Denislava Deleva

Serum ganglioside patterns in multiple sclerosis.

The relative distribution of gangliosides was determined in the serum of 37 patients with multiple sclerosis (MS) and of 30 healthy subjects. There was a significant increase of GM1 and GD1a, and a decrease of GM3 proportion in the serum of relapsing-remitting MS patients (RRMS) during their first MS attack. The RRMS patients in relapse with a long duration of the disease had a significant decrease of GM1 and an increase of GD1a portion in the serum. An increase of GD1a, one of the major brain neuron ganglioside fraction, suggested the neuron injury in the early and with a long duration RRMS. The finding of an increase of GM1, the main human myelin ganglioside, during the first MS attack in RRMS patients confirms previous evidence for the possible involvement of gangliosides in the early pathological course of demyelination in MS.

Developmental study of tripeptidyl peptidase I activity in the mouse central nervous system and peripheral organs

Tripeptidyl peptidase I (TPPI) — a lysosomal serine protease — is encoded by the CLN2 gene, mutations that cause late-infantile neuronal ceroid lipofuscinosis (LINCL) connected with profound neuronal loss, severe clinical symptoms and early death at puberty. Developmental studies of TPPI activity levels and distribution have been done in the human and rat central nervous systems (CNS) and visceral organs. Similar studies have not been performed in mouse. In this paper, we follow up on the developmental changes in the enzyme activity and localization pattern in the CNS and visceral organs of mouse over the main periods of life — embryonic, neonate, suckling, infantile, juvenile, adult and aged — using biochemical assays and enzyme histochemistry. In the studied peripheral organs (liver, kidney, spleen, pancreas and lung) TPPI is present at birth but further its pattern is not consistent in different organs over different life periods. TPPI activity starts to be expressed in the brain at the 10th embryonic day but in most neuronal types it appears at the early infantile period, increases during infancy, reaches high activity levels in the juvenile period and is highest in adult and aged animals. Thus, in mice TPPI activity becomes crucial for the neuronal functions later in development (juvenile period) than in humans and does not decrease with aging. These results are essential as a basis for comparison between normal and pathological TPPI patterns in mice. They can be valuable in view of the use of animal models for studying LINCL and other neurodegenerative disorders.

Axon reactions precede demyelination in experimental models of multiple sclerosis.

nating disease are currently known. Among these, multiple sclerosis is a widespread disease in Europe and the US. Despite the fact that the etiology of multiple sclerosis remains unknown, it has been demonstrated that an autoimmune mechanism plays an important role in the pathogenesis of demyelination [25]. Chronic recurrent experimental allergic encephalomyelitis (crEAE) is a valuable model for studies of the pathogenesis of multiple sclerosis in animals, as the clinical and pathological changes in this condition correspond to those seen in human patients with multiple sclerosis [4, 12, 13]. Multiple sclerosis is regarded as a primary demyelinating disease in the early stages of which the myelin sheath is damaged while the axon is preserved. However, data have been obtained indicating axon damage also occurs at the early stages of multiple sclerosis. On the other hand, it has been demonstrated that formation of the myelin sheath (myelination) involves various complex interactions between neurons and oligodendrocytes in the CNS, these interactions in the peripheral nervous system occurring between neurons and Schwann cells during several phases of myelinogenesis [7, 9, 11, 14, 18]. Recent data [18–20] show that during periods of active myelination, some of the myelin phospholipids are synthesized in neuron bodies. These are transported with the axon flow and only then enter the myelin sheath of the fiber [10]. We thus suggested that demyelination, which occurs in multiple sclerosis, is not the result of primary damage to the myelin sheath but is associated with damage to the neuron [21, 22]. It is therefore of interest to use electron microscopy to obtain detailed data on the structural responses of axons and glio-axonal interactions during demyelinating processes in experimental models of multiple sclerosis, which was the aim of the present work.

Ganglioside spinal cord changes in chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rats.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Lewis rats by inoculation with guinea-pig myelin and complete Freunds adjuvant followed by treatment with low-dose cyclosporin A. Rats were sacrified at different phases of the disease (just before the onset of clinical signs, during the first clinical episode of CREAE and during the first recovery). Gangliosides were extracted from the spinal cord, analysed after purification by two-dimensional chromatography and quantified densitometrically. An increase of GM1, the main rat myelin ganglioside, and a decrease of GT1b, suggested to play a role in mediating the interactions between oligodendroglia and axons, were observed during the development of the CREAE. These findings indicating significant ganglioside changes in CREAE give further support to the concept concerning the involvement of gangliosides in autoimmune demyelination.

Neurodegenerative changes in rat produced by lithium treatment.

Lithium is extensively used in psychiatric practice for the prevention and treatment of manic-depressive disorders. However, neurotoxicity attributed to lithium salts within therapeutic doses was also reported in patients, manifested by transient or persistent neurological deficits. In this study, morphological changes were examined in rats treated acutely and chronically with lithium. Pathological changes were observed in different brain regions including cerebral cortex, cerebellum, medulla oblongata, mesencephalon, thalamus, and pons, using a silver–copper impregnation technique for neurodegeneration. Vacuolization of brain tissue with subsequent formation of spongiosis was the prominent morphological feature following lithium administration. The zones of spongiosis were irregularly distributed throughout the brain. More intensive compact areas with spongiform changes were found in the cerebral cortex and medulla oblongata. Less pronounced vacuolization was noted in the pons and thalamic region. The cerebellum and mesencephalon appeared least affected. Vacuolization in the cerebellar cortex was found at loci with Purkinje cells, but the classical picture of spongiosis was not apparent. Data indicate that both acute and chronic lithium intoxication accelerated neurodegenerative changes normally seen with normal brain aging.

A Variant for Isolation of Serum Gangliosides

Abstract The serum gangliosides are of great diagnostic importance. A step-by-step procedure for their isolation from serum is described and it includes the following stages: a) dehydration of the sample; b) total lipids extraction; c) non-polar lipids removal by preparative TLC; d) elimination of the blood sugar by Sep-Pak technique; e) TLC of the ganglioside fractions. Azeotropic distillation of the mixture of serum water/n-propanol was used for sample dehydration. Triple extraction was carried out: with cyclohexane (I); chloroform : methanol = 1 : 1 (v/v) (II) and chloroform : methanol = 1 : 2 (v/v) (III). The extracts were combined and the non-polar lipids were removed. The eluate composition was checked out by HPTLC with a modified mobile phase: chloroform:methanol:0.1 M sodium lactate = 55 : 40 : 10 (v/v/v). Comparative data for the Rf values of the ganglioside fractions as well as recovery data are presented. This method is also applicable for milk samples.

A method for determination of lipid-bound sialic acid after chromatographic isolation of brain gangliosides

Abstract The lipid-bound sialic acid determination gives an indirect idea of the ganglioside composition. An attempt at adapting the ‘resorcinol method’ of Svennerholm using thin-layer chromatography is described in this article. An aliquot of a lipid extract from bovine brain was chromatographed and the ganglioside carrying area was visualised. The sorbent was scrapped and eluted. The sialic acid was hydrolysed with a mixture of resorcinol/hydrochloric acid and the color product formed was extracted with butanol/butylacetate. The color intensity was measured at 580 nm. It was found that the lipid-bound sialic acid in bovine brain was about 780 μg/g fresh tissue. The recovery data calculated by means of a standart solution were 97–102%. This method is particularly useful for some routine diagnostic studies.

Analysis of the effects of antibodies to gangliosides on the electrical activity of Retzius neurons in the leech and on the functional activity of influx sodium current channels.

The effects of anti-ganglioside antibodies on the functional states of two types of influx Na+ current channels were studied. Experiments used 20% anti-ganglioside antiserum prepared by standard methods by immunizing rabbits with total bovine brain gangliosides. These experiments showed that incubation of neurons in physiological saline containing antiserum induced discordance in the operation of the two types of influx current Na+ channels responsible for spike generation. This reaction was found to be associated with the slowed rate of activation of TTX-sensitive Na+ channels. Synaptic stimulation of cells in the presence of antiserum induced blockade of TTX-insensitive influx Na+ current channels. High-frequency synaptic activation of cells (10 Hz) showed that apart from blockade of TTX-insensitive Na+ channels, anti-ganglioside antibodies prevented plastic rearrangements in the gate system of TTX-sensitive Na+ channels. This resulted in impairment of the development of the acclimation process – the response of the neuron to high-frequency stimulation seen in normal conditions.

Antibodies against gangliosides GM1 of patients with multiple sclerosis alter the response of neurons to synaptic activation

Earlier, we showed in electrophysiological experiments performed with a demyelinated preparation (neural abdominal chain ganglion of the leech) that a 40-min incubation in the presence of rabbit antiganglioside serum caused significant changes in the electric activity of the Retzius neurons [3, 5]. We found that these changes resulted from the interaction between gangliosides contained in the Retzius neuron membrane and antibodies against gangliosides, leading to disturbance of the functional activity of Na-channels for incoming current, which are responsible for the generation of spikes by this cell [5]. As serum of patients with multiple sclerosis often has a high titer of antibodies against gangliosides GM1 [7, 8, 11–13], we assumed that this serum, as well as serum of immunized rabbits, which contains a mixture of four major gangliosides (GM1, GD1a, GD1b, and GT1b), may have a similar effect of neuronal electrogenesis.

Ganglioside Changes in Brain in Chronic Relapsing Experimental Allergic Encephalomyelitis Induced in the Lewis Rat

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Lewis rats by inoculation with guinea-pig myelin and complete Freunds adjuvant followed by treatment with low-dose cyclosporin A. Rats were sacrified at different phases of the disease (just before the onset of clinical signs, during the first clinical episode of CREAE and during the first recovery). Gangliosides were extracted from the brain, analysed after purification by HPTLC fractionation and quantified densitometrically. An increase of GM1, the main rat myelin ganglioside, and a decrease of GT1b, suggested to play a role in mediating the interactions between oligodendroglia and axons, were observed during the development of the CREAE. These findings indicating significant ganglioside changes in CREAE give further support to the concept concerning the involvement of gangliosides in autoimmune demyelination.