primary-cultures-rat-astrocytes-microglia-their-use-study-amyotrophic

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

University of Belgrade Faculty of Biology

Magnetic resonance imaging (MRI) is the only noninvasive technique that provides structural information on both cell loss and metabolic changes. After reviewing all the results obtained in clinical studies, reliable biomarkers in neurological diseases are still lacking. Diffusional MRI, MR spectroscopy, and the assessment of regional atrophy are promising approaches, but they cannot be simultaneously used on a single patient. Thus, for further research progress, reliable animal models are needed. To this aim, we have used the clinical MRI to assess neurodegenerative processes in the hSOD-1(G93A) ALS rat model and in the trimethyltin (TMT)-treated model of Alzheimer's-like disease. T2-weighted (T2W) hyperintensive neurodegenerative foci were found in the brainstem of the ALS rat with apparent lateral ventricle dilation (T1W-hypointensity vs. T2W-hyperintensity). Degenerative processes in these areas were also confirmed by confocal images of GFAP-positive astrogliosis. MRI after i.v.i. of magnetic anti-CD4 antibodies indicated an accumulation of inflammatory cells near dilated ventricles. TMT-treated rats also revealed the dilation of lateral ventricles. Expected deterioration in the hippocampus was not observed by clinical MRI, but immunocytochemistry could reveal significant redistribution of macro- and microglia in this structure. In both models, Gd-DTPA contrast revealed a compromised blood brain barrier that may serve as the passage for inflammatory immune cells in the vicinity of dilated lateral ventricles. Moreover, in both models the midbrain region of the dorsal hippocampus was the target of BBB compromise, thus revealing a potentially vulnerable point that can be the primary target of neurodegeneration in the central nervous system.

In vivo morphological changes in animal models of amyotrophic lateral sclerosis and Alzheimer's-like disease: MRI approach.

L&#39;imagerie par r&eacute;sonance magn&eacute;tique (IRM) est la technique seulement non invasive qui fournit des informations structurelles sur la perte de cellules et des changements m&eacute;taboliques. Apr&egrave;s avoir examin&eacute; tous les r&eacute;sultats obtenus dans les &eacute;tudes cliniques, les biomarqueurs fiables dans les maladies neurologiques manquent encore. Par diffusion MRI, M. spectroscopie et l&#39;&eacute;valuation de l&#39;atrophie r&eacute;gionale sont des approches prometteuses, mais ils ne peuvent pas &ecirc;tre utilis&eacute;s simultan&eacute;ment sur un seul patient. Ainsi, &agrave; de nouveaux progr&egrave;s de recherche, des mod&egrave;les animaux fiables sont n&eacute;cessaires. Dans ce but, nous avons utilis&eacute; l&#39;IRM clinique pour &eacute;valuer des processus neurod&eacute;g&eacute;n&eacute;ratifs dans le mod&egrave;le de rat de hSOD-1(G93A) ALS et dans le trim&eacute;thyl&eacute;tain (TMT) - mod&egrave;le trait&eacute; d&#39;Alzheimer - comme la maladie. Pond&eacute;r&eacute;es en T2 (T2W) hyperintensive neurod&eacute;g&eacute;n&eacute;ratives foyers ont &eacute;t&eacute; trouv&eacute;s dans le tronc c&eacute;r&eacute;bral du rat ALS avec dilatation des ventricules lat&eacute;raux apparents (T1W-hypointensity vs T2W-hypersignal). Les processus d&eacute;g&eacute;n&eacute;ratifs dans ces domaines ont &eacute;galement &eacute;t&eacute; confirm&eacute;es par des images confocales d&#39;astrogliosis GFAP-positive. IRM apr&egrave;s que i.v.i. des anticorps anti-CD4 magn&eacute;tique indique une accumulation de cellules inflammatoires pr&egrave;s de ventricules dilat&eacute;s. Chez les rats trait&eacute;s TMT a &eacute;galement r&eacute;v&egrave;lent la dilatation des ventricules lat&eacute;raux. D&eacute;t&eacute;rioration attendue dans l&#39;hippocampe n&#39;a pas &eacute;t&eacute; observ&eacute;e par IRM clinique, mais immunocytochimie pourrait r&eacute;v&eacute;ler une redistribution significative des macronutriments et la microglie dans cette structure. Dans les deux mod&egrave;les, le contraste de Gd-DTPA a r&eacute;v&eacute;l&eacute; une barri&egrave;re de cerveau de sang infect&eacute; qui peut servir de passage pour les cellules immunitaires inflammatoires dans le voisinage des ventricules lat&eacute;raux dilat&eacute;s. En outre, dans les deux mod&egrave;les la r&eacute;gion m&eacute;senc&eacute;phalique de l&#39;hippocampe dorsal a &eacute;t&eacute; la cible d&#39;un compromis BBB, r&eacute;v&eacute;lant ainsi un point potentiellement vuln&eacute;rable qui peut &ecirc;tre la cible principale de la neurod&eacute;g&eacute;n&eacute;rescence dans le syst&egrave;me nerveux central.

Les changements morphologiques in vivo dans des mod&egrave;les animaux de scl&eacute;rose lat&eacute;rale amyotrophique et maladie de type Alzheimer : approche de MRI.

Risonanza magnetica (MRI) &egrave; la tecnica solo non invasiva che fornisce informazioni strutturali sulla perdita di cellule sia cambiamenti metabolici. Dopo aver esaminato tutti i risultati ottenuti negli studi clinici, biomarcatori affidabili in malattie neurologiche sono ancora carenti. Diffusione MRI, signor spettroscopia e la valutazione dell&#39;atrofia regionale sono promettenti approcci, ma non possono essere utilizzati contemporaneamente su un singolo paziente. Cos&igrave;, per ulteriori progressi della ricerca, sono necessari modelli animali affidabili. A questo scopo, abbiamo usato la risonanza magnetica clinica per valutare processi neurodegenerativi nel modello di ratto hSOD-1(G93A) ALS e in trimetilxtina (TMT) - modello di trattato del morbo di Alzheimer - come malattia. T2-pesate (T2W) hyperintensive neurodegenerative focolai sono stati trovati nel tronco cerebrale del ratto ALS con dilatazione ventricolo laterale apparente (T1W ipointensit&agrave; vs T2W-iperintensit&agrave;). Processi degenerativi in queste aree sono stati confermati anche da immagini confocal di GFAP-positivo astrogliosis. MRI dopo i.v.i degli anticorpi anti-CD4 magnetico indicato un accumulo di cellule infiammatorie nei pressi di ventricoli dilatati. TMT-trattato ratti ha rivelato anche la dilatazione dei ventricoli laterali. Atteso deterioramento nell&#39;ippocampo non &egrave; stata osservata da MRI clinica, ma immunocytochemistry potrebbe rivelare significativa ridistribuzione di macro - e microglia in questa struttura. In entrambi i modelli, il contrasto di Gd-DTPA ha rivelato un compromesso emato-encefalica che pu&ograve; servire come il passaggio per le cellule immunitarie infiammatorie in prossimit&agrave; dei ventricoli laterali dilatati. Inoltre, in entrambi i modelli regione del mesencefalo dell&#39;ippocampo dorsale &egrave; stato il bersaglio di compromesso BBB, rivelando cos&igrave; un punto potenzialmente vulnerabile che pu&ograve; essere l&#39;obiettivo primario di neurodegenerazione del sistema nervoso centrale.

Cambiamenti morfologici in vivo in modelli animali di sclerosi laterale amiotrofica e la malattia di Alzheimer-come: approccio MRI.

The founder of physiology studies in the Balkans and the pioneer of research on hypothermia, Ivan Djaja (Jean Giaja) was born 1884 in L'Havre. Giaja gained his PhD at the Sorbonne in 1909. In 1910 he established the first Chair of Physiology in the Balkans and organized the first Serbian Institute for Physiology at the School of Philosophy of the University of Belgrade. He led this Institute for more than 40 subsequent years. His most notable papers were in the field of thermoregulation and bioenergetics. Djaja became member of the Serbian and Croatian academies of science and doctor honoris causa of Sorbonne. In 1952 for the seminal work on the behavior of deep cooled warm blooded animals he became associate member of the National Medical Academy in Paris. In 1955 the French Academy of Sciences elected him as associate member in place of deceased Sir Alexander Fleming. Djaja died in 1957 during a congress held in his honor. He left more than 200 scientific and other papers and the golden DaVincian credo "Nulla dies sine experimento". His legacy was continued by several generations of researchers, the most prominent among them being Stefan Gelineo, Radoslav Andjus and Vojislav Petrović.

Ivan Djaja (Jean Giaja) and the Belgrade School of Physiology.

To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011).

Increased survival after irradiation followed by regeneration of bone marrow stromal cells with a novel thiol-based radioprotector.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1 ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1 rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of changes in BBB permeability and for the unprecedented in vivo monitoring of the brain tissue redox status, as early markers of ALS.

In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1 ALS rat model.

National Brain Councils - common challenges and regional cooperation.

S100A4, belonging to a large multifunctional S100 protein family, is a Ca-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-κB (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying  pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions.

The S100A4 Transcriptional Inhibitor Niclosamide Reduces Pro-Inflammatory and Migratory Phenotypes of Microglia: Implications for Amyotrophic Lateral Sclerosis.

Article	Year
In vivo morphological changes in animal models of amyotrophic lateral sclerosis and Alzheimer's-like disease: MRI approach. Anatomical record (Hoboken, N.J. : 2007)\| PubMed ID: 19943341	2009
Ivan Djaja (Jean Giaja) and the Belgrade School of Physiology. Physiological research\| PubMed ID: 21777022	2011
Increased survival after irradiation followed by regeneration of bone marrow stromal cells with a novel thiol-based radioprotector. Croatian medical journal\| PubMed ID: 24577826	2014
The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment. Brain structure & function\| PubMed ID: 27089885	2017
In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1 ALS rat model. Free radical biology & medicine\| PubMed ID: 28366802	2017
National Brain Councils - common challenges and regional cooperation. Croatian medical journal\| PubMed ID: 31483126	2019
The S100A4 Transcriptional Inhibitor Niclosamide Reduces Pro-Inflammatory and Migratory Phenotypes of Microglia: Implications for Amyotrophic Lateral Sclerosis. Cells\| PubMed ID: 31623154	2019

Pavle R. Andjus

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