Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase.

Ca2+-ATPase cytochemistry frequently uses the incubation medium of Ando et al. that was introduced in 1981. Some studies, however, have suggested that this medium localizes ecto-ATPase in addition to Ca2+-ATPase and that Ca2+-ATPase is sensitive to fixation. Strong activity of the enzyme on the luminal surface of the blood-brain barrier (BBB) also is considered indicative of immature or pathological microvessels. We address here five questions. 1) Is the incubation medium of Ando et al. specific for BBB Ca2+-ATPase or does it also localize ecto-ATPase? 2) How are the two enzymes distributed in the BBB? 3) How would data interpretation be prone to error if the cytochemical study does not use controls identifying ecto-ATPase? 4) Does the amount of reaction product of both enzymes vary significantly when the cortical tissue is exposed to different fixatives? 5) Does the presence of Ca2+-ATPase on the luminal membrane of the BBB necessarily indicate immature or abnormal brain endothelial cells? Adult male Sprague-Dawley rats were perfused with one of two different fixatives and vibratome slices of the brain cortex were incubated in the medium of Ando et al. The controls used were those demonstrating the ecto-ATPase and those that do not. The results indicate that the incubation medium is not specific for Ca2+-ATPase, because it also localizes the ecto-ATPase. Ca2+-ATPase appears to be localized primarily on the luminal surface of the BBB, while ecto-ATPase is localized on both the luminal and abluminal surfaces. The portion of the reaction product contributed by Ca2+-ATPase would not have been identified if the controls uniquely identifying the ecto-ATPase had not been used. The amount of reaction product formed by Ca2+-ATPase is strongly dependent on the type of fixative used. The strong localization of Ca2+-ATPase on the luminal surface of the BBB is not only normal, but also better accounts for the physiological homeostasis of Ca2+ across the blood-brain interface and should not be interpreted as indicative of immature or pathological microvessels.

Use of image analysis for estimation of the numerical densities of neurons and synapses in cerebral cortex.

Relating to the protocol by Mikki et al. [Brain Res. Protocols 2 (1997) 9-16], the use of an image analysis system is recommended in place of micrographs and photoprints for the counting and measuring of neuronal nuclei.

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells.

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

An in situ cytochemical evaluation of blood-brain barrier sodium, potassium-activated adenosine triphosphatase polarity.

It is presently believed that sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is localized on the abluminal plasma membrane of brain endothelial cells. But there have been contrary reports from some cytochemical studies. We examined the localization of the enzyme in rat cerebral microvessel endothelium using the in situ model originally employed to establish the abluminal polarity concept. Alterations in fixation and incubation media from the original reports were conducted to determine the effect on localization pattern. With the Ernst indirect incubation method as originally used, three types of localization patterns were obtained: abluminal only, luminal only, and on both surfaces of endothelial cells. With the direct incubation method of Mayahara, reaction product was seen on both surfaces. Reduction in fixation time followed by the use of the indirect incubation method resulted in a complete loss of the reaction product. The same reduction in fixation time followed by the use of the direct method did not alter the localization pattern of the enzyme. Our results demonstrated that Na+, K+-ATPase is localized on both surfaces of brain endothelial cells. The localization pattern of Na+, K+-ATPase is significantly dependent upon fixation and the incubation medium used in the in situ model. Data discrepancies for the enzyme as reported in the literature appear to be caused by differences in cytochemical protocols, rather than the biological reasons advocated by other investigators. We conclude that past cytochemical reports of blood-brain barrier (BBB) Na+, K+-ATPase abluminal localization were incomplete. The currently held abluminal polarity theory of the enzyme needs to be reexamined. Past basic and clinical cytochemical studies of BBB Na+, K+-ATPase should be viewed and interpreted with caution.

The oligodendroglial reaction to brain stab wounds: an immunohistochemical study.

Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5, 15, 28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 2'3'-cyclic nucleotide 3'-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 2'3-cyclic nucleotide 3'-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.

Ultrastructural localization of phosphatase activity in the guinea pig pineal gland by the cerium technique.

Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.

Ultrastructural localization of acetylcholinesterase in the guinea pig pineal gland.

The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland.

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.

Improved procedures for pineal gland fixation for electron microscopy.

The common procedures used for preparing some organs and tissues for electron microscopy, in which a fixative with the buffer portion adjusted to near-isotonicity to plasma is perfused in vivo, causes intolerable shrinkage of rat pineal cells. The present study was undertaken to optimize the parameters involved in the fixation of the pineal gland. The buffer and its concentration and the aldehyde or aldehydes used were among the variables investigated. The buffers tried were phosphate, cacodylate, PIPES, and HEPES. Decreasing the buffer concentration prevented shrinkage with all four buffers. The optimum concentrations were 0.05 M phosphate, 0.07 M cacodylate, 0.05 M or 0.057 M PIPES, and 0.1 M HEPES. PIPES and HEPES were clearly superior in retaining cytoplasmic density when compared with phosphate or cacodylate. The use of lithium PIPES and HEPES instead of the sodium equivalents enhanced membrane detail. A small volume of more concentrated aldehyde fixative perfused ahead of the main perfusate (a strong prewash) definitely helped prevent shrinkage. Using a mixture of aldehydes consisting of glutaraldehyde, formaldehyde, and acrolein reduced the tendency for shrinkage when compared with glutaraldehyde only. Some of the shrinkage space artefacts could be easily misinterpreted as normal features. Since the pineal gland commonly contains degenerating structures, a dependable fixation procedure is particularly needed. Also, accurate preservation is essential in the evaluation of physiological changes.

Ultrastructural characterization of glial cells in the rat pineal gland with special reference to the pineal stalk.

In the present study the "interstitial" cells of the superficial pineal gland and the nonparenchymal cells of the pineal stalk in Sprague-Dawley rats were examined ultrastructurally with the aim of defining the cells more closely. The "interstitial" cells of the superficial pineal gland do not represent a homogeneous cell population. The most abundant cell type is the mononuclear phagocyte, most easily recognized by its dark appearance and its content of primary and conspicuous secondary lysosomes. Astrocytes can be distinguished by the typical appearance of their nuclei (i.e., a thin continuous rim of heterochromatin adjacent to the nuclear membrane), identical to that of astrocytes in the CNS. Depending on the absence or presence of glial filaments and their amount, a spectrum of astrocytic cells is present. Mature astrocytes with filaments throughout their cytoplasm are rare. Immature glial cells with few or no filaments predominate. In the vicinity of blood vessels pericytes are present. In view of the fact that the "interstitial" cells could generally be identified it is suggested to abandon the term interstitial for the cells in question. In the pineal stalk mature astrocytes predominate; they have some features in common with pinealocytes, i.e., the presence of intergrade endoplasmic reticulum and grumose bodies (lysosomes). Other unusual features are a relative abundance of coated pits and vesicles. Oligodendrocytes are restricted to the proximal part of the stalk, near the deep pineal, where myelinated axons are abundant. More distally a few Schwann cells were seen.

The ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk.

In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1,132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense-cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.

Ribosome structure and chylomicron formation in rat intestinal mucosa.

Sucrose gradient sedimentation and electron micrographic studies were made on the ribosomes of the cells of intestinal mucosa isolated from control, bile fistula, and puromycin-treated rats. In comparison to controls, there was a 40 to 60 per cent decrease in polysome content of the cells following administration of puromycin or deprivation of luminal choline by creation of a bile fistula. Feeding of lysolecithin of choline to the bile fistula rats or addition to the isolated cells in vitro resulted in a complete restoration of the polysome profile along with lipprotein synthesis and chylomicron release. Addition of lysolecithin to isolated cells treated with puromycin in vitro also brought about a reaggregation of the ribosomes and a reactivation of phospholipid biosynthesis. Choline had no detectable effect on phospholipid synthesis or ribsosme aggregation when fed to ppuromycin-treated rats or when added to puromycin-treated cells in vitro. The results suggest that chylomicron formation and the release by the muscosal cells depend upon intact rough endoplasmic reticulum and an active protein adn phospholipid biosynthesis. The role of lysolecithin in this process is ratonalized on the basis of its ability to supply a precursor of lecithin as well as a surfactant which affects the aggregation, or membrane rebinding of ribosomes, or both.