Maria Claudia Vigliani

Reactive cell proliferation and microglia following injury to the rat brain

The non–astrocytic cells which proliferate in the rat brain after the induction of an area of necrosis have been characterized and counted by means of combined in vivo bromodeoxyuridine (BrdU) administration and immuno–histochemical demonstration of glial fibrillary acid protein (GFAP), vimentin, Ricinus communis agglutinin 120 (RCA–1), Griffonia simplicifolia B4 isolectin (GSI–B4), keratan sulphate (KS), carbonic anhydrase C (CA.C), transferrin (TF) and ferritin. Two days after the injury, 7.5% of the proliferating cells were GFAP–positive reactive astrocytes, 5.7% were RCA–1–positive cells and 17.4% were GSI–B4–positive cells. Lectin–binding cells had the microscopic and ultrastructural aspects of microglia; they proliferated around the needle track and in the corpus callosum. Microglia represented a large fraction of the proliferating cells. Evidence is presented for the origin of at least a proportion of perilesional astrocytes and microglia from the periventricular matrix, and of microglia from blood precursors. Other non–proliferating microglia cells transiently appeared in the normal brain around the wound, in agreement with the existence of two different microglia cell populations reacting with different modalities to an area of necrosis.

Ependymoma: internal correlations among pathological signs: the anaplastic variant.

In a series of 298 cases of ependymoma, survival analysis identified some prognostic histological factors but failed to demonstrate a worse survival for the anaplastic variant diagnosed with the common criteria used for assessing anaplasia in primitive brain tumors. This finding suggests that either anaplastic ependymoma does not exist, or that the established criteria are not useful in its identification. To solve these problems, the association of histological, immunohistochemical, and ultrastructural signs in 173 intracranial cases was investigated and analyzed by means of contingency tables. Many signs had only focal distribution. Some signs, meaningful for anaplasia, such as very high cell density and number of mitoses, were found to be associated, whereas other signs usually considered indicative of anaplasia, such as endothelial hyperplasia, glomeruli, and necrosers, were not. In addition, pseudorosettes, mesodermic areas, and incomplete formation of perivascular pseudorosettes were signs associated with very high cell density and number of mitoses. Distribution of glial fibrillary acidic protein and vimentin, as well as other immunohistochemical and ultrastructural features, were not helpful, with the exception of microsettes found by electron microscopy. Our conclusion is that the anaplastic variant of ependymoma is recognizable only when some histological prognostic factors, such as cell density and number of mitoses, are maximally expressed.

Immunohistochemistry of glial reaction after injury in the rat: double stainings and markers of cell proliferation.

The astrocytic reaction in the rat after brain injury has been studied immunohistochemically for intermediate filaments (GFAP and vimentin), also with double staining procedures, and for markers of proliferation (BrdU and PCNA). GFAP‐positive reactive astrocytes appeared around the lesion, where they were vimentin‐positive and at a distance. BrdU and PCNA showed a high labelling index around the wound at day 2 and scattered positive nuclei were also found at a distance in the ipsilateral side. BrdU‐positive astrocytes represented a minor fraction of GFAP‐ and vimentin‐positive astrocytes. The expression of vimentin persisted at least 15 days after the lesion. Our results could suggest that distant reactive astrocytes originate through hypertrophy while those close to lesion arise by hyperplasia from mature or immature glial cells. The hypothesis is formulated that cells of the periventricular matrix contribute to the post‐traumatic proliferative activity.

Ubiquitin in Motor Neuron Disease: Study at the Light and Electron Microscope

Several neurodegenerative diseases, including motor neuron disease (MND), are characterized by formation of abnormal cytoskeleton-derived inclusions which contain ubiquitin (Ubq). We have studied the distribution of Ubq in 26 cases of MND with light and electron microscopic immunocytochemistry. Ubiquitin-positive inclusions were found in neurons of anterior horns in most cases of amyotrophic lateral sclerosis (ALS) but were not present in other forms of MND. Ubiquitin immunoreactivity was observed in 10–15 nm intraneuronal filaments, which were not stained by antibodies to neurofilaments, and on dense bodies of dystrophic neurites throughout the neuropil of anterior horns and pyramidal tracts. Data analysis showed a trend toward lower percentage of Ubq-positive neurons in cases with longer duration of illness or lower number of neurons. A high percentage of Ubq-positive inclusions occurred in cases with an aggressive clinical course, suggesting that ubiquitination takes place at early stages of the disease.

Anti-Hu-associated brainstem encephalitis

Objective: A series of patients with anti-Hu-associated brainstem encephalitis is reviewed to better define the clinical presentation and to improve its recognition. Methods: Data were collected from 14 patients diagnosed by members of the Paraneoplastic Neurological Syndromes Euronetwork, and eight patients from the literature who presented with isolated brainstem encephalitis and had anti-Hu antibodies. Results: The median age of the 22 patients was 64 years (range 42–83), and 50% were men. All patients developed a subacute neurological syndrome, in days or weeks. Brain MRI was always normal. Mild cerebrospinal fluid pleocytosis was reported in only two patients. The following syndromes were identified on admission: A medullary syndrome was seen in 11 (50%) patients. Seven of them presented with dysphagia, dysarthria and central hypoventilation. The other four in addition of bulbar symptoms, without central hypoventilation, presented pontine manifestations. Six (27%) patients developed a pontine syndrome with paresis of the VI or VII cranial nerves, nystagmus, usually vertical, and gait ataxia. There was a rapid downward progression to the medulla in all patients. Five (23%) patients presented a ponto-mesencephalic syndrome with uni- or bilateral palsy of the III and VI cranial nerves and gait ataxia, but rapidly progressed to complete gaze paresis and medullary dysfunction. Conclusions: The study confirms the predominant medullary involvement but also shows that half of the patients present with clinical features that indicate an upper, mainly pontine, dysfunction before downward progression.

Proliferating cell nuclear antigen expression in brain tumors, and its prognostic role in ependymomas: an immunohistochemical study

SummaryProliferating cell nuclear antigen (PCNA)/cyclin is currently often investigated immunohistochemically in tumors as a marker of cell proliferation, but many problems remain open concerning its reliability as a prognostic factor. PCNA has been studied in a series of 123 brain tumors using the monoclonal antibody PC10. A clear intra-and inter-tumor variability of PCNA-positive nuclei has been found, but taking into account the tumor areas with the highest number of positive nuclei, a positive correlation between this number and the histological malignancy of tumors has been demonstrated. The staining intensity of nuclei was variable; very-intensely positive nuclei, counted separately, are hypothesized to represent nuclei in S-phase of the cell cycle. In ependymomas the investigation included a quantitative statistical analysis. The number of PCNA-positive nuclei correlated with cell density and mitotic index, but only very intensely positive nuclei showed a significant statistical correlation with survival. In spite of the many possibilities of wrong interpretation of PCNA expression, the most important of which is its deregulation, the method is useful in the practice for prognostic purposes. Its important advantages are the possibility of a retrospective application and a visual analysis of the proliferation potential of tumors.

The vascular response to tumor infiltration in malignant gliomas

SummaryNeo-vascularization and endothelial hyperplasia have been shown to be very active in malignant gliomas. In this contribution the vascularization of the cortex infiltrated by malignant gliomas is morphometrically studied and the endothelial proliferations are immunohistochemically investigated and reconstructed by a three-dimensional computer-assisted procedure. Vessel density increases after tumor infiltration in some cases only. The diameter of vessels increases and so does the number of nuclei/vessel after the complete invasion of the cortex when vascular glomeruli develop. In completely infiltrated cortex with development of glomeruli and circumscribed necroses, vessel density is very low. No neoformation of vessels takes place before the complete infiltration of the cortex by the tumor. The hyperplastic formations, usually arranged parallel to the deep or outer cortical layers, take origin from the radially penetrating vessels from the meninges and their lateral branching. The hyperplasia deforms the vascular network, making it often inadequate to supply tumor cells. Immunohistochemically, the cells composing the hyperplastic structures are variably positive for factor VIII/RAg and, at a lesser extent, for α-smooth muscle actin. The poorness of the vascular network in many instances of completely infiltrated cortex is responsible for the development of circumscribed necroses.

Ubiquitinated filamentous inclusions in spinal cord of patients with motor neuron disease

The ultrastructural localization of ubiquitin (Ubq) in spinal cord of 2 cases of amyotrophic lateral sclerosis was studied, using immunoperoxidase and immunogold labeling on Vibratome sections or on sections cut from paraffin blocks. Two different types of ubiquitinated cytoplasmic inclusions were observed: (1) bundles composed of 10-15 nm filaments; (2) small rounded bodies without a limiting membrane. A panel of antibodies to neurofilaments (NFs) did not stain the ubiquitinated bundles, and decorated only axonal swellings. Ubq-positive filamentous deposits could be identified with inclusions previously described ultrastructurally. The absence of staining with antibodies to NFs might be due to abnormalities of the filaments, possibly because of a dysfunction of the ubq proteolytic system.

Prognostic factors in adult medulloblastoma. A clinico-pathologic study.

Aims and background. Medulloblastoma in adults is a rare tumor. The small number of cases in the reported series has not permitted a definite assessment of the prognostic role of clinical, pathologic and cell kinetics factors. The largest series of medulloblastoma in adults treated in a single institution is herein reported. Methods. The clinical, therapeutic, pathologic and proliferation features of medulloblastoma in 44 adult patients (> 18 years) were analyzed retrospectively with regard to postoperative survival. The proliferation potential of each tumor was evaluated by the immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) and Ki-67, clone MIB-1, in paraffin sections. Results. The overall 5- and 10-year survival rates were 40% and 35.6%, respectively. Significant factors in predicting a longer postoperative survival were: age < 37 years, decade of management (1977-1990), radiotherapy (50-55 Gy on the posterior fossa and 30-35 Gy on the spinal cord) and nuclear isomorphism. When corrected for adequacy of radiotreatment, desmoplastic type and differentiation were significantly correlated with a shorter survival. The PCNA-labelling index (LI) ranged from 34.5 to 82.2%, the MIB-1-LI ranged from 9.6 to 64.7%. No association was found between PCNA- or MIB-1-LI values and microscopic features, or between LI values and prognosis. Conclusions. Contrary to a general assumption, desmoplastic medulloblastoma and differentiated medulloblastoma are negative prognostic factors in adequately radiotreated adult patients. This may possibly be referred to lower radiosensitivity of these tumor variants. The LI with PCNA or Ki-67 is of no help in identifying aggressive tumors.

Dystrophic neurites around amyloid plaques of human patients with Gerstmann-Sträussler-Scheinker disease contain ubiquitinated inclusions

Dystrophic neurites have been previously observed around prionic protein-derived amyloid plaques of Gerstmann-Sträussler-Scheinker (GSS) disease. Ubiquitin (Ubq) immunohistochemistry reveals the presence of dot-like stainings around many of these plaques. In order to determine the nature of ubiquitinated deposits, we performed an immunogold electron microscope study on autoptic samples from the cerebellum of a GSS patient. Both pre- and post-embedding staining methods showed Ubq-positive dense bodies and filamentous structures, belonging to dystrophic neurites. They are analogous to ubiquitinated neuritic processes described around cerebellar amyloid plaques of Alzheimers disease (AD). These results suggest that amyloid deposition is responsible for the degeneration of adjacent axon terminals in both AD and GSS.