Interaction between APOE4 genotype and environmental risk factors in Alzheimer's disease

Alzheimer's disease (AD) is probably caused by both genetic and environmental risk factors. The major genetic risk factor is the E4 variant of apolipoprotein E gene called apoE4. Several risk factors for developing AD have been identified including lifestyle, such as dietary habits. The mechanisms behind the AD pathogenesis and the onset of cognitive decline in the AD brain are presently unknown. In this study we wanted to characterize the effects of the interaction between environmental risk factors and apoE genotype on neurodegeneration processes, with particular focus on behavioural studies and neurodegenerative processes at molecular level. Towards this aim, we used 6 months-old apoE4 and apoE3 Target Replacement (TR) mice fed on different diets (high intake of cholesterol and high intake of carbohydrates). These mice were evaluated for learning and memory deficits in spatial reference (Morris Water Maze (MWM)) and contextual learning (Passive Avoidance) tasks, which involve the hippocampus and the amygdala, respectively. From these behavioural studies we found that the initial cognitive impairments manifested as a retention deficit in apoE4 mice fed on high carbohydrate diet. Thus, the genetic risk factor apoE4 genotype associated with a high carbohydrate diet seems to affect cognitive functions in young mice, corroborating the theory that the combination of genetic and environmental risk factors greatly increases the risk of developing AD and leads to an earlier onset of cognitive deficits. The cellular and molecular bases of the cognitive decline in AD are largely unknown. In order to determine the molecular changes for the onset of the early cognitive impairment observed in the behavioural studies, we performed molecular studies, with particular focus on synaptic integrity and Tau phosphorylation. The most relevant finding of our molecular studies showed a significant decrease of Brain-derived Neurotrophic Factor (BDNF) in apoE4 mice fed on high carbohydrate diet. Our results may suggest that BDNF decrease found in apoE4 HS mice could be involved in the earliest impairment in long-term reference memory observed in behavioural studies. The second aim of this thesis was to study possible involvement of leptin in AD. There is growing evidence that leptin has neuroprotective properties in the Central Nervous System (CNS). Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. However, there are still numerous questions, regarding the molecular mechanism by which leptin acts, that remain unanswered. Thus, given to the importance of the involvement of leptin in AD, we wanted to clarify the function of leptin in the pathogenesis of AD and to investigate if apoE genotype affect leptin levels through studies in vitro, in mice and in human. Our findings suggest that apoE4 TR mice showed an increase of leptin in the brain. Leptin levels are also increased in the cerebral spinal fluid of AD patients and apoE4 carriers with AD have higher levels of leptin than apoE3 carriers. Moreover, leptin seems to be expressed by reactive glial cells in AD brains. In vitro, ApoE4 together with Amyloid beta increases leptin production by microglia and astrocytes. Taken together, all these findings suggest that leptin replacement might not be a good strategy for AD therapy. Our results show that high leptin levels were found in AD brains. These findings suggest that, as high leptin levels do not promote satiety in obese individuals, it might be possible that they do not promote neuroprotection in AD patients. Therefore, we hypothesized that AD brain could suffer from leptin resistance. Further studies will be critical to determine whether or not the central leptin resistance in SNC could affect its potential neuroprotective effects.

Studio in vitro della suscettibilità genetica degli ovini alle EST: il PMCA come alternativa agli studi di trasmissione in vivo

Le encefalopatie spongiformi trasmissibili (EST), o malattie da prioni, sono malattie neurodegenerative che colpiscono l'uomo e gli animali. Le più note tra le EST animali sono la scrapie della pecora e della capra, l’encefalopatia spongiforme bovina (BSE), la Sindrome del dimagrimento cronico (CWD) dei cervidi. Negli uomini ricordiamo la malattia di Creutzfeldt-Jakob (CJD) nelle sue diverse forme (sporadica, genetica, iatrogenica e variante). La dimostrazione che la variante della CJD (vCJD) sia causata dallo stesso agente eziologico della BSE, ha evidenziato il potenziale zoonotico di queste malattie. Le EST sono caratterizzate da tempi di incubazione estremamente lunghi ed esito invariabilmente fatale. Il momento patogenetico centrale comune a tutte queste malattie è rappresentato dalla modificazione conformazionale di una proteina cellulare denominata PrPC (proteina prionica cellulare) in una isoforma patologica denominata PrPSc, insolubile e caratterizzata da una parziale resistenza alle proteasi, che tende a depositarsi sotto forma di fibrille amiloidee nel SNC dei soggetti colpiti. La suscettibilità degli ovini alla scrapie è largamente influenzata dal genotipo del gene dell’ospite che codifica per la PrP (PRNP), e più precisamente da tre polimorfismi presenti ai codoni 136, 154 e 171. Questi si combinano in cinque principali alleli, ARQ, VRQ, AHQ, ARH e ARR, correlati a differenti gradi di suscettibilità alla malattia. Risultati ottenuti da un precedente studio d’infezione sperimentale di ovini di razza Sarda con scrapie classica (Vaccari G et al 2007), hanno suggeriscono l’ordine di suscettibilità ARQ>AHQ>ARH. L’allele ARR, è risultato invece associato ai più alti livelli di protezione dalla malattia. Dallo stesso studio di trasmissione sperimentale e da uno studio epidemiologico di tipo caso-controllo, è inoltre emerso che nella razza Sarda, ovini con l’allele ARQ, con sostituzione amminoacidica al codone 137 Metionina (M)/Treonina (T) (AT137RQ) o al 176 Asparagina (N)/Lisina (K) (ARQK176) in eterozigosi sono protetti dalla scrapie. Inoltre studi di trasmissione sperimentale della BSE in ovini della stessa razza con tre differenti genotipi (ARQ/ARQ, ARQ/ARR e ARR/ARR), hanno dimostrato come la BSE abbia un targeting genetico molto simile a quello della scrapie, evidenziando il genotipo ARQ/ARQ come il più suscettibile. L’obbiettivo della seguente tesi è stato quello di verificare se fosse possibile riprodurre in vitro la differente suscettibilità genetica degli ovini alle EST evidenziata in vivo, utilizzando il PMCA (Protein Misfolding Cyclic Amplification), la metodica ad oggi più promettente e di cui è stata dimostrata la capacità di riprodurre in vitro diverse proprietà biologiche dei prioni. La tecnica, attraverso cicli ripetuti di sonicazione/incubazione, permette la conversione in vitro della PrPC presente in un omogenato cerebrale (substrato), da parte di una quantità minima di PrPSc (inoculo) che funge da “innesco” della reazione. Si è voluto inoltre utilizzare il PMCA per indagare il livello di protezione in omozigosi di alleli rari per i quali, in vivo, si avevano evidenze di protezione dalla scrapie solo in eterozigosi, e per studiare la suscettibilità degli ovini alla BSE adattata in questa specie. È stata quindi testata in PMCA la capacità diversi substrati ovini recanti differenti genotipi, di amplificare la PrPSc dello stesso isolato di scrapie classica impiegato nel precedente studio in vivo o di un inoculo di BSE bovina. Inoltre sono stati saggiati in vitro due inoculi di BSE costituiti da omogenato cerebrale di due ovini sperimentalmente infettati con BSE (BSE ovina) e recanti due differenti genotipi (ARQ/ARQ e ARR/ARR). Per poter descrivere quantitativamente il grado di correlazione osservato i risultati ottenuti in vitro e i quelli riscontrati dallo studio di sperimentazione con scrapie, espressi rispettivamente come fattori di amplificazione e tempi d’incubazione registrati in vivo, sono stati analizzati con un modello di regressione lineare. Per quanto riguarda la scrapie, i risultati ottenuti hanno evidenziato come i genotipi associati in vivo a suscettibilità (ARQ/ARQ, ARQ/AHQ and AHQ/ARH) siano anche quelli in grado di sostenere in PMCA l’amplificazione della PrPSc, e come quelli associati a resistenza (ARQ/ARR and ARR/ARR) non mostrino invece nessuna capacità di conversione. Dall’analisi di regressione lineare è inoltre emerso come l’efficienza di amplificazione in vitro dei differenti genotipi testati sia inversamente proporzionale ai tempi d’incubazione registrati in vivo. Inoltre nessuna amplificazione è stata riscontrata utilizzando il substrato con genotipo raro ARQK176/ARQK176 suggerendo come anche questo possa essere associato a resistenza, almeno nei confronti dell’isolato di scrapie classica utilizzato. Utilizzando come inoculo in PMCA l’isolato di BSE bovina, è stato possibile riscontrare, nei tre genotipi analizzati (ARQ/ARQ, ARQ/ARR e ARR/ARR) un evidente amplificazione per il solo genotipo ARQ/ARQ, sottolineando anche in questo caso l’esistenza di una correlazione tra suscettibilità riscontrata in vivo e capacità di conversione in PMCA. I tre i substrati analizzati mostrano inoltre una buona efficienza di amplificazione, per altro simile, se si utilizza la PrPSc dell’inoculo di BSE sperimentalemente trasmessa agli ovini. Questi genotipi sembrerebbero dunque ugualmente suscettibili se esposti a BSE adattata alla specie ovina. I risultati di questa tesi indicano dunque una correlazione diretta tra la capacità di conversione della PrPC con il PMCA e la suscettibilità osservata in vivo per i differenti genotipi analizzati. Mostrano inoltre come il PMCA possa essere una valida alternativa agli studi di trasmissione in vivo e un rapido strumento utile non soltanto per testare, ma anche per predire la suscettibilità genetica degli ovini a diversi ceppi di EST, rappresentando un valido aiuto per l’individuazione di ulteriori genotipi resistenti, così da incrementare la variabilità genetica dei piani di selezione attuati per gli ovini per il controllo di queste malattie.

Studio di popolazione sull'associazione tra sindrome delle gambe senza riposo ed emicrania

Introduzione: La sindrome delle gambe senza riposo (RLS) è un disturbo caratterizzato da sensazione spiacevole disestesica generalmente agli arti inferiori, che si presenta o peggiora nelle ore serali-notturne e che migliora con il movimento. Studi clinici hanno mostrato una maggiore prevalenza di RLS negli emicranici, mentre mancano studi condotti su popolazione generale non selezionata. Lo scopo di questo studio era quello di valutare la associazione tra emicrania e RLS in una popolazione italiana adulta. Inoltre è stata valutata l’associazione tra RLS e cefalea fenotipizzata attraverso metodica di principal components analysis (PCA). Materiali e metodi: la presenza di RLS e di emicrania è stata determinata attraverso questionari basati sui criteri diagnostici correnti in un campione di 1567 partecipanti di un fase preliminare di uno studio in corso sulla popolazione adulta della Val Venosta (BZ). Risultati: gli emicranici hanno presentato un significativo maggior rischio di soffrire di RLS rispetto ai non emicranici, anche dopo aggiustamento per fattori confondenti come età, sesso, depressione, ansia e qualità del sonno (p = 0.049). Questa associazione non era modificata dalla presenza di aura emicranica, di cause possibili di RLS secondaria e dalla frequenza di attacchi emicranici. Inoltre la RLS non era risultata significativamente associata alla cefalea di tipo tensivo (TTH). Dall’analisi di associazione tra RLS e cefalea fenotipizzata attraverso PCA era emerso che la componente 1, caratterizzata da sintomi di sensitivizzazione del sistema nervoso centrale (SNC), correlava significativamente con la presenza di RLS (p = 0.021). Conclusioni: RLS ed emicrania sono risultate associate nel nostro campione di popolazione adulta; inoltre la RLS ha mostrato una correlazione significativa con i sintomi di sensitivizzazione del SNC legati agli attacchi di cefalea. Questa associazione potrebbe risiedere in una possibile base patogenetica comune.

La fibroina della seta come biomateriale per la medicina neurorigenerativa

Una delle grandi sfide della medicina moderna e dell’ingegneria biomedica è rappresentata dalla rigenerazione e il recupero dei tessuti nervosi. I danni al Sistema Nervoso Centrale (SNC) e Periferico (SNP) provocano effetti irreversibili e influiscono sulla qualità della vita dei pazienti. L’ingegneria tissutale è stata definita come “un campo interdisciplinare che applica i principi dell’ingegneria e delle scienze della vita per lo sviluppo di sostituti biologici che ripristinino, mantengano, o migliorino la funzione di un tessuto o di un intero organo” (Langer R et al, 1993). Lo sviluppo dei biomateriali, i progressi scientifici nel campo delle cellule staminali e dei fattori di crescita, nonché le migliorie nelle tecniche di differenziazione e del rilascio dei farmaci offrono nuove opportunità di sviluppo terapeutico. Sono stati infatti creati tessuti in laboratorio attraverso la combinazione di matrici extracellulari ingegnerizzate, comunemente definite scaffold, cellule e molecole biologicamente attive. Tali “impalcature”, forniscono un supporto fisico e biochimico alla crescita delle cellule nervose. In quest’ottica si configura come essenziale il contributo della seta e di una sua particolare molecola: la fibroina. Quest’ultima grazie alle specifiche caratteristiche di biocompatibilità, lenta degradabilità e alle notevoli proprietà meccaniche, è stata ampiamente studiata, in anni recenti, per nuove applicazioni in ambito biomedico, come nel caso dell’ingegneria dei tessuti e del rilascio di farmaci. La fibroina della seta utilizzabile in vari formati quali film, fibre, reti, maglie, membrane, gel e spugne supporta l'adesione, la proliferazione e la differenziazione in vitro di diversi tipi di cellule. In particolare studi recenti indicano che la seta ha una buona compatibilità per la crescita di cellule neuronali dell'ippocampo. In questo elaborato saranno presentate le caratteristiche della fibroina della seta come biomateriale, con particolare riferimento all’ingegnerizzazione e al processo di fabbricazione degli scaffold finalizzati al supporto della rigenerazione cellulare – neuronale in caso di insulti traumatici, acuti e/o cronici del Sistema Nervoso.

Cancer, a disease of aging (part 1) - trends in older adult cancer mortality in Switzerland 1991-2008

It is crucial for aging societies to evaluate trends in cancer mortality rates of older adults. This study examined socio-demographic and regional characteristics specifically focused on the cancer mortality experience of older adults in Switzerland.

Cancer, a disease of aging (part 2) - risk factors for older adult cancer mortality in Switzerland 1991-2008

Cancer is disease of aging that disproportionately affects older adults and often results in considerable public health consequences. This study evaluated gender-age-specific cancer mortality risk factors in older adults in Switzerland with attention to the most common types of cancer.

Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

Impact of unlinked deaths and coding changes on mortality trends in the Swiss National Cohort

BACKGROUND Results of epidemiological studies linking census with mortality records may be affected by unlinked deaths and changes in cause of death classification. We examined these issues in the Swiss National Cohort (SNC). METHODS The SNC is a longitudinal study of the entire Swiss population, based on the 1990 (6.8 million persons) and 2000 (7.3 million persons) censuses. Among 1,053,393 deaths recorded 1991-2007 5.4% could not be linked using stringent probabilistic linkage. We included the unlinked deaths using pragmatic linkages and compared mortality rates for selected causes with official mortality rates. We also examined the impact of the 1995 change in cause of death coding from version 8 (with some additional rules) to version 10 of the International Classification of Diseases (ICD), using Poisson regression models with restricted cubic splines. Finally, we compared results from Cox models including and excluding unlinked deaths of the association of education, marital status, and nationality with selected causes of death. RESULTS SNC mortality rates underestimated all cause mortality by 9.6% (range 2.4%-17.9%) in the 85+ population. Underestimation was less pronounced in years nearer the censuses and in the 75-84 age group. After including 99.7% of unlinked deaths, annual all cause SNC mortality rates were reflecting official rates (relative difference between -1.4% and +1.8%). In the 85+ population the rates for prostate and breast cancer dropped, by 16% and 21% respectively, between 1994 and 1995 coincident with the change in cause of death coding policy. For suicide in males almost no change was observed. Hazard ratios were only negligibly affected by including the unlinked deaths. A sudden decrease in breast (21% less, 95% confidence interval: 12%-28%) and prostate (16% less, 95% confidence interval: 7%-23%) cancer mortality rates in the 85+ population coincided with the 1995 change in cause of death coding policy. CONCLUSIONS Unlinked deaths bias analyses of absolute mortality rates downwards but have little effect on relative mortality. To describe time trends of cause-specific mortality in the SNC, accounting for the unlinked deaths and for the possible effect of change in death certificate coding was necessary.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

Core formation and mantle differentiation on Mars

Geochemical investigation of Martian meteorites (SNC meteorites) yields important constraints on the chemical and geodynamical evolution of Mars. These samples may not be representative of the whole of Mars; however, they provide constraints on the early differentiation processes on Mars. The bulk composition of Martian samples implies the presence of a metallic core that formed concurrently as the planet accreted. The strong depletion of highly siderophile elements in the Martian mantle is only possible if Mars had a large scale magma ocean early in its history allowing efficient separation of a metallic melt from molten silicate. The solidification of the magma ocean created chemical heterogeneities whose ancient origin is manifested in the heterogeneous 142Nd and 182W abundances observed in different meteorite groups derived from Mars. The isotope anomalies measured in SNC meteorites imply major chemical fractionation within the Martian mantle during the life time of the short-lived isotopes 146Sm and 182Hf. The Hf-W data are consistent with very rapid accretion of Mars within a few million years or, alternatively, a more protracted accretion history involving several large impacts and incomplete metal-silicate equilibration during core formation. In contrast to Earth early-formed chemical heterogeneities are still preserved on Mars, albeit slightly modified by mixing processes. The preservation of such ancient chemical differences is only possible if Mars did not undergo efficient whole mantle convection or vigorous plate tectonic style processes after the first few tens of millions of years of its history.

Reproducibility of effects of homeopathically potentised gibberellic acid on the growth of Lemna gibba L. in a randomised and blinded bioassay

BACKGROUND: Reproducibility of basic research investigations in homeopathy is challenging. This study investigated if formerly observed effects of homeopathically potentised gibberellic acid (GA3) on growth of duckweed (Lemna gibba L.) were reproducible. METHODS: Duckweed was grown in potencies (14x-30x) of GA3 and one time succussed and unsuccussed water controls. Outcome parameter area-related growth rate was determined by a computerised image analysis system. Three series including five independent blinded and randomised potency experiments (PE) each were carried out. System stability was controlled by three series of five systematic negative control (SNC) experiments. Gibbosity (a specific growth state of L. gibba) was investigated as possibly essential factor for reactivity of L. gibba towards potentised GA3 in one series of potency and SNC experiments, respectively. RESULTS: Only in the third series with gibbous L. gibba L. we observed a significant effect (p = 0.009, F-test) of the homeopathic treatment. However, growth rate increased in contrast to the former study, and most biologically active potency levels differed. Variability in PE was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. CONCLUSIONS: Gibbosity seems to be a necessary condition for reactivity of L. gibba to potentised GA3. Further still unknown conditions seem to govern effect direction and the pattern of active and inactive potency levels. When designing new reproducibility studies, the physiological state of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

The use of plant-based bioassays in homeopathic basic research.

OBJECTIVES The objective was to evaluate homeopathic basic research studies that use plant-based bioassays. With this in view, a compilation was made of the findings of three systematic literature reviews covering plant-based bioassays in the three fields of healthy, abiotically, or biotically stressed plants. This compilation focused on investigations using advanced experimental methods and detailed descriptions, also with the aim of supporting the design of future experiments. METHODS Publications included had to report on studies into the effects of homeopathic preparations on whole plants, seeds, plant parts and cells. Outcomes had to be measured by established procedures and statistically evaluated. A Manuscript Information Score (MIS) was applied using predefined criteria to identify publications with sufficient information for adequate interpretation (MIS ≥ 5). Additional evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations, and on the use of systematic negative control (SNC) experiments to ensure the stability of the bioassay. Only a fraction of the studies reported here were performed with 'ultra high' dilutions, whereas other studies were performed with moderate or high dilutions. RESULTS A total of 157 publications were identified, describing a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS ≥ 5, thus allowing adequate interpretation. 29 studies had adequate controls to identify specific effects of homeopathic preparations, and reported significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro's number. 10 studies reported use of SNC experiments, yielding evidence for the stability of the experimental set-up. CONCLUSION Plant models appear to be a useful approach for investigating basic research questions relating to homeopathic preparations, but more independent replication trials are needed in order to verify the results found in single experiments. Adequate controls and SNC experiments should be implemented on a routine basis to exclude false-positive results.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

Evaluación de metales pesados en suelos de los oasis irrigados de la Provincia de Mendoza : concentraciones totales de Zn, Pb, Cd y Cu

En Mendoza, Argentina, no existen antecedentes respecto de la presencia de elementos trazas, totales y disponibles, relacionados con el uso de los suelos en los oasis irrigados. El objetivo del trabajo, en esta etapa, fue determinar los contenidos totales de plomo (Pb), cadmio (Cd), cinc (Zn) y cobre (Cu) en la capa superficial de suelos (0-25 cm), diferenciados en siete tipos según una clasificación utilitaria: suelos vírgenes (SV); suelos de banquina (SB); suelos vecinos a banquina (SVB); suelos de agricultura intensiva (SAI); suelos de agricultura protegidos por lucha antigranizo (SLAG); suelos afectados por actividad industrial (SI) y suelos urbanos (SU). Sobre un total de 200 muestras se efectuó una digestión ácida en caliente y en los extractos se determinaron los metales en su fracción total, mediante espectrofotometría de absorción atómica (AAS). El análisis estadístico de los datos muestra que los niveles más elevados de Cu y Cd se detectan en SAI, con medias de 39,3 y 2,5 mg kg-1 respectivamente. En SI se observan los mayores valores de Pb, con una media de 80,6 mg kg-1 y en SU los mayores tenores de Zn, con un valor medio de 740 mg kg-1. La Ciudad de Mendoza, con mayor densidad poblacional, presentó los mayores contenidos de Zn, Pb y Cd. Las concentraciones encontradas se ubican por debajo de las exigencias de la legislación argentina y la mayoría de los suelos pueden clasificarse como no contaminados (SNC) o ligeramente contaminados (SLC). Se prevé completar este estudio con la determinación de las fracciones disponibles de los elementos estudiados, correlacionándolos con variables edáficas físico-químicas como textura, pH y materia orgánica.