Cellular expression of tumor necrosis factor a and its receptors in human ischemic stroke.

We evaluated by immunocytochemistry cellular localization and time-dependent expression of tumor necrosis factor a (TNF-alpha) and its receptors p55 (TNF-RI) and p75 (TNF-R2) in human ischemic brains. We observed them in microglia, neurons, astrocytes, macrophages and blood vessels. Since TNF-alpha expression was very intense and prolonged in microglia, it probably constitutes the main cellular source of the cytokine following cerebral ischemia in humans. Constitutive expression of TNF-alpha receptors was observed in neurons and blood vessels while in other cells it was induced by ischemia. In macrophages, dominant immunolabeling for TNF-R2 was seen. In other cells, immunoreactions for both types of TNF-alpha receptors were similar but the pattern of immunostaining was different: homogenous for TNF-R1 and granular for TNF-R2. Beneficial and detrimental role of TNF-alpha in cerebral ischemia and supposed mechanisms of action are discussed.

Pathomorphological variations of the AIDS-associated progressive multifocal leukoencephalopathy.

Pathological analysis of 20 cases of the progressive multifocal leukoencephalopathy (PML) appearing in the course of acquired immune deficiency syndrome (AIDS) is presented. PML occurred in 10% of all AIDS cases, collected in the period from 1987 to 1999. PML appeared either as the only brain pathology or accompanied HIV-related brain alterations isolated or concomitant with one or several opportunistic infections and/or neoplastic growth (malignant lymphoma). Basing on the pathomorphological picture and clinical symptomatology early, atypical and severe forms of the disease were distinguished. All of them were characterized by typical PML demyelination with oligodendroglial and astrocytic pathology. The group with early changes revealed widespread, multifocal myelin alterations of a moderate intensity with predominant oligodendroglial abnormalities and less advanced astrocytic changes. Atypical form of the disease was represented by cases with unifocal changes, although containing all key elements of PML pathology. The leading pathological feature of the severe form of the disease consisted in a particular intensity of the demyelination, resulting in tissue destruction often with its cavitation, with typical glial reaction and intense macrophage and lymphocytic infiltration. The other distinguishing feature consisted in strong topographic prevalence of the pathological process either to brain hemispheres or cerebellum. Differences of PML pathology in the course of AIDS as compared with non-AIDS cases are discussed. Due to the relatively high frequency of cases of isolated or strongly predominant involvement of cerebellum, separation of the cerebellar form of the disease has been suggested.

Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.

Immunoultrastructural expression of intercellular adhesion molecule-1 in endothelial cell vesiculotubular structures and vesiculovacuolar organelles in blood-brain barrier development and injury.

Blood vessels from the vasculature of mouse brains during postnatal development and from human brain tumors (hemangiomas) removed at biopsy were examined immunocytochemically by transmission electron microscopy (TEM) or high-voltage transmission electron microscopy (HVEM) to determine the expression of intercellular adhesion molecule-1 (ICAM-1). In the mouse brains, ICAM-1 was shown to be initially expressed on the luminal and abluminal endothelial cell (EC) surfaces on day 3 after birth. ICAM-1 intensity increased on the luminal EC surfaces and labeled vesiculotubular profiles (VTS, defined in the present report) between days 5 and 7. After 2 weeks and at 6 months after birth, ICAM-1 labeling was weak or absent on the luminal EC surfaces. The hemangiomas presented a strong ICAM-1 reaction product on the luminal EC surfaces of small and large blood vessels associated with the VTS, with a weaker labeling of the abluminal or adventitial aspects of larger blood vessels. TEM of vesiculovacuolar structures (VVOs) within ECs from arteries and veins also demonstrated reaction product for ICAM-1 labeling. Three-dimensional stereo-pair images in the HVEM enhanced the visualization of gold particles that were attached to the inner-delimiting membrane surfaces of EC VTS, and VVOs, respectively. These observations raise the possibility that the neonatal leukocytes and tumor cells may utilize these endothelial structures as a route across the developing and injured blood-brain barrier (BBB).

Opportunistic infections of the central nervous system in the course of acquired immune deficiency syndrome (AIDS). Morphological analysis of 172 cases.

A neuropathological analysis of 172 cases of AIDS in adults was carried out, to determine the occurrence and nature of the opportunistic infections of the central nervous system (CNS). The material comprised 155 cases of men, and 17 women. Mean age of patients was 38 years. Collection under study originated from the period between 1987 and 1997. Opportunistic infections were present in 57.5 percent of cases being in 38.4 percent the only pathological process, whereas in 19.1 percent they coexisted with HIV-dependent pathology or with neoplastic growth. Cytomegalovirus infection (22.7%), toxoplasmosis (16.3%), cryptococcosis (8.1%) and progressive multifocal leukoencephalopathy (9.3%) were the most common opportunistic infections of CNS. The remaining viral (herpetic encephalitis, tick-borne encephalitis and herpes zoster multifocal encephalitis), bacterial (lues, metastatic encephalitis connected with heart valvular changes) and fungal (candidiasis) infections were present only in single cases. It is worth mentioning 3 cases of brain aspergillosis and 5 cases of leptomeningeal tuberculosis. Great morphological variability in the most common opportunistic infections found in our material (cytomegaly, toxoplasmosis, cryptococcosis and PML) was the most striking phenomenon. Neuropathological abnormalities in cases of toxoplasmosis and cryptococcosis revealed remarkable dependence on clinical medication used. Cases of PML were characterized by strong variances of the type and intensity of demyelination, ranging from disseminated foci of various size to diffuse complete myelin loss in the white matter involving uni- or bilaterally cerebral or cerebellar hemispheres. The coexistence of opportunistic infections with HIV-dependent cerebral pathology or other types of opportunistic processes was a very characteristic feature. Concomitance of HIV-dependent pathology with viral opportunistic processes was common. The frequency of this concomitance and more severe HIV-dependent pathology in cases with other viral cerebral infections may suggest pathogenetic interaction of viral infections. Cerebral tuberculosis was less frequent as compared with other neuropathological collections, especially those from the United States. However, it seems worth mentioning that 3 of 5 cases occurred in the last year of observation.

Cerebral lymphomas in AIDS. Neuropathological study.

A morphological analysis was done of 15 cases of malignant cerebral lymphomas selected from the material of 160 brains of patients, who died in the course of full-blown acquired immune deficiency syndrome (AIDS) during the period of 1987-1997. Cases with cerebral lymphomas comprised 9.4% of the whole collection. There were 13 males and 2 females in the studied group. The patients age ranged from 25 to 61 years. In 10 cases lymphomas were localized solely in the central nervous system, and in further 4 they were accompanying systemic neoplastic process. In one case lack of clinical and autopsy data did not permit classification of neoplasm to the primary or to the secondary group. In 13 cases immunophenotype of the lymphomas was characterized by immunohistochemical methods. In 11 cases neoplastic cells originated from B cells line and in 2--from T cells line. In 10 cases lymphomas were found in macroscopic examination, in the remaining 5 cases they were disclosed at the brain histopathology. The dynamics and extensiveness of the neoplastic process were different in particular cases. In most of them the process was multifocal and manifested in the form of diffuse proliferation, formed tumors with changing nature of their delineation and as multilayer perivascular cuffs. The characteristic feature of diffuse neoplasmatic growth was the appearance of large coagulative necroses in the central parts of tumors. Neoplastic foci were localized most often in the cerebral hemispheres (white matter, basal ganglia, periventricular regions), less frequently in the brain stem and cerebellum. In one case diffuse lymphoid growth involved selectively leptomeninges. In most of the cases leptomeningeal infiltrations accompanied large parenchymal neoplastic foci. The most striking feature of our collection consisted in concomitance of cerebral lymphomas with HIV-specific brain pathology and/or opportunistic infections mostly of viral etiology. Their frequency was much higher than in cases of AIDS without cerebral lymphomas. Another finding which seems to be worth mentioning was the appearance of morphological exponents of various pathological processes such as for instance multinuclear giant cells, CMV inclusions within neoplastic tissue. The relatively frequent presence of numerous HIV-specific giant cells on the periphery of lymphomatous tumors suggests pathogenetic participation of immune deficiency virus in the blastomatous transformation of lymphoid cells within the central nervous system.

Cerebral accumulation of beta-amyloid following ischemic brain injury with long-term survival.

Deposits that are recognized by antibodies specific for the C-terminal and beta-amyloid peptide (beta A) but not the N-terminal sequences of the amyloid precursor protein (APP) fragments are present in the extra- and intracellular space in ischemic rat brain with 1 year survival. The immunohistochemical profile indicates that the APP in these deposits is truncated between the N-terminal and beta A and terminates at the C-terminal. This process probably is reaching into the extracellular space.

Endothelin-like immunoreactivity in hippocampus following transient global cerebral ischemia. II. The blood-brain interphase.

The effect of transient, global cerebral ischemia on the distribution of endothelin (ET) in blood-brain barrier (BBB) in CA1 area of hippocampus long-time after ischemia was estimated using post-embedding immunogold technique. ET-like immunoreactivity as a gold particles was localized in all compartments of the blood-brain barrier e.g. in endothelial cells, in pericytes, in periendothelial space including basement membrane, and in astroglial processes. In control animal the density of labelling in all elements of BBB in CA1 area of hippocampus was moderate. ET-like immunoreactivity (ET-like IR) was estimated 1 week-12 months after ischemia. Intense ET-like IR in all elements of BBB was noted 2 and 6 months after ischemia. A potential pathophysiological role of endothelin in cerebral vasospasm in long-time after ischemia is well documented.

Neuropathological syndromes in the course of full blown acquired immune deficiency syndrome (AIDS) in adults in Poland (1987-1995).

Morphological analysis of the brains from 100 cases of full blown AIDS patients observed in the course of 1987-1995 years was performed. The material comprised 96 males, 3 females and 1 infant, 11 months old. Early material consisted almost exclusively of homo- and bisexuals, while in the last years heterosexual drug addicts prevailed. Gross brain examination revealed focal changes in 25% of cases, most of them being connected either with opportunistic infections or primary proliferating malignancies. Brain atrophy with an evident regional differences was observed macroscopically in 35 cases. Microscopic examination allowed detection of pathological changes in the brains of 87 cases, although in the remaining 13 cases there occurred some slight abnormalities taking the form of non-specific neuronal degeneration and loss, considered as resulting from perimortal cardio-pulmonary insufficiency or bleeding. Specific HIV-related changes in the form of HIV-encephalitis, HIV-encephalopathy or coexistence of both and HIV-leptomeningitis as well as HIV-vasculitis were present in 35 cases. They were accompanied by HIV-associated changes (vacuolar myelopathy, vacuolar leukoencephalopathy and selective poliodystrophy). Very seldom they appeared as independent pathological features and were characterized by very low frequency. Opportunistic infections composed the largest group of 59 cases. Proliferative malignancies occurred altogether in eleven cases, 10 of which were primary and secondary brain lymphomas. One case of Kaposi sarcoma completed the neoplastic series. Sixteen cases revealed various types of brain pathology such as hepatogenic encephalopathy, traumatic cortical scars, central pontine myelinolysis etc. The 59 cases of opportunistic infections consisted of a wide spectrum of viral and bacterial as well as fungal and protozoan infections. Among viral infections cytomegalovirus encephalitis was the most common, way ahead the progressive multifocal leukoencephalopathy. The second in frequency among opportunistic infections was brain toxoplasmosis and some fungal infections such as cryptococcosis and aspergillosis. Bacterial infections were in fact limited to tuberculosis, taking the form of granulomatous leptomeningitis with severe vascular pathology and/or tuberculoma formation. Many pathological processes appearing in a single case was characteristic feature of our collection. There was coexistence of HIV-specific CNS pathology and opportunistic infections, malignant neoplastic growth and other types of pathology. Various opportunistic infections coexisted without HIV-specific changes as well as malignant proliferation with opportunistic infections. Similarities and differences of our series were compared with data characterizing other, earlier collections of NeuroAIDS.

Ultrastructural studies of PECAM-1/CD31 expression in the developing mouse blood-brain barrier with the application of a pre-embedding technique.

The aim of this study was to investigate the time of expression of the adhesion molecule platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in the developing mouse blood-brain barrier (BBB). Ultrastructural studies employing a preembedding technique described herein demonstrate that PECAM-1 is initially expressed on the luminal and abluminal endothelial cell surfaces in the newborn animals. This adhesion molecule expression appears to increase in intensity at 7-10 days post partum and then decreases to a weak labelling of the luminal endothelial cell surfaces at two weeks after birth. Our results present immunocytochemical detection of active angiogenesis during early brain development in the mouse. Moreover, because of the important role that adhesion molecules play in immune responses in the central nervous system, upregulation of PECAM-1 prior to structural maturity of the BBB may suggest that the development of an "immune BBB" manifests prior to anatomical closure of the BBB in the mouse.

Endothelial nitric oxide synthase in vascular endothelium of rat hippocampus after ischemia: evidence and significance.

Electron microscopy immunocytochemical study was performed to clarify ultrastructural localization and role of endothelial nitric oxide synthase (EC-NOS) in the endothelial cells (EC) of rat hippocampal vessels after transient cerebral ischemia. EC-NOS immunoreactivity was found in the endothelial cells in association with plasma membrane, sub-plasmalemmal vesicles, basal membrane and in cytosol (cytoplasm free of subcellular organelles). A sharp transient increase in immunoreactivity of NOS was observed at 10 min up to 1 hour after ischemia. The results of the present study indicate that NO, as a potent vasodilator, may play a protective role in ischemic brain damage.

Mitochondrial damage following exposure of organotypic cultures of human malignant gliomas to 2-chloro- and 2-bromo-2'-deoxyadenosine.

The effects of 2-chloro-2'-deoxyadenosine (cladribine, 2-CdA) and a closely related compound 2-bromo-2'-deoxyadenosine (2-BdA) on organotypic cultures of human malignant gliomas were studied with the use of electron microscopy. The cultures grown from surgical biopsies included six glioblastomas, three anaplastic astrocytomas and low-grade fibrillary astrocytoma. After 6 to 10 days of the in vitro growth the cultures were exposed to 0.3-10 microM 2-CdA or 2-BdA for 1 to 10 days. Mitochondrial swelling and disappearance of cristae following exposure to the tested substances were observed, but only in highly anaplastic (low-differentiated) tumor cells. The mitochondrial toxicity was dose- and time-dependent, and no difference was found between the effect of 2-CdA and 2-BdA.

2-chloro- and 2-bromo-2'-deoxyadenosine have no effect on the morphology of rat neural and glial cells in organotypic culture.

Organotypic cultures of hippocampus and cerebellum, established from brains of 1-3 days old rats, were exposed at different stages of development (3, 14 and 21 DIV) to 2-chloro-2'-deoxyadenosine (cladribine, 2-CdA) and 2-bromo-2'-deoxyadenosine (2-BdA) at concentrations up to 10 microM, for up to 10 days. Normal pattern and dynamics of differentiation and maturation of both neurons and glial cells was found with the use of light and electron microscopy. No ultrastructural abnormalities were induced by the substances tested. We conclude that 2-CdA and its sister compound 2-BdA do not exert cytotoxic effects toward normal rat central nervous system tissues in organotypic culture.

Intercellular adhesion molecule-1 (ICAM-1) upregulation in human brain tumors as an expression of increased blood-brain barrier permeability.

The distribution of intercellular adhesion molecule (ICAM-1) binding sites was studied in the microvasculature of several types of human brain tumor biopsies (angioma, glioblastoma multiforme and meningioma). Immunoelectron microscopy was performed with the application of immuno-HRP or -gold probes using a pre-embedding technique. Ultrastructural analysis demonstrated a pronounced ICAM-1 upregulation on the luminal EC and/or perivascular surfaces. Reaction product for ICAM-1 was observed associated with some but not all blood vessels of the tumors examined. The strongest reaction product was noted associated with the angioma cases with lesser expression observed on the glioblastoma multiforme and meningioma cases. The reaction product using immuno-HRP probe was observed most pronounced on the luminal endothelial cell surface and also within vesiculo-tubular structures. Concentrated immunosignals with gold label were often expressed on EC microvilli. These data suggest that several types of brain tumors are actively involved in the process of upregulating ICAM-1, presumably for tumor cell adhesion and trafficking, the process of angiogenesis or both. We suggest that the ICAM-1-positive vesiculo-tubular structures reflect specialized, targeted regions on the ECs for tumor cell adhesion and eventual trans-BBB passage. Further, our studies also provide evidence that adhesion molecules may be a useful tool for the study of blood-brain barrier injury.

Upregulation of bax protein levels in neurons following cerebral ischemia.

The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.

Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II. Extracellular and intracellular accumulation of apolipoproteins E and J in the brain.

The distribution of apolipoprotein E (apo E) and apolipoprotein J (apo J) was investigated immunocytochemically in rats at various time intervals after 10 min global cerebral ischemia (GCI) induced by cardiac arrest. Strong apo E and weaker apo J immunoreactivity was found extracellularly in multiple deposits located close to the microvessels. These deposits appeared 3 h after GCI and were present, but not in all the animals, at all time intervals studied post-GCL. In some rats, apo E immunoreactivity was also found in small necrotic foci. Widespread, neuronal apo E immunostaining appeared 6 h post-GCI. However, the strongest neuronal apo E immunoreactivity was found 7 days post-GCI in those neurons, most often observed in the CA1 hippocampal region, exhibiting signs of ischemic cell damage. These ischemically damaged neurons displayed weaker immunoreactivity to apo J, despite its increase in the response to GCI in the various brain regions examined. Our data show that mechanisms operating in ischemia are able to supply large amounts of apo E and apo J to the brain tissue and suggest involvement of both apo E and apo J in a complex series of events occurring in the ischemic brain. Perivascular deposits of apo E/apo J colocalized with amyloid beta protein precursor epitopes that have been disclosed by us previously in this model. Whether this phenomenon is limited to postischemic brain tissue, or can be encountered also in other pathological conditions will require further elaboration.

DNA protein flow cytometry of dissociated cultures of human anaplastic gliomas. Pattern of proliferation and differentiation, and the effect of a new cytostatic drug cladribine (2-CdA).

A technique of protein-DNA flow cytometry was applied to characterize cell cycling, and to assess the cytotoxicity of cladribine (2-chloro-2'deoxyadenosine) toward seven dissociated cultures of human primary brain tumors (anaplastic astrocytoma and glioblastoma multiforme) grown in vitro from surgical biopsies. Control cytograms were suggestive of that a clonogeneic fraction of the cell population consists mainly of cells with low protein content, which do not require increase in protein content before entering the S phase of the cell cycle. Following 24 or 48 hours exposure to cladribine, 1 nM approximately 1 microM, no cytotoxic effect was evident in 4 cultures, whereas in two cases dose-dependent progressive block of the phase of the cell cycle was noted. In one case a massive cytotoxic effect resulted in disintegration of culture exposed to 100 nM of the drug. However, the treatment with cladribine was ineffective in a patient bearing the tumor which was the source for the last culture, suggesting that cytotoxicity in vitro may not be predictive of clinical response.

Ultrastructural studies of cerebral vascular spasm after cardiac arrest-related global cerebral ischemia in rats.

The present investigation was undertaken to study the ultrastructural morphology of brain blood vessels during vasospasm following total cerebral ischemia. Global cerebral ischemia was produced in rats by compression of the cardiac vessel bundle (i.e., cardiac arrest) using a metal hook that was introduced into the mediastinum. Ischemia lasted for 10 min with blood recirculation for 6, 12 and 24 h. Rat brains were perfusion-fixed and regions from the cerebral cortex and associated leptomeningeal vessels were evaluated by scanning and transmission electron microscopy. We noted three general vasoconstrictive responses in vessels of various sizes including veins and arteries. These alterations related to the smooth muscle cell arrangement associated with each constricted vessel including a circumferential, and longitudinal arrangement, or a combination of both types. Other features in the three types of vasoconstricted vessels included thickening of the vessel basement membranes with increased endothelial microfilaments and vesicular profiles. Our studies present evidence that ischemia of 10-min duration with blood reflow for 6, 12 and 24 h produces profound and variable vasospastic changes in some but not all vessels. These vascular alterations are thought to be caused in part by vasoactive substances released both by endothelial and blood cells and by perivascular cellular elements in response to the ischemic episode.