Paola Cavalla

Grey matter pathology in multiple sclerosis.

Abstract The aim of our study is to evaluate the extent and distribution of grey matter demyelinating lesions in multiple sclerosis (MS), addressing also neuronal loss and synaptic loss. Whole coronal sections of 6 MS brains and 6 control brains were selected. Immunohistochemistry was performed for myelin basic protein, neurofilaments, synaptophysin, ubiquitin, and activated caspase-3. Neuronal density and optical density of synaptophysin staining were estimated in cortical lesions and compared with those observed in corresponding areas of normal (i.e. nondemyelinated) cortex in the same section. Demyelinating lesions were observed in the cerebral cortex, in the thalamus, basal ganglia, and in the hippocampus. The percentage of demyelinated cortex was remarkable in 2 cases of secondary progressive MS (48% and 25.5%, respectively). Neuronal density was significantly reduced in cortical lesions (18-23% reduction), if compared with adjacent normal cortex, in the 2 cases showing the higher extent of cortical demyelination; in the same cases, very rare apoptotic neurons expressing caspase-3 were observed in cortical lesions and not in adjacent normal cortex. No significant decrease in optical density of synaptophysin staining was observed in cortical lesions. Grey matter demyelination and neuronal loss could contribute to disability and cognitive dysfunctions in MS.

A Study of Apoptosis in Normal and Pathologic Nervous Tissue After In situ End-Labeling of DNA Strand Breaks

Abstract. Programmed cell death (PCD) via apoptosis is characterized by nuclear pyknosis and fragmentation, and biochemically by oligonucleosomal cleavage of DNA. Apoptosis occurs in the developing nervous system, whereas its role in neurodegenerative diseases is still debated. Recognition of apoptotic cells has recently been facilitated by in situ end-labeling (ISEL) techniques which identify DNA strand breaks through incorporation of labeled nucleotides. We have applied two ISEL assays to physiological and pathological conditions affecting the nervous system in which PCD is likely to occur. Terminal transferase assay was more sensitive than DNA polymerase assay and allowed the recognition of a larger number of cells than conventional histology. Apoptotic cells were readily found in the developing spinal cord and dorsal root ganglia. Medulloblastomas, gliomas, brain lymphomas and metastases showed abundant apoptotic cells either isolated or grouped in small foci. Labeling was also found in cells without a clearcut apoptotic morphology. Apoptotic cells were not found in Alzheimers disease, amyotrophic lateral sclerosis and human and mouse prionic encephalopathies. Our results show that ISEL is a useful technique for demonstrating apoptotic cells in nervous tissue during development and in brain tumors. Lack of staining in neurodegenerative diseases suggests that other types of PCD might be involved.

Reactive astrogliosis of the spinal cord in amyotrophic lateral sclerosis

Many observations have been carried out on astrogliosis in the cerebral cortex in amyotrophic lateral sclerosis (ALS), whereas little attention has been paid to astrogliosis in the spinal cord. Twenty autopsy cases of sporadic, common form of ALS have been studied. Spinal cords have been examined at the cervical, thoracic and lumbar levels by histological methods and immunohistochemistry for GFAP, Vimentin, Tau-protein, Neurofilaments, PCNA. A gliosis was found in the ventral horns, in dorsal horns and at the transition between gray matter and anterior and lateral funiculi, especially close to laminae VII, VI and V as being due to secondary gliosis. The findings cannot be interpreted on the only basis of the substitutive role of reacting glia. The proposed pathogenetic mechanisms of ALS are evaluated as possible responsible stimuli; the coincidence of the distribution of reactive astrocytes with the entering points of the corticospinal tracts into the gray matter is considered of primary importance. Of special interest are reactive astrocytes at the transition between laminae VII, VI and V and the lateral funiculus, where dystrophic neurites are known to concentrate.

Demyelination, Inflammation, and Neurodegeneration in Multiple Sclerosis Deep Gray Matter

Gray matter (GM) lesions are recognized as important components of the pathology of multiple sclerosis (MS), and involvement of the deep gray matter (DGM) is suggested by magnetic resonance imaging. The aims of this study were to determine the frequency and distribution of lesions and characterize the inflammatory and neurodegenerative changes in DGM of MS patients. Histochemistry, immunohistochemistry, and morphometry were performed on whole coronal sections of 14 MS and 12 control (6 normal, 6 from amyotrophic lateral sclerosis patients) brains. Demyelinating lesions were frequent in MS DGM; most often in the thalamus and caudate, but they were also seen in the putamen, pallidum, claustrum, amygdala, hypothalamus, and substantia nigra. Most DGM lesions involved both GM and white matter. Inflammation in active DGM lesions was similar to that in lesions only in white matter but was less intense, and there was a preponderance of activated microglia, scarce myelin-laden macrophages, and a lesser extent of axonal damage. Neuronal loss was observed both in DGM lesions and nondemyelinated DGM with neuron atrophy in nondemyelinated DGM. In conclusion, demyelination and neurodegenerative changes are common in MS DGM and may contribute to clinical impairment. Inflammation in DGM lesions is intermediate between the destructive inflammation of white matter lesions and the minimal inflammation of cortical lesions. We hypothesize that alterations of glutamate reuptake mechanisms may contribute to these differences.

Altered glutamate reuptake in relapsing-remitting and secondary progressive multiple sclerosis cortex: correlation with microglia infiltration, demyelination, and neuronal and synaptic damage.

Cortical involvement in multiple sclerosis (MS) is emerging as an important determinant of disease progression. The mechanisms responsible for MS cortical pathology are not fully characterized. The objective of this study was to assess the role of excitotoxicity in MS cortex, evaluating excitatory amino acid transporter (EAAT) expression and its relationship with demyelination, inflammation, gliosis, and neuronal and synaptic pathology. EAATs are essential in maintaining low extracellular glutamate concentrations and preventing excitotoxicity. Ten MS brains (3 relapsing-remitting MS cases and 7 secondary progressive MS cases) were evaluated by immunohistochemistry for myelin basic protein, CD68, HLA-DR, EAAT1, EAAT2, glial fibrillary acidic protein, phosphorylated c-Jun N-terminal kinase (pJNK), synaptophysin, and neurofilaments. Cortical lesions were frequently observed in MS brains in variable numbers and extensions. In cortical lesions, activated microglia infiltration correlated with focal loss of EAAT1, EAAT2, and synaptophysin immunostaining, and with neuronal immunostaining for pJNK, a protein involved in response to excitotoxic injury. No reduction of EAATs or synaptophysin immunostaining was observed in demyelinated cortex in the absence of activated microglia. Alterations of the mechanisms of glutamate reuptake are found in cortical MS lesions in the presence of activated microglia and are associated with signs of neuronal and synaptic damage suggestive of excitotoxicity. Excitotoxicity may be involved in the pathogenesis of demyelination and of neuronal and synaptic damage in MS cortex.

Network-Based Multiple Sclerosis Pathway Analysis with GWAS Data from 15,000 Cases and 30,000 Controls

Multiple sclerosis (MS) is an inflammatory CNS disease with a substantial genetic component, originally mapped to only the human leukocyte antigen (HLA) region. In the last 5 years, a total of seven genome-wide association studies and one meta-analysis successfully identified 57 non-HLA susceptibility loci. Here, we merged nominal statistical evidence of association and physical evidence of interaction to conduct a protein-interaction-network-based pathway analysis (PINBPA) on two large genetic MS studies comprising a total of 15,317 cases and 29,529 controls. The distribution of nominally significant loci at the gene level matched the patterns of extended linkage disequilibrium in regions of interest. We found that products of genome-wide significantly associated genes are more likely to interact physically and belong to the same or related pathways. We next searched for subnetworks (modules) of genes (and their encoded proteins) enriched with nominally associated loci within each study and identified those modules in common between the two studies. We demonstrate that these modules are more likely to contain genes with bona fide susceptibility variants and, in addition, identify several high-confidence candidates (including BCL10, CD48, REL, TRAF3, and TEC). PINBPA is a powerful approach to gaining further insights into the biology of associated genes and to prioritizing candidates for subsequent genetic studies of complex traits.

Real-life impact of early interferonβ therapy in relapsing multiple sclerosis

Recent findings support greater efficacy of early vs. delayed interferon beta (IFNβ) treatment in patients with a first clinical event suggestive of multiple sclerosis (MS). We aimed to evaluate the effectiveness of early IFNβ treatment in definite relapsing‐remitting MS (RRMS) and to assess the optimal time to initiate IFNβ treatment with regard to the greatest benefits on disability progression.

Apoptosis and cell proliferation in human neuroepithelial tumors

Apoptosis and cell proliferation were studied in 180 human neuroepithelial tumors (30 medulloblastomas, 30 intracranial ependymomas, 30 oligodendrogliomas and 90 astrocytic tumors, including 30 astrocytomas, 30 anaplastic astrocytomas and 30 glioblastomas). Apoptotic nuclei were detected by morphology and in situ end-labeling (ISEL) of DNA breaks. The frequency of apoptotic nuclei varied from oncotype to oncotype and their distribution in each oncotype was uneven. An apoptotic index (AI) was calculated; this was high in malignant tumors and in tumors of embryonal origin and lower in tumors of the glial series. The AI/mitotic index (MI) ratio was lower in malignant tumors and higher in benign tumors, suggesting a relationship between apoptosis and cell proliferation. There was no significant correlation of either AI or AI/MI ratio with either labeling index (LI) of Ki-67 clone MIB-1 or with survival. A trends towards low AI/MI ratio in tumors with high LI and short survival was observed. Apoptosis expresses cell loss in tumors, but it did not appear to be a prognostic factor.

Prognostic factors in oligodendroglioma.

BACKGROUND A reliable marker for tumor oligodendroglial cells is not yet available, so that the histological recognition of the tumor still encounters uncertainties. There is no general agreement also on prognostic factors in oligodendroglioma. The inconsistency concerns mainly the histopathological factors. The aim of the study was recognition of prognostic factors in oligodendroglioma. METHODS In a series of ninety-eight oligodendrogliomas, including twenty mixed oligoastrocytomas, clinical [sex, age at surgery, tumor location, symptoms at presentation], therapeutic [extent of resection, year of surgery, post-operative Karnofsky score, post-operative radiotherapy, post-operative chemotherapy], histological [cell density, nuclear pleomorphism, vascular endothelial proliferation, necrosis, microcysts, mitoses, mitotic index (MI), apoptosis, apoptotic index (AI)] and immunohistochemical parameters [MIB-1 and PCNA Labeling Indexes (LIs), staining for GFAP, positivity for p53] were correlated with survival in uni- and multivariate analysis in order to identify their prognostic significance. RESULTS Age at surgery, extent of surgical resection, year of surgery, post-operative Karnofsky score and MIB-1 LI were associated with survival in both uni- and multivariate analysis. Location, symptoms at presentation, mitoses, MI, AI, and PCNA LI showed a significant correlation with survival in uni- but not in multivariate analysis. The twenty cases of oligoastrocytomas did not show any difference in survival from pure oligodendrogliomas. CONCLUSIONS Some clinical and therapeutic factors together with MIB-1 LI play a prognostic role. MIB-1 LI is prognostic with a cutoff of 8%. Histology gives a limited contribution to the prognosis. Oligoastrocytomas had the same outcome and prognostic factors as pure oligodendrogliomas.

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.