C. Cavallotti

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.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus

The aim of this study was to examine rat thymus innervation using denervation techniques and to explore the related microanatomical localization of dopamine, D1, D2 receptors and dopamine membrane transporter (DAT). In the thymus subcapsular region, the parenchymal cholinergic fibers belong exclusively to phrenic nerve branching. No somatic phrenic nerve branching was detected in any other analysed thymus lobule regions. In rats subjected to sympathetic or parasympathetic ablation, it was observed that catecholaminergic and cholinergic nerve fibers respectively contributed to forming plexuses along vessel walls. In the subcapsular and septal region, no parenchymal nerve branching, belonging to sympathetic or parasympathetic nervous system was noted. Instead, in the deep cortical region, cortico-medullary junction (CM-j) and medulla, catecholaminergic and cholinergic nerve fibers were detected along the vessels and parenchyma. Dopamine and dopamine receptors were widely diffused in the lobular cortico-medullary junction region and in the medulla, where the final steps of thymocyte maturation and their trafficking take place. No variation in dopamine and DAT immune reaction was observed following total or partial parasympathectomy or phrenic nerve cutting. After chemical or surgical sympathectomy however, neither dopamine nor DAT immune reaction was noted again. Instead, D1 and D2 dopamine receptor expression was not affected by thymus denervation. In rats subjected to specific denervation, it was observed the direct intraparenchymal branching of the phrenic nerve and sympathetic and parasympathetic fibers into thymus parenchyma along vessels. These findings on the dopaminergic system highlight the importance of neurotransmitter receptor expression in the homeostasis of neuroimmune modulation.

A Morphometric Study of Age Changes in the Rat Optic Nerve

Age-related changes of the optic nerve fibres were studied in 3-month-old (young), 12-month-old (adult) and 24-month-old (aged) male Sprague-Dawley rats. The optic nerve was harvested with particular care from the intracranial portion. Cross sections from the optic nerve of animals of different age groups were stained with toluidine blue and examined under a light microscope at low and high magnification. Other sections were stained for the demonstration of glial cells using the method described by Holzer. A third group of sections were stained by the immunohistochemical method to detect glial fibrillary acidic protein, which is a marker for localising and characterising astrocytes. All these morphological results were subjected to the quantitative analysis of images and to statistical analysis of the values to identify significant morphometric data. Biochemical dosages of proteins were also performed on homogenised fragments of the optic nerve. Our results demonstrate that the following age-related changes can be observed: (1) an increase in meningeal membranes, (2) an increased number of astrocytes, (3) an increase in areal density of glial fibrillary acidic protein immunoreactivities, (4) an increased thickness of the entire optic nerve and an increased area of the nerve, (5) a decreased number of nerve fibres and (6) a decrease in the nerve fibre/meningeal membrane ratio from 3:1 to 1:1. Moreover, the amount of protein does not change with age. The rat optic nerve, therefore, appears sensitive to aging processes.

Analysis of Nerve Supply Pattern in Thoracic Duct in Young and Elderly Men

BACKGROUND Analysis of the innervation pattern of the thoracic duct in young and elderly human subjects has been performed. The subdivision of the vessels in cervical and lumbar region were taken in consideration. METHODS AND RESULTS Immunostaining for general nerve fibers with a PGP 9.5 marker disclosed a diffuse innervation of the thoracic duct in young subjects, which was strongly reduced in elderly subjects. In young subjects, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) immunoreactive fibers, markers of noradrenergic postganglionic sympathetic fibers, were frequent; choline acetyltransferase (ChAT) immunoreactive fibers, marker of cholinergic parasympathetic nerve fibers, were also well represented. Therefore, the influence of sympathetic and parasympathetic nerve systems on the thoracic duct can be confirmed. The immunoreactivity of vasoactive intestinal peptide (VIP), a neuropeptide frequently present in cholinergic parasympathetic nerve fibers, was scarcely present. Dopamine-positive fibers were observed in few short nerve fibers. Substance P (SP)-positive fibers were widely distributed in the medial and intimal smooth muscle layers, suggesting their involvement as contractile modulating fibers and sensitive fibers. In elderly subjects, an evident reduction of all specific nerve fibers analyzed was detected, the ChAT-positive fibers being the most affected. CONCLUSIONS The lymphatic vessel thoracic duct is able to regulate hydrodynamic lymph flow by intrinsic contraction of its smooth muscle layer. Therefore, analysis of the thoracic duct innervation pattern may be important in assessing the regulation of vessel contraction. These findings called attention to the reduction of lymphatic drainage functionality affecting fluid balance in the elderly.

Age-related changes in the human thymus studied with scanning electron microscopy

This investigation describes some morphological, age‐related changes in different compartments and cells of the human thymus. Scanning electron microscopic observations were done on human thymus samples obtained from four young and eight elderly patients during thoracic surgery and/or diagnostic biopsy of the thymus, after receiving authorization from the Ethical Committee of our university. The morphological data were submitted to quantitative image analysis so as to obtain quantitative results. Subsequently, the related values were used for statistical analysis. Our findings demonstrate that (1) all thymus compartments (subcapsular spaces, cortical, medullar, thymus microenvironment) contain numerous thymocytes even after the thymus has aged. (2) In older humans, residual thymus lymphoid islets contain, in addition to fatty cells and/or fibrous cells, also the same types of resident and nonresident cells (permanent and moving cells) that are found in young and adult subjects. (3) Endothelial cells of thymus microvessels contain numerous gaps. These gaps are tight in young subjects and become loose with age. (4) Thymocytes, in older subjects, are always found near these loose endothelial gaps of thymus microvessels. (5) While thymus cortical microvessels are provided with pericytes and/or periarteriolar spaces, microvessels of the thymus medullar are free of such spaces. Our morphological and quantitative results lead us to consider the possibility that the thymus fraction of resident and permanent cells (including thymocytes and reticular epithelial cells) is larger in younger subjects compared with older ones. The endothelial loose gaps of thymus microvessels, in older subjects, can allow the bidirectional transit of thymocytes through the wall of the said microvessels. Microsc. Res. Tech., 2008.

Distribution of peptidergic nerve fibres in the guinea pig trabecular meshwork.

A quantitative analysis of peptidergic nerve fibres located in the trabecular meshwork of the guinea pig has been performed. Our results confirm that this structure contains VIP‐, NPY‐ and substance P‐like immunoreactivity as major neurotransmitters. These findings were obtained using immunohistochemical techniques. For this purpose serial sections of the eye were stained by immunohistochemistry for each of three neurotransmitters and stained sections were analysed by quantitative image analysis. Our findings demonstrate that SP‐positive, NPY‐positive and VIP‐positive nerve fibres occupy 11.2, 4.9 and 2.4%, respectively, of the observed area (expressed as conventional units, C.U.) in the trabecular meshwork of the guinea pig eye. It is relevant to emphasize that the area containing these three types of peptidergic nerve fibres appears to be large (18.5 ± 6.6 C.U.) in proportion to the total observed area. The innervation of the drainage angle of the guinea pig eye has been well described by many authors. This is the first study to report quantitative measurements of three types of peptidergic nerve fibres identified and measured in this area. The presence of these three neurotransmitters in the trabecular meshwork of guinea pig eye suggests their possible participation in aqueous humor regulation.

Neuropeptides of human thymus in normal and pathological conditions.

Human thymus of healthy subjects and patients affected by thymoma-associated Myastenia Gravis were studied in order to visualize and compare the morphological distributive pattern of four neuropeptides: vasoactive intestinal peptide, substance P, neuropeptide Y, and neurotensin. Based on our observations, we formulated hypotheses on their relations in neuro-immunomodulation under physiological and pathophysiological conditions. Immuno-histochemical staining for neuropeptides was performed and morphological and morphometrical analyses were conducted on healthy and diseased thymus. In normal thymus, a specific distributive pattern was observed for the several neuropeptide-positive nerves in different thymus lobular zones. In particular substance P-positive fibers were observed in subcapsular zone, specifically located into parenchyma, where they represent the almost total amount of fibers; neurotensin-positive fibers were observed primarily located in parenchyma than perivascular site of several thymus lobular zones, and more abundant the cortico-medullary and medullary zones. Instead VIP- and NPY-positive fibers were widely distributed in perivascular and parenchymal sites of several thymus lobular zones. In thymoma, the distribution of neuropeptide-positive fibers was quantitatively reduced, while cells immunopositive to VIP and substance P were quantitatively increased and dispersed. Observation of the perivascular and parenchymal distribution of the analyzed neuropeptides suggests evidence that a regulatory function is performed by nerves and cells that secrete neuropeptide into the thymus. The alteration of neuropeptide patterns in thymoma suggests that these neurotransmitters play a role in autoimmune diseases such as Myastenia Gravis.

Cholinergic Innervation of Balt (Bronchus Associated Lymphoid Tissue) in Rat

The presence and distribution of acetylcholinesterase (AChE) and cholineacetyl transferase activities (Chat) were examined in the bronchusassociated lymphoid tissue (BALT) of juvenile, adult and old rats. Histoenzymatic and immunochemical methods were used in association with quantitative analysis of images and statistical analysis of the data. Our results showed that both AChE and Chat activities were primarily confined to the BALT lymphoid cells. Only a low level of activity was observed in the sub-pleural parenchyma of the lung and in the wall of the bronchus. Moreover, both AChE and Chat activities in the BALT are specifically located in the lymphoid cells. Histoenzymatic staining and corresponding values of quantitative analysis of images confirmed morphological and immunochemical results. Finally, the intensity of histoenzymatic staining for AChE and of immunochemical staining for Chat in BALT of rats strongly decreases with age. On the basis of our results we hypothesize that both AChE and Chat activities may play an important role in BALT and both these enzymes undergo specific age-related changes.

Distribution of acetylcholinesterase and cholineacetyl‐transferase activities in the human pulmonary vessels of younger and older adults

Background:  Age‐related changes were studied in younger and older humans, by staining of acetylcholinesterase and cholineacetyl‐transferase activities on autopsy samples of pulmonary vessels (arteries, veins and micro‐vessels).