Extensive DNA fragmentation in oxyphilic cell lesions of the thyroid.

The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown.

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum.

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

Isochromosome 17q is a constant finding in medulloblastoma. An interphase cytogenetic study on tissue sections.

Isochromosome 17q (i[17q]) is the most frequent chromosomal abnormality in medulloblastoma, occurring in 30-60% of cases by karyotype analysis. In the present study i[17q] was demonstrated in routinely processed tissue sections of 20 medulloblastomas by in situ hybridization (ISH), using a chromosome 17 centromeric alpha satellite DNA probe. All medulloblastomas showed the i[17q] specific signal, i.e. two hybridization spots slightly apart from each other. The specific hybridization signal was not observed in ependymomas, cerebellar astrocytomas, haemangioblastomas, supratentorial neuroblastomas and ependymoblastomas. The constant finding of i[17q] in medulloblastoma depends on the much higher number of nuclei which can be analysed by ISH compared with cytogenetic techniques. Molecular data on medulloblastoma are consistent with the present results. The number of cells with i[17q] in medulloblastoma cases ranged from 3% to 9%; these figures are underestimated because of nuclear truncation in tissue sections. The percentage was not linked to patients' age, location of tumour, MIB-1 labelling index and histological type (classical vs desmoplastic). The present results indicate that i[17q] is a key event in the pathogenesis of medulloblastoma, and suggest a genetic difference between medulloblastoma and other primitive neuroectodermal tumours.

Diverse cell death pathways result from a single missense mutation in weaver mouse.

Neuronal death affects selectively granule cell precursors of the cerebellum and the dopaminergic neurons of midbrain in the weaver mutant mouse. The weaver phenotype is associated with a missense mutation in the gene coding for the GIRK2 potassium channel, which results in chronic depolarization. Using DNA gel electrophoresis, electron microscopy (EM), the in situ end-labeling (ISEL) technique at the light and EM level, and immunohistochemistry for apoptosis-related proteins c-Jun and proliferating cell nuclear antigen (PCNA), we have investigated the mechanisms of cell death in cerebellum and substantia nigra. Between postnatal day P1 and P21, in the external germinal layer of the cerebellum, most degenerating granule cell precursors were found to aggregate to form clusters. Degenerating cells exhibited strong nuclear staining for ISEL, c-Jun, and PCNA and had a typical apoptotic morphology by EM. Increased c-Jun and ISEL staining were also occasionally seen in Purkinje cells. Between P14 and P21, when dopaminergic neurons start to degenerate, staining for ISEL, c-Jun, and PCNA in weaver substantia nigra was the same as in controls. By EM, however, we found only in weaver mice numerous dopaminergic cells that showed extensive vacuolar and autophagic changes of cytoplasm, preservation of membrane and organelle integrity, and absence of chromatin condensation or DNA fragmentation by EM-ISEL. The combination of vacuolar and autophagic changes identifies a novel type of non-necrotic, nonapoptotic cell death. After biochemical analysis of DNA, a clear-cut laddering, suggestive of oligonucleosomal fragmentation, was present in samples from weaver cerebellum. Cell death diversity appears to be influenced by specific features of target cells. These findings may be relevant for understanding the mechanisms of cell death in neurodegenerative diseases.

c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis.

There is increasing evidence that oxidative damage plays a major role in amyotrophic lateral sclerosis (ALS), but how it contributes to motor neuron degeneration and astrocytic gliosis, two pathologic hallmarks of the disease, is unknown. A few studies have suggested that ALS motor neurons die via apoptosis and show upregulation of c-jun, an immediate early gene that is necessary for neuronal apoptosis. In order to elucidate the mechanisms of cell damage induced by oxidant stress, we have studied in ALS and control spinal cord the immunohistochemical expression of c-Jun, of JNK/SAPK, a kinase that activates c-Jun following various types of stress, and of NF-kappa B, a transcription factor that is induced by oxidant stress and has prominent neuroprotective functions. An in situ end-labeling assay was performed for detecting apoptotic cells. We show that (a) the JNK/SAPK-c-Jun pathway is dramatically overexpressed in ALS spinal cord; (b) the strongest activation occurs in astrocytes, while motor neurons show unusually low expression of the pathway; (c) increased JNK/SAPK expression in glial cells is accompanied by NF-kappa B activation, indicating the presence of a protective response to oxidant sress, which is deficient in motor neurons; (d) activation of JNK/SAPK, c-Jun and NF-kappa B is unrelated to apoptotic cell death. These results support the view that astrocytes are directly involved in the pathologic process of ALS, and might explain the selective vulnerability of motor neurons by their relative lack of antioxidant defenses.

Role of apoptosis in the prognosis of oligodendrogliomas.

Prognostic factors in oligodendrogliomas are not well defined, even considering the labeling index of proliferation markers. As in other neuroepithelial tumors, the difficulty in calculating cell loss may contribute to this uncertainty. Proliferation markers Ki-67/MIB.1 and PCNA, mitoses, apoptotic nuclei, p53 and bcl-2 expression were investigated in 98 oligodendrogliomas. Apoptosis was assessed by the aspect of nuclei, by in situ end-labeling (ISEL) technique and by c-Jun immunohistochemical demonstration. The Bcl-2 also was immunohistochemically studied for its anti-apoptotic role. Mitotic index (MI), labeling index (LI) for MIB.1 and PCNA and apoptotic index (AI) were calculated and compared among themselves and with histology and survival. It was found that AI correlated with MI (p = 0.001) and was significantly higher in anaplastic than in classic oligodendrogliomas (p = 0.001). Apoptosis occurred only slightly more frequently in cases with high LIs for proliferation markers (MIB.1 and PCNA) (p = non-significant) and it was definitely higher in p53-positive cases (p = 0.008). It did not correlate with bcl-2 which was poorly expressed in oligodendrogliomas, with the exception of cells with astrocytic features. Apoptotic index correlated very weakly with survival (p = 0.05); therefore, it cannot be considered a highly reliable prognostic factor in oligodendrogliomas.

RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.

Bcl-2 distribution in neuroepithelial tumors: an immunohistochemical study.

Bcl-2 proto-oncogene prevents apoptosis in many conditions. First detected in lymphomas, it has been also described in non-lymphoid tissues. The immunohistochemical distribution of bcl-2 protein in 100 neuroepithelial tumors is presented. Bcl-2 was positive in some neurons of normal nervous tissue, in reactive astrocytes and variably in all neuroepithelial tumor. The reaction product was either diffuse or granular, due to bcl-21 protein localization on cytoplasmic, nuclear and mitochondrial membranes. The positivity was high in medulloblastomas and in astrocytic tumors. In the latter, the strongest staining was found in cells retaining the astrocytic aspect. Oligodendroglial cells were minimally stained. No correlation of bcl-2 staining with survival was found in each tumor type. The interpretation of the results is based on the one side on the constitutive role played by bcl-2 in the nervous tissue and its neoplastic derivatives. On the other side, in tumors bcl-2 acts by preventing tumor cells from undergoing apoptosis. BCl-2 expression in brain tumors, therefore, receives a dual interpretation. For this reason and for the lacking of correlation with survival, bcl-2 expression cannot be regarded as a prognostic factor.

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.

Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques.

Recently developed techniques based on 'in situ end-labelling' (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold-embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin-labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM-ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly 'pre-apoptotic' cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material.

A study of apoptosis in normal and pathologic nervous tissue after in situ end-labeling of DNA strand breaks.

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 Alzheimer's 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.

bcl-2 protein expression in aged brain and neurodegenerative diseases.

The proto-oncogene bcl-2 is involved in the regulation of cell death and is able to block apoptosis in neurones through reduced generation of reactive oxygen species (ROS). We have studied the immunohistochemical expression of bcl-2 protein in the aged brain and in various human neurodegenerative diseases. In all cases, bcl-2 was strongly enriched within lipofuscin and autophagic vacuoles of neurones, glial and vascular cells. Our data show that accumulation of bcl-2 is not disease-specific and represents a general cellular response which accompanies the increased formation of lipofuscin. Since oxidative stress is directly involved in lipofuscinogenesis, accumulation of bcl-2 may reflect a mechanism for counterbalancing ROS-mediated damage, or it might represent the impairment of bcl-2-dependent protection from ROS.

Tumor cell proliferation and apoptosis in medulloblastoma.

The distribution of proliferating cell nuclear antigen (PCNA)-(clone PC 10)- and Ki-67-(clone MIB-1)-positive nuclei was investigated in 60 medulloblastomas of childhood. Although the labeling index of the two markers did not coincide, both showed a wide range of parallel variations. The percentage of positive nuclei was similar in both classic and desmoplastic tumors. A variable proliferation capacity was found in the different tumor structures. Areas with neuronal and glial differentiation showed very few positive nuclei; these were very abundant in the infiltration areas, and along penetrating vessels from subarachnoidal growths. Pale islands were negative or positive only in their peripheral part. Large-cell areas were richer in positive nuclei than classic ones, accounting for their more malignant character. Hyperchromatic round nuclei, not belonging to necrotic foci and called lymphocyte-like nuclei, differently interpreted in the past, were variably found in every case. They are known, from previous experience, to stain orange with Acridine Orange fluorochroming, like single-stranded DNA. They were not easily distinguishable from mitoses and were stained by in situ end-labeling of DNA strand breaks, as demonstrated by incorporation of labeled nucleotides. They were regarded as possible apoptotic nuclei, representing either a peculiar type of cell death or the preservation of the cell deletion capacity, typical of the embryonal tissue of origin.

Monoclonal antibodies to keratan sulfate immunolocalize ramified microglia in paraffin and cryostat sections of rat brain.

We used six monoclonal antibodies (MAb) recognizing epitopes within keratan sulfate (KS) chains for an immunocytochemical study of adult rat brain. One of the MAb selectively stained microglia and their ramified processes. KS-positive cells were found throughout the CNS in both paraffin-embedded and cryostat sections; the greatest number were present in hippocampus and brainstem. In the cortex the positive processes of some cells surrounded neuronal somata. In the white matter the processes were both parallel and perpendicular to the axon bundles. Double staining showed that KS-positive cells did not express astrocytic or oligodendroglial markers. By immunoelectron microscopy, the positivity was localized around the perikarya and cell processes of small cells with peripheral chromatin clumps and dark cytoplasm, which often contained secondary lysosomes. The KS-positive cells did not contribute to myelin sheaths and were not surrounded by a basal membrane. In addition to the cellular staining, three other MAb stained the white matter diffusely. Anti-KS MAb are therefore proposed as immunohistochemical markers for ramified microglia in both paraffin and cryostat sections of adult rat brain.

Is polar spongioblastoma a tumor entity?

The distribution of cells in a parallel fashion with palisades of nuclei is common in neuroepithelial tumors. The authors have selected 16 such tumors from their series for study, as examples of different neuroepithelial oncotypes containing palisades of nuclei: three ependymomas, three hemispheric pilocytic astrocytomas, three oligodendrogliomas, three medulloblastomas, three cerebellar astrocytomas, and one central neuroblastoma. In two additional tumors, affecting a 12-year-old girl and a 51-year-old woman, this feature was present in the entire surgical specimen and the diagnosis was consistent with a polar spongioblastoma. This diagnosis applies in the literature to rate tumors of childhood and adolescence, both malignant and with embryonal features. In one specimen, a clear ependymomatous feature was found in a remote area of the tumor and in the other there were ultrastructural characteristics of neuroblastoma. Nuclear palisades can be found as local architectural features in many neuroepithelial tumors. The rare tumors diagnosed as polar spongioblastoma, according to published criteria, correspond to ependymomas and neuroblastomas. Polar spongioblastoma does not exist as a tumor entity.

Age-related ubiquitin deposits in dystrophic neurites: an immunoelectron microscopic study.

Widespread neuritic dystrophy is a hallmark of Alzheimer's disease (AD) and, in a less severe form, of brain ageing in various mammalian species. By immunohistochemistry, diffuse dot-like staining for ubiquitin (Ubq), a polypeptide involved in the degradation of abnormal and short-lived proteins, has been associated with human brain ageing. The nature of the Ubq deposits was investigated by immunogold electron microscopy on autopsy samples from aged human and dog brains. Most of the dot-like staining was localized to the white matter and corresponded to myelinated dystrophic neurites filled by Ubq-labelled lysosomal dense bodies. They did not contain paired helical filaments or multilamellar bodies. A minority of Ubq deposits was represented by amorphous densities in focal enlargements of the myelin sheaths. Our findings show that the spectrum of Ubq changes in ageing brain is wider than formerly recognized, and support the hypothesis that a defective regulation of the lysosomal system might be involved in the pathogenesis of structural abnormalities both in the ageing brain and in Alzheimer's disease.

Immunohistochemical observations on rat radial glia: relationship with the origin of ethylnitrosourea-induced tumors.

Gliomas induced in the rat by transplacental administration of ethylnitrosourea (ENU) are intensely immunoreactive for vimentin and scarcely for glial fibrillary acidic protein (GFAP). Since tumoral transformation takes place during the late fetal and early postnatal period, the sequential expression of the two glial antigens has been investigated in this age period in ENU-treated and control rats. Immunohistochemical and immunoelectron microscopical methods have been employed. Vimentin was widely expressed starting from embryonal day 14 (E 14) in the processes of radial glia; as long as radial glia was present, vimentin decorated it. GFAP was, at earliest, observed at E 20 and expressed by glial cells with a stellate, i.e., mature shape. No GFAP-positive radial process was observed. No difference was found between ENU-treated and control rats. Since ENU is most effective in producing tumors when administered at the 16-17th day of fetal life, vimentin-positive radial glia is a candidate target of ENU. The similarity of intermediate filament pattern between radial glia in the late fetal life and tumors induced by transplacental ENU suggests that radial glia might be the cell of origin.

Histologic prognostic factors in ependymoma.

The prognostic value of a series of histologic signs and clinical features was studied in a series of 298 ependymomas, collected from different institutions. The distribution of tumor sites varied in relation to patient age, with infratentorial cases prevailing under 4 years. Life table univariate analysis demonstrated as highly significant prognostic factors: (1) the number of mitoses; (2) endothelial hyperplasia; (3) necrosis; (4) intracranial site; (5) age less than 4 years. Multivariate analysis by tumor site revealed mitoses cell density, age greater than 16 years in supratentorial cases, and subependymoma in infratentorial cases to be prognostically important. Comparison of the anaplastic variant with the other tumor types in intracranial cases did not show a significant difference in survival even though the median survival time of anaplastic cases was shorter. The main conclusion is that the histological criteria employed to diagnose anaplasia in ependymomas. The number of mitoses is a very important prognostic factor in supratentorial cases, whereas endothelial proliferations and necroses are much less important as prognostic factors than in gliomas.

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.