IOP in the rat eye with the aim of impeding the flow of aqueous humour out of the eye. Chronic ocular hypertension in rats induces morphofunctional changes in the optic nerve head and retina. Death of ganglion cells is thought to follow an apoptotic pathway. Changes have also been reported in neuronal and non-neuronal cells, levels of cyclooxygenase, and nitric oxide synthase, endothelin 1 and brain derived neurotrophic factor. Other mechanisms include intracellular electrolyte imbalance, microglial phagocytosis and elevated glutamate levels. Neuroprotection is the treatment strategy by preventing neuronal death. Hypotensive drugs (beta-blockers, alpha-agonists and prostaglandins), Ca++ channel blockers, NMDA antagonists and nitric oxide synthase inhibitors have been used as neuroprotective drugs in experimental models of glaucoma.

Ultrastructural changes in thyroid perifollicular capillaries during normal postnatal development and after infrared laser radiation.

This study was carried out on the perifollicular capillaries of the thyroid gland during postnatal (PN) development in normal conditions and after irradiation (46.8 J/cm(2)) with an infrared (IR) laser (904 nm). This was done using 11-, 21-, and 35-day-old Wistar rats. The changes in the capillaries were determined using quantitative methods as well as electron microscopy. During normal PN development the most relevant changes were an increase in the size of the capillaries, especially the lumen, thinning of the endothelium and an increase in the size of pinocytotic vesicles. Our results suggest that during PN development, the capillaries undergo some growth and maturation processes until they reach the optimal morphological conditions for their exchange functions. IR laser irradiation seems to stimulate the growth and maturation of endothelial cells in the youngest rats, while in older ones it causes irregular thickening of the endothelium and a reduction of the capillary lumen. These changes could be a sign of functional alterations in follicular cells caused by exposure to IR laser.

A quantitative investigation of microvascular changes in the thyroid gland after infrared (IR) laser radiation.

We present an ultrastructural study of thyroid capillaries in which 50-day-old rats Wistar rats, were irradiated with an infrared (IR) laser, (total dose, 46.80 J/cm2), the tissue quantified 1 day after ending treatment and a quantitative capillary analysis carried out by light and electron microscopy. Light microscopy was used to calculate capillary volume density revealing a significant increase in the irradiated rats. The quantitative measurement of parameters by electron microscopy required a two stage analysis: Level I, Electron Microscopy (Magnification x5,000); and Level II, Electron Microscopy (Magnification x26,000). At Level I, the following parameters were measured in each capillary: capillary area, capillary diameter, luminal area, luminal diameter, endothelial area, nuclear area and mean endothelial thickness. At Level II, pinocytotic vesicle diameter and their numerical density in endothelial cells were evaluated. Electron microscopic analysis revealed an increased luminal area in the capillaries of the irradiated rats. They also presented a decrease in endothelial cell thickness and vesicular diameter and an increase in vesicle numerical density. This latter increase is indicative of presumptive changes in capillary permeability, but the possible functional significance of these morphological changes in the endothelial cells requires further investigation.

Stereological age-related changes in neurons of the rat dorsal lateral geniculate nucleus.

Quantitative methods were used to compare the changes taking place in the volume of the dorsal lateral geniculate nucleus (dLGN) and corresponding neurons of young, adult and old rats. The study was carried out on male albino rats aged 3, 18, 24 and 28 months. In order to estimate the volume of the dLGN, neuronal volume density, numerical density and total number of neurons, we used serial sections stained according to the Klüver-Barrera technique and stereological methods. We found that dorsal lateral geniculate nucleus volume increases between 3 and 28 months, with a larger increase between 24 and 28 months. Neuronal volume density and numerical density of neurons are greater at 3 months and undergo a significant decrease between 24 and 28 months. Finally, the total number of neurons is shown to be smaller in adult and old animals than in younger ones, even though no significant variations are found between 18 and 28 months. Furthermore, this study confirms the need to analyze the total number of neurons and not just neuronal density if we want to correctly evaluate some of the microscopic changes occurring during senescence.

Relation between RNA content and ageing in neurons of the dorsal lateral geniculate nucleus.

We carried out a study to establish ribonucleic acid (RNA) content in the nucleus and cytoplasm of single neuronal cells from the dorsal lateral geniculate nucleus (dLGN) of 3-30 month-old rats. Mean RNA content was calculated as the product of RNA concentration and nuclear or cytoplasmic surface. The analysis of neuronal nuclei revealed no significant differences in RNA concentration, nuclear area, and RNA content from 3-18 months. However, a significant decrease in RNA concentration (18.73%) was found from the 18th-24th month, although no changes were observed in nuclear area and RNA content. The oldest rats, 24-30 months old, presented a significant increase in nuclear area and RNA content. As regards to the neuronal cytoplasm, no significant differences were found in any of the parameters at the ages from 3-18 months and 18-24 months. In contrast, a significant increase in RNA concentration (26.26%), cytoplasm area (18%), and RNA content (52%) takes place from the 24th-30th month. The increase in RNA content could be related to neuronal hypertrophy.

Prognosticating tools in primary neuroendocrine (Merkel-cell) carcinomas of the skin: histopathological subdivision, DNA cytometry, cell proliferation analyses (Ki-67-immunoreactivity) and NCAM immunohistochemistry. A clinicopathological study in 25 patients.

Histopathologically, 18 of our patients had classical Merkel-cell carcinomas (MCC); seven had neuroendocrine (NE) carcinomas with features different from MCC, here called "aberrant MCC". These patients showed a progressive neoplastic disease with a fatal outcome in four of them. The cytometric DNA distribution pattern of the tumor cell nuclei of all the aberrant MCCs was found to be of the aneuploid type. By contrast, the neoplastic disease of the majority of patients with classical MCC ran a milder course; a fatal outcome occurred in only one of them. Here, the DNA ploidy pattern was of the euploid (diploid or tetraploid) type in eight cases and of the aneuploid type in another eight. Our recently described "proliferation cell index" (PCI), based on nuclear immunoreactivity (IR) with the proliferation "marker" antigen Ki-67, was significantly lower in those five MCCs of the classical "DNA-diploid" type than in the seven "DNA-aneuploid" ones. These five patients presented a mild neoplastic disease; only one had a local recurrence and none had metastases. Otherwise, neither the PCI values nor the NCAM IR of the MCC cells were found to be of any prognostic significance.

Quantitative morphological changes in neurons from the dorsal lateral geniculate nucleus of young and old rats.

BACKGROUND: We studied the morphological changes occurring in neurons from the dorsal lateral geniculate nucleus (dLGN) during aging by analysing the size and shape of cell bodies and nuclei. METHODS: Male albino Wistar rats, aged 3, 18, 24, and 30 months, were used. After appropriate tissue preparation and following the usual histological procedure, the profiles of 1,920 neuronal bodies and nuclei were drawn using a camera lucida. Data was later recorded and processed with a semiautomatic image analyser. RESULTS AND CONCLUSIONS: We observed that dLGN neurons do not change in size from the age of 3-24 months. Between 24 and 30 months, the soma and nucleus of the cell undergo hypertrophy, 32.8% and 35.6%, respectively, when compared to those from 3-month-old animals (P < 0.01). Furthermore, we found a high correlation between cell body size/nucleus size, which does not disappear with age. The r values (correlation coefficient) were 0.7998, 0.8662, 0.8433 and 0.7304, and R2 (determination coefficient) was equal to 0.6397, 0.7504, 0.7112, and 0.5335. These latter values show that in 63.97%, 75.04%, 71.12%, and 53.35% of cases, respectively, modifications in somata size were accompanied by similar changes in nucleus size, and vice-versa. The study of the shape of the soma and nucleus of the cell revealed that both structures have a rounded-oval configuration that does not change in a significant way from adulthood to old age.

DNA ploidy changes in rhino mouse skin induced by all-trans retinoic acid and retinol.

OBJECTIVE: In order to assess the proliferative changes induced by all-trans retinoic acid (RA) and retinol (ROL), we have carried out a study of the DNA content of basal and suprabasal keratinocytes after epicutaneous application on the rhino mouse. STUDY DESIGN: Skin sections were analyzed stereologically and cytophotometrically using the Feulgen technique. The diploid DNA value (2C) was obtained from hepatocyte nuclei of control animals. Whereas cells in phase G0-G1 will show a 2C content, cells during phase S and in phase G2-M will show DNA values ranging from 2C to 4C and 4C, respectively. RESULTS: Although epidermal thickness (ET) increased significantly in all treated animals, surface density only increased in animals treated with all-trans RA. Quantification of DNA content of basal keratinocytes showed reduction of 2C and 2C-4C populations with a commensurate increase in proportions of cells with 4C and > 4C in the animals treated with 0.025% all-trans RA and ROL. Suprabasal keratinocytes of mice treated with 0.025% all-trans showed a decrease of the 2C population and an increased proportion of cells with 4C. Whereas 0.025% all-trans RA induced an increase of both basal and suprabasal DNA indices, ROL enhanced only the basal DNA index significantly. CONCLUSION: Animals treated with 0.025% ROL showed a significant increase in the basal proliferative index (PI) while the suprabasal PI remained constant; treatment with 0.025% all-trans RA produced a significant increase of both basal and suprabasal PIs and parakeratotic hyperkeratosis probably due to incomplete differentiation.

The effect of ageing on RNA content in neurons from the thalamic reticular nucleus visual sector.

In this paper we investigate nucleic acid content in neurons from the dorsocaudal region of the thalamic reticular nucleus in ageing Wistar rats. Nucleic acid per surface unit was analysed by calculating mean extinction using cytophotometric methods. Once the mean extinction and nuclear and cytoplasmic areas were known, nucleic acid total content was calculated. There was an increase in nucleic acid total content and in nuclear and cytoplasmic areas from the age of 3 months onwards. We interpreted these findings as a compensatory response, by 'neuronal hypertrophy', to the deterioration process occurring in the ageing rats. Between the 24th and 30th month, i.e. old age, nucleic acid per surface unit and total content in the cytoplasm exhibited a considerable decrease.

Quantitative histochemical study of cytochrome oxidase in the dLGN of aging rats.

We carried out a quantitative histochemical study of the enzyme cytochrome oxidase (CO) in neurons of the dorsal lateral geniculate nucleus (dLGN) of male Wistar rats aged 3, 18, 24 and 28 months. The results show that the activity of cytochrome oxidase decreases significantly between 24 and 28 months. We also checked whether a correlation existed between neuronal size and enzymatic activity. Low correlation coefficients were obtained which were between 0.4139 at 3 months 0.2092 at 28 months. Nevertheless, we observed a certain relationship between both parameters, and therefore we classified the neurons as light, moderate and dark according to their optical density, which correlates with enzyme cytochrome oxidase activity, and as small, medium and large depending on their size. We found that light neurons were scarcely represented in the dLGN. At the age of 3 months, the most frequent neurons were moderate, medium-size ones, and dark, small ones. The population of moderate neurons increased with age, reaching 74.5% at the 28th month, 52.2% of which corresponded to medium-size neurons. In the same group dark neurons decreased, falling to a total of 15.3% made up of medium and large-size ones. These results could be interpreted as reflecting a decrease in the bioenergetic competence of the neurons of this nucleus in old age.

The effect of ageing on neurones in the visual sector of the thalamic reticular nucleus.

This paper studies the quantitative morphological changes occurring during ageing in neurones of the dorsocaudal or visual sector of the thalamic reticular nucleus. Male Wistar rats aged 3, 6, 18, 24 and 30 months were used in this study which applied morphometric methods. We have observed an increase in the size of neurones from this sector between the 3rd and 24th month and a decrease between the 24th and 30th month. In all the ages studied the majority of neurones are fusiform.

Evolution of neuronal density in the ageing thalamic reticular nucleus.

In this paper we present an analysis of the visual sector of the Thalamic Reticular Nucleus (TRN) from 3, 6, 18, 24 and 30 month old Wistar rats using stereological methods. The volume density (Vv), the number of neurones per surface unit (Na) and the neurone numerical density (Nv) showed a progressive decrease between the 3rd and the 24th months as the animals aged, whereas a significant increase was observed between the 24th and the 30th month, the period at which these rodents have fully entered old age.

Study of certain clinical variables in patients with psoriasis and their relation to DNA content of keratinocytes.

BACKGROUND: In a previous study of 26 patients with psoriasis we analyzed cytophotometrically the nuclear DNA content of the germinative compartment of involved and uninvolved skin by means of the Feulgen technique. These subjects were classified into three groups according to their DNA profile. Group 1 had a monomodal diploid profile, group 2 showed a significantly increased 2C-4C population, and group 3 demonstrated high proportions of 4C and hyperdiploid keratinocytes. OBJECTIVE: Our purpose was to analyze clinical variables implicated in the development of psoriasis in reference to the three groups. METHODS: Nuclear DNA content of each group by quantitative histochemical studies was analyzed and correlated with variables such as chronologic age, sex, age at onset, duration of flare during the study, stress, and the Koebner phenomenon. RESULTS: No significant differences in DNA profile were observed in the involved epidermis among the clinical variables. The only differences in the uninvolved skin pertained to the duration of the flare, where a statistically significant difference was observed between groups 1 and 3 in the basal (p < or = 0.0459) and suprabasal keratinocytes (p < or = 0.06), and in the Koebner phenomenon, which was induced in all subjects (100%) in groups 2 and 3 and in only 44% of subjects in group 1. CONCLUSION: Uninvolved skin of patients with psoriasis should be included in analysis of the clinical behavior of the disease. Furthermore, the Koebner phenomenon is a good clinical indicator of the DNA profile of these subjects.

DNA determination of human hair bulbs in normal and androgenetic alopecia.

We carried out a cytophotometric study of the DNA content of matrix cells from normal hair follicles and those affected by androgenetic alopecia (early and established baldness), using the Feulgen technique. We thereby obtained a 2C reference value from lymphocyte nuclei from normal skin, from which we established a 2C-4C and 4C DNA content. The results obtained showed 71.75% of matrix cells in normal scalp to have a 2C content, while the remaining 28.25% was distributed between the values of 2C-4C and 4C. However, in early baldness, the number of 2C cells dropped to 48%, with a notable increase in cell populations with a 2C-4C and 4C content, which varied from 29.45% and 22.55% respectively. Finally, in established baldness, the 2C values decreased to as low as 29.56%, with the percentage of cells with a 2C-4C content reaching 38.71% and those of 4C, 31.73%.

Proacrosin as a marker of meiotic and post-meiotic germ cell differentiation: quantitative assessment of human spermatogenesis with a monoclonal antibody.

A quantitative immunohistochemical study of human spermatogenesis was performed using the 4D4 anti-proacrosin monoclonal antibody (mAb 4D4) as a marker of meiotic and post-meiotic germ cell differentiation. Cells from 15 testicular biopsies with normal spermatogenesis, 18 with slight and nine with marked hypospermatogenesis and six with maturation arrest were assigned to spermatogenic stages according to both nuclear maturation and proacrosin labelling patterns. The results showed that four spermatogenesis steps (mid- and late-pachytene primary spermatocytes, early and late spermatids) have to be separately considered for the classification of a given biopsy. Conversely, data from primary spermatocytes in the metaphase, anaphase and telophase stages and secondary spermatocytes did not show significant differences between biopsies. We conclude that: (1) slight hypospermatogenesis is due only to fewer cells entering meiosis, whereas in marked hypospermatogenesis there is also germ cell loss during the later meiotic steps and spermiogenesis; (2) the sloughing of germ cells from the epithelium could be of pathological significance; and (3) immunodetection with mAb 4D4 improves the assessment of spermatogenesis because it can label a protein expressed as early as meiotic prophase. In addition, mAb 4D4 labels a protein which is a marker of the Golgi complex allowing the detection of disturbances of cytoplasmic events during meiosis or spermiogenesis. Such an analysis is facilitated by mAb 4D4 labelling of paraffin-embedded sections.

Cytochrome oxidase activity in the lateral geniculate nucleus of postnatal rats.

We have studied the cytochrome oxidase activity and its pattern of distribution in the dorsal lateral geniculate nucleus of the rat during postnatal development. Between the 1st and the 8th postnatal days, the geniculate nucleus is seen to have a homogeneous enzymatic pattern with high neuronal density and moderately reactive neuropil. On the 15th postnatal day, different levels of neuronal enzymatic activity are found, and adult morphology is attained as of the 21st. The densitometric study has revealed that moderate and lightly reactive neurons are predominant between the 1st and the 8th postnatal days, whereas dark neurons are more numerous and optical density maximum on the 15th postnatal day. No variation in the enzymatic pattern was observed between the 21st and the 42nd days.

Effect of IR laser radiation on germ cell DNA content after one cycle of the seminiferous epithelium.

The effect of IR laser radiation on rat germ cells was investigated by cytophotometric quantification of the nuclear DNA content after a 15-day resting period. Two different doses of radiation energy were applied: 28.05 J/cm2 and 46.80 J/cm2. Both doses were found to increase the percentages of both spermatogonia with a 4c DNA content and primary spermatocytes with a > 4c DNA content. The elongating and elongated spermatids had a c DNA nuclear content, except for a small proportion (5%) of elongating cells that displayed a 2c DNA content at 28.05 J/cm2. Results revealed an increase in the germ cell DNA content after one cycle of the seminiferous epithelium after in vivo exposure to IR laser light. The laser sensitivity of the germ cells appeared to be related to their stage of differentiation and to the radiation energy applied, and the spermatogonia were the most sensitive. In all cases the lowest dosage induced the highest alteration.

Quantitative study of the human hair follicle in normal scalp and androgenetic alopecia.

We carried out a quantitative study of the matrix and dermal papilla of the human hair follicle of the scalp, both normal and in various degrees of androgenetic alopecia. A stereological study showed the measured parameters to decrease with increase in the degree of alopecia, particularly as regards the total volume of the matrix and its papilla. The ratio of the two volumes increased by 30%, indicating a much more marked decrease in size of the papilla than in the matrix size. The number of cell nuclei in the matrix and the papilla of alopecic scalp was found to be 30% and 50% smaller, respectively, than those of normal scalp. Finally, a morphometric study revealed enlarged nuclei in the matrix and papilla, as shown by their increased areas, perimeters, and maximal diameters.

Study of the DNA content of basal and suprabasal keratinocytes in psoriatic patients.

We carried out a cytophotometric study of the DNA content of basal and suprabasal keratinocytes using involved and uninvolved histological sections of epidermis from psoriatic patients using the Feulgen technique. The reference 2C value of the DNA content was obtained from lymphocyte nuclei in a sub-epidermal infiltrate from normal skin. According to the DNA content profiles obtained for the keratinocytes of psoriatic patients, individuals were classified into three groups: Group 1, composed of individuals with a monomodal diploid profile and preferential keratinocytes with a 2C DNA content and, hence, a low proliferation activity; Group 2, consisting of patients with significantly increased 2C-4C and 4C populations; and Group 3, made up of individuals with high proportions of 4C and hyperdiploid (6C, 8C, and > 8C) keratinocytes. The results obtained in this work and the observed changes in the proliferative activity of the keratinocytes in the three groups of patients led us to suggest that the cytophotometric determination of the DNA content of basal and suprabasal keratinocytes may be useful as a prognostic criterion for the classification of psoriatic patients in terms of the extent of alteration of their proliferative epidermal activity.

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.