Igor Klatzo

Experimental Production of Neurofibrillary DegenerationI. Light Microscopic Observations

1. Intracerebral or intratheeal administration of alum phosphate in rabbits resulted in the development of convulsive seizures, as well as of characteristic neuronal changes throughout the CNS. 2. The neuronal change consisted in a hydropic alteration of the cytoplasm and in a conspicuous neurofibrillary degeneration. 3. Chemical analysis of brain obtained from animals sacrificed during the terminal convulsive state demonstrated significant decreases in brain water and chloride space, and a relatively large increase in the thiocyanate space. 4. The effect of alum phosphate which is described may provide an experimental model for further investigations of the morphological substrate of epileptic neuronal activity, as well as of the pathomechanism of neurofibrillary degeneration.

Experimental cerebral ischemia in Mongolian gerbils

SummaryBehaviour of biogenic amines was studied in the brains of Mongolian gerbils subjected to unilateral occlusion of the common carotid artery. Assays on the hemispheres ipsilateral to occlusion revealed in symptom-positive animals a progressive decrease in norepinephrine and dopamine, and an increase in serotonin throughout the duration of an ischemic insult. In post-ischemic periods following the release of the clip, changes in biogenic amine levels generally conformed to the principles of a previously described “maturation” phenomenon, with delayed reactions occurring after the shorter ischemic insults.

The blood-brain barrier to proteins under normal and pathological conditions

Abstract The sites of various barriers within the brain to the extracellular movement of exogenous protein have been examined electron-microscopically in several classes of vertebrates. In mice, intravenously administered peroxidase (M.W. 43,000) cannot leave the capillary lumen because the extracellular clefts between adjacent endothelial cells are closed by uninterrupted, circumferential belts of tight junctions. The same kind of junction between the epithelial cells lining the choroid plexus, median eminence, and area postrema prevents the entry of protein into the ventricular cerebrospinal fluid. If peroxidase is injected directly into the cerebral ventricles, the protein percolates through the open cerebral interspaces by passing around the discontinuous gap junctions between ependymal cells and glial cells, even filling the perivascular basement membranes. However, the passage of peroxidase from here into the capillary lumen is prevented by the same endothelial tight junctions that block movement from blood to parenchyma. In the shark brain, the endothelial junctions are open to peroxidase which is then prevented from entering the parenchymal interspace by some structure, presumably glial, lying beyond the endothelium. In pathological conditions the damage to the BBB can be expressed in different patterns of abnormal passage of protein tracers. In brain edema of the vasogenic type, locally-extravasated proteins spread extensively and preferentially through the white matter, migrating via enlarged intercellular spaces. The rate of this migration is greatly influenced by systolic blood pressure. It was shown that an acute hypertension alone can produce extensive disturbances of the BBB to proteins. On the other hand, a remarkable resistance to the development of post-traumatic brain edema was observed in sharks in which brain tissue appears also to be greatly resistant to the effects of radiation. In conditions associated with an acute oxygen deprivation of the mammalian brain, there is frequently a striking pattern of protein tracer accumulation in the nerve cells. Such distribution of the markers might be related to assumed selective vulnerability of the neurons in hypoxia.

The effects of 5-minute ischemia in Mongolian gerbils: II. Changes of spontaneous neuronal activity in cerebral cortex and CA1 sector of hippocampus

SummarySpontaneous neuronal activity was recorded in the cerebral cortex and the CA1 sector of the hippocampus in gerbils during and after 5-min ischemia, produced by bilateral clamping of the common carotid arteries. It was found that spontaneous activity in both cortical and CA1 neurons ceased within 60 s after the onset of ischemia and that it began to reappear 10–20 min after the recirculation. During the next 24 h most CA1 neurons which were recorded showed hyperactivity. This was evident primarily by an increase in spike discharges, whereas recordings from the cerebral cortex were within the preocclusion ranges. On the 2nd day after ischemia, functioning CA1 neurons could not be found, as if they were in a state of functional death, although histological sections showed a general preservation of their cellular structure at that time.

The biphasic opening of the blood-brain barrier to proteins following temporary middle cerebral artery occlusion

SummaryThe behavior of the blood-brain barrier (BBB) was studied in cats following release after 1-h middle cerebral artery (MCA) occlusion. The regional cerebral blood flow (rCBF) was determined by hydrogen clearance method in the caudate nucleus and the cerebral cortex. The BBB was assayed with Evans blue (EB) tracer and by immunohistochemical peroxidaseantiperoxidase (PAP) method. Following release of MCA occlusion, there were two openings of the BBB, separated by a refractory period. The first opening, occurred shortly after recirculation; this was associated with rCBF below 15 ml/100 g/min during the ischemic period and a pronounced reactive hyperemia promptly following release of MCA occlusion. A refractory period of the BBB was indicated by the absence of EB leakage in cats injected with the tracer 30 min before killing at 3 h after recirculation, although the rCBF values in these animals were even lower (6±1 ml/100 g/min) during occlusion, and all of them showed a pronounced hyperemia after recirculation. The occurrence of the previous BBB opening in these animals was confirmed by the PAP staining. The second opening of the BBB was observed at 5 and 72 h after recirculation in cats which were injected with EB 30 min before killing, and which showed rCBF below 15 ml/100 g/min during occlusion, followed by a pronounced reactive hyperemia. No EB extravasations were observed at any time in cats in which the rCBF during occlusion was above 15 ml/100 g/min and which failed to show a marked reactive hyperemia.

The effects of 5-minute ischemia in mongolian gerbils: I. Blood-brain barrier, cerebral blood flow, and local cerebral glucose utilization changes

SummaryChanges in morphology, behavior of the blood-brain barrier (BBB), regional cerebral blood flow (rCBF), and local cerebral glucose utilization (LCGU) were assessed and correlated in Mongolian gerbils following 5 min cerebral ischemia, produced by bilateral clamping of the common carotid arteries. The morphological changes were confined to the hippocampus and revealed a conspicuously delayed destruction of the CA1 neurons, occurring after 3 days. Following release of carotid occlusions, there were two separate openings of the BBB. One, occurring shortly after recirculation, was associated with focal hyperemia in the cerebral cortex, hippocampus and basal ganglia; the second opening was observed after several days and was associated with severe neuronal destruction in the CA1 sector. Correlation of quantitative and qualitative rCBF assays with14C-deoxyglucose autoradiographic observations indicated an uncoupling between blood flow and glucose metabolism, observed in the hippocampus at 10 min after recirculation. The described changes provide a further insight into the post-ischemic events which determine the outcome of ischemic injury.

Selective Vulnerability of the Blood-Brain Barrier in Chemically Induced Lesions*

1. Vulnerability of the blood-brain barrier to unilateral intracarotid perfusion with chemical injurious agents was studied with the application of various fluorescent and radioactive tracers. 2. Evans blue when bound to albumin fluoresces brightly red in formalin-fixed frozen sections as viewed under ultraviolet light provides a useful and convenient tool for microscopic localization of this tracer. 3. Using fluorescent labeled albumin as a single indicator, several patterns of its abnormal penetration through the damaged cerebral vessels have been elucidated and discussed. 4. Tracing the BBB damage by concurrent intrasystemic administration of albumin and γ-globulin labeled with contrasting fluorescent color markers revealed dissociation features in their extravascular penetration. The albumin indicator spreads, as a rule, more extensively. 5. Differences in extent of penetration were also observed when fluorescent albumin or sodium fluorescein was concurrently used with radioactive inulin or sucrose. 6. A pronounced inhibition of C14methyl-O-glucose brain uptake was observed on the side of a slight mercurial blood-brain barrier damage, which otherwise failed to produce any abnormal penetration of sodium fluorescein. 7. The features of selective vulnerability of the blood-brain barrier arc discussed with regard to possible mechanisms involved.

The effect of acute arterial hypertension on the blood-brain barrier to protein tracers

SummaryA study was made on the effect of acute hypertension on the blood-brain barrier to proteins. The arterial blood pressure was raised by intravenous injections of metaraminol bitartrate (Aramine) and Evans Blue was used as protein tracer.Multiple foci Evans Blue extravasation became evident in the gray matter of the brain as early as 10 min after the sudden increase of blood pressure by 90 mm Hg or higher. As observed under the fluorescence microscope, the tracer had penetrated the walls of intracerebral vessels and accumulated in numerous adjacent neurons. EEG recordings from blue stained areas of the cerebral cortex showed changes in neuronal activity.ZusammenfassungDie Wirkung akuter Hypertension auf die Blut-Hirnschranke für Proteine wurde untersucht. Die arterielle Blutdrucksteigerung wurde durch. i.v. Verabreichung von Metaraminol-bitartrat (Aramine®) erzeugt. Als Schrankenindikator wurde Evansblau verwendet.Multiple Herde von Evansblau-Extravasaten fanden sich in der grauen Hirnsubstanz frühestens 10 min nach plötzlicher Steigerung des Blutdruckes um 90 mm Hg oder höher. Fluorescenzoptische Untersuchungen ergaben, daß der Indicator durch die Wand der intracerebralen Gefäße ausgetreten war und sich in zahlreichen benachbarten Nervenzellen angesammelt hatte. EEG-Ableitungen der blau angefärbten Rindenareale ergaben Veränderungen der neuronalen Aktivität.

The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain

Summary1.Vascular permeability in normal and edematous brain tissue was studied by application of the fluorescein labeled serum proteins (FLSP) as well as by the use of free fluorescein isothiocyanate (FITC) marker.2.The electrophoretic studies demonstrated that the binding capacity of the FITC to albumin was of such degree that morphological observations made after injection of the free tracer can be considered depending primarily on the visualization of the fluorescent serum albumin in the brain tissue.3.The normal brain, with the exception of the several regions described below, showed no evidence of FLSP passage beyond the vascular endothelial lining.4.The choroid plexus, area postrema and the tuber cinereum exhibited green FLSP fluorescence in the connective tissue investitures of the blood vessels but no parenchymatous penetration of the FLSP could be detected in the last two mentioned structures.5.Edema of the white matter, produced by the cortical cold injury in the cat, was associated with the extravascular passage of FLSP which was studied at various stages.6.The appearance of discrete FLSP inclusions in glial cells is interpreted as resulting from pinocytotic uptake of extravasated serum proteins and the significance of pinocytosis in the nervous tissue is discussed.7.The involvement of the microglia in the transport of FLSP from the area of edema towards the pial and ependymal surfaces suggests the importance of this glial element with regard to the transport of soluble substances in the brain tissue.8.It is suggested that the area of edema in the white matter resulting from cold injury may be associated with the presence of an appreciable extracellular compartment.Zusammenfassung1.Unter Verwendung von fluorescein-markierten Serumproteinen (FLSP) und von freiem Fluorescein-Isothiocyanat (FITC)-Tracer wurde die vasculäre Permeabilität in normalem und ödematösem Hirngewebe untersucht.2.Die elektrophoretischen Untersuchungen ergaben, daß die Bindungskapazität des FITC an Albumin so stark ist, daß man morphologische Beobachtungen nach Injektion mit der freien Tracersubstanz als hauptsächlich von der Sichtbarmachung des fluoreszierenden Serumalbumins im Hirngewebe abhängig betrachten kann.3.Mit Ausnahme der unten angeführten Regionen konnte im normalen gehirn kein Nachweis für die FLSP außerhalb der Gefäßendothelschicht gefunden werden.4.Das Adergeflecht, die Area postrema und das Tuber cinereum zeigten grüne FLSP-Fluorescenz in den bindegewebigen Hüllen der Blutgefäße; jedoch konnte in den beiden letztgenannten Strukturen kein Eindringen der FLSP in das Parenchym festgestellt werden.5.Ein bei der Katze durch eine corticale Kälteschädigung verursachtes Ödem der weißen Substanz ging mit extravasalem Durchtritt von FLSP einher, der in verschiedenen Phasen beobachtet wurde.6.Das Auftreten von geringfügigen FLSP-Einschlüssen in Gliazellen wird auf die pinocytotische Aufnahme von extravasalen Serumproteinen zurückgeführt; die Bedeutung der Pinocytose im Nervengewebe wird erörtert.7.Die Beteiligung der Mikroglia am FLSP-Transport von der Ödemregion zu den pialen und ependymalen Oberflächen läßt auf die wichtige Rolle schließen, die dieses Gliaelement beim Transport löslicher Substanzen im Hirngewebe spielt.8.Die Annahme wird vorgebracht, daß das durch Kälteschädigung entstandene Ödem der weißen Substanz mit dem Vorhandensein eines beträchtlichen extracellulären Raumes in Verbindung steht.

Experimental Model for Repetitive Ischemic Attacks in the Gerbil: The Cumulative Effect of Repeated Ischemic Insults

An experimental model for repeated ischemic attacks, which allows easy induction of cerebral ischemia of any desired duration and frequency, has been developed in the gerbil. With this procedure, a pronounced cumulative effect on development of edema and tissue injury was observed using 3 separate, 5-min bilateral occlusions of the common carotid arteries spaced at various time intervals. This effect was most evident when the occlusions were carried out at 1-h intervals, i.e., during the period of marked postischemic hypoperfusion. Such animals, killed after 24 h of recirculation, showed significantly more severe edema and brain tissue injury in the areas exposed to ischemia than was observed in animals killed 24 h after single 5- or 15-min occlusions. The changes of regional CBF, assayed with a [3H]nicotine method, indicated a relatively rapid onset of hypoperfusion of similar degree after each release of arterial occlusion. The hypoperfusion recovered significantly within 6 h of recirculation following either single or multiple occlusions, and no residual hypoperfusion was observed in animals which, when killed at 24 h, showed severe edema and brain tissue injury. This model should prove useful in elucidating the pathophysiological mechanisms operative in repetitive cerebral ischemia.