Clinico-pathological correlation in case of BRAT1 mutation.

The clinical picture of BRCA1-associated protein required for ATM activation-1 (BRAT1) comprises retractable early-onset epileptic encephalopathy, progressive microcephaly, and early demise. Both, inter- and intrafamilial variations of features of BRAT1-associated disease have been described. Here, the familial case of a brother and sister with homozygous pathogenic variants in BRAT1 is presented with special emphasis on differences in seizure type/onset and central nervous system lesions. The neuropathology is extensively discussed and hypotheses put forward that may shed light on etiology of brain symptomatology within the context of BRAT1 mutations.

Commitment of protein p53 and amyloid-beta peptide (Aß) in aging of human cerebellum.

Protein p53 is known to induce the cell cycle arrest and apoptosis in response to a variety of cellular distress signals and DNA damage. A recent study has demonstrated that in blood cells of aging subjects, p53 may induce early pathological changes that precede the amyloidogenic cascade. However, it is not clear whether p53 participates in the local deposition of amyloid-beta peptide (Aß) in the nerve tissue of normal aging subjects. Therefore, in the present study, we analyse the distribution of both (Aß and p53) proteins in the cerebellum of individuals without any history of dementia or other neurological illness who died suddenly in traffic accidents. We found that in the subjects at the beginning of their aging process (60-65 years of age) Aß deposits were localized in subependymal areas of the cerebellar cortex and such deposits were not linked to the presence of p53 in the nerve tissue. In groups of subjects over 65 years of age, numerous Aß diffuse plaques were scattered throughout the cerebellar cortex. In these subjects, p53 protein was detected in the cytoplasm or in the nucleus of the cerebellar nerve cells. All the results lead to the conclusion that in nerve tissue p53 participates in the process of neurodegeneration and then it is involved in the deposition of A in the nerve tissue.

Toll-like receptor 2 (TLR2) is a marker of angiogenesis in the necrotic area of human medulloblastoma.

Angiogenesis plays a key role in the progression of malignant tumors. In recent years, anti-angiogenic drugs have been shown to be effective against tumors. However, some tumors are able to adopt escape mechanisms, suggesting that the vascular network in these tumors may be formed or may function in a different way. Medulloblastomas are tumors characterized by poor prognosis and low patient survival rates. These tumors rarely metastasize, but the reason why they almost always recur locally is not known. Central to mediating neoplastic changes is the interaction between cell surface receptors and their cognate ligands, which through intracellular signaling induce alternations in gene expression. In this context, the aim of our present study was to examine in medulloblastoma the distribution of Toll-like receptor 2 (TLR2) and receptor for advanced glycosylation end-product (RAGE), and mast cells associated with the tumor neovascularization process. Immunohistochemical study with a battery of specific antibodies was used. The results show that in the tumor necrotic area, TLR2 participates in all steps of vascular network formation, but in regions where the tumor was not affected by necrosis, the capillary network was TLR2 immunonegative. The TLR2 vascular network of the necrotic area was not associated with RAGE and mast cells. However, in the region of the medulloblastoma not affected by necrosis, the RAGE receptor was present in the endothelium of all capillaries, and mast cells were numerous only in the perivascular space of large brain and meningeal vessels at the border of the tumor. In conclusion, our results show that the receptor of innate immunity TLR2 plays an important role in recognition of ligands delivered by dying necrotic medulloblastoma cells and participates in tumor neovascularization. Moreover, the results show that the RAGE receptor and mast cells operate in different medulloblastoma regions and influence different parts of the tumor vascular network.

Crosstalk in human brain between globoid cell leucodystrophy and zinc-α-2-glycoprotein (ZAG), a biomarker of lipid catabolism.

Zinc-alpha-2-glycoprotein (ZAG) is a protein identified as a lipid-mobilizing factor participating in a lipid catabolism. In spite of intensive studies conducted during last five decades, the role of this protein in processes of neurodegeneration remains unclear. The aim of our study was to examine the presence of ZAG protein in the brain of patients with Krabbe's disease, which is considered as a psychosine lipidosis caused by a mutation of a known gene. We found intracellular and extracellular localization of ZAG in the brain of Krabbe's disease patients but in the brain of control age-matched patients, ZAG was not detected. Distribution of ZAG in the brain suggests that the influx of ZAG into the brain involved a blood-brain barrier mechanism and adenoreceptors localized on astrocytes and some neurons.

Morphological evidence of the beneficial role of immune system cells in a rat model of surgical brain injury.

The blood-brain barrier prevents infiltration of peripheral immunocompetent cells into the CNS under physiological conditions. Following brain trauma there is reported a rapid and massive immunological response. Our earlier data indicated that surgical brain injury causes breaking of brain parenchyma integrity and results in cell changes and death, astrogliosis and disruption of blood vessels. The aim of the present studies was to investigate and characterize immunocompetent cells entering brain damaged parenchyma in the early period following the injury in a rat model of surgical damage. In the investigations we used light and electron microscopy techniques. Four days following the lesion many monocytes and macrophages were detected in the injured parenchyma. We also found many activated microglial cells with phagosomes within the cytoplasm. The phagocytes digest the cellular debris and clean up the parenchyma. The data suggest the beneficial role of immunocompetent cells following surgical injury.

Congenital glioblastoma coexisting with vascular developmental anomaly.

Congenital central nervous tumours form a unique group of neoplasms. They are different from other tumour groups not only due to the onset time but also to their histopathology, anatomic location, and biologic behaviour. Congenital glioblastoma is one of the rarest types of congenital brain tumours and is uncommon in the prenatal period. We report a rare case of congenital glioblastoma detected prenatally by ultrasound examination and magnetic resonance imaging at 26 gestational weeks. Based on MRI findings and consultation of a team of specialists, pregnancy was terminated at 28 weeks. The newborn presented hydrops foetal. The child died shortly after birth due to cardiorespiratory insufficiency. At autopsy a large tumour with a spongy-like appearance was found. The tumour involved nearly the whole right cerebral hemisphere and led to marked hydrocephalus. In the histological and immunohistochemical examination, the tumour presented features of glioblastoma. Neoplastic cells were immunopositive for GFAP, S-100 protein and negative for neuronal markers. Frequent mitoses and high MIB-1 labelling index were seen in the tumour areas. The coexistence of tumour and vascular developmental anomaly was stated. The conglomerates of numerous, distended, thin-walled foetal-like blood vessels were located beside the tumour tissue, which presented disturbance in differentiation and maturation of the vascular net. Such coexistence of malignant glioma with vascular developmental anomaly is unique.

Toll-like receptors as an innate immunity bridge to neuroinflammation in medulloblastoma.

The relationship between inflammation, immunity and cancer is widely accepted but mechanisms mediating this relationship remain unknown. Our present study was undertaken to examine the presence and distribution of Toll-like receptors (TLRs) in necrotic areas of medulloblastoma. These receptors fulfil the criteria postulated for the receptors of innate immunity and signalling from TLRs induces synthesis of various pro-inflammatory cytokines, enzymes and mediators. The study was performed on human medulloblastoma samples containing areas of necrosis within the tumour and/or within the normal nerve tissue at the periphery of the tumour. Proteins of four TLRs: TLR 2, 3, 4 and 9 were detected in the tissue with the immunohistochemical method using the specific antibodies. Two types of necrotic areas were found. In the first type, the area of dead cells was surrounded by undifferentiated medulloblastoma cells. A lot of these cells expressed TLR 2 and TLR 3 antigens. TLR 2 was also expressed on the wall of de novo formed blood vessels that fill tumour regions already cleared from dead cells. The second type of necrotic areas were found at the periphery of the tumour and composed of normal nerve tissue cells. TLR 2, TLR 3 and TLR 9 were detected in hypertrophic glia cells. Our findings show a new function of TLRs as sensors of pathogens released by medulloblastoma dead cells. This new function may provide a key link connecting innate immunity, neuroinflammation and angiogenesis in the tumour.

Immunodistribution of amyloid beta protein (Aß) and advanced glycation end-product receptors (RAGE) in choroid plexus and ependyma of resuscitated patients.

RAGE (receptor for advanced glycation end-products) participates in the influx transport of glycated Aß (amyloid beta) from the blood to the brain. Because little is known of the RAGE operating in brain barriers such as those in the choroid plexus and ependyma, the aim of the present study was to examine the immunodistributions of RAGE and Aß peptides in the choroid plexus where the blood-cerebrospinal fluid barrier (B-CSF) is located, and in ependyma of the brain ventricles associated with functions of the cerebrospinal fluid-brain barrier (CSF-B). The study was performed on patients over 65 years successfully resuscitated after cardiac arrest with survival a few weeks. The control group consisted of age-matched individuals who were not resuscitated and died immediately after cardiac arrest. In resuscitated patients, but not in controls, RAGE receptors were localized in choroid plexus (CP) epithelial cells and in ependymal cells bordering the brain ventricles. These cells form the B-CSF and CSF-B barriers. The presence of Aß was detected within the CP blood vessels and in the basement membrane of the CP epithelium. In numerous cytoplasmic vacuoles of CP epithelial and ependymal cells Aß protein was found and our observations suggest that the contents of those vacuoles were undergoing progressive digestion. The results demonstrated that CP epithelium and ependymal cells, equipped with RAGE receptors, not only play an important role in the creation of amyloid deposits in the brain but are also places where Aß may be utilized. The RAGE transportation system should be a main target in the therapy of brain amyloidosis, a well-known risk factor of Alzheimer disease.

Association of mast cells with calcification in the human pineal gland.

Increased pineal calcifications and decreased pineal melatonin biosynthesis, both age related, support the notion of a pineal bio-organic timing mechanism. The role of calcification in the pathogenesis of pineal gland dysfunction remains unknown but the available data document that calcification is an organized, regulated process, rather than a passive aging phenomenon. The cellular biology and micro-environmental conditions required for calcification remain poorly understood but most studies have demonstrated evidence that mast cells are strongly implicated in this process. The aim of the present study was to examine the phenotype of mast cells associated with early stages and with the progressive development of calcification in the human pineal gland. The study was performed on pineal samples of 170 fetuses and children whose brains were autopsied and diagnosed during 1998-2002. The representative cerebral and pineal specimens were stained with haematoxylin and eosin or the von Kossa staining technique and for the distribution of mast cell tryptase, mast cell chymase, histamine H4 receptor and vascular network using biotinylated Ulex europaeus agglutinin. Tryptase mast cells were found in all stages of pineal gland development independently of the presence of local tissue lesions. All of them were always localized in the close vicinity of the blood vessels and expressed immunoreactivity to histamine H4 receptor antibody. Immunolocalization of mast cells by chymase antibody (and following dual immunostaining with both chymase and tryptase antibodies) demonstrated that these cells were few in number and were located in the subcapsular region of the gland. In our study, all functional mast cells that underwent activation and were co-localized with deposits of calcium did not contain chymase. All of them were stained with tryptase and represent the MC-T phenotype. Tryptase mast cells and extracellular tryptase were often associated with areas of early and more advanced stages of calcification. Our results lead to the conclusion that the tryptase mast cells play a major role in the pineal calcification process as sites where this process starts and as a source of production of numerous biologically active substances including tryptase that participate in calcification.

Calbindin positive Purkinje cells in the pathology of human cerebellum occurring at the time of its development.

Development of cerebellum continues over an extremely long period of time extending from the early embryonic phase until the first postnatal years. During an extended time of maturation the cerebellum is vulnerable to harmful agents. A group of cytoplasmic proteins that may protect cells against injury are the calcium binding proteins, among others calbindin. The distribution of this protein is not well known in cerebellar pathology, thus in the present study the localisation and appearance of calbindin expressing Purkinje cells in different pathological conditions occurring at the time of cerebellar development was examined. The investigations were carried out on human maturing cerebellar cortex (age range 30 gestational weeks - 2 years) of cases with paraneoplastic cerebellar degeneration and cerebellar neuronal migration disturbances. The Purkinje cells located in cerebellar heterotopias and dysgenesias were morphologically well developed and strongly immunostained with calbindin antibody. In paraneoplastic cerebellar degeneration the progressive decrease of calbindin content and disintegration of Purkinje cells were observed. Our results show that intrauterine harmful agents that disturb migration of the cerebellar neurons do not affect the content of calbindin in misoriented neurons and that this protein may play a role in development of Purkinje cells located in heterotopias and cerebellar dysgenesias. The progressive decrease of calbindin content in the Purkinje cells undergoing degeneration and death during paraneoplastic changes in the cerebellum also supports the hypothesis that this protein is very important component of intracellular homeostasis in cerebellar neurons.

Astroglia and microglia in cerebellar neuronal migration disturbances.

Between the neuronal and glial cells there is a close relationship conditioning a tight morphological correlation and proper functional interplay. Disturbed interaction between glial and neuronal components leads to inappropriate neural circuits. The reflection of the failure of neural circuit organisation is the picture of morphological changes of neurons and glia. The appearance of microglia and astroglia was analysed in a defectively formed cellular network due to cerebellar neuronal migration disturbances. Focal disruption of neuron migration leads to their differentiation in an abnormal position manifested as heterotopias and cortical anomalies. Neurons that had lost their proper migratory way and heterotopically settled in the white matter were encircled by GFAP-positive astrocytes, with morphology appropriate for surrounding white matter. The microglial cells infiltrated the parenchyma within the heterotopic neurons playing a role in their elimination. In the cerebellar cortical malformations astrocytes were grouped near the Purkinje cells. In the minimal cortical dysplasia the increased number of astrocytes supported the neurons. Impaired morphological components of the glial-pial barrier were observed. In the massive cortical malformations a few degenerated astrocytes followed the disarranged Purkinje cells, while microglia and Bergmann glia fibres were not present. Absence of cells supporting and organizing the cerebellar cortex had an effect on loss of Purkinje cell shape, their disorientation and abnormal position. The appearance and localisation of the astroglia and microglia in the abnormal cerebellar circuitry due to migration disturbances is dependent on the pathomechanism of the anomalies.

Effects of ischaemia and hypoxia on the development of the nervous system in acardiac foetus.

The twin-reversed arterial perfusion (TRAP) sequence and development of an acardius are rare and severe complications in monozygotic twin pregnancy. Haemodynamic disturbances in placental perfusion via abnormal vascular anastomoses allow inter-twin transfusion to occur. Because of blood perfusion, one of the twins is poorly oxygenated and contains metabolic waste products. Retrograde placental perfusion leads to the formation of a non-viable malformed acardiac foetus. We studied the effects of haemodynamic disturbances in acardiac foetus on the development of the nervous system. The acardius was a product of a 32-weeks pregnancy. Caesarean section yielded a skin covered ovoid mass (size, 10 x 8 cm; weight, 220 g). The dissection of the acardiac twin showed a skin with hair and appendages, rudimentary lower limbs, vertebral column and brain mass. The rudimentary brain tissue was considerably disorganised structurally. We distinguished two main morphological forms of various appearances. In the centre, we observed a scarcely vascularised mass of tissue containing mature and immature neurones, glial cells and randomly distributed fibres. The mass of tissue appeared poorly differentiated, although there were some arrangements reminiscent of cerebral structures. Clusters of neurones provided a slight suggestion of nuclear or fibre structure. The cerebellar cortex was the only well recognisable structure. In the other fragment of the tissue, we found a slit cavity with ependymal outline and well-developed choroid plexus, which seemed to represent the 3rd ventricle. The scarcely vascularised disorganised tissue was surrounded by the highly vascularised one. It included many thin-walled sinusoid vessels. In some places, they were so concentrated that they resembled cavernous haemangioma. The spinal cord appeared comparatively well organised with a slightly dilated central canal. The morphological picture of the rudimentary brain tissue was similar to the picture of the cerebrovasculosa area. The effect of ischaemia in the presented case is the anomalous formation of the cerebral structures. The morphological features imply that the failure occurred after neurulation and before the prosencephalic began to grow. The failure of neural tube formation occurred on the 22nd-25th day of gestation. The malformed formation of the nervous system might be caused by impaired induction due to altered gene expression or to the interference of exogenous agents that interrupt normal development. The haemodynamic abnormal placental circulation, which induced lack of oxygen supply and nutritional deficiency, implies the morphological pattern of the anomaly.

Morphology of pineal glands in human foetuses and infants with brain lesions.

The pineal gland is an organ involved in regulation of homeostasis and body rhythms. It plays an important role in the growth foetuses and adaptation of newborns to new environmental conditions. The requirements of foetuses and newborns progressively change during development. The purpose of the study was to evaluate morphological changes of pineal glands in foetuses and infants with brain lesions. The results of our study showed that parenchyma of developing pineal glands was susceptible to injury in most autopsied foetal and infantile cases. Morphological changes in pineal glands were found in 90% of autopsied brains but 100% of the cases had infections. The lesions in the pineal included mainly haemorrhagic, necrotic and cystic changes. In our autopsied foetuses and children, morphological changes in pineal glands were concomitant with various lesions of brain parenchyma. All results of our study lead to the conclusion that the pineal gland during its development is very susceptible to injury. The failure of normal pineal gland development and subsequent impaired production of melatonin decrease resistance of newborns and children to various environmental harmful agents.

Proinflammatory cytokines in injured rat brain following perinatal asphyxia.

In contrast to astrogliosis, which is common to injuries of the adult CNS, in the developing brain this process is minimal. Reasons postulated for this include the relative immaturity of the immune system and the consequent insufficient production of cytokines to evoke astrogliosis. To explore this hypothesis, the study was undertaken to detect the presence of some proinflammatory cytokines in the injured rat brain following perinatal asphyxia (ischaemia/hypoxia). The localisation of TNF-alpha, IL-15, IL-17 and IL-17 receptors was visualised by means of immunohistochemistry. In numerous neurones of the rat brain, the IL-17 appeared to be constitutively expressed. In the early period of inflammation the IL-15 was produced mainly by the blood cells penetrating the injured brain but later it was synthesised also by reactive astrocytes surrounding brain cysts and forming dense astrogliosis around necrotic brain regions. The direct effect on astrogliosis of other estimated cytokines seems to be negligible. All the results lead to the conclusion that from all cytokines identified in the injured immature rat brain the IL-15 plays the most important role during inflammatory response and participates in the gliosis of reactive astrocytes.

Discrete glioneuronal malformative lesions in the foetal and infantile cerebral cortex.

Microdysgenesis is a term describing microscopic cortical cytoarchitectural abnormalities. Histologically this change shows an irregular glioneuronal tissue combination forming an abnormal structure of the cortex. The pathological features of this malformation are subtle and less well defined than other more distinctive cortical malformations. The clinical significance of these discrete glioneuronal malformations is controversial. Microscopic dysgenetic changes have been reported in cases with intractable epilepsy but similar changes may be seen in neurologically normal adults. The purpose of our study was the investigation of microdysgenetic lesions in the developing nervous system with regard to normal neuronal migration, differentiation and maturation. The post-mortem routine investigated foetal and infantile brains which were analysed histologically for the presence of discrete cortical malformations. A wide spectrum of cytoarchitectural glioneuronal malformations was found in the investigated material. We observed leptomeningeal glioneuronal heterotopias, subpial bands of heterotopic neurones, nests of ectopic neurones in the first cortical layer, neuronal and glial clusters, small foci with irregularity of laminar structure of the cortex. Microdysgenetic changes arose from an insult occurring in the later stages of cortical development and influencing the normal fate of neuroglial cells. Various types of focal morphological and cytoarchitectonial developmental abnormalities have been associated with behavioural and neuropsychological deficits in older infants.