Lysosome abnormalities and lipofucsin content of nerve cells of oedematous human cerebral cortex

Lysosome alterations and lipofucsin content of nerve cells, capillary endothelial cells and pericytes were examined in the anoxic-ischaemic brain parenchyma of thirty two patients with congenital hydrocephalus, complicated brain traumatic injuries, brain tumours and vascular anomalies. Cortical biopsies of frontal, parietal and temporal cortex were processed for transmission electron microscopy. In oedematous non pyramidal and pyramidal nerve cells, lysosomes showed fragmentation of their limiting membranes and an associated dense granulation. Areas of cytoplasmic focal necrosis were observed surrounding the lysosomes. Lipofucsin granules were also observed in neonate and infant patients with congenital hydrocephalus, suggesting that lipofucsin formation is a life span process. Lysosomes coexisting with an increased amount of lipofucsin granules were observed in young and adult patients with brain trauma, tumours and vascular anomalies. Phagocytic astrocytes and activated oligodendroglial cells showed the overall spectrum of an altered endosomal/lysosomal system. Lipofucsin granules and multivesicular bodies also were distinguished in endothelial and pericyte cells. The role of released and activated lysosomal enzymes is discussed in relation with the cytoplasmatic focal necrosis of nerve cells and the genesis of moderate and severe oedema.     

Restricted cyclooxygenase-2 expression in the central nervous system following acute and delayed-type hypersensitivity responses to bacillus Calmette-Guérin.

The expression of cyclooxygenase-2, a key enzyme in prostaglandin and thromboxane synthesis in inflammation, was studied immunohistochemically in in vivo models of acute and chronic inflammatory responses in rat central nervous system. In the acute inflammatory response to intracranial injection of heat-killed bacillus Calmette-Guérin as well as in the immune-mediated, delayed-type hypersensitivity response to the same pathogen, cyclooxygenase-2 expression was restricted to major infiltrating haematogenous cell populations such as neutrophils and mononuclear phagocytes, while the expression of the enzyme by brain non-neuronal resident cells (astrocytes, microglia, perivascular cells) appeared to be limited to perivascular cells of the blood vessels in the vicinity of the lesion and in the surrounding area. On the basis of their morphology and location, these perivascular cells were identified as perivascular macrophages, but we could not rule out the possibility that some endothelial cells also expressed cyclooxygenase-2. The constitutive neuronal cyclooxygenase-2 was not affected by the ongoing inflammation. Interestingly, in spite of the extensive astrocyte and microglial reaction occurring over a broad area surrounding the inflammatory lesions, there was no obvious cyclooxygenase-2 staining in these cells. These data indicate that the up-regulation of cyclooxygenase-2 expression in acute and chronic, immune-mediated lesions in the brain parenchyma is remarkably restricted to the lesion site. Since cyclooxygenase metabolites can regulate important functions of resident as well as infiltrating cells, the increased synthesis of prostaglandins and thromboxanes, which is likely to occur as a consequence of the expression of cycloxygenase-2 at the lesion site, might represent an important component of the inflammatory processes within the brain.     

脑内和肌内接种小鼠胶质母细胞瘤株后神经末梢的观察

本实验应用Nonidez及Glees二种镀银法,对诱发的小鼠胶质母细胞瘤株(G 422)进行了观察。脑内及肌内接种胶质母细胞瘤后,在肿瘤边缘可见大小不等的神经束,伴随或不伴随血管伸入瘤内。有的在血管周围间隙形成血管周围神经丛。这些神经纤维与肿瘤周围宿主的脑组织、皮下、毛囊和肌肉间隙的神经纤维相联系。因此我们推测肿瘤内的神经是由肿瘤周围宿主的器官组织的神经延伸来的。肿瘤边缘的神经纤维的数量多于核心区,走行于肿瘤的间质或实质,沿途不断分支,终末分布到肿瘤细胞的表面。我们观察到球形、游离分叉状、梭形、环形、树枝状、杵状及丛刷状等类型的神经末梢。以上观察表明,恶性肿瘤——小鼠胶质母细胞瘤是受神经支配的。     

Microvascular and Cellular Responses in the Retina of Rats with Acute Experimental Allergic Encephalomyelitis (EAE)

The microvascular and cellular responses in the retina during acute EAE were characterized using whole-mount preparations. The earliest detectable event was the accumulation of monocytes and T cells within veins on day 7 postinduction (pi). Mild breakdown of the blood-retinal barrier (BRB), activation of microglia and infiltration of monocytes and T cells into the retinal parenchyma were first evident on days 7 to 8 pi. Monocyte adhesion to the vessel wall and breakdown of the BRB were colocalized in the same vessel segments and occurred predominantly in veins. The marked difference in response observed in the retina versus the myelinated region of the optic nerve suggests that two types of inflammatory cascades are initiated. A mild response, characterised by very low numbers of T cells and monocytes and an absence of expression of MHC class II by resident microglia, is initiated when only small amounts of the encephalitogenic antigen are present in the perivascular space or associated with perivascular antigen-presenting cells. A full blown inflammatory reaction, as observed in the optic nerve, is initiated in the presence of substantial amounts of encephalitogenic antigen. This severe response is characterised by the infiltration of large numbers of CD4⁺, CD8⁺T cells and ED1⁺ monocytes, and by abundant MHC class II expression by resident microglia as well as other cell types. Thus, MHC class II expression by resident microglia may be a possible effective amplifier mechanism if the encephalitogenic antigen is encountered in the tissue parenchyma.     

Intracerebral schwannoma in a child with infiltration along perivascular spaces resembling meningioangiomatosis

Schwannoma arising within brain parenchyma is a rare lesion, usually found in children. Reported herein is a case of intracerebral schwannoma in a 5-year-old boy, with a review of the English-language literature on the subject, in which 47 cases were found. Few detailed histological reviews of intracerebral schwannoma exist. The tumor had a distinctive plexiform growth pattern, and small aggregates of Schwann cells spread extensively into the surrounding brain tissue along perivascular spaces adjacent to the tumor nodule. Histological differential diagnoses included perivascular schwannosis and meningioangiomatosis. A few intratumoral axons, seen on immunostaining for neurofilament protein, were trapped at the periphery of the main lesion, but there was no evidence of intralesional axons in the multiple nodules of Schwann cell proliferations that extended into the perivascular spaces, suggesting that the lesions are neoplastic. Because Schwann cells are not a natural component of the central nervous system, the origin of intracerebral schwannomas remains unknown. The histology suggests that Schwann cells of the perivascular nerve plexus are a likely site of origin.     

Formation of transendothelial channels in traumatic human brain edema

The formation of incomplete transendothelial channels is reported in four cases of traumatic human brain injury complicated with subdural or epidural hematoma or hygroma. Such structures were observed coexisting with increased transendothelial vacuolar and vesicular transport. They appeared in the following manners: as electron lucent corridors formed by deep invaginations of luminal endothelial plasma membranes, as chained spheroidal or elongated vacuoles connected with the luminal and abluminal endothelial membranes, as a result of a combined process of membrane fusion and fission, as a large protein containing vacuole or a micropinocytotic vesicle occupying the entire width of peripheral endothelial cytoplasm, as abluminal profiles of dilated basement membrane expansions, extending their course to the vicinity of endothelial luminal plasma membrane. The endothelial junctions appeared intact in most cases. The transendothelial channels apparently represent a transcytosis response to the traumatic brain injury. They may provide specific cytoplasmic corridors of facilitated transport for edema formation, which incorporate the intraluminal capillary content, ferry it across the peripheral zone of endothelial cytoplasm and empty it at the abluminal surface, into the surrounding basement membrane, from where it floods the intercellular spaces of neighbouring neuropile. The albuminal transendothelial channels, formed by the dilated basement membrane bifurcations, might be a reverse shuttle for endogeneous substances moving from parenchymal clefts to blood as a capillary mechanism of edema resolution.     

Intercellular Communication by Extracellular Vesicles with Emphasis on the Roles of Cordocytes in the Human Brain. An Ultrastructural Study

We describe in this work the presence of extracellular vesicles (EVs) along different cell types, especially cordocytes, in various clinical conditions of the human brain (atherothrombotic disease, cerebral tumors, hygroma durae matris, intracerebral cysts, Moyamoya disease and parenchymatous hematoma) using transmission electron microscopy (TEM). EVs, illustrated as exosomes and microvesicles, were causally related to cell-to-cell communication, and other vital functions of resident cells around the brain parenchyma, either around the cortical vessels or into the subarachnoid space and the reticular arachnoid. Our direct demonstration by TEM of these information transporters in all locations and situations where the cordocytes play coordinating and regulating roles, producing and delivering a significant number of EVs to their targets, remains to be better documented in future studies. This first study on this topic showed clearly that EVs can be important modulators of cell functions with roles in cell activation, differentiation, phenotypic change, cancer progression, from precursor/stem cells to tumoral phenotypes, because EVs are released en masse during key interactions and certain moments.     

Defining antigen-dependent stages of T cell migration from the blood to the central nervous system parenchyma

In experimental autoimmune encephalomyelitis (EAE), intravenous transfer of activated CD4(+) myelin-specific T cells is sufficient to induce disease. Transferred T cells access the CNS parenchyma by trafficking across the blood brain barrier (BBB) vascular endothelium into the perivascular space, and then across the glial limitans that is made up of astrocytes and microglia. Flow cytometry analysis of cells isolated from CNS tissue does not distinguish between T cell populations at the various stages of migration. In this study, we have used GK1.5 (anti-CD4) treatment along with immunohistochemistry to distinguish between populations of T cells that are associated with the vasculature, T cells that have migrated into the perivascular space, and T cells in the parenchyma. We have also re-evaluated antigen specificity requirements of T cells as they are recruited to the CNS parenchyma. Activated myelin-specific T cells are restricted to the CNS vasculature for at least 24 h post transfer. MHC class II expression on the recipient is required for cells to traffic across the CNS vascular endothelium. Further, Con A-stimulated or non-CNS-specific (ovalbumin-specific) T cells fail to migrate into the perivascular space, and only enter the CNS parenchyma when co-transferred with myelin-specific T cells. Our results indicate that Th1 populations cannot accumulate in the perivascular (subarachnoid, Virchow-Robbins) space without a CNS antigen-specific signal.     

Neuromuscular biopsy and diagnosis of vasculitis

One characteristic histological lesion on biopsy specimens is mandatory to establish the diagnosis of vasculitis. Combined nerve and muscle biopsies, by the same cutaneous incision, improve significantly the percentage of positive results. Nerve fragments should be taken in every patient presenting sensory manifestations. Such vasculitic lesions are present in medium-sized arterioles and/or small vessels, and correspond mainly to 4 necrotizing vasculitis: panarteritis nodosa (PAN), microscopic polyangiitis (MPA), Churg and Strauss syndrome and Wegener granulomatosis. Microvasculitis should be added to these classical entities, because it corresponds to small vessel wall infiltration by inflammatory cells, as observed in PAN and MPA, but without any necrosis. Microvasculitis has to be differentiated from the inflammatory cell infiltrates surrounding small vessels. However, such perivascular inflammatory cell infiltrates enable the diagnosis of probable vasculitis when associated with clusters of neo-vessels, hemosiderin deposits, or a focal damage of nerve fibers. Grossly, one third of vasculitis diagnosis is confirmed on muscle fragments, a second third on nerve fragments, and the last third on both nerve and muscle fragments. Moreover, in the search for vasculitis, an unpredicted diagnosis of lymphoma or amyloidosis is occasionally established on the neuro-muscular biopsy.     

Detection of measles virus RNA in lymphocytes from peripheral-blood and brain perivascular infiltrates of patients with subacute sclerosing panencephalitis

To clarify the relation between lymphocytes and measles virus in subacute sclerosing panencephalitis, we used in situ hybridization and a cloned measles virus DNA probe, specific for nucleocapsid protein, to detect measles virus RNA sequences in circulating lymphocytes and brain perivascular cuffs of patients with subacute sclerosing panencephalitis. Seventy to 90 per cent of peripheral mononuclear cells from three such patients were found to contain measles virus RNA sequences. In contrast, only a few infected cells were observed in four seropositive adults (0.1 to 5 per cent) and three age-matched children (10 to 15 per cent) used as controls. In one sample of brain tissue from a patient with subacute sclerosing panencephalitis, viral RNA sequences were also detected in nerve cells and in numerous cells from the perivascular infiltrates. In contrast, no hybridization was observed in brain tissue from a patient with herpetic encephalitis and from a patient with postlymphoma encephalitis. We conclude that measles virus has a strong tropism for lymphocytes and nerve cells in subacute sclerosing panencephalitis and that lymphocytes may be involved in the pathogenesis of the disease.     

Lymphokines and immunoregulatory molecules in subacute sclerosing panencephalitis

Frozen brain specimens from six patients with subacute sclerosing panencephalitis (SSPE) were analyzed immunohistochemically for the presence of leukocyte subpopulations and specific cytokines. In brain regions demonstrating perivascular cell infiltration and gliosis, CD4 and CD8 positive cells were identified within the brain parenchyma. Cytokine analysis revealed cells staining positively for tumor necrosis factor-alpha and interferon-gamma. These results were similar to those observed in multiple sclerosis (MS) and progressive rubella panencephalitis tissue and were different from other predominantly noninflammatory neurologic diseases and normal controls. Although SSPE and MS differ significantly in their etiology and histopathology, the similarities in leukocyte and cytokine staining patterns suggest a common mechanism of disease progression.     

Wide range of lineages of cells expressing nerve growth factor mRNA in the nerve lesions of patients with vasculitic neuropathy: an implication of endoneurial macrophage for nerve regeneration

In situ localization of nerve growth factor (NGF) mRNA was examined in the nerve lesions of patients with vasculitic neuropathy. Double labeling of in situ hybridization for NGF mRNA and immunohistochemistry for cell markers showed that NGF mRNA was expressed in a wide range of lineages of cells: Schwann cells, infiltrating macrophages, T cells and perivascular cells. Round-shaped macrophages with early-phase features expressed high levels of NGF mRNA, in contrast to late-phase polymorphic macrophages, which expressed low levels of NGF mRNA. NGF mRNA was also expressed universally in T cells with various cell surface markers. Epineurial macrophages surrounding vasculitic lesions and endoneurial T cells expressed high levels of NGF mRNA in the damaged nerves. Moreover, the extent of endoneurial NGF expression level in macrophages was closely related to the degree of axonal regeneration. These results suggest that NGF is expressed in a wide range of lineages of cells but is differentially expressed spatially in vasculitic nerve lesions, and that the expressed NGF, particularly in macrophages, may play an important role in the nerve regeneration process.     

Granular cell traumatic neuroma: a lesion occurring in mastectomy scars

BACKGROUND: Granular cell changes can be observed in a variety of benign and malignant tumors, and are seen more commonly in granular cell tumors, which in about 5% of cases develop in the breast. Granular cells also have been observed in sites of previous trauma, such as surgery, and are found to be inflammatory reactions of histiocytic origin. METHODS AND RESULTS: We investigated, morphologically and immunohistochemically, 2 granular cell lesions occurring in mastectomy scars after surgery for carcinoma. Both lesions were composed of strands and nests of large granular cells, haphazardly set in a background of fibrous tissue, with sparse inflammatory infiltrates. Several tortuous hypertrophic nerve bundles were also embedded in the fibrous tissue. A few of these nerve bundles showed degenerative changes and contained granular cells. Immunohistochemically, granular cells were positive for S100 protein, neuron-specific enolase, vimentin, and CD68 antigen. CONCLUSIONS: We consider these proliferative lesions of peripheral nerves to have the features of both granular cell tumor and traumatic neuroma. These cases indicate that traumatic neuroma can undergo extensive granular cell changes and constitute a previously unrecognized entity, which we provisionally label granular cell traumatic neuroma. Granular cell traumatic neuroma has to be taken into consideration when evaluating lesions occurring at mastectomy scars and should be differentiated from malignant tumors with granular cells, such as apocrine carcinoma and alveolar soft part sarcoma.     

大鼠脑内大分子物质的引流通路及方法学研究

为探讨脑内大分子物质的引流通路,本研究微量注射示踪剂墨汁到大鼠右侧尾壳核,Ⅰ组动物使用传统的脑内注射方法,Ⅱ组动物采取改良方法防止示踪剂从进针处进入蛛网膜下腔,术后1、3、7、14、21 d处死动物,分别用肉眼、光镜及电镜观察墨汁在脑内、蛛网膜下腔、颈总动脉及颈部淋巴结的分布。结果显示:墨汁在两组动物脑实质内的分布趋势相同,即在白质内沿神经纤维弥漫性分布,7 d后在灰质内选择性地沿血管周围间隙分布,部分碳颗粒被管周细胞和巨噬细胞吞噬;Ⅱ组动物中观察到墨汁从大脑顶部进入蛛网膜下腔后沿其中的血管周围间隙分布并引流到脑的底部和嗅球及筛板区域,在耳蜗、前庭蜗神经和视神经等的脑神经鞘以及颈总动脉壁和颈部淋巴结有碳颗粒沉积;Ⅰ组动物没有这种分布。以上结果表明,大鼠脑实质内的大分子物质在白质和灰质中的引流方式不同;进入脑脊液的大分子物质可由颈部淋巴系统引流;墨汁不是很好的研究脑实质内大分子物质引流的示踪剂。     

Ultrastructural Basis of the Vasculopathy in and Around Brain Tuberculomas: Possible Significance of Altered Basement Membrane

The fine structure of small blood vessels in and around ten brain tuberculomas was examined. In the peripheral reactive zone of the tuberculomas, examination of 1-μ-thick survey sections established the chronic inflammatory process and the vasculitis characterized by infiltration of the vasomurium (vessel wall) by large and small mononuclear cells. This reaction was typical of chronic epithelioid cell granuloma. Electron microscopic examination of the reactive zone confirmed the vascular proliferation and vasculitis, the venule being the most frequently involved type of blood vessel. It showed the infiltrating cells to lie amidst osmiophilic, concentrically proliferated basement membrane laminae, which formed the greater part of the thickened vessel wall, generally surrounding the endothelial cells directly, the pericytes having disappeared. This appearance, together with the results of Gomori's reticulin stain on paraffin sections, suggested that the altered basement membrane material was reticulin. The possibility is discussed that the altered basement membrane material could be antigenic and that it might be responsible for perpetuating the necrotic vascular and perivascular reaction in tuberculous meningitis and tuberculomas. The above change in the basement membrane was not encountered in the blood vessels of the surrounding edematous brain. The endothelial cells and tight junctions were relatively well-preserved. Intact arterioles could be recognized even in severely edematous brain tissue. At both sites the fine structure of the blood vessels was typical of that expected in the central nervous system. Fenestrated vessels were not seen. The perivascular astrocytic end-feet were destroyed in the reactive zone and either distended or ruptured in the overtly edematous brain tissue also. In the central caseous part of the tuberculoma, there were few blood vessels, and they were in a state of advanced necrosis, but ghost outlines of proliferated basement membrane could be seen.     

Comparison of metastatic brain tumour models using three different methods: the morphological role of the pia mater

As methods of cancer diagnosis and treatment progress, interest in metastatic brain tumours continues to increase. There are many studies using various methods of animal model and we considered that each model reflects different pathological processes because of the unique composition of the brain. We prepared metastatic brain tumour models using three different methods. In this study, we attempted to elucidate the roles of the pia mater in brain metastasis. The metastatic foci showed an angiocentric pattern, forming collars of neoplastic cells, and were designated 'perivascular proliferations'. Furthermore, we observed neoplastic cells that infiltrated the brain parenchyma, the border of which had become indistinct. These were labelled 'invasive proliferations'. The internal carotid artery injection model reflects haematogenous metastasis. In this model, both perivascular and invasive proliferations were observed. The intrathecal injection model reflects leptomeningeal carcinomatosis. In this model, metastasis to the meninges was observed. In the stereotactic injection model, the tumour proliferation at the injection site and the infiltration into the brain parenchyma were observed. The pia-glial membrane serves as a scaffold when neoplastic cells spread to the perivascular space forming angiocentric pattern. The pia-glial membrane is found between the brain parenchyma and blood vessels. Blood vessels penetrate the brain through tunnels known as perivascular spaces that are covered by pia mater. Three different methods which we prepared reflect three different pathological processes. Our findings suggest that the pia mater is a critical factor in brain metastasis.     

Nerve cell death types in the edematous human cerebral cortex

Cortical biopsies of 18 patients with clinical diagnosis of congenital hydrocephalus, brain trauma, and vascular anomaly were examined with the transmission electron microscope to study the distinct and overlapped morphological cell types of nerve cell death in the human edematous cerebral cortex. The nerve cells showed lobulated and shrunken nucleus, irregular enlargement and fragmentation of perinuclear cistern, with areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. The nucleolus appears unaltered in moderate edema and with distorted nucleolar subcompartments in severe edema. Most nonpyramidal nerve cells, astrocytes and oligodendrocytes underwent an oncotic-apoptotic-necrotic continuum featured by swollen nucleoplasm, cytoplasm, and cell organelles, chromatin condensation and marginalization, and formation of apoptotic bodies. In a lesser proportion other nonpyramidal nerve cells, astrocytes and oligodendrocytes only showed apoptosis or oncosis. Autophagic cell death characterized by presence of autophagic vacuoles of lysosomal origin was rarely seen. The above findings suggest that different mechanisms of nerve cell death occur in the human edematous cerebral cortex related with brain trauma, congenital hydrocephalus, vascular anomaly, and their anoxic-ischemic conditions. An oncotic-apoptotic continuum process leading to necrosis predominates in human cerebral cortex nerve cell populations. The nerve cell death is discussed in relation with the severity of brain edema, anoxic-ischemic conditions of brain parenchyma, oxidative stress, glutamate excitotoxicity, calcium overload, and caspase dependent and independent mechanisms.     

Ultrastructural and permeability features of microvessels in the hippocampus, cerebellum and pons of senescence-accelerated mice (SAM)

We previously reported that the accumulation of blood-borne radiolabelled serum albumin in brain parenchyma increased with aging, especially in senescence-accelerated mice (SAMP8), which showed age-related deficits in learning and memory. In this study, in order to examine morphological events related to the age-related increase of the brain accumulation of serum albumin, the transvascular passage of blood-borne horseradish peroxidase (HRP) and ultrastructural features of microvessels were examined in the hippocampus, cerebellum and pons of SAMP8 and SAMR1 (control) mice. Ultrastructural examination of the hippocampus showed that the staining for HRP was occasionally spreading throughout the parajunctional cytoplasm of the endothelial cell of aged SAMP8 mice, but not in young SAMP8 mice nor in SAMR1 mice. The number of vessels showing the staining reaction for HRP in the parajunctional cytoplasm of the endothelial cells in aged SAMP8 mice increased significantly compared with that in the others. Electron microscopic morphometry showed that there were no significant differences among the number of HRP-positive vesicles per unit area of the endothelial cell cytoplasm in young and old mice of both strains. The staining reaction for HRP was not seen in the basal lamina of microvessels and the perivascular neuropil in all mice examined. Perivascular lipofuscin-like granules and collagen deposits, swelling of astroglial perivascular endfeet and perivascular cells containing foamy, lipid-like droplets were frequently found in several brain regions of aged SAMP8 mice. The perivascular cells with a few lipid-like droplets and more electron-homogeneous lysosomes were occasionally seen in SAMR1 and young SAMP8, while the other findings were scarcely observed in SAMR1 and young SAMP8 mice.These findings suggest that the blood-brain barrier to HRP was preserved in microvessels in three brain regions of SAM mice but the blood microvessels showed some age-related ultrastructural alterations in SAMP8 brains. Uncontrolled passage of HRP through the parajunctional cytoplasm of the endothelial cells may partly contribute to the age-related increase of accumulation of serum albumin in SAMP8 brains.