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Marin F, Boya J, Calvo JL, Lopez‐Munoz F, Garcia‐Maurino JE. Immunocytochemical localization of basic fibroblast growth factor in the rat pineal gland. J. Pineal Res. 1994: 16: 44–49. The immunocytochemical localization of basic fibroblast growth factor (b‐FGF) during the postnatal development of the rat pineal gland was studied using a polyclonal antibody against the fraction 1–24 of bovine recombinant b‐FGF. Basic FGF immunoreactivity was evident from day 20 after birth in the endothelial cells and perivascular spaces of the gland. Although b‐FGF immunostaining showed its maximal expression at 30–45 days, it was maintained throughout the entire study period (up to 6 months), mainly in the distal zone of the gland. Pinealocytes did not show b‐FGF immunoreactivity at any time. There were no differences in the localization patterns or the intensity of b‐FGF immunostaining after the prenatal denervation with DSP‐4, a neurotoxic amine. The physiological role of b‐FGF in the adult pineal gland remains unknown; however, it does not seem to play a major role during the cytodifferentiation period of the parenchymal cells, or during the neovascularization in the early postnatal days. Furthermore, its immunocytochemical expression is not affected by the prenatal sympathetic denervation with DSP‐4, in contrast with other neutrophic factors. Copyright © 1994, Wiley Blackwell. All rights reserved

The immunohistochemical expression of glial fibrillary acidic protein (GFAP) and vimentin (VIM) was studied in reactive astrocytes of the rat cerebral cortex 5 days after a brain injury. Seriated Epon semithin sections were immunostained alternatively for GFAP or VIM. Thereafter, both antigens were detected in consecutive sections of the same cell. Bordering the wound, an inner reactive glial layer 300–350 μm thick, showed positive astrocytes with the two immunohistochemical techniques. In this layer, about 60% of the GFAP positive astrocytes were also positive for VIM. Outside the inner layer, only GFAP positive astrocytes could be found.

Microglial cells were selectively demonstrated in the central nervous system of adult rabbits and rats using lectin histochemistry. Biotinylated Ricinus communis agglutinin-120 (RCA-1) and biotinylated Griffonia simplicifolia B4 isolectin (GSA I-B4) were used as histochemical markers on sections of Bouin-fixed paraffin-embedded cerebrum and cerebellum. Results were quite similar using both lectins and both species. GSA I-B4 resulted in a better staining in the rat, while RCA-1 labelling was superior in the rabbit. Neither neurons nor glial cells other than microgha were stained with our technique. Lectin histochemistry applied for the detection of microglial cells appears to be of sufficient selectivity and may be considered as an important tool in the morphological and neurobiological study of these cells.

The ultrastructure of the dog pineal gland from the first postnatal day to the seventh month is described. In the first postnatal stages, pineal parenchyma only shows immature proliferative cells with abundant cytoplasmic glycogen. Nerve fibers are seen in the pineal connective tissue spaces. The differentiation of the dog pineal cell types begins in the first postnatal week. Both pinealocytes and pigmented cells are first seen on the fourth postnatal day. The pineal astrocytes are observed on the tenth day. Immature cells are still found in the pineal gland of 1 mo-old dogs. The differentiation of the dog pineal cell types is completed by the second postnatal month.

The localization of vimentin (VIM) and glial fibrillary acidic protein (GFAP) was studied immunohistochemically in adult rat optic nerve. Consecutive Epon semithin tissue sections were immunostained respectively for VIM or GFAP. With this method, both antigens were detected in consecutive sections of the same cell. According to our results, most astrocytes in the adult rat optic nerve showed coexpression of VIM and GFAP.

The cellular proliferative activity in the adult rat pineal gland was studied using bromodeoxyuridine immunohistochemistry during the light and dark phases of an artificial 12L:12D photoperiod. The results showed statistically significant differences in the number of labeled cells between the light and dark phases, with the labeled cells being almost threefold more abundant in the light period. Minor changes were also found in the pineal gland volume between both periods. The decrease in the number of labeled cells in the dark phase of the cycle could be related to the well-documented antimitotic action of melatonin.

The ultrastructure of the pineal gland in the adult cat is described and compared with that of other mammals. Connective tissue spaces showed capillaries with nonfenestrated endothelia and numerous unmyelinated nerve fibers. In the proximal region of the gland, myelinated nerve fibers coming from the anterior commissure were also found. Cat pinealocytes showed a nucleus with prominent nucleoli, a well developed Golgi apparatus, centrioles, granular endoplasmic reticulum, ribosomes, abundant microtubuli and enlarged mitochondria. Pinealocytes showed several long processes with bulbous endings filled with clear vesicles and scarce "synaptic" ribbons. Pineal astrocytes and their processes were characterized by the presence of abundant filaments.

The expression of glial antigens vimentin (VIM) and glial fibrillary acidic protein (GFAP) is described in the pineal gland of cats and dogs from the first postnatal days to adulthood. VIM immunopositive cells were observed from the first postnatal days in both species. GFAP expression starts from the second postnatal week. In adults, a notable population of stellate cells immunopositive for GFAP and VIM was found dispersed throughout the gland. According to their immunocytochemical profile, these cells could be identified as astrocytes.

The developmental expression of the glial antigens, vimentin (VIM), glial fibrillary acidic protein (GFAP), and S-100 protein is described in the rat pineal gland from the first postnatal day to adulthood. Thick VIM immunopositive cell cords forming a network throughout the pineal gland were observed from the first postnatal days. These cords progressively disappeared during the first postnatal month as their cells dispersed into the pineal parenchyma. From 20 to 25 postnatal days, pineal glial cells appeared as isolated star-shaped VIM immunopositive cells. Immunostaining for GFAP and S-100 protein showed a similar developmental expression pattern. Both antigens appeared later than VIM (15–20 postnatal days) and were restricted to the pineal glial cells located in the proximal third of the gland, close to the pineal stalk.

Sympathetic nerve fibers arising from the superior cervical ganglia are the main innervation of the rat pineal gland. Since most organs innervated by these ganglia contain nerve growth factor (NGF), the hypothetical existence of NGF in the pineal gland was investigated. The peroxidase anti-peroxidase technique was applied for the immunohistochemical demonstration of NGF using a polyclonal antiserum on Bouin-fixed, paraffin-embedded pineal glands from adult, young and 6-hydroxydopamine (6-OHDA)-treated rats. Few immunopositive cells were observed in the adult pineal gland. A more conspicuous population of immunoreactive cells was noted in young animals (20-45 days old), especially in those chemically denervated with 6-OHDA. NGF immunoreactive cells displayed a stellate shape resembling the interstitial or glial cells previously described in the rat pineal gland. Since NGF plays a trophic effect on sympathetic neurons during development and adulthood, we postulate that its presence in the pineal gland may exert a trophic role on its sympathetic innervation.

In the present work, coexpression of vimentin (VIM) and glial fibrillary acidic protein (GFAP) is demonstrated in the glial cells of the adult rat pineal gland. Serial consecutive Epon semithin sections (0.5 microns thick) were alternately immunostained for VIM and GFAP. GFAP positive cells and processes were found in the proximal region of the pineal gland, near the pineal stalk. Most of these cells were also immunostained for VIM in adjacent semithin sections. The significance of the coexpression VIM-GFAP and the restricted location of GFAP positive cells is discussed in relation with the maturation of pineal glial cells.

Light- and electron-microscopic features of pigmented cells in the cat pineal gland are described. These cells were observed throughout postnatal life from the second postnatal day to the oldest cats studied (up to 13 years old). No apparent relationship was observed among the amount of pigment and the animal age or sex. Pigmented cells showed a preferential localization at the ventral surface of the pineal gland near its distal end. The pineal pigment was histochemically identified as melanin. Pineal pigment granules showed ultrastructural features similar to melanocyte melanin granules.

The development of microglial cells in the postnatal rat retina is described using histochemical techniques for acid phosphatase and peroxidase as well as silver impregnations for microglia. On the second postnatal day, round acid phosphatase-positive macrophages appeared on the vitreal surface of retina, locating themselves close to developing blood vessels. Later, microglial precursors invaded retinal tissues, reaching the outer plexiform layer by the tenth postnatal day. In all stages studied, microglia or their precursors were peroxidase-negative. The transformation of round microglial precursors into adult ramified microglia is also described. Owing to the relation found between developing microglia and blood vessels, a vascular origin is proposed for the retinal microglial cells.

The adult dog pineal gland was studied with the electron microscope. Pineal connective tissue spaces were poorly developed and showed capillaries with nonfenestrated endothelial cells. Two cell types, pinealocytes and astrocytes, could be identified in pineal parenchyma. Dog pinealocytes showed microtubules, centrioles, occasional cilia, and well-developed Golgi complexes. These cells showed thin processes with bulbous endings packed with vesicles. Astrocytes were characterized by the presence of numerous filaments. Their processes finished forming a glial layer bordering connective tissue spaces. The presence of myelinated and unmyelinated nerve fibers was also described.

An ultrastructural study of the origin of microglial cells has been performed in albino rat brains taken from 17-day-old embryos up to 35-day-old rats. Invasion of the nervous parenchyma by macrophagic cells which appear in mesodermal sources is described. Although the two main microglial sources are the meningeal membranes and the vascular adventitia, pericytes may also participate in the formation of microglial cells.

The pineal gland of rats 12-28 months old was studied with light and electron microscopes. All pineal components exhibited regressive changes of different intensity with age. In type I pinealocytes, there was a marked increase in dense bodies as well as the occasional appearance of wide cell profiles full of vesicles. Type II pinealocytes showed nuclear infoldings and cytoplasmic deposits of lipofuscin. Pineal stroma displayed an increase in connective tissue fibers, both collagen and oxytalan, as well as remains of basement membranes and other materials of unknown nature. Calcareous concretions were also found, mostly in the pineal capsule. All regressive changes were more intense with increasing age.