Caracterització de les estructures granulars patològiques presents a l’hipocamp dels ratolins amb senescència accelerada SAMP8

[cat] La soca de ratolins amb senescència accelerada SAMP8 és utilitzada habitualment com a model de senescència; i el seu ús per a l’estudi de la malaltia d’Alzheimer s’ha estès degut a la manifestació espontània dels seus trets histopatològics distintius. En estudis recents es va identificar la presència de grànuls amiloides a l’hipocamp dels ratolins SAMP8. Aquests clústers de grànuls augmenten amb l’edat i s’estenen al llarg de l’hipocamp. A més a més, les característiques morfològiques dels grànuls amiloides són molt similars als grànuls tenyits amb Periodic Acid Schiff (PAS), descrits anteriorment en els SAMP8 i en ratolins envellits d’altres soques. L’objectiu principal d’aquesta tesi ha estat estudiar la composició, l’origen i el desenvolupament dels grànuls que apareixen amb l’edat a l’hipocamp dels ratolins senescents SAMP8 amb la finalitat d’aportar nova informació sobre la neuropatologia d’aquests ratolins per al seu ús com a model de la malaltia d’Alzheimer. En aquest treball s’ha identificat la correspondència entre els grànuls amiloides i els grànuls PAS de l’hipocamp d’aquests animals. Aquests grànuls són el resultat d’un procés degeneratiu present en els astròcits i que pot afectar també zones properes, i estan formats per fragments membranosos provinents de l’acumulació de restes d’estructures i orgànuls, com els mitocondris. A més a més, s’ha identificat la presència d’un neo-epítop en el nucli dels grànuls que és, almenys parcialment, de naturalesa glucídica. Aquest neo-epítop és reconegut per anticossos IgM contaminants presents en anticossos comercials produïts en ascites de ratolí o sèrum de ratolí i conill. Aquestes IgM contaminants són probablement anticossos naturals, i algunes IgM hemaglutinen de forma positiva i específica els eritròcits humans de tipus A. La presència tant del neo-epítop com de les IgMs han estat la causa de falses tincions positives que han generat interpretacions errònies del significat fisiopatològic dels grànuls. És el cas, per exemple, de la presència de β-amiloide o la proteïna tau en aquestes estructures. Finalment, les lesions cerebrals de pacients associades a malalties neurodegeneratives i a l’envelliment no corresponen als clústers de grànuls de l’hipocamp d’aquests ratolins, tot i que seria interessant estudiar la presència del neo-epítop en aquestes condicions fisiopatològiques.[eng] SAMP8 mice present an accelerated senescence and are usually used as a model of aging. Because this strain of mice spontaneously expresses several histopathological features of Alzheimer’s disease (AD), it has been recently used as a model for studying this disease. Alterations in the expression of β-amyloid (Aβ) peptides, tau hyperphosphorilation, an increase of oxidative stress and gliosis suggest that SAMP8 mice could be a relevant model for studying the first stages of sporadic AD and its relation to aging. Recent studies in our group identified the presence of Aβ clustered granules in the hippocampus of SAMP8 mice when immunohistochemical stainings were performed with antibodies directed against Aβ peptides. These clustered granules increase in number and extension with age, and they spread throughout the hippocampus. The features of Aβ clustered granules are very similar to the granules stained with Periodic Acid Schiff (PAS) already described in SAMP8 mice, as well as in aged mice of other strains. On the other hand, human brain lesions present in aging and other neurodegenerative diseases, such as AD, may vary their chemical composition with time, suffering substantial alterations in the principal components and the addition of secondary components. The main objective of this thesis is to study the composition, origin and development of the granules that appear with age in the hippocampus of SAMP8 mice, with the aim of further describing the neuropathology of these animals and provide new information for using them as a murine model of AD. Therefore, in this thesis we studied the composition and order of appearance of the constituents of the hippocampal granules of SAMP8 mice, and the possible coincidence between Aβ and PAS granules. The morphology, ultrastructure, cellular origin and formation process of these structures has been also ascertained; and a comparison between these structures and human brain lesions of neurodegenerative diseases have been performed. The results obtained in this thesis allowed reaching several conclusions. The pathological clustered granules of the hippocampus of aged SAMP8 mice correspond to the PAS granules described in aged mice of several mouse strains. These granules are the result of a degenerative process located in astrocytes that could affect the surrounding neuropil. In the granules formation process, several abnormal membranous structures and cellular organelles, like mitochondria, produce membranous waste that finally accummulates forming the core of the granules. On the other hand, a neo-epitope absent before the granules formation appears in the membranous residues. This neo-epitope is located in the nucleus of the mature granules and contains at least some structures of glycosidic nature. The neo-epitope is recognized by contaminant IgM antibodies present in a wide range of commercial antibodies produced in mouse ascites or mouse and rabbit sera, which are probably natural antibodies, and some of these IgMs haemmagglutinate A-type human erythrocytes, but not B and O subtypes. The neo-epitope could be considered an A-like epitope because monoclonal antibodies anti-A-type human erythrocytes do not recognize it. Both the neo-epitope presence in the granular structures and the IgMs contained in commercial antibodies have been the cause of false-positive stainings, and as a result, they have generated misunderstandings about the composition and physiopathological significance of the hippocampal granules of aged mice, as is the case of Aβ peptides and tau protein, which are not contained in these granules. Finally, human brain lesions described to date in patients of neurodegenerative diseases or aged individuals do not correspond to the clustered pathological granules present in the hippocampus of aged mice. The study of the presence of the neo-epitope in diseased human brain could be interesting in the understanding of the neuropathology of aging and neurodegenerative diseases

Trafficking of Gold Nanoparticles Coated with the 8D3 Anti-Transferrin Receptor Antibody at the Mouse Blood-Brain Barrier

Receptor-mediated transcytosis has been widely studied as a possible strategy to transport neurotherapeutics across the blood-brain barrier (BBB). Monoclonal antibodies directed against the transferrin receptor (TfR) have been proposed as potential carrier candidates. A better understanding of the mechanisms involved in their cellular uptake and intracellular trafficking is required and could critically contribute to the improvement of delivery methods. Accordingly, we studied here the trafficking of gold nanoparticles (AuNPs) coated with the 8D3 anti-transferrin receptor antibody at the mouse BBB. 8D3-AuNPs were intravenously administered to mice and allowed to recirculate for a range of times, from 10 min to 24 h, before brain extraction and analysis by transmission electron microscope techniques. Our results indicated a TfR-mediated and clathrin-dependent internalization process by which 8D3-AuNPs internalize individually in vesicles. These vesicles then follow at least two different routes. On one hand, most vesicles enter intracellular processes of vesicular fusion and rearrangement in which the AuNPs end up accumulating in late endosomes, multivesicular bodies or lysosomes, which present a high AuNP content. On the other hand, a small percentage of the vesicles follow a different route in which they fuse with the abluminal membrane and open to the basal membrane. In these cases, the 8D3-AuNPs remain attached to the abluminal membrane, which suggests an endosomal escape, but not dissociation from TfR. Altogether, although receptor-mediated transport continues to be one of the most promising strategies to overcome the BBB, different optimization approaches need to be developed for efficient drug delivery. Keywords: blood−brain barrier; drug delivery; electron microscopy; monoclonal antibodies; receptor-mediated transport; transferrin receptor

Neo-epitopes emerging in the degenerative hippocampal granules of aged mice can be recognized by natural IgM autoantibodies

Background: Degenerative granular structures appear progressively with age in the hippocampus of most mouse strains. We recently reported that these granules contain a neo-epitope that is recognised by IgM antibodies present as contaminants in many commercial antibodies obtained from mouse ascites and mouse or rabbit serum. We hypothesise that these anti-neo-epitope IgMs are in fact natural auto-antibodies that are generated spontaneously during the foetal stage without previous contact with external antigens and whose repertoire and reactivity pattern have been determined through evolution, being remarkably stable within species and even between species. Findings: In the present work we found that mice from the ICR-CD1, BALB/C and SAMP8 strains have anti-neoepitope IgM antibodies in their plasma at all ages tested and even when maintained under specific opportunistic pathogen-free conditions. Moreover, we determined that these anti-neo-epitope IgMs are also present in rabbit, goat and rat serum. We also found that, in each mouse that presented hippocampal granules, the anti-neo-epitope IgMs contained in its plasma recognised the neo-epitopes in its own granules. Conclusions: This study led to the conclusion that anti-neo-epitope IgMs are widespread natural auto-antibodies contained in the plasma of mice and other species. The presence of these natural auto-antibodies not only explains why they are frequently found as contaminants in commercial antibodies, but also paves the way for a new approach to a treatment and diagnosis of pathological brain processes based on natural IgMs and neo-epitopes

Astrocytes and neurons produce distinct types of polyglucosan bodies in Lafora disease

Lafora disease (LD), the most devastating adolescence‐onset epilepsy, is caused by mutations in the EPM2A or EPM2B genes, which encode the proteins laforin and malin, respectively. Loss of function of one of these proteins, which are involved in the regulation of glycogen synthesis, induces the accumulation of polyglucosan bodies (PGBs) known as Lafora bodies (LBs) and associated with neurons in the brain. Ageing and some neurodegenerative conditions lead to the appearance of another type of PGB called corpora amylacea , which are associated with astrocytes and contain neo‐epitopes that can be recognized by natural antibodies. Here we studied the PGBs in the cerebral cortex and hippocampus of malin knockout mice, a mouse model of LD. These animals presented not only LBs associated with neurons but also a significant number of PGBs associated with astrocytes. These astrocytic PGBs were also increased in mice from senescence‐accelerated mouse‐prone 8 (SAMP8) strain and mice with overexpression of Protein Targeting to Glycogen (PTGOE), indicating that they are not exclusive of LD. The astrocytic PGBs, but not neuronal LBs, contained neo‐epitopes that are recognized by natural antibodies. The astrocytic PGBs appeared predominantly in the hippocampus but were also present in some cortical brain regions, while neuronal LBs were found mainly in the brain cortex and the pyramidal layer of hippocampal regions CA2 and CA3. Our results indicate that astrocytes, contrary to current belief, are involved in the etiopathogenesis of LD

HISTOFLASH: una eina per al reconeixement de teixits que millora l'aprenentatge

Podeu consultar la Vuitena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/66524Els estudiants de Fisiologia i Fisiopatologia III del Grau de Farmàcia realitzen pràctiques de laboratori entre d’altres activitats complementàries. El curs 2011-12, l’Equip Docent de l’assignatura va posar a punt una pràctica d'histologia que té com a objectiu que els estudiants, per mitjà de l'observació al microscopi òptic, identifiquin estructures en diferents sistemes orgànics. El temps dedicat a aquesta pràctica depenia, en gran mesura, de les habilitats de l’estudiant i de la presència del professor, que havia de confirmar o corregir la identificació que feia l'alumne i donar les pautes per a una correcte interpretació de les preparacions. En una enquesta realitzada durant el curs 2012-2013, un 84% dels estudiants van opinar que seria útil disposar de les imatges de les preparacions, tant per preparar la pràctica com per repassar els continguts posteriorment. En aquest context, l’Equip Docent de l’assignatura ha creat un programa interactiu, l’Histoflash, que permet a l’alumne estudiar els teixits a l’ordinador a través de la simulació de l’observació al microscopi. El desenvolupament i l’aplicació del programa pretén facilitar l’aprenentatge autònom dels estudiants per a millorar de forma no presencial les habilitats d’observació al microscopi, prèviament i posteriorment a les que adquireixen al laboratori de pràctiques. Durant el curs 2013-2014, per mitjà d’una enquesta, es va observar que els estudiants valoren molt positivament aquesta eina ja que poden dedicar a cada preparació el temps necessari segons les seves necessitats. A més a més, en les activitats d’avaluació del curs 2013-14, el nombre d’estudiants que supera aquesta pràctica ha augmentat significativament després de la implementació d’aquesta eina. Es pot concloure que l’Histoflash és un bon complement a l’activitat presencial i facilita el procés de ensenyament-aprenentatge

Creació i aplicació del programa HISTOFLASH a les classes pràctiques d'histologia de l'assignatura Fisiologia i Fisiopatologia III del Grau de Farmàcia de la Universitat de Barcelona. Programa interactiu com a eina d'aprenentatge

Els estudiants de Fisiologia i Fisiopatologia III del Grau de Farmàcia realitzen pràctiques de laboratori entre d'altres activitats complementàries. Una de les pràctiques es basa en l'observació i identificació de teixits humans sans i patològics de diferents sistemes orgànics. L'equip docent de l'assignatura ha creat un programa interactiu, l'Histoflash, que permet a l'alumne estudiar els teixits a l'ordinador a través de la simulació de l'observació al microscopi

Periodic acid-Schiff granules in the brain of aged mice: from amyloid aggregates to degenerative structures containing neo-epitopes

Brain ageing in mice leads to the progressive appearance and expansion of degenerative granular structures frequently referred as "PAS granules" because of their positive staining with periodic acid-Schiff (PAS). PAS granules are present mainly in the hippocampus, although they have also been described in other brain areas such as piriform and entorhinal cortices, and have been observed in other mammals than mice, like rats and monkeys. PAS granules have been identified as a wide range of brain deposits related to numerous neurodegenerative diseases, such as amyloid deposits, neurofibrillary tangles, Lafora bodies, corpora amylacea and polyglucosan bodies, and these identifications have generated controversy and particular theories about them. We have recently reported the presence of a neo-epitope in mice hippocampal PAS granules and the existence of natural IgM auto-antibodies directed against the neo-epitope in the plasma of the animals. The significance of the neo-epitope and the autoantibodies is discussed in this review. Moreover, we observed that the IgM anti-neo-epitope is frequently present as a contaminant in numerous commercial antibodies and is responsible of a considerable amount of false positive immunostainings, which may produce misinterpretations in the identification of the granules. Now that this point has been clarified, this article reviews and reconsiders the nature and physiopathological significance of these degenerative granules. Moreover, we suggest that neo-epitopes may turn into a useful brain-ageing biomarker and that autoimmunity could become a new focus in the study of age-related degenerative processes

Neo-epitopes emerging in the degenerative hippocampal granules of aged mice can be recognized by natural IgM autoantibodies

Background: Degenerative granular structures appear progressively with age in the hippocampus of most mouse strains. We recently reported that these granules contain a neo-epitope that is recognised by IgM antibodies present as contaminants in many commercial antibodies obtained from mouse ascites and mouse or rabbit serum. We hypothesise that these anti-neo-epitope IgMs are in fact natural auto-antibodies that are generated spontaneously during the foetal stage without previous contact with external antigens and whose repertoire and reactivity pattern have been determined through evolution, being remarkably stable within species and even between species. Findings: In the present work we found that mice from the ICR-CD1, BALB/C and SAMP8 strains have anti-neoepitope IgM antibodies in their plasma at all ages tested and even when maintained under specific opportunistic pathogen-free conditions. Moreover, we determined that these anti-neo-epitope IgMs are also present in rabbit, goat and rat serum. We also found that, in each mouse that presented hippocampal granules, the anti-neo-epitope IgMs contained in its plasma recognised the neo-epitopes in its own granules. Conclusions: This study led to the conclusion that anti-neo-epitope IgMs are widespread natural auto-antibodies contained in the plasma of mice and other species. The presence of these natural auto-antibodies not only explains why they are frequently found as contaminants in commercial antibodies, but also paves the way for a new approach to a treatment and diagnosis of pathological brain processes based on natural IgMs and neo-epitopes

Modelling middle Pliocene warm climates of the USA

The middle Pliocene warm period represents a unique time slice in which to model and understand climatic processes operating under a warm climatic regime. Palaeoclimatic model simulations, focussed on the United States of America (USA), for the middle Pliocene (ca 3 Ma) were generated using the USGS PRISM2 2º x 2º data set of boundary conditions and the UK Meteorological Office’s HadAM3 General Circulation Model (GCM). Model results suggest that conditions in the USA during the middle Pliocene can be characterised as annually warmer (by 2º to 4ºC), less seasonal, wetter (by a maximum of 4 to 8 mm/day) and with an absence of freezing winters over the central and southern Great Plains. A sensitivity experiment suggests that the main forcing mechanisms for surface temperature changes in near coastal areas are the imposed Pliocene sea surface temperatures (SST’s). In interior regions, reduced Northern Hemisphere terrestrial ice, combined with less snow cover and a reduction in the elevation of the western cordillera of North America, generate atmospheric circulation changes and positive albedo feedbacks that raise surface temperatures. A complex set of climatic feedback mechanisms cause an enhancement of the hydrological cycle magnifying the moisture bearing westerly wind belt during the winter season (Dec., Jan., Feb.). Predictions produced by the model are in broad agreement with available geological evidence. However, the GCM appears to underestimate precipitation levels in the interior and central regions of the southern USA

Histoflash del pulmó

Eina desenvolupada en el marc del Projecte d'Innovació Docent 012PID-UB/59Projecte: 012PID-UB/59Podeu consultar la versió actualitzada a: http://hdl.handle.net/2445/171737Eina interactiva per a l'aprenentatge autònom de l'adquisició d'habilitats d'identificació de les estructures que conformen el pulm