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The actin-binding protein ezrin is associated with cellular shape changes, motility, tumor invasion, and lymphocyte activation. We have earlier shown that ezrin immunoreactivity (IR) is faintly present in normal astrocytes but increased in malignant human astrogliomas. We studied the role of ezrin in astrocyte activation, applying immunostaining on serial paraffin sections from human autopsied brain tissues (51 cases). Cerebral HIV infection was chosen as a model displaying consistent exemplary astrocyte activation. Semiquantitative ezrin-IR was compared with the common glial markers GFAP, ferritin, and HLA-DR in relation to clinical and morphologic criteria of HIV encephalopathy. In all cases with HIV infection, GFAP-, HLA-DR-, and ferritin-IR were elevated in comparison to normal brain tissues. In contrast, high ezrin-IR in HIV infection strictly correlated with additional HIV encephalopathy. HIV encephalopathy with particularly high ezrin-IR was correlated with neuronal apoptosis (TUNEL). Combined ezrin-IR and GFAP-IR thus reveals 2 distinct states of astrocytic activation. Normal ezrin-IR, when paralleled by upregulated GFAP, reflects astroglial activation not associated with neuronal apoptosis. High ezrin-IR indicates specific astrocyte stressors related to cellular damage within the central nervous system. Ezrin-IR might also provide a diagnostic tool for the classification of HIV encephalopathy.

In contrast to the growing interest in proton-MR-spectroscopy (1HMRS) for preoperative examination of patients with brain tumors, there is nearly no knowledge about a correlation between data from 1HMRS and histomorphology as confirmed by quantitative morphological methods. Whether a correlation can be confirmed between data from 1HMRS and quantitative histomorphology of glioblastomas representing the most frequent type of brain tumors was investigated in the present study. Furthermore, it was of interest, whether correlations between spectroscopic data and histomorphology can be confirmed for proliferating and non-proliferating tumor cell nuclei independently. Using stringent inclusion criteria for this study, 24 patients were investigated by means of preoperative 1HMRS and by means of digital image analysis of paraffin sections from the surgical specimen. Proliferating and non-proliferating tumor cell nuclei were investigated separately in the region with the highest proliferative activity in each tumor using immunohistological staining for the proliferation marker Ki67. Main results showed highly significant correlations between total creatine and variables of nuclear size, as well as correlations between choline and variables of nuclear shape. These results were confirmed for both proliferating and non-proliferating tumor cell nuclei. A significant correlation between N-acetyl-aspartate level and topometric variables (number of neighbors per nucleus, variables describing distances between tumor cell nuclei) was confirmed for proliferating tumor cell nuclei. Discriminant analysis provided a good separation of cases with high and with low values for these spectroscopic variables based on histomorphometric data. In conclusion, the results confirm a direct correlation between data from preoperative 1HMRS and histomorphological characteristics of glioblastomas supporting the biological relevance of spectroscopic data for the examination of brain tumor patients.

We report on 7 cases of a nonnecrotizing type of autoimmune myopathy found in suspected connective tissue disease and related disorders. In muscle biopsy, these cases showed myopathic alterations characterized by irregular outlines of muscle fibers in cross and longitudinal section nearly or totally lacking inflammatory cell infiltrations or macrophages and no necroses of myofibers. They revealed deposition of immunoglobulins and of collagen IV antibodies attached to fiber surface. The number of capillaries per myofiber was enlarged. Capillaries of pipestem type with thickened wall were found at the electron microscopic level. This type of myopathy was seen especially in subacute cases presenting with muscle pain, weakness and increased unspecific inflammatory laboratory parameters. No evidence for polymyositis, dermatomyositis or inclusion body myositis could be found. In all cases, steroid therapy was of striking benefit.

Although tumour cell nuclei are important histological structures for grading of astrocytomas according to the WHO-classification of brain tumours, there is no reported morphometric study of astrocytomas which describes quantitatively the four main morphologic criteria of tumour cell nuclei: size, shape, texture (densitometric characteristics) and spatial relationships between the nuclei (topometric analysis). Using a set of morphometric parameters describing these criteria as well as the Ki67-proliferation index, 74 astrocytomas from 74 patients were studied by means of a digital image analysis system. The objective of the study was to test, if these morphometric parameters were sufficient for statistical discrimination between pilocytic astrocytomas WHO-grade I, astrocytomas grade II and anaplastic astrocytomas grade III. Our results showed a correct reclassification of 97.3% (72/74) of the cases with respect to the tumour grade by means of cross-validated discriminant analysis. Morphometric parameters characterizing nuclear shape (shape factors, Fourier-amplitudes) showed the most prominent differences between the three groups of cases, followed by topometric parameters (number of neighbours per nucleus, distances between the nuclei). Less pronounced differences between the tumour grades were found for parameters characterizing nuclear size, nuclear texture and the Ki67-proliferation index. In conclusion, the present morphometric procedure provided good discrimination between the tumour grades, supporting the view that histomorphometry of tumour cell nuclei could be a valuable tool for grading of astrocytomas.

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.

The actin-binding protein ezrin has been associated with motility and invasive behavior of malignant cells. To assess the presence of this protein in human glial cells of the brain and its potential role in benign and malignant glial tumors, we studied ezrin immunoreactivity (IR), proliferation (MIB-1-IR), and apoptosis (terminal dUTP nick-end labeling) in normal human brain tissues from 10 autopsies and tissues from 115 cases of human glial tumors including astro-cytomas, ependymomas, oligodendrogliomas, and glioblastomas. We found weak staining of peripheral processes in normal human brain astrocytes and in World Health Organization grade II benign astrocytomas. Staining was markedly increased in anaplastic astrocytomas (World Health Organization grade III) and clearly strongest in glioblastomas (World Health Organization grade IV). The increase of ezrin-IR correlated significantly with increasing malignancy of astrocytic tumors (P < 0.0001). Statistical analysis revealed a stronger association with increasing malignancy for ezrin-IR than for MIB-1-IR or terminal dUTP nick-end labeling staining. Ezrin-IR was absent in normal oligodendrocytes and in oligodendrogliomas, but pronounced in normal ependymal cells and ependymomas. Ezrin-IR seems to be specific for astrocytes and ependymal glia in the normal brain. Our results indicate that ezrin-IR may provide a useful tool for the distinction of oligodendrogliomas and astrocytomas and for the grading of astrocytic tumors.

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human transmissible spongiform encephalopathies (prion diseases) are proposed for the following disease entities: CJD--sporadic, iatrogenic (recognised risk) or familial (same disease in 1st degree relative): spongiform encephalopathy in cerebral and/or cerebellar cortex and/or subcortical grey matter; or encephalopathy with prion protein (PrP) immunoreactivity (plaque and/or diffuse synaptic and/or patchy/perivacuolar types). Gerstmann-Sträussler-Scheinker disease (GSS) (in family with dominantly inherited progressive ataxia and/or dementia): encephalo(myelo)pathy with multicentric PrP plaques. Familial fatal insomnia (FFI) (in member of a family with PRNP178 mutation): thalamic degeneration, variable spongiform change in cerebrum. Kuru (in the Fore population). Without PrP data, the crucial feature is the spongiform change accompanied by neuronal loss and gliosis. This spongiform change is characterised by diffuse or focally clustered small round or oval vacuoles in the neuropil of the deep cortical layers, cerebellar cortex or subcortical grey matter, which might become confluent. Spongiform change should not be confused with non-specific spongiosis. This includes status spongiosus ("spongiform state"), comprising irregular cavities in gliotic neuropil following extensive neuronal loss (including also lesions of "burnt-out" CJD), "spongy" changes in brain oedema and metabolic encephalopathies, and artefacts such as superficial cortical, perineuronal, or perivascular vacuolation; focal changes indistinguishable from spongiform change may occur in some cases of Alzheimer's and diffuse Lewy body diseases. Very rare cases might not be diagnosed by these criteria. Then confirmation must be sought by additional techniques such as PrP immunoblotting, preparations for electron microscopic examination of scrapie associated fibrils (SAF), molecular biologic studies, or experimental transmission.

The topographical distribution of adrenocorticotropic hormone, growth hormone and prolactin was studied in nine multihormonal pituitary adenomas and two normal adenohypophyses using quantitative immunohistochemical methods. For each pituitary hormone paraffin sections stained by antihormone antibodies were scanned with a light microscope equipped with a projection and scanning device. The number of immunoreactive cells of each square in the grid was counted and transformed into pseudocolors. In this way, the distribution of each hormone was documented. A chi-square test was applied to these distribution patterns to prove randomness. A nonrandom distribution of immunoreactive cells was observed in all pituitary adenomas and normal pituitary glands. Cluster formations of immunoreactive cells were found for each bormone. The clusters were surrounded by halo-like areas of decreasing number of immunoreactive cells from center to periphery. The cluster-halo type of immunoreactive cells was found not only in the adenomas but also in the normal pituitary gland. This may be explained by the similarity of biological processes during cell proliferation either in normal development or in adenoma formation. It can be concluded from our studies that pituitary adenomas in most cases develop by multicentric but not diffuse proliferation of cell clones of different endocrine activity.

The understanding of delayed hippocampal death as a therapeutic window for post-ischemic treatment of the brain has led to numerous investigations focusing upon underlying cellular mechanisms and pharmacological potentials in gerbils and rats. Nevertheless, studies on the occurrence of delayed neuronal death in the human brain have been singular and dealt with only small files of patients. To complement these limited data, in the present study 26 adult patients with a history of a single cardiac arrest were included. Following successful resuscitation, individual survival ranged from less than 1 h to 186 days (mean = 11 days). The severity of the resultant ischemic injury in hippocampus CA1, among Purkinje cells, or in frontal neocortex, respectively, was quantified by direct counting of necrotic neurons. Additionally, hippocampal specimens were immunostained for neuron-specific enolase. The data obtained demonstrate the occurrence of delayed neuronal death in human hippocampus and, in a minor form, in cerebellar Purkinje cells. This is in contrasts to the immediate manifestation of ischemic neuronal necrosis in the neocortex. Unlike previous findings in experimental animals and in humans, the delay of CA1 cell death could be defined as lasting about 7 days following cardiac arrest. Moreover, the immunohistochemical results indicate delayed neuronal recovery in CA1, which in the time course reciprocally corresponds to delayed manifestation of hippocampal neuronal death. Interpretation of the results must consider the lack of information about the exact individual duration of cardiac arrest and resuscitation, as well as missing data concerning pre-ischemic physiological variables.

Focal and widespread intense hyperplasia of piloid astrocytes was found in the brain of a 51 year old woman with multiple sclerosis and typical demyelinating lesions. The piloid astrocytosis was confined to older plaques in brain stem, cerebellum and periventricular white matter, but could not be found in telencephalic plaques distant from the ventricular system. The piloid astrocytes and accompanying glial fiber bundles were oriented in alternating direction, thus imitating structural characteristics of spongioblastomas. In the pons, an especially high density of astrocytosis was found with lobulated and giant nuclei and binuclear cells. One large demyelinated plaque in the central white matter of the cerebellum contained numerous Rosenthal fibers between the piloid astrocytes. By their intense glial fiber production, the piloid astrocytes took part of the formation of glial scars. The term spongiocyte is proposed for piloid astrocytes of this highly differentiated type, in order to separate them from the immature spongioblasts of gliogenesis. The pathogenetic mechanism is not clear. It is supposed that during intense reparative astrocytic proliferation the development of atypic reactions is favoured. A primary evocation of this type of reaction by the demyelinating factor itself must also be taken into consideration. The topographic predilection, in our case, of piloid astrocytosis to brain stem and cerebellum indicates a disposition of regions formed by the embryogenetic side plate to reactions of this type.

Light and electron microscopic observations are reported on a brain biopsy of a man of 59 with a rare familial disease of the CNS and a 5-year clinical course. Electron micrographs of the frontal biopsy reveal plaque-like deposits composed of amyloid cores, often multicentric in the cortex and subcortical white matter. They are localized between enlarged astrocytic processes. In the neuropil they are sometimes associated with abnormal neuritic processes, in the white matter with processes of fibrous astroglia and basement membranes. There are no signs of primary neuritic or synaptic involvement in the plaque formation which is more obviously associated with altered astrocytic processes. Moreover, degenerative alterations in the cortical vessels and slight astroglial spongiform changes as well as oligodendroglial proliferation can be found. Plaques are considered to be mainly of the kuru type; the relationship with transmissible spongiform encephalopathies is discussed.

Die drusige Entartung der Arterien der Hirnrinde im Senium wird an einem besonders ausgepragten Fall eines im 65. Lebensjahr unter dem Bild der senilen Demenz Verstorbenen polarisationsoptisch und elektronenmikroskopisch untersucht. Die Resultate der beiden Untersuchungsmethoden erganzen sich gegenseitig. In den degenerierenden kongophilen Gefaswanden sind bei voller Ausbildung des Prozesses polarisationsoptisch vier Schichten unterscheidbar, in denen abwechelnd senkrecht zueinander orientierte anisodiametrische Teilchen vorliegen; ihnen entsprechen elektronenmikroskopisch in gleicher Weise angeordnete 100 A breite Filamente. Sie sind in der Tunica media parallel zur Gefaslangsachse sowie zirkular ausgerichtet, in einer inneren und auseren, lichtmikroskopisch gestreiften Schicht sind sie senkrecht zur Gefasachse — radiar zum Lumen —, in einer zwischen diesen gelegenen Zone wieder parallel zur Gefasachse orientiert. In den wesentlichen tinktoriellen Resultaten, in den polarisationsoptischen und feinstrukturellen Befunden stimmen die entarteten Hirngefasewande im Senium mit den Ablagerungen in den Korperorganen bei allgemeiner Amyloidose uberein; das spricht fur ihre Amyloidnatur. Die vonScholz gewahlte Bezeichnung “drusige Entartung der Hirnarterien” wird dem feinstrukturellen Bild gerecht; gemeinsames Charakteristicum der gestreiften Schichten der kongophilen Gefaswand und der “Kerne” der senilen Drusen ist die keiner ortsstandigen Struktur folgende Formierung der filamentaren Amyloidkomponente in parallel oder radiar geordneten Bundeln, also die eigengesetzliche, kristallartige Ausfallung an diesen Orten. Strauchartig uber die Gefaswand hinaus sich erstreckende Filamentbundel entsprechen dem Ubertritt kongophiler Substanz in das Gewebe. Ein Kohlenhydratbestandteil des Amyloid ist wahrscheinlich an die filamentare, die Eiweiskomponente gebunden. Uber die zweite, amorphe Komponente des Amyloid lassen sich keine Angaben machen. Die Beziehung der drusigen Gefasentartung zu den unter dem Terminus “kongophile Angiopathie” beschriebenen Wandveranderungen und die Verwendung dieses Terminus werden diskutiert.

Nach Durchtrennung der Hinterstrange am Ruckenmark der weisen Ratte kommt es peritramatisch in den Axonstumpfen zu einem strukturellen Umbau im Cytoplasma der Axone bei meist erheblicher lokaler Vergroserung des Axondurchmessers (Axonauftreibungen). Im Axoplasma, das normalerwiese verhaltnismasig arm an geformten Strukturelementen ist, treten Zisternensysteme unterschiedlicher Auspragung und Dichte (endoplasmatische Reticula), Mitochondrien verschiedener Form und Grose in betrachtlicher Anzahl, Vesikel in wechselnder Verteilung sowie filamentoses Material auf. Die Veranderungen sind 12 Std nach der Durchschneidung bereits deutlich ausgepragt, der Hohepunkt wird zwischen dem 3. und 7. Tag erreicht. Die Beobachtungen lassen den Schlus zu, das die vorgefundenen Strukturen und Organellen vorwiegend an Ort und Stelle entstehen, sie sind aus einer Anstauung oder Wanderung praexistenter Elemente nicht allein erklarbar. Die als Reaktion auf die exogene Schadigung rasch in Gang kommenden anabolen Vorgange in den lasionsnahen Axonabschnitten, die der “primaren retrograden Reaktion” vonSpatz entsprechen, stellen den Versuch des Neurons dar, von hier aus den verlorengegangenen distalen Anteil des Zellfortsatzes wieder aufzubauen. In den Axonauftreibungen treten jedoch nach wenigen Tagen regressive Veranderungen auf, die sich in einer Dichtezunahme im Grundcytoplasma und in der Matrix der Mitochondrien zeigen und bis zur Aggregation der vorher aufgebauten Strukturen fortschreiten. Die Axonauftreibungen gewinnen schlieslich Fremdkorpercharakter und konnen, wie aus lichtmikroskopischen Untersuchungen hervorgeht, monate- bis jahrelang im Gewebe persistieren. Isolierte Axonkugeln sind vermoge ihres Mitochondrienreichtums in der Lage, uber mehrere Tage einen gewissen Stoffwechsel aufrechtzuerhalten, bevor sie der Nekrobiose verfallen. Anzeichen fur eine Bildung junger Axone innerhalb der Axonauftreibungen waren nachweisbar.

1. Das Endothelcytoplasma der Tumorgefase von Gliomen — Spongioblastomen, Astrocytomen, Oligodendrogliomen, Ependymomen und Glioblastomen — enthalt oft reichlich filamentares Material. Die Filamente gleichen den Filamenten im Cytoplasma der Geschwulstzellen selbst sowie den Filamenten der normalen und reaktiven Astroglia (100 A Durchmesser, kompakter Bau, unterschiedliche Langenausdehnung, unverzweigt). Die Bedeutung derFilamente im Endothel der Gliomgefase ist noch unklar. Im Gegensatz zu den Gefasen der Korperorgane treten im ZNS, soweit bisher bekannt, weder normalerweise noch im Rahmen mesenchymaler Reaktionen filamentare Elemente im Gefasendothel auf. 2. In einem Spongioblastom wurde ein disseminiert-herdformiger Zerfall der Gliafilamente beobachtet, der mit einer Anhaufung osmiophilen Materials an den zerfallenden Filamenten und im interfilamentaren Grundcytoplasma der betroffenen Bezirke einhergeht. Der Prozes spielt sich in den filamentreichen Zellfortsatzen der Spongioblasten ab, seltener in ihren Pericarya. Durch Konfluenz der Herde entstehen ausgedehnte langliche osmiophile Korper von granularer bis amorpher Feinstruktur und hoher Dichte, die die Fortsatze ausfullen und oft auftreiben. Sie entsprechen den sogenanntenRosenthalschen Fasern der Spongioblastome. Die Gebilde werden als Ergebnis einerStapelungsdystrophie der Spongioblasten angesehen. Gestapelt werden Proteine, fakultativ Kohlenhydrate, in spaten Stadien auch Lipide. Die Rosenthalschen “Fasern” gehen also aus faserhaltigen Zellteilen hervor, sind aber selbst keine Fasern.

In Achsenzylinderauftreibungen, wie sie in grauer und weiser Substanz des menschlichen Gehirns nach exogenen Schadingungen (Trauma, kreislaufbedingte Erweichungen) vorkommen, wurden mit dem Phasenkontrastverfahren darstellbare saurefuchsinophile und eisenhamatoxylin-farbbare Substanzanhaufungen festgestellt, die sich nach Anwendung histochemischer Methoden als glykolipoid-und proteidhaltig erwiesen. Es wird angenommen das sie durch Aufbau mitochondrienreicher cytoplasmatischer Strukturen in den aufgetriebenen Axonabschnitten zustande kommen, das es sich also hier nicht, wie bisher beschrieben, vorwiegend um Flussigkeitsaufnahme (Quellung) handelt. Die so gekennzeichneten Achsencylinderveranderungen sind Ausdruck der am proximalen Stumpf ladierten Nervenfasern ablaufenden “primaren retrograden Reaktion”. Sie stellen den Versuch der Nervenzelle dar, durch Intensivierung des Stoffwechsels und Aufbau neuer Cytoplasma bestandteile in weiter Entfernung vom Perikaryon den verlorengegangenen peripheren Anteil des Axons von der Lasionsstelle aus wieder aufzubauen. Am Inhalt der Axonauftreibungen kommt es jedoch schon nach kurzer Zeit zu Stoffumwandlungen, es laufen regressive Veranderungen ab; infolge des entstandenen fremdkorperartigen Charakters konnen isolierte Axonauftreibungen dann jahrelang unabgeraumt im nervosen Gewebe liegenbleiben.

9 Std nach experimentellen traumatischen Lasionen am Ruckenmark der Ratte wurden peritraumatisch in der grauen Substanz der Hinterhorner die Folgen einer Plasmaexsudation in das Gewebe elektronenmikroskopisch beobachtet. Es kommt zu plasmatischer Durchtrankung des Extracellularfugensystems bei Erhaltung der dicht gefugten Lagerung der Zellteile im Neuropil. Es folgen Einrisse der Zellmembranen und Eintritt von Plasmabestandteilen in die Zellfortsatze. Im Cytoplasma akkumulieren Blutplasmabestandteile in rundlichen membranumgrenzten Korpern, die aus dem endoplasmatischen Reticulum stammen. Fibrin wird in Form fein verteilter Faden eingelagert. Zwischen Gefaswanden und Neuropil finden sich Fibrinaggregate mit typischer Querstreifung. Die Zellfortsatze zerfallen schlieslich, in den lockeren Nekrosebezirken restieren dichte Korper und Doppelmembranbruchstucke. Die plasmatische Infiltrationsnekrose (Scholz) unterscheidet sich somit nicht nur pathogenetisch, sondern auch feinstrukturell von den primar hypoxischen Gewebsschaden (Kolliquations- und Koagulationsnekrose). In der Umgebung der Nekrosezonen tritt ein kollaterales Odem mit Schwellung der Zellfortsatze auf. Aus den Befunden geht in ubereinstimmung mit Beobachtungen anderer Autoren hervor, das die extracellularen Fugen im ZNS in vivo existieren und von einem leicht permeablen Medium erfullt sein mussen. Sie erlauben nicht nur die Verteilung ionaler Losungen, sondern auch kolloidal geloster Stoffe in der Grosenordnung der Plasmaproteine.

One postnatal and one prenatal case (same family) of a neurovisceral lipidosis compatible with a diagnosis of Niemann-Pick disease type C were studied. The postnatal case, aged 4 and 6/12 at death, was characterized morphologically (foamy cells in the bone marrow; storage histiocytes in rectal submucosa and extraneural viscera and ballooned neurons, the two types of cells containing pleomorphic and oligomenbranous inclusion bodies, respectively; central demyelination) as well as biochemically (elevated spleen and liver content of sphingomyelin, cholesterol, glucosyl ceramide and lysobisphosphatidic acid). Sphingomyelinase activity (SM) was not significantly lowered and showed no greatly abnormal electrofocused pattern of activity; its extractability from brain, liver and spleen was distinctly hindered, a finding interpreted as expression of a reduced bioavailability of the enzyme. — The prenatal case was diagnosed by low SM in amniotic fluid. Diminished SM was confirmed in cultured amniotic cells and in tissues of the aborted fetus which, additionally, showed an elevated sphingomyelin and cholesterol content in the liver. A prenatal diagnosis of Niemann-Pick disease type C was made for the first time. The phenotypical variation of the disease may reflect genetic heterogeneity and, there-fore, a prenatally lowered SM need not be a constant finding. — The apparent normalization of SM in the postnatal case was accompanied by a decrease of visceromegaly raising the question of a causal relationship between the two phenomena.

Despite many sensational and intimidating reports in the mass media, transmissible spongiform encephalopathies (prion disease) are not contagious in the usual sense. Successful transmission requires both specific material (an affected individual's tissue, from or adjacent to CNS) and specific modes (mainly penetrating contact with the recipient). Nevertheless, specific safety precautions are mandatory to avoid accidental transmission and to decontaminate any infectivity. Autopsy is essential for definite diagnosis of these disorders. Recommendations are given here for performance of the autopsy, for neuropathology service and appropriate decontamination; they are based on the current literature and on precautions taken in most laboratories with experience in handling tissue from transmissible spongiform encephalopathies. In particular, special care must be taken to avoid penetrating wounds, possible contamination should be kept to a minimum, and potential infectious material must be adequately decontaminated by specific means.