Catecholamines and indolalkylamines of the pia mater and spinal cord in vertebrates

Fluorimetric methods were used to determine adrenaline, dopamine, noradrenaline, serotonin and tryptamine in the pia mater of the brain and spinal cord of various vertebrates (fishes, birds, mammals) and of man. The histochemical method using glyoxylic acid showed the presence of biogenic monoamines in adrenergic nerve fibres and in the monoaminocytes. Their total amount in the pia mater is roughly the same, except in man, in whom it is significantly lower. From the higher adrenergic axon concentration on the one hand and the lower number of monoaminocytes on the other, it can be concluded that the neuronal factor has a more important role in the regulation of brain haemodynamics in man.     

Catecholamine and serotonin content in the lymph nodes following deafferentation

Using Falck fluorescent technique in combination with microspectrofluorometry the measurement of catecholamine and 5-hydroxytriptamine level was performed in popliteal lymph nodes of normal dogs and 2 weeks after deafferentation. Maximal catecholamine and 5-hydroxytriptamine content was observed in adrenergic nerve fibers, elements of trabecular-elastic complex and monoaminocytes of medullary substance of intact animals. Biogenic amine level decreased deafferented limb lymph nodes. Monoamine imbalance according to divergent type was registered in a colateral limb, i. e. 5-hydroxytriptamine level was increased and catecholamine level was decreased.     

Histochemical studies of the microvascular effectors regulating blood supply to the cerebral cortex

The innervation of the pial arteries as well as the activity of enzymes (phosphorylase I, II, III, succindehydrogenase, lactate dehydrogenase, ATPase, GTPase and CTPase) responsible for vascular smooth muscle function were studied histochemically on total microscopic preparations of rabbit pia matter. An especially rich adrenergic and cholinergic innervation was found around the active microvascular effectors - sphincters of pial and precortical arterial off-shoots. The nerve fibers followed the radial arteries entering the cerebral cortex. No differences were detected between the pial arteries and active microvascular effectors in the enzyme activity.     

Cholinergic mechanisms in pial vessels

Summary Plexuses of cholinergic nerve terminals were demonstrated (acetylcholinesterase staining) in pial arteries (down to a diameter of about 15) at the base of the brain and on the brain convexities of mice, rats, rabbits, hamsters, guinea-pigs, and cats. The pial veins were less well supplied than the arteries. Consecutive formaldehyde gas treatment (to visualize adrenergic nerves) and acetylcholinesterase staining revealed that the adrenergic and cholinergic plexuses followed each other closely, the axon terminals running together in the same Schwann cell strands. This was confirmed by electron microscopy after KMnO₄ fixation or 5-hydroxydopamine treatment. The varicosities of cholinergic and adrenergic axons were sometimes seen as close as 250 Å. In the neuro-effector area, the terminals of both nerve types (naked or surrounded by an incomplete Schwann cell covering) approached the smooth muscle cells as close as 800–1100 Å, and they were separated from the latter only by the fused neuronal and muscular basement membranes. In this area axo-axonal contacts were observed. The adrenergic, but not the cholinergic, nerves disappeared after bilateral removal of the superior cervical sympathetic ganglia. Isolated cat middle cerebral artery contracted strongly with acetylcholine, and the effect was inhibited by atropine.With regard to the cholinergic neural control of the intracranial arteries, it may have particular functional implications: (1) that these vessels do have a cholinergic parasympathetic innervation in contrast to most other vascular systems, for example, in the mesenterium, (2) that this cholinergic nerve supply was found to be about equally prominent as the adrenergic (sympathetic) innervation which, in some pial vessels, is even better developed than in the mesenteric arteries, and (3) that the adrenergic and cholinergic systems in the intracranial arteries may interact, even at the level of the neuro-muscular contacts, a complex situation which may be partly responsible for the previous difficulties in defining the autonomic neural influence on the brain circulation.Part of the findings were reported at Journées Internationales de Circulation Cérébrale, Toulouse, April 21–22, 1972.     

Localization of biogenic monoamines in the dura mater of the rat brain

Quantitative and qualitative composition of catecholamines (noradrenaline and dophamine) and indolalkylamines (serotonin and tryptamine) and their localization have been studied in cells and neural fibers of the rat dura mater. Luminiscent-cytophotometric and electron microscopic methods have revealed two types of cells depositing biogenic monoamines. As demonstrate the experiments with rausedil injection, monoaminocytes of the first type (mast cells) contain predominantly indolalkylamines and a small amount of dophamine. Monoaminocytes of the second type (chromaffin cells) synthesize mainly serotonin and, perhaps, tryptamine. In adrenergic neural fibers only noradrenaline has been found to be present.     

Vascular monoamine oxidase activity in the rat brain: variation with the substrate and the vascular segment

Brain vascular monoamine oxidase (MAO) was assayed in order to determine (a) whether microvessel MAO is more or less specific for certain substrates and (b) if the extraparenchymal, pial arteries possess an MAO activity as high as that in the microvessels. Rat brain microvessels were prepared by gentle homogenisation of grey matter, followed by filtration and differential centrifugation of the matter retained. Pial arteries were carefully freed of the meninges and cut into small segments. For comparison, rat mesenteric arteries were also dissected out and cut up. MAO was assayed by measuring the rate of oxygen consumption in a small cell with a Clark electrode. Although a high microvessel MAO activity (2.2 +/- 0.3 nmol min-1 mg prot.-1) was found using noradrenaline as substrate, significantly higher rates were found with tyramine, serotonin and beta-phenyl-ethylamine. By contrast, both pial and mesenteric arteries showed a 6-7 fold lower activity (substrate tyramine). These results indicate first, that a certain specialisation of the microvessel MAO activity exists which is apparently independent of the classical A or B-form category of the substrates, and second, that the extraparenchymal vessels (pial arteries) appear to possess significantly lower MAO activity, in accordance with the concept that blood-brain properties are induced by the cerebral parenchyma.     

THE REGIONAL DISTRIBUTION OF THE POLYAMINES SPERMIDINE AND SPERMINE IN BRAIN

Abstract— The distribution in brain of the polyamines spermidine and spermine is described in the rat, dog, sheep, rabbit and in man. The distribution pattern was about the same in all the species, spermidine concentration being highest in areas rich in white matter. The concentration of spermine was lower than that of spermidine and showed less variation from area to area. Rat brain polyamine content was the same in rats killed by decapitation as in those killed by rapid freezing in liquid nitrogen and was also unchanged up to 48 h after the death of the animal.     

Adrenergic innervation of the arteries of different diameters in the human and animal pia mater

The adrenergic nervous plexuses of the pial arteries from 450- to 50 micron in diameter have been studied in dogs, cats and humans from 4 age groups (22-44 years, 55-64 years, 65-74 years and 75-86 years old). It has been found that the decrease in the vessel diameter was accompanied by a marked decline in the absolute number of nervous fibers in the nervous plexuses, however the concentration of the nerve fibers has not revealed any significant differences between human arteries from 450 to 100 micron in diameter and animal arteries from 300 to 80 micron in diameter. The number of varicosities-thickness along the nerve fiber--was the greatest in 200-100 micron human arteries and in 80-60 micron animal arteries. With ageing, the number of varicosities in the adrenergic nervous plexus of human pial vessels decreased faster than in the nerve fibers.     

Banding of the pulmonary artery in infancy: selection of patients and results of main pulmonary artery banding in the treatment of congenital heart disease.

Patterns of blood flow were examined in the surface vessels of the surgically exposed brain by intracarotid injection of 1% fluorescein and rapid serial photographs timed by a photo-cell signal. Matching colour filters were used for black and white or Ektachrome film.As developed in cats and monkeys, and applied in five patients during craniotomy, the technique gave a picture of flow patterns in the pial and cortical vascular bed, demonstrating “water-shed” areas bordering major arterial territories, laminar flow in veins, and, in particular, the details of filling and clearing in the fine pial vessels, the superficial cortical capillary bed and in the vascular beds of tumours.Since these features are rendered in finer detail and sharper contrast than by standard x-ray angiography, the method affords a new means of more adequately examining the epicerebral circulation in man during craniotomy for a variety of lesions.     

Adrenergic innervation of various sections of the heart in laboratory animals and man

By means of incubating slices in 2% glyoxylic acid solution, distribution of adrenergic fibers in the myocardium of various cardiac parts has been studied in the white rat, rabbit, cat, guinea pig and in the man. Both in the animals and in the man the distribution density of the adrenergic fibers of the myocardial plexuses in the auricle is higher than in the ventricle, and in the left half of the heart it is lower than in the right one. There are certain species differences in distribution of the adrenergic fibers. The density of the adrenergic fibers in the guinea pig myocardium is the highest, and in the white rat is is the lowest.     

Structural and functional units in the pial microvascular system

A study was made of the pial arterial microcircles formed upon successive branching and anastomosing of the terminal pial vessels on the brain cortex surface at different levels of the phylogenetic development of the vertebrata. It was discovered that the pial arterial microcircles progressively become more complicated in the following order: chicken, rabbit, cat, dog, monkey. The morphological signs of the microcircles undergo progressive development: 1) they are formed primarily from small pial arterial branches possessing high vasomotor activity; 2) the area of each circle becomes less and less and their amount per unit of the brain surface increases respectively; 3) the quantity of the feeding arterial branches rises despite the reduction of the circle size; 4) the number of outgoing precortical and radial arteries entering the brain cortex increases; 5) the areas of the brain cortex supplied by individual radial arteries become less and less. This ensures increasingly delicate regulation of adequate blood supply of the smallest areas of the brain cortex.     

Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BK(Ca) and Kir channels

We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ～30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.     

IMMUNOCHEMICAL AND IMMUNOFLUORESCENCE STUDIES OF BETA-TRACE PROTEIN IN DIFFERENT SPECIES AND ORGANS, WITH SPECIAL REFERENCE TO THE CENTRAL NERVOUS SYSTEM

—The localization of beta-trace protein in different human organs and in the brains of other mammals has been investigated with immunochemical and immunofluorescence methods. With the double immunodiffusion technique, the immunological identity between beta-trace protein in human CSF, brain, femoral nerve, testis, epididymis, spermatic cord, ovary and fallopian tube and in monkey brain could be demonstrated. By means of single radial immunodiffusion on agar, the highest concentrations of beta-trace protein were found in preparations of water-soluble proteins of brain and genital organs, especially in white CNS matter, testis and epididymis. With the indirect immunofluorescence technique, bright fluorescence was found in CNS, especially in white matter, and in the stroma of epididymis. The content of beta-trace protein in femoral nerve was found to be considerably lower than that in the CNS. The results indicate a connexion between beta-trace protein and the CNS, probably in the myelin or glial cells, and also between beta-trace protein and the genital system, possibly to cells involved in steroid metabolism. A specificity for highly developed mammals, i.e. man and monkey, is proposed for beta-trace protein, as no precipitation could be found when brain extracts from calves, rabbits or mice were incubated with anti-serum against human beta-trace protein.     

Experimental endotoxemia induces leukocyte adherence and plasma extravasation within the rat pial microcirculation

Disturbance of capillary perfusions due to leukocyte adhesion, disseminated intravascular coagulation, tissue edema is critical components in the pathophysiology of sepsis. Alterations in brain microcirculation during sepsis are not clearly understood. The aim of this study is to gain an improved understanding of alterations through direct visualization of brain microcirculations in an experimental endotoxemia using intravital microscopy (IVM). Endotoxemia was induced in Lewis rats with Lipopolysaccharide (LPS, 15 mg/kg i.v.). The dura mater was removed via a cranial window to expose the pial vessels on the brain surface. Using fluorescence dyes, plasma extravasation of pial venous vessels and leukocyte-endothelial interaction were visualized by intravital microscopy 4 h after LPS administration. Plasma cytokine levels of IL1-beta, IL-6, IFN-gamma, TNF-alpha and KC/GRO were evaluated after IVM. A significant plasma extravasation of the pial venous vessels was found in endotoxemia rats compared to control animals. In addition, a significantly increased number of leukocytes adherent to the pial venous endothelium was observed in septic animals. Endotoxemia also induced a significant elevation of plasma cytokine levels of IL1-beta, IL-6, IFN-gamma, TNF-alpha and KC/GRO. Endotoxemia increased permeability in the brain pial vessels accompanied by an increase of leukocyte-endothelium interactions and an increase of inflammatory cytokines in the plasma.     

Increased Cerebral Water Content in Hemodialysis Patients

Little information is available on the impact of hemodialysis on cerebral water homeostasis and its distribution in chronic kidney disease. We used a neuropsychological test battery, structural magnetic resonance imaging (MRI) and a novel technique for quantitative measurement of localized water content using 3T MRI to investigate ten hemodialysis patients (HD) on a dialysis-free day and after hemodialysis (2.4±2.2 hours), and a matched healthy control group with the same time interval. Neuropsychological testing revealed mainly attentional and executive cognitive dysfunction in HD. Voxel-based-morphometry showed only marginal alterations in the right inferior medial temporal lobe white matter in HD compared to controls. Marked increases in global brain water content were found in the white matter, specifically in parietal areas, in HD patients compared to controls. Although the global water content in the gray matter did not differ between the two groups, regional increases of brain water content in particular in parieto-temporal gray matter areas were observed in HD patients. No relevant brain hydration changes were revealed before and after hemodialysis. Whereas longer duration of dialysis vintage was associated with increased water content in parieto-temporal-occipital regions, lower intradialytic weight changes were negatively correlated with brain water content in these areas in HD patients. Worse cognitive performance on an attention task correlated with increased hydration in frontal white matter. In conclusion, long-term HD is associated with altered brain tissue water homeostasis mainly in parietal white matter regions, whereas the attentional domain in the cognitive dysfunction profile in HD could be linked to increased frontal white matter water content.     

Mutations in LAMB1 Cause Cobblestone Brain Malformation without Muscular or Ocular Abnormalities

Cobblestone brain malformation (COB) is a neuronal migration disorder characterized by protrusions of neurons beyond the first cortical layer at the pial surface of the brain. It is usually seen in association with dystroglycanopathy types of congenital muscular dystrophies (CMDs) and ocular abnormalities termed muscle-eye-brain disease. Here we report homozygous deleterious mutations in LAMB1, encoding laminin subunit beta-1, in two families with autosomal-recessive COB. Affected individuals displayed a constellation of brain malformations including cortical gyral and white-matter signal abnormalities, severe cerebellar dysplasia, brainstem hypoplasia, and occipital encephalocele, but they had less apparent ocular or muscular abnormalities than are typically observed in COB. LAMB1 is localized to the pial basement membrane, suggesting that defective connection between radial glial cells and the pial surface mediated by LAMB1 leads to this malformation.     

Change in monoamine content and monoamine oxidase activity in brain structures during experimental allergic encephalomyelitis]

The serotonin, noradrenaline, adrenaline concentration in the blood, cerebrospinal fluid and some brain structures, and tissue monoaminoxidase activity (MAO) were investigated in dogs during experimental allergic encephalomyelitis. During the perparalytic period there was a tendency to reduction of the serotonin concentration and to the elevation of the catecholamine content. The stage of clinical manifestation of encephalomyelitis was accompanied by reduction of the serotonin level in the white matter of the cerebral hemispheres, and by genralized inhibition of the adrenergic structures. In this case the MAO activity displayed no significant change; this permitted to consider the disturbance of the monoamine synthesis as a result of immune aggression, as a possible cause of inhibition of the monoaminergic system.     

Pial arteriole dilation during somatosensory stimulation is not mediated by an increase in CSF metabolites

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.     

Topographic determination of zinc in human brain by atomic absorption spectrophotometry

Atomic absorption spectroscopy was used for measuring the zinc content, in ppb (μg/1), of brain tissue. A new method for determining the correction factor of atomic absorption interference is described. Measurements of the zinc content of twenty-four regions of adult human brains showed the maximum zinc content in resistent sector and endplate of the Amnion's horn, corroborating the histochemical data. The distribution of zinc in other regions was relatively uniform, but white matter showed lower values than gray matter. The zinc content of seventeen regions of human newborn brains was below that in adult brains, for all regions. The blood content of brain tissue contributed only insignificantly to its zinc content.     

Dolichol in Human Brain: Regional and Developmental Aspects

Distinct regional differences in dolichol content were defined in human brain from 15 to 76 years of age. Concerning the regional distribution of dolichol, levels were: higher in cortical gray matter than in subcortical white matter, highest among cortical regions in temporal gray matter, highest among all brain regions in thalamus, and lowest among all brain regions in lower brain stem and spinal cord. The developmental changes in the contents of dolichol were found to be different among brain regions. For example, among regions with the highest levels of dolichol, in thalamus there was a six to sevenfold increase, but in parietal gray matter, only a 2.5-fold increase. Regional and developmental changes in the proportions of the individual molecular species (isoprenologues) of dolichol were also observed. The findings indicate that the metabolism of dolichol is not uniform among regions of developing and aging human brain and may have implications for the role of dolichol in normal and diseased human brain.