The human neurosecretory neurones under perinatal hypoxia: a quantitative immunohistochemical study of the supraoptic nucleus in autopsy material.

In the rat, experimental manipulations that cause activation of the magnocellular neurosecretory neurones result in the synthesis, in addition to vasopressin (AVP) and oxytocin (OXY), of other neurotransmitters or peptides, including tyrosine hydroxylase (TH), the first and rate limiting enzyme for catecholamine biosynthesis. In the human neonate, our previous study showed that TH was selectively increased in AVP neurones of subjects that died from prolonged perinatal hypoxia. The purpose of the present study was to quantitatively investigate the expression of TH, AVP, OXY and neurophysin in magnocellular neurones of the human neonate in relation to the severity/duration of perinatal hypoxia, as estimated by neuropathological criteria. Autopsy was performed after obtaining parental written consent for diagnostic and research purposes. The intensity of the immunohistochemical reactions and the cellular/nuclear size were measured in the dorsolateral supraoptic nucleus using a computerised image analysis system. We showed that prolonged perinatal hypoxia resulted in the activation of the magnocellular neuroendocrine neurones of the human neonate, as indicated by their increased neuronal and nuclear size. OXY neurones appeared larger than the AVP ones at birth, possibly indicating an active role of foetal OXY during labour or even earlier. The gradual increase in the duration of the insult resulted in the reduction of intracellular AVP content, in parallel with a dramatic increase in the expression of TH, indicating a functional interaction of these peptides under neuronal activation. Ιsolated evidence in our series, obtained from an infant of a diabetic mother, raises the probability that in the case of hyperglycaemia the above pathogenetic mechanisms are diversified.

Simultaneous detection of tyrosine hydroxylase-immunoreactivity and vasopressin mRNA in neurons of the human paraventricular and supraoptic nucleus.

Our purpose was to investigate the proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons expressing vasopressin (VP) mRNA in the human paraventricular and supraoptic nuclei by combining in situ hybridization with immunohistochemistry on the same tissue section. A variability in the proportion of TH-IR neurons synthesizing VP mRNA was observed in adults which was usually more than 50%. In neonates almost all the TH-IR neurons appeared to contain VP mRNA.

Colocalization of tyrosine hydroxylase with oxytocin or vasopressin in neurons of the human paraventricular and supraoptic nucleus.

In the developing and adult human paraventricular (PVN) and supraoptic (SON) nucleus, a large proportion of neurons contains the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). In the present study we investigated the possible colocalization of TH with oxytocin (OXT) or vasopressin (VP) in the adult and neonatal PVN and SON. Adjacent paraffin sections were incubated simultaneously with two antibodies: a polyclonal against TH and a monoclonal against OXT or VP and stained with a double peroxidase-antiperoxidase/alkaline phosphatase method. We observed that TH-immunoreactive(IR) perikarya in the human PVN and SON were also positive for OXT or VP. A clear difference between the neonates and adult cases of our sample was observed in the proportion of TH-IR neurons that colocalize OXT or VP. In the neonates the majority of the TH-IR perikarya was also stained for VP, while only few TH-IR neurons were also positive for OXT. The opposite was observed in the adults, where the majority of the double-stained TH-IR neurons colocalizes OXT while only few TH-IR perikarya appear to contain VP. Our study establishes the colocalization of TH with OXT or VP in the adult and neonatal PVN and SON and indicates that antemortem factors such as perinatal hypoxia might increase TH-immunoreactivity of the VP neurons in man.

Development of tyrosine hydroxylase-immunoreactive neurons in the human paraventricular and supraoptic nucleus.

Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were found in the developing human paraventricular and supraoptic nucleus. In the preterm infants only few, small, incompletely differentiated TH-IR neurons were evident in a minority of the cases. In the full-term infants a considerable but strongly variable population of morphologically mature TH-IR perikarya was observed in these neuroendocrine nuclei in most subjects.

Tyrosine hydroxylase-immunoreactive neurons in paraventricular and supraoptic nuclei of the human brain demonstrated by a method adapted to prolonged formalin fixation.

We studied the distribution of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the adult human hypothalamus using a modification of the peroxidase-antiperoxidase immunohistochemical method which can be applied on autopsy brain material following prolonged formalin fixation. We observed that most of the TH-IR perikarya localized within the paraventricular (PVN) and supraoptic (SON) nuclei were large and showed homogeneous staining over the entire cytoplasm and processes. These results show that in the human brain a large population of neurons within the neurosecretory nuclei are able to synthesize a catecholamine.

Lewy bodies in parkinsonism share components with intraneuronal protein bodies of normal brains.

Histochemical characteristics of the Lewy bodies, in catecholamine neurons of 10 Parkinsonian patients, were compared to those of the spherical protein bodies, the basic protein-rich markers of catecholamine neurons in man. Special methods for proteins and lipids showed that the core of the Lewy bodies, in the neurons of the locus coeruleus and the substantia nigra, contains basic proteins and lipids normally found in the protein bodies. Acid fuchsin and the lipid-soluble fluorescent dye rhodamine B stained the entire core of the Lewy body in the parkinsonian brains and the entire sphere of the protein body in the control brains. Bromsulfophthalein, another acidic dye, which selectively binds to the enzyme gluthathione-S-transferase, had affinity only for a ring-like lamina at the outer layer of the core of the Lewy body and for the outer rim of the protein body. These results demonstrate that Lewy bodies and protein bodies contain similar macromolecular components, that is lipids and two different types of proteins, which also show similar stratification in the two structures. On the other hand, the presence in several neurons of the Parkinsonian patients, of aggregates representing transitional forms between protein bodies and Lewy bodies, indicates that abnormalities of protein bodies precede, and are somehow linked to Lewy body production.

Similarities between neuronal Lewy bodies in parkinsonism and hepatic Mallory bodies in alcoholism.

The aim of the present study was to identify components of the Lewy body, which is a characteristic neuronal lesion in idiopathic Parkinsonism, using histochemical methods that selectively stain the Mallory body, a characteristic lesion of the hepatocyte in alcoholism. Our observation that Lewy bodies stain with phosphotungstic acid hematoxylin, the dye originally used for demonstrating alcoholic hyaline (Mallory bodies), promoted this study. The material consisted of formalin-fixed, brain stem tissue from Parkinsonian subjects, and of similarly preserved liver tissues from alcoholic individuals. The methods selected were Roque's chromotrope 2R-aniline blue, and Liisberg's rhodamine B, which stains Mallory bodies due to its affinity for sites of tissue keratinization. Hence, skin was also included in this study as control tissue. Our results showed that Lewy bodies in the brain, Mallory bodies in the liver and stratum corneum in the skin have identical staining properties with the dyes used, indicating the presence of histochemically similar components. Taking into account the reactions of these dyes with model substances, we suggest that the similar components shared by Lewy bodies and Mallory bodies are arginine-rich proteins and lipids associated with keratinization. Similar findings in both, a toxin-induced lesion of the liver, and a spontaneous lesion of the brain may offer clues for understanding the latter's mode of formation.

Localization of phospholipids in human catecholamine neurons with Baker's acid haematein.

Protein bodies in catecholamine neurons of the normal human brain contain arginine-rich proteins similar to those present in the core of Lewy bodies in Parkinsonian brains. Lewy bodies are known also to contain phospholipids in their core, demonstrable with Baker's acid haematein. We used Baker's procedure on catecholamine neurons of normal brains to test whether the protein bodies also contain phospholipids. Postmortem tissues of three control individuals were used in this study. Locus coeruleus and substantia nigra were dissected out from the fresh brains and two sets of blocks were made from each area. One set was fixed in formol-calcium for the preservation of lipids, the second was fixed in Bouin's solution and treated with hot pyridine for the extraction of lipids. Finally, both sets were subjected to the same chroming and staining procedure according to Baker. In catecholamine neurons the protein bodies were stained with acid haematein, indicating the presence of phospholipids. Since this staining resisted pyridine extraction we conclude that these phospholipids are firmly bound to the basic proteins. Thus, protein bodies in healthy catecholamine neurons give the same positive reaction for phospholipids as that reported for the core of Lewy bodies in damaged neurons in Parkinsonism.

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification.

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.

Histochemical protein markers of monoamine cell bodies in man.

Neurons in brainstem and hypothalamus of postmortem, formaldehyde-fixed tissue of 25 healthy controls were studied with a double-staining procedure for the consecutive demonstration of Nissl material and of cytoplasmic spherical bodies, which are rich in basic proteins. The investigation established that the protein bodies are located in cells that correspond to the aminergic neurons demonstrable by histofluorescence. Protein bodies were also found in the neurons of the inferior olive, which are not as a rule included in the aminergic cell groups. The localization of protein bodies is perikaryal and dendritic and, thus, parallels the distribution of monoamines and monoamine-synthesizing enzymes reported in experimental animals. The invariable presence of protein bodies in all aminergic perikarya of control human brains and their absence from nonaminergic neurons permits us to consider them markers of monoamine neurons in man.

Histochemical changes of peripheral blood leukocytes during lithium treatment.

The effect of lithium on the histochemistry of blood granulocytes was studied in 16 manic-depressive patients during lithium prophylaxis. Blood smears obtained before, at 2 months and at 12 months after initiation of treatment showed that lithium caused condensation of the chromatin in granulocytes, conformation changes in eosinophilic granules and the disappearance of basic proteins found in the cytoplasm of eosinophils in the drug-free patients. The latter findings are interpreted as indicating the involvement of lithium in histamine metabolism.