Robert S. Bourke

EFFECTS OF NOREPINEPHRINE ON THE MORPHOLOGY AND SOME ENZYME ACTIVITIES OF PRIMARY MONOLAYER CULTURES FROM RAT BRAIN

Addition of norepinephrine or isoproterenol to primary cultures started from the brains of 1‐3 day old rats caused up to 200‐fold increases in cAMP levels, which reached a maximum by 5‐10 min and then declined. This effect was studied in detail for norepinephrine. The rise in cAMP levels was followed by morphological changes, in which up to 65% of the cells exhibited an astrocyte‐like morphology, and 2‐3 fold increases in carbonic anhydrase and (Na+‐K+) ATPase activities. However, morphological transformation also occurred after much smaller increases in total cAMP levels. These effects on cell morphology and enzyme activities reached a maximum 1‐2 h after addition of norepinephrine and then declined. Carbonic anhydrase activity was found both in the particulate and post 100,000 g supernatant fractions from homogenates of these cultured cells, and in the latter case the activity was activated 3‐fold by addition of cAMP. The significance of these obscrvations on the cellular localization of, and functional role for similar increases in cAMP in brain tissue is discussed.

Cation transport and membrane potential properties of primary astroglial cultures from neonatal rat brains

This paper describes K+ and Na+ content and transport in primary monolayer cultures from dissociated newborn rat brains, considered to consist predominantly of astroglial cells. Net changes in cation content after addition of ouabain, and steady state fluxes using 86Rb+ as a marker for K+ and 22Na+ as a marker for Na+, were measured. The results found indicate that the cells maintained a conventional pattern of cation homeostasis with net efflux of K+ being balanced by its active uptake and net uptake of Na+ balanced by active extrusion mediated by a ouabain sensitive (Na+K) pump. These processes maintained internal measured K+:Na+ ratios of 12--25:1. The cells were normally flat but addition of DBcAMP caused them to round up and form numerous processes, an appearance resembling that of astroglial cells in vivo. DBcAMP treatment also reduced the steady state levels of K+ measured with 86Rb+ by 15--30%, and had no effect on initial rates of 86Rb+ and 22Na+ uptake. The membrane potentials of cells treated with DBcAMP were studied, since only these were easily impaled. The membrane potentials of separate groups of cells gave means ranging from --65 to --75 mV at 35 degrees C, at an external K+ concentration ([K+]o) of 4.5 mM. The dependence of the membrane potentials of individual cells and groups of cells on [K+]o was studied. The slope of the potential per 10-fold change in [K+]o was 55--57 mV, at concentrations of K+ greater than 10--20 mM K+, and diverged from this slope at concentrations below this. This shows that these cells had some permeability to ions other than K+. Assuming that Na+ was the only other ion affecting the membrane potential, it was calculated that the permeability to Na+ was about 30 times less than K+. A similar result was obtained based on estimates of Na+ and K+ permeability from transport experiments on cells also treated with DBcAMP. The results obtained from these cells are compared to those found for other cultured glial cells and glial cells in vivo. We conclude that the membrane potentials of the cultured cells used in the present study show the closest resemblance so far to glia in vivo, since they are large and negative and are determined mainly by K+. However, the cultured cells have different properties from those reported in some studies for glial cells in vivo by showing free permeability to ions other than K+.

Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture

Abstract Primary cultures of normal astroglia started from the cerebral hemispheres of neonatal rats took up dopamine (DA) and norepinephrine (NE) in the concentration range of 10 −7 to 10 −4 M and metabolized each to their respective principal central nervous system products by the actions of both catechol-0-methyl transferase and monoamine oxidase. At 10 −7 M, uptake of 3 H labelled DA and NE was inhibited by omission of Na + , addition of ouabain or lowered temperatures. Uptake at 10 −4 M was considerable but was Na + -independent. Only Na + -independent uptake was seen in primary cultures started from the meninges of neonatal rats. These data suggest that astroglial cells in the CNS have a high affinity uptake system for catecholamines, and such uptake is followed by catecholamine metabolism.

ATPase and carbonic anhydrase activities of bulk-isolated neuron, glia and synaptosome fractions from rat brain.

The (Na+ + K+) ATPase, carbonic anhydrase and HCO3−-stimulated ATPase activities of bulk-isolated neuronal perikarya, glial and synaptosome fractions from 20–26-day-old rats were studied. The effects of varying K+, Na+ and ATP concentrations were investigated to determine limits on how these enzymes might respond to changes in the levels of these substances in vivo. The (Na+ + K+) ATPase activity of all three fractions had a similar high affinity for K+, withKm values in the range of0.7–1.7mM. TheKm for Na+ was around 10-fold higher, in the range of10–15mM.Km values for ATP were also not markedly different between the different fractions, being1.2mM for the neurons and1.8mM for the glia and synaptosome fractions, respectively. The Vmax at infinite [ATP] in the presence of10mM K+ was 2.9-fold higher for the glia as compared to the neuron fraction and 1.5-fold higher than the synaptosome fraction. At infinite K+ in the presence of3.3mM ATP the corresponding figures were 3.0 and 2.1. Arrhenius plots of (Na+ + K+) ATPase activity were different for the neuron and glia fractions as compared to the synaptosome fraction,, suggesting subtle differences in themembrane environment and/or the enzyme molecule itself. The HCO3−-stimulated ATPase activity was only 14% higher in the glial fraction compared to the neurons and was variably stimulated by added K+ at concentrations< 10mM. The glial-enriched cell fraction had a 2-fold higher specific carbonic anhydrase activity than the neuron fraction, but the combined activity of these fractions only represented< 2% of the total brain activity. Total brain carbonic anhydrase activity was not stimulated by added K+ in the range of5–50mM K+. These data indicate that none of the enzymes studied are likely to directly respond to extracellular K+ levels in excess of10mM, and therefore would only be secondarily involved in cerebrocortical swelling caused by increasing K+ concentrations20mM. The relation of these findings to previous work on increased (Na+ + K+) ATPase and carbonic anhydrase activity in glial cells, the role of glial cells in passive or mediated transport processes related to increased levels of extracellular K+ and the suitability of bulk-isolated glia and neuron-enriched fractions as experimental models are discussed.

Ultrastructural features of a brain injury model in cat

SummaryWe present qualitative and quantitative ultrastructural observations on the changes induced in neuroglia and blood vessels of gray matter of cat brain by an experimental acceleration-deceleration injury which, when used alone, causes negligible morbidity and mortality, but, when combined with systemic hypoxia, leads to coma and delayed death in approximately 50% of experimental subjects. An increase in the proportion of neuropil occupied by astrocytic cytoplasm is detectable qualitatively in layer Vb of pericruciate cortex 20 min after injury without hypoxia, and is maximal (22%, as measured morphometrically, vs 11.4% in controls) 40 min afterward. Near-normal values (14.1%) are obtained 100 min following the insult. If trauma is succeeded 40 min later by a 60-min period of hypoxia, there is prolongation of astrocytic edema and other neuroglial accompaniments of the traumatic lesion, such as aggregation of nuclear nucleoprotein granules and, in astrocytes, fusion of rosette ribosomes and enlargement of mitochondria. A decrease in luminal area occurs in capillaries 40 min after trauma applied alone. Hypoxia without trauma leads to a significant increase in capillary luminal area, which, however, is abolished when trauma precedes the hypoxic interlude. Intravenous injection of a non-diuretic, fluorenyl derivative (L-644,711) of (aryloxy)alkanoic acid loop diuretics, completely prevents the astrocytic swelling ordinarily present 40 min after acceleration-deceleration injury. Also, L-644,711 improves mortality and morbidity scores in cats subjected to trauma with hypoxia. We suggest that astroglial swelling may be a critical step in the evolving pathology of this head injury model and its prevention, as by L-644,711 administration, may have relevance to the treatment of cerebral edema in human head injury and other clinical disorders accompanied by astrocytic swelling.

THE EFFECT OF HCO3− ON THE SWELLING AND ION UPTAKE OF MONKEY CEREBRAL CORTEX UNDER CONDITIONS OF RAISED EXTRACELLULAR POTASSIUM

Abstract— Increasing the HCO3− concentration of incubation media containing raised K+ concentrations (18‐71 mm) caused increased swelling of monkey cerebral cortex slices. This swelling was mainly associated with increased intracellular levels of Na+ and Cl− ions. It was independent of the type of buffer used and was not a result of the increased Na+ concentration in the media due to added HCO3− or the increased osmolarity. The levels also were unaffected by alteration of the pH in the range of 6·9‐ 7·8 or pCO2 in the range of 3–81 mm Hg.

Swelling and ion uptake in cat cerebrocortical slices: control by neurotransmitters and ion transport mechanisms.

Cat cerebrocortical slices incubating in medium containing normal K+ concentrations were exposed to a number of different transmitters. Norepinephrine, histamine and adenosine or 2-chloroadenosine caused increased swelling of the slices associated with an increased Na+ and Cl− content. These effects were seen only when both Cl− and HCO3− were present in the medium, and were inhibited by a number of anion transport inhibitors. These characteristics were identical to those of the HCO3−-dependent component of the swelling induced by high K+ levels in the medium. Other transmitters, namely 5-hydroxytryptamine, dopamine, and γ-amino butyric acid, were ineffective. The effects of norepinephrine, histamine and 2-chlorcadenosine were antagonised by propranolol and phentolamine, chlorpheniramine and diphenhydramine, and theophylline respectively. These antagonists also inhibited HCO3−-dependent, K+-stimulated swelling. The transmitters which induced swelling also stimulated the carbonic anhydrase activity of cerebrocortical slices. We conclude from these data that the HCO3−-dependent component of K+-stimulated swelling may be due to K+-stimulated release of transmitters. Furthermore, the fact that the transmitters which induce swelling have also been reported to be most effective in increasing cAMP content in both brain slices or cultured astrocytes is consistent with the swelling response being mediated via cAMP-induced changes and being predominantly localized to astrocytes.

The effects of temperature and inhibitors on HCO3−- stimulated swelling and ion uptake of monkey cerebral cortex

In the presence of high concentrations of K+, additions of HCO3- as low as 0.35 mM caused a 23% increase in swelling, and concomitant increases in the chloride content of incubating monkey cerebrocortical slices. The uptake of chloride was accompanied by increased uptake of sodium and was highly temperature dependent, showing a marked activation at approximately 30 degrees C. A similar temperature activation was also found for a Mg2+-dependent, HCO3-stimulated ATPase activity in monkey cerebral cortex, consistent with a possible role for this enzyme in the K+ and HCO3-dependent swelling process and its associated ion movements. K+-dependent, HCO3-stimulated cerebrocortical tissue swelling with uptake of Na+ and Cl- was inhibited by acetazolamide indicating that carbonic anhydrase was also involved. The addition of ouabain also inhibited swelling and K+ and Cl- uptake at low concentrations, but led to increased swelling at higher concentrations ( greater than 10 mum). A similar biphasic effect on swelling was also seen following addition of ethacrynic acid.

Alterations in cat cerebrocortical capillary morphometrical parameters following K+-induced cerebrocortical swelling

SummaryHistochemical, electron microscopic, and morphometrical techniques were employed in the determination of the effects attributed to K+-induced cerebrocortical swelling on cat cerebrocortical capillary diameter, length, surface area, volume, and minimal intercapillary distance.Bilaterally exposed and intact temporoparietal cerebral cortices of 4 conditioned adult cats were simultaneously superfused with isotonic, artificial CSF containing 3.5 mM K+ (control) and 54 mM K+ (experimental) for 1 h at 37°C with monitoring of systemic vital function, hematocrit, arterial blood gases, and determination of cerebrocortical tissue water content.The mean values for cerebrocapillary diameter were 5% (P<0.05) greater in swollen tissues when compared with comparable mean values determined for controls. The values for minimal intercapillary distance determined from control and experimental animals plotted as relative frequency histograms represented two distinct populations (P<0.0005). The significance of altered capillary morphometric parameters are discussed in relation to K+-induced cerebrocortical swelling.

Artificial Embolization of the Middle Cerebral Artery in Primates Description of an Experimental model with Extracranial Technique

Artificial embolization of the middle cerebral artery (MCA) was produced in the primate, with a technique similar to that described by Luessenhop and Spence (1960) for the treatment of an inoperable arteriovenous malformation in the territory of the MCA. Silicone spheres (1 to 1.5 mm in diameter) were introduced into the internal carotid artery (ICA) via the external carotid artery (ECA). Emboli (1 to 1.3 mm) passed into the anterior cerebral artery (ACA) in 12%, and into the MCA in 50%. Emboli (1.2 to 1.5 mm) stopped at the ICA bifurcation in 54%. In all primates (82.35%) in which the emboli occluded the ICA bifurcation or the MCA, immediate contralateral hemiplegia developed. The correlation of the anatomical characteristics of the intracranial vasculature of the ICA bifurcation, the diameter of the emboli, and the anatomical localization of the silicone spheres suggests that this experimental model can produce a selective acute “point” occlusion of the MCA in at least 75% of the cases without violating the cranium, in which the resultant changes in the distribution of water and electrolytes in the brain during the acute ischemic event in the territory of the MCA of the primate can be studied.