Sporadic amyotrophic lateral sclerosis: is SMN-Gemins protein complex of importance for the relative resistance of oculomotor nucleus motoneurons to degeneration?

Lower motoneurons (MNs) show varied vulnerability in amyotrophic lateral sclerosis (ALS): those of non-ocular brainstem nuclei and most of those of the spinal cord are highly vulnerable, while those of extraocular brainstem nuclei are quite resistant. Results of our former study on the immunoexpression of the survival of motor neuron protein (SMN) and Gemins 2-4 in cervical spinal cord anterior horn -MNs of sporadic ALS patients suggested that a relative deficit in Gemin2 may play some role in the pathomechanism of the disease. Here, we tested this idea further by comparing immunoexpression patterns of SMN and Gemins 2-8 between MNs of the oculomotor nucleus and -MNs of the cervical spinal cord anterior horns in autopsy material from sALS patients and controls. In the latter, no considerable difference in any studied protein was found between these structures except that SMN expression was slightly but significantly lower (p < 0.01) in the oculomotor MNs. In the sporadic ALS patients, the expression of SMN, Gemin4 and Gemin7 was significantly weaker (p < 0.05, p < 0.05 and p < 0.01, respectively), while that of Gemin8 was stronger (p < 0.001) in the MNs of the oculomotor nucleus than in the examined cervical spinal cord anterior horn -MNs. The immunoexpression of Gemin3 and Gemin6 in the spinal cord correlated strongly negatively with ALS duration (Spearman's correlation coefficient: RS = -0.84, p < 0.001, and RS = -0.86, p = 0.002, respectively). In the oculomotor nucleus MNs, no studied protein immunoexpression correlated significantly with ALS duration, but there was a tendency for such negative correlation for Gemin2 (RS = -0.56, p = 0.07). There was an apparent relative deficit of Gemin2 and Gemin8 in the spinal cord -MNs and of Gemins 2, 4 and 7 in the oculomotor nucleus MNs. These data do not support the hypothesis that the diverse ALS vulnerability of the two MN subsets is related to their disparate expression patterns of SMN and Gemins 2-8. The differences in these patterns may result from ALS-related epiphenomena, or from intrinsic differences in the structure and function between the MN subsets, or both.

Nanofiber mat spinal cord dressing-released glutamate impairs blood-spinal cord barrier.

An excessive glutamate level can result in excitotoxic damage and death of central nervous system (CNS) cells, and is involved in the pathogenesis of many CNS diseases. It may also be related to a failure of the blood-spinal cord barrier (BSCB). This study was aimed at examining the effects of extended administration of monosodium glutamate on the BSCB and spinal cord cells in adult male Wistar rats. The glutamate was delivered by subarachnoidal application of glutamate-carrying electrospun nanofiber mat dressing at the lumbar enlargement level. Half of the rats with the glutamate-loaded mat application were treated systemically with the histone deacetylase inhibitor valproic acid. A group of intact rats and a rat group with subarachnoidal application of an 'empty' (i.e., carrying no glutamate) nanofiber mat dressing served as controls. All the rats were euthanized three weeks later and lumbar fragments of their spinal cords were harvested for histological, immunohistochemical and ultrastructural studies. The samples from controls revealed normal parenchyma and BSCB morphology, whereas those from rats with the glutamate-loaded nanofiber mat dressing showed many intraparenchymal microhemorrhages of variable sizes. The capillaries in the vicinity of the glutamate-carrying dressing (in the meninges and white matter alike) were edematous and leaky, and their endothelial cells showed degenerative changes: extensive swelling, enhanced vacuo-lization and the presence of vascular intraluminal projections. However, endothelial tight junctions were generally well preserved. Some endothelial cells were dying by necrosis or apoptosis. The adjacent parenchyma showed astrogliosis with astrocytic hypertrophy and swelling of perivascular astrocytic feet. Neurons in the parenchyma revealed multiple symptoms of degeneration, including, inter alia, perikaryal, dendritic and axonal swelling, and destruction of organelles. All the damage symptoms were slightly less severe in the rats given valproic acid treatment, and were absent from both the intact rats and the rats with 'empty' nanofiber mat dressing. These results demonstrate that glutamate-loaded nanofiber mat dressing can locally create glutamate levels capable of damaging BSCB and that the resulting damage can be mitigated with concurrent systemic valproate treatment.

Protective effect of valproic acid on cultured motor neurons under glutamate excitotoxic conditions. Ultrastructural study.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that involves the upper and lower motor neurons and leads to the patient's death within 5 years after diagnosis. Approximately 2 per 100,000 people worldwide are affected every year. The only FDA-approved drug available for medical treatment is riluzole. It slows the disease progression and improves limb function and muscle strength for 3-4 months. Thus, looking for new therapeutic agents is a pressing challenge. Valproic acid (VPA) is a short-chain fatty acid, widely used for the treatment of seizures and bipolar mood disorder. The beneficial effect of VPA has been documented in different neurodegenerative experimental models, including amyotrophic lateral sclerosis (ALS). The real mechanisms underlying numerous beneficial effects of VPA are complex, but recently it has been postulated that the neuroprotective properties might be related to direct inhibition of histone deacetylase (HDAC). The aim of this ultrastructural study was to evaluate the beneficial effect of VPA on the spinal cord motor neurons (MNs) in a glutamate (GLU)-induced excitotoxic ALS model in vitro. It had been previously documented that chronic GLU excitotoxicity resulted in various MN injuries, including necrotic, apoptotic and autophagic modes of cell death. The present results demonstrated the neuroprotective properties of VPA associated with inhibition of apoptotic and autophagic changes of spinal MNs in a model of neurodegeneration in vitro.

NF-κB deficit in spinal motoneurons in patients with sporadic amyotrophic lateral sclerosis--a pilot study.

Amyotrophic lateral sclerosis (ALS) is a fatal incurable neurodegenerative disease whose etiology is unknown and pathogenesis is still not fully understood. A great majority of its cases are sporadic. Clinical ALS signs are caused by damage and dying-out of the lower and upper motor neurons. This study was aimed at identifying possible sporadic ALS-associated aberrations in the spinal cord expression of the transcription nuclear factor κ light-chain-enhancer of activated B cells (NF-κB). NF-κB is widely distributed among various cell types, including those specific for the central nervous system (CNS), and is involved in the control of many physiological and pathological processes, including, inter alia, inflammatory response, proliferation, angiogenesis, and cell survival and death. It is constitutively expressed and its inactive form resides in the cytoplasm. After activation, it enters the cell nucleus and promotes the transcription of target genes. NF-κB is a dimer and its most common form is a heterodimer made of subunits p50 and p65. In this study, we estimated and compared by immunohistochemical means the contents of these subunits in spinal cord motoneurons in a few archival cases of sporadic ALS of varying disease duration and the respective age-matched control cases with no CNS pathology. The major goal of the study was to seek possible changes in the expression of these proteins in the course of the disease. The control cases showed a strong expression of both p50 and p65 in spinal cord motoneurons, with both cytoplasmic and nuclear localization. In contrast, the ALS cases studies revealed a considerably lower and varying intensity of specific immunohistochemical staining for the two subunits, which suggested an increased deficit of their expression linked to longer disease duration. Moreover, there was an apparent shift toward mostly cytoplasmic localization of the two subunits. These preliminary data suggest that the changes in the expression of theses NF-κB subunits may be involved in pathogenesis of sporadic ALS.

Diverse expression of selected SMN complex proteins in humans with sporadic amyotrophic lateral sclerosis and in a transgenic rat model of familial form of the disease.

BACKGROUND AND OBJECTIVE: There is circumstantial evidence linking sporadic amyotrophic lateral sclerosis (ALS) cases to a malfunction or deficit of a multimeric SMN complex that scrutinizes cellular RNAs; the core of this complex is survival motor neuron (SMN, or gemin 1) protein. We intended to verify this hypothesis by comparing the expression of both SMN and several other functionally associated gemins in the anterior horn motoneurons of patients who died of sporadic ALS (sALS), of transgenic rats with overexpression of the mutated human superoxide dismutase 1 gene (SOD1(G93A)) that represent a model of familial ALS (fALS), and of the respective controls. METHODS: Using archival material of paraffin blocks with samples of human and rat spinal cords, immunohistochemical reactions with antibodies against SMN and gemins 2, 3, and 4 were performed and assessed by light microscopy. RESULTS: The expression of SMN and all other studied gemins was observed in motoneurons of sALS patients, fALS rats, and in all controls, although the intensity varied. The immunolabeling was most intense in sALS patients with relatively fast disease course, and decreased with increasing disease duration in both the human sALS and rat fALS material. Irrespective of the disease stage, sALS material showed no or very low gemin 2 immunoreactivity, while clear gemin 2 immunoreactivity was observed in all fALS rats and control material. CONCLUSION: The deficient expression of gemin 2 in spinal cord motoneurons in human sALS may lead to a dysfunction and loss of neuroprotective action of the SMN complex.

Effects of liposomes with polyisoprenoids, potential drug carriers, on the cardiovascular and excretory system in rats.

BACKGROUND: The unpredictable side effects of a majority currently used drugs are the substantial issue, in which patients and physicians are forced to deal with. Augmenting the therapeutic efficacy of drugs may prove more fruitful than searching for the new ones. Since recent studies show that new cationic derivatives of polyisoprenoid alcohols (APrens) might exhibit augmenting properties, we intend to use them as a component of liposomal drug carriers. In this study we investigate if these compounds do not per se cause untoward effects on the living organism. METHODS: Male Sprague-Dawley rats received for four weeks daily injections (0.5 ml sc) of liposomes built of dioleoyl phosphatidylethanolamine (DOPE), liposomes built of DOPE and APren-7 (ratio 10:1) or water solvent. Weekly, rats were observed in metabolic cages (24h); blood and urine were sampled for analysis; body weight (BW) and systolic blood pressure (SBP) were determined. After chronic experiment, kidneys and heart were harvested for histological and morphometric analysis. RESULTS: The 4-week BW increments were in the range of 97 ± 4 to 102 ± 4%, intergroup differences were not significant. Microalbuminuria was the lowest in the group receiving liposomes with APren-7 (0.22 ± 0.03 mg/day). Water and food intake, plasma and urine parameters were similar in all groups. CONCLUSIONS: Newly designed liposomes containing APren-7 did not affect functions of the excretory and cardiovascular systems, and renal morphology; therefore we find them suitable as a component of liposomal drug carriers.

Non-woven nanofiber mats - a new perspective for experimental studies of the central nervous system?

(Sub)chronic local drug application is clearly superior to systemic administration, but may be associated with substantial obstacles, particularly regarding the applications to highly sensitive central nervous system (CNS) structures that are shielded from the outer environment by the blood-brain barrier. Violation of the integrity of the barrier and CNS tissues by a permanently implanted probe or cannula meant for prolonged administration of drugs into specific CNS structures can be a severe confounding factor because of the resulting inflammatory reactions. In this study, we tested the utility of a novel way for (sub)chronic local delivery of highly active (i.e., used in very low amounts) drugs to the rat spinal cord employing a non-woven nanofiber mat dressing. To this end, we compared the morphology and motoneuron ( + ) counts in spinal cord cervical and lumbar segments between rats with glutamate-loaded nanofiber mats applied to the lumbar enlargement and rats with analogical implants carrying no glutamate. Half of the rats with glutamate-loaded implants were given daily valproate treatment to test its potential for counteracting the detrimental effects of glutamate excess. The mats were prepared in-house by electrospinning of an emulsion made of a solution of the biocompatible and biodegradable poly(L-lactide-co-caprolactone) polymer in a mixture of organic solvents, an aqueous phase with or without monosodium glutamate, and sodium dodecyl sulfate as an emulsifier; the final glutamate content was 1.4 µg/mg of the mat. Three weeks after mat implantation there was no inflammation or considerable damage of the spinal cord motoneuron population in the rats with the subarachnoid dressing of a glutamate-free mat, whereas the spinal cords of the rats with glutamate-loaded nanofiber mats showed clear symptoms of excitotoxic damage and a substantial increase in dying/damaged motoneuron numbers in both segments studied. The rats given systemic valproate treatment showed significantly lower percentages of damaged/dying motoneurons in their lumbar enlargements. These results demonstrate the capacity of nanofiber mats for generation of neurotoxic glutamate in the rat CNS. However, the tested nanofiber mats need further improvements aimed at extending the period of effective drug release and rendering the release more steady.

Blood vessel ultrastructural picture in a CADASIL patient diagnosed at an advanced age.

We report the case of an 84-year-old male patient afflicted by cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) showing minimal symptoms of disease. The patient was diagnosed on the basis of ultrastructural and genetic examinations. Ultrastructurally, a typical vascular pathology was found. However, in abnormal capillary vessel walls no granular osmiophilic material (GOM) was found. In the arteriole there were only a few GOM deposits that revealed various structures, of which only some resembled typical round GOM. The arteriolar walls showed severe damage, including fragmentation, degeneration and loss of vascular smooth muscle cells (VSMCs) with numerous deposits of elastin, mucosubstances, different granular debris, as well as collagen fibres in the basement membrane. Lysosomal inclusions with fingerprint morphology, atypical for CADASIL, were located in some of the VSMCs. Very old age at the onset of the disease may suggest that morphological changes in blood vessels, described in this report, may be due to both the disease and the patient's age. To our best knowledge it is the first description of pathology of blood vessels and GOM morphology in a CADASIL patient diagnosed at an advanced age.

Enlargement of the Nissl substance as a manifestation of early damage to spinal cord motoneurons in amyotrophic lateral sclerosis.

AIMS: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motoneurons. Recent studies indicate that in ALS, degeneration of motoneuron body is late in comparison to degeneration of axons. The morphological consequence of the axonal damage is chromatolysis. Therefore, loss of tigroid in motoneurons as a morphological manifestation of chromatolysis should be a prominent feature seen in an early stage of the disease. To verify that assumption we examined morphologically spinal cord motoneurons in patients with sporadic ALS. MATERIAL AND METHODS: In anterior horn motoneurons of 33 patients tigroid were assessed at light microscopy and morphometrically analyzed. Material was divided into an "acute" ALS group with a duration of the disease of up to 1 year, and a "chronic" ALS group with a clinical course lasting for 4 - 9 years. RESULTS: In the "acute" ALS group, loss of motoneurons was slight, and only a part of them showed central chromatolysis. Instead of chromatolysis the enlargement of the tigroid was found. This phenomenon was observed only in "acute" ALS and confirmed by morphometric analysis. CONCLUSIONS: In ALS, enlargement of the tigroid seems to be an early morphological feature - occuring earlier than central chromatolysis. Its presence may be connected with endoplasmic reticulum stress, disturbed axonal transport or functional compensation of the neuronal deficit.

Immunoexpression of gemins 2 and 4 in the rat spinal cord. Is the SMN complex a new target in investigations of sporadic amyotrophic lateral sclerosis pathogenesis?

Sporadic amyotrophic lateral sclerosis (sALS) is a neurodegenerative disease leading to degeneration and loss of motoneurons in different structures of the nervous system. Although aetiology of the disease is unknown, it is hypothesized that the survival motor neuron (SMN) protein which protects motoneurons in spinal muscular atrophy, may play a similar role in ALS. Relatively little is known about normal expression and functions of the SMN complex compounds, i.e. SMN protein and the related gemins. Therefore, we have decided to examine the physiological expression of SMN and gemins 2 and 4 in spinal cords of healthy Wistar rats at different age using immunofluorescence and immunohistochemical methods. Our study revealed that (1) in rat spinal cord neurons, the immunoexpression of SMN and gemins 2 and 4 is present through the whole animal lifespan although the reactive cells reveal different intensity of the immunolabeling, (2) both SMN and gemin 2, and SMN and gemin 4 are present in the same motoneurons, (3) immunoexpression of gemin 2 and 4 decreases slightly with aging.

Survival motor neuron: motor neuron insurance for a whole lifespan?

The SMN (survival motor neuron) gene plays an important role in ontogenesis and its dysfunction leads to immatu-rity of skeletal muscles and motor neurons in the spinal cord. As a result of SMN mutations the affected cells die and clinical symptoms of spinal muscular atrophy (SMA) develop. Physiologically, SMN together with gemins is part of a multiprotein complex of particular importance to motor neuron development. Since the SMN gene is necessary for normal motor neuron maturity, a question arises whether its expression is preserved in postnatal life or finishes with the end of ontogenesis. To answer this question we examined expression of SMN and gemins 2, 3 and 4 in spinal cords of Wistar rats at age 1-350 days using immunofluorescence and immunohistochemical methods. In the examined animals expression of SMN appeared in neurons in 20-day old rats and increased with animal age. In rats aged 30-350 days SMN immunoreactivity was similar in all the examined animals. The same phenomenon was observed in assessment of gemin expression. Our study revealed that in rat spinal cord expression of SMN and gemins 2, 3 and 4 is present through a whole animal lifespan and not only in motor but also in sensory and autonomic neurons.

Inflammatory cerebral amyloid angiopathy: the overlap of perivascular (PAN-like) with vasculitic (Aß-related angiitis) form: an autopsy case.

Beside advanced age, cerebral amyloid angiopathy (CAA) and hypertension (HTA) are the two most important risk factors for haemorrhagic stroke. Inflammatory changes of amyloid-laden vessels have been reported only in rare sporadic CAA cases. We present the case of a 78-year-old woman with a history of hypertension, dementia and haemorrhagic stroke of the right frontal lobe 2 years before admission. She was admitted with recurrence of symptoms of transient aphasia and central, right-side facial paresis that occurred an hour before her arrival at the hospital. In the admission unit, she was only slightly confused, with no other neurological deficits. An urgent CT scan revealed a recent haemorrhagic stroke in the left frontal lobe. In an hour her condition suddenly deteriorated. After a generalized seizure she presented with right-side hemiparesis with signs of uncal herniation and remained unconscious. A control CT scan showed a large haemorrhagic expansion that comprised the whole left brain hemisphere with 2 cm midline shift. She died about 10 hours after the onset of symptoms. At autopsy chronic inflammation of the thyroid gland, bronchopneumonia, fibrous and fatty heart degeneration and kidney haemorrhagic infarcts were documented. Amyloid deposition and systemic immune disorders in the inner organs were not demonstrated. In neuropathological examination we diagnosed inflammatory form of CAA with coexistence (the overlap) of two, perivascular and vascular, subtypes of CAA-related inflammation.

Capillary vessel wall in CADASIL angiopathy.

The study was aimed at investigating the morphology of capillaries in four skin and muscle biopsy specimens obtained from CADASIL patients. In all cases diagnosis confirmed at the ultrastructural level, and additionally in three cases, the genetic test revealed the Notch3 gene mutations. Using histological and immunohistochemical (IHC) markers for components of capillary vessel wall we showed the reduction and loss of pericytes and and fibrous vessel wall including the thickened basement membrane. The thorough ultrastructural study revealed the presence of widespread GOM deposits in capillary wall, but less numerous than in arterioles. They were located in the vicinity of pericytes and also in pericyte infolding like vascular smooth muscle cells (VSMC) in arterioles. Sometimes GOM deposits were observed near endothelial cells. The endothelial cells, damaged but not lost, were also observed while most of capillaries revealed only residual pericytes. The destruction and loss of pericytes in capillary wall, like those of VSMC in arteriole wall, was the main vascular pathology in our the examined cases consistent to that pericytes functionally correspondent to VSMC. The Notch3 receptor is expressed on VSMC and pericytes, the results of our study confirm that in capillaries devoid of VSMC, pericytes are the primary morphological target of the Notch3 gene mutation. It should be indicated that in diagnostic ultrastructural examinations of skin and/or skeletal muscle biopsies, not only arterioles but also capillaries, occurring in a larger amount, should be thoroughly analysed, because such an approach may facilitate the detection of GOM deposits - the pathognomonic feature of CADASIL.

Neuroprotective effect of erythropoietin in amyotrophic lateral sclerosis (ALS) model in vitro. Ultrastructural study.

Erythropoietin (EPO) is a chemokine hormone that is widely distributed throughout the body including nervous system. For last years its role as cytokine involved in many physiological processes out of the bone marrow has been suggested. Moreover, it plays a very important role in CNS as potential neuroprotective agent. There is much evidence that EPO protects neuronal cells in vitro and in vivo models of brain injury, independently of its erythropoietic action. The aim of this study was to determine the potential neuroprotective effects of erythropoietin on the glutamate-mediated injury of motor neurons (MNs) in vitro. The study was performed on organotypic cultures of the rat lumbar spinal cord subjected to glutamate uptake blocker, DL-threo-beta-hydroxyaspartate (THA) and pretreated with EPO. Ultrastructural study evidenced that the spinal cord cultures pretreated with EPO exhibited less severe neuronal injury. The cultures exposed to EPO + THA showed inhibition of early MNs degeneration, including various mode of degenerative changes caused by THA, whereas in the later period the typical postsynaptic necrotic changes of neuronal cells occurred. However, the ultrastructural characteristics of apoptotic MNs changes were not observed during the whole period of observation. The results of this study indicate that, in the model of chronic glutamate excitotoxicity, EPO exhibits the neuroprotective ability mainly through prevention of apoptotic neuronal changes.

Is the spinal cord motoneuron exclusively a target in ALS? Comparison between astroglial reactivity in a rat model of familial ALS and in human sporadic ALS cases.

OBJECTIVE: Motoneurons are the focus of most investigations of amyotrophic lateral sclerosis (ALS), while the astrocyte reaction is regarded as a phenomenon secondary to neuron degeneration. Since astroglial reactivity differed in different studies of human and animal ALS models and often varied from case to case, we examined and compared astrocyte reactivity within the anterior horns of the spinal cord in a transgenic rat model of familial ALS and in human sporadic ALS (sALS) cases. METHODS: Routine histological staining and immunohistochemical reactions to cytoskeletal proteins [neurofilaments, glial fibrillary acidic protein (GFAP), vimentin and tau] and proliferative markers (proliferating cell nuclear antigen and Ki-67). RESULTS: In human sALS cases and in rats at the early pre-symptomatic and symptomatic stages of the disease, the astroglial reaction was very weak, although there was visible evidence of the morphological manifestations of motoneuron degeneration. Poor immunoreactivity to the GFAP and vimentin antigens and increased expression of tau protein were observed in astrocytes, particularly in the rat model. The astrocyte reaction was evident during a short 'transient' phase of the disease in animals, between the asymptomatic and paretic stages. Proliferating cell nuclear antigen immunoreactivity in glial and neuronal nuclei was observed only in animal material. CONCLUSIONS: Abnormalities in astrocyte cytoskeletal proteins are characteristic features for ALS, both in acquired and congenital forms of the disease. The cytoskeletal aberrations may lead to astroglial dysfunction and disturbances in glutamate uptake that may in turn increase the degeneration of motoneurons.

Unexpected morphological changes within hippocampal structures in a photochemical ring model of cerebral ischaemia.

A photochemical ring model of ischaemia was introduced in the middle of the nineteen eighties. Irradiation by a laser or arc lamp followed by intravenous injection of rose bengal resulted in thrombosis of pial and superficial cortical vessels. This ring model imitated focal ischaemic damage in humans. In our experiment twenty-seven Wistar rats of both sexes weighing 250-300 grams were examined. A photochemical ring model based on irradiation of the area of parietal bone 4 mm posteriorly to the bregma and 4 mm laterally from the sagittal suture was applied. A ring-shaped light beam with a wavelength of 510-540 nm with 5 mm diameter was generated by a high pressure discharge lamp at a power of 400 W. Two groups of rats treated and untreated with MK-801 and two rings of the thickness of 0.35 mm and 0.5 mm were used in the experiment. Morphological examination was performed in animals sacrificed 1 and 4 days after the irradiation. On formalin-fixed and paraffin-embedded slices HE staining method and immunoreaction with antibodies to ubiquitin were applied. Our material confirmed well known information about the dynamics of infarct breakdown, ischaemic-induced angiogenesis, glial reaction and other typical changes described previously in handbooks and numerous papers. In the experiment, morphological changes were more intensive after the irradiation by 0.5 mm than 0.35 mm irradiating rings and 4 days than one day after the irradiation. A surprising finding observed in some of the examined animals was more intensive neuronal damage after treatment with MK-801. Another unpredicted discovery was intensive morphological alterations found in CA4 and CA3 hippocampal sectors. Moreover, these alterations were not limited to the damaged hemisphere, but were also observed contralaterally. In some of the rats, ischaemic and necrotic cells were additionally found within both parasagittal areas. We connect this atypical localization of the ischaemic changes with dispersion of light emitted by the used lamp. Dispersed light also leads to thrombotic occlusion of the meningeal arteries in the parasagittal area. Among these arteries, thrombosis in pericallosal and penetrating arteries was present. Our experiment demonstrated that if a non-laser lamp is used, brain areas distant from the necrotic ring must be carefully investigated.

CDP-choline protects motor neurons against apoptotic changes in a model of chronic glutamate excitotoxicity in vitro.

Cytidine-5-diphosphocholine (CDP-choline, citicoline) is an endogenous nucleoside involved in generation of phospholipids, membrane formation and its repair. It demonstrates beneficial effects in certain central nervous system injury models, including cerebral ischaemia, neurodegenerative disorders and spinal cord injury. Defective neuronal and/or glial glutamate transport is claimed to contribute to progressive loss of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Our previous ultrastructural studies, performed on an organotypic tissue culture model of chronic glutamate excitotoxicity, documented a subset of various modes of MN death including necrotic, apoptotic and autophagocytic cell injury. The aim of this ultrastructural study was to determine the potential neuroprotective effect of CDP-choline on neuronal changes in a glutamate excitotoxic ALS model in vitro. Organotypic cultures of the rat lumbar spinal cord subjected to 100 microM DL-threo-beta-hydroxyaspartate (THA) were pretreated with 100 microM of CDP-choline. The exposure of spinal cord cultures to CDP-choline and THA distinctly reduced the development of typical apoptotic changes, whereas both necrotic and autophagocytic THA-induced MN injury occurred. These results indicate that CDP-choline treatment might exert a neuroprotective effect against neuronal apoptotic changes in a model of chronic excitotoxicity in vitro.

Clinical, biochemical, neuropathological and molecular findings of the first Polish case of adenylosuccinase deficiency.

Adenylosuccinase (ADSL) deficiency is an autosomal recessive disorder affecting mainly the nervous system. The disease causes psychomotor retardation, frequently with autistic features and epilepsy. ADSL deficiency may be diagnosed by detection of two abnormal metabolites in body fluids--succinyladenosine (S-Ado) and succinylaminoimidazole carboxamide riboside (SAICAr). It is assumed that the former metabolite is neurotoxic. We present clinical, biochemical and neuropathological findings of a child affected by a severe form of ADSL deficiency. She had progressive neurological symptoms that started immediately after birth and died at 2.5 months of age. Macroscopically the brain showed signs of moderate atrophy. Histological examination of all grey matter structures showed widespread damage of neurons accompanied by microspongiosis of neuropile. Cerebral white matter showed lack of myelination in the centrum semiovale and diffuse spongiosis of neuropile. Myelination appropriate for the age was visible in posterior limb of internal capsule, in striatum, thalamus and in brain stem structures but diffuse destruction of myelin sheets was seen with severe marked astroglial reaction with signs of destruction of the cells and their processes. Ultrastructural examination showed enormous destruction of all cellular elements, but astonishingly mitochondria were relatively spared. The neuropathological changes can be considered as the neurotoxic result of metabolic disturbances connected with adenylosuccinase deficiency.

Morphological changes in the brain during experimental hyponatraemia. Do vasopressin and gender matter?

Hyponatraemia is the most common electrolyte balance disorder occurring in hospitalized patients. The disease results frequently from inappropriate secretion of vasopressin (SIADH). It has been evidenced that the brain consequences of hyponatraemia are more dramatic in young females than in men or postmenopausal women. Since both vasopressin and oestrogen have been reported to inhibit ion fluxes essential for the adaptation of the brain to the lowering of serum sodium concentration, we sought to study the effect of acute and chronic hyponatraemia or hyponatraemia associated with vasopressin on brain morphology in male and female rats. Hyponatraemia was induced with vasopressin (AVP) or with desmopressin (dDAVP) in 12 male and 12 female adult Wistar rats for either 3 hours (acute) or 3.5 days (chronic). The brains of the animals with diagnosed hyponatraemia were fixed in 10% formalin and, following the standard procedure, stained with haematoxylin and eosin. Acute hyponatraemia resulted in white matter oedema with no obvious differences between genders or between groups with AVP- or dDAVP-induced hyponatraemia. Although in chronic hyponatraemia most neurons and astrocytic nuclei appeared to be normal, some neurons were swollen or ischaemic ("dark" neurons) and astrocytes showed a weak reaction. The most spectacular differences between males and females were found in the appearance of blood vessels. Swollen endothelial cells were observed more frequently in female than in male brains and in AVP- than in dDAVP-induced hyponatraemia. The widened Virchow-Robin spaces indicated perivascular oedema and blood-brain barrier damage. The results point to limited vascular adaptation to AVP-associated hyponatraemia in female gender.

Small cerebral vessel disease in familial amyloid and non-amyloid angiopathies: FAD-PS-1 (P117L) mutation and CADASIL. Immunohistochemical and ultrastructural studies.

Three patients (of two unrelated Polish families) with early-adult onset dementia were subjects of the study. Two cases, previously diagnosed as familial Alzheimer's disease (FAD) with cerebral amyloid angiopathy (CAA), were confirmed by genetic and neuropathological studies, and one case of CADASIL was ultrastructurally confirmed by the presence of vascular granular osmiophilic material. Now the brain autopsy material has been reinvestigated using immunohistochemical (IHC) markers for vascular smooth muscle cells, paying special attention to collagen markers for extracellular matrix components and ultrastructural microvascular changes. In both diseases, IHC examination showed a reduction or loss of expression of smooth muscle actin (SMA) in tunica media of the cerebral arterioles. Fibrous thickening of the wall of the small meningeal arteries, intracerebral arterioles and numerous capillaries, with amyloid or granular deposits, drew our attention. In these vessels, marked expression of fibrillar collagen type III as well as strong immunoreactivity of the basement membrane (BM) component collagen type IV were found. The most damage was observed in the FAD/CAA double-barrel vessel wall and in some CADASIL arterioles changed by fibrinoid necrosis. The fibrous changes of the small vessels were more distinct in CADASIL t han in FAD/CAA. In FAD,electronmicroscopic examination revealed both amyloid and collagen fibres within the thickened BM of capillaries and the small arterioles. Clusters of collagen fibres between lamellae of BM, frequently in a pericyte position,were observed,and some were seen in the degenerated pericytes as well. Typical changes of the pericytes were accumulation of lipofuscin-like material and their degeneration. The mitochondria of the pericytes and of the endothelium were rare and swollen, with damaged and reduced cristae. The VSMCs of the arteriolar walls exhibited degenerative changes with atrophy of the cellular organelles. The fibrous,collagen-richCADASILsmallcerebralvessels,despite the weakness of the vessel wall due to reduction of VSMCs, appeared to be stronger than in FAD/CAA. These findings may suggest an accelerated process of transformation of the small cerebral vessels in which early onset of VSMCs loss is a predominant feature of the vascular changes in both presented diseases.