Alicia García-Culebras

Aging increases microglial proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia.

BackgroundAging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke.MethodsWe have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months).ResultsAging increased both microglial proliferation, as shown by a higher number of BrdU+ cells and BrdU/Iba1+ cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1+/epidermal growth factor receptor (EGFR)+/nestin+/glial fibrillary acidic protein (GFAP)+ cells) and type-C cells (prominin-1+/EGFR+/nestin−/Mash1+ cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX+ cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN+/BrdU+ and GAD67+ cells) in the peri-infarct cortical area 14 days after stroke.ConclusionsOur data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and decreases the number of new interneurons in the cortical peri-infarct area. Thus, our results highlight the importance of using aged animals for translation to clinical studies.

Genetic Diversity and Geographic Population Structure of Bovine Neospora caninum Determined by Microsatellite Genotyping Analysis

The cyst-forming protozoan parasite Neospora caninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among N . caninum isolates based on microsatellite sequences (MSs). MSs may be suitable molecular markers for inferring the diversity of parasite populations, molecular epidemiology and the basis for phenotypic variations in N . caninum , which have been poorly defined. In this study, we evaluated nine MS markers using a panel of 11 N . caninum -derived reference isolates from around the world and 96 N . caninum bovine clinical samples and one ovine clinical sample collected from four countries on two continents, including Spain, Argentina, Germany and Scotland, over a 10-year period. These markers were used as molecular tools to investigate the genetic diversity, geographic distribution and population structure of N . caninum . Multilocus microsatellite genotyping based on 7 loci demonstrated high levels of genetic diversity in the samples from all of the different countries, with 96 microsatellite multilocus genotypes (MLGs) identified from 108 N . caninum samples. Geographic sub-structuring was present in the country populations according to pairwise F ST. Principal component analysis (PCA) and Neighbor Joining tree topologies also suggested MLG segregation partially associated with geographical origin. An analysis of the MLG relationships, using eBURST, confirmed that the close genetic relationship observed between the Spanish and Argentinean populations may be the result of parasite migration (i.e., the introduction of novel MLGs from Spain to South America) due to cattle movement. The eBURST relationships also revealed genetically different clusters associated with the abortion. The presence of linkage disequilibrium, the co-existence of specific MLGs to individual farms and eBURST MLG relationships suggest a predominant clonal propagation for Spanish N . caninum MLGs in cattle.

Serological diagnosis of bovine neosporosis: A comparative study of commercially available ELISA tests

Bovine neosporosis control programs are currently based on herd management and serodiagnosis because effective treatments and vaccines are unavailable. Although a wide variety of serological tools have been developed, enzyme-linked immunosorbent assays (ELISAs) are the most commonly commercialized tests. Partial comparative studies have been performed in the past, and the panel of available ELISAs has notably changed in the last few years. Therefore, diagnostic laboratories are requesting updated information about the performance of these tests. Accordingly, the aim of this study was to compare all of the commercially available ELISAs (n=10) by evaluating their performance and to re-standardize them based on TG-ROC analyses when necessary. For this purpose, a well-characterized serum panel from experimentally and naturally infected bovines and non-infected bovines (n=458) was used. Two different definitions of gold standard were considered: (i) the result of the majority of tests and (ii) pre-test information based on epidemiological, clinical and serological data. Most of the tests displayed high sensitivity (Se) and specificity (Sp) values when both gold standard criteria were considered. Furthermore, all the tests showed near perfect agreement, with the exception of the pair-wise comparisons that included the VMRD and SVANOVIR. The best-adjusted ELISAs were the HIPRA-CIVTEST, IDVET, BIOVET and IDEXX Rum (Se and Sp>95%). After the TG-ROC analyses, higher Se and Sp values were obtained for the BIO-X, LSI Bov, LSI Rum and IDEXX Bov, though the increases were more significant for the SVANOVIR and VMRD. The Kappa values also increased with the new adjusted cut-offs. This is the first study that offers updated performance evaluations of commercially available ELISAs. Such analyses are essential for diagnostic laboratories and are valuable to the companies that develop and distribute these tests.

Toll-Like Receptor 4 Mediates Hemorrhagic Transformation After Delayed Tissue Plasminogen Activator Administration in In Situ Thromboembolic Stroke

Background and Purpose— Hemorrhagic transformation is the main complication of revascularization therapies after stroke. Toll-like receptor 4 (TLR4) is implicated in cerebral damage and inflammation in stroke. This study was designed to determine the role of TLR4 in hemorrhagic transformation development after tissue plasminogen activator (tPA) administration. Methods— Mice expressing (TLR4+/+) or lacking functional TLR4 (TLR4−/−) were subjected to middle cerebral artery occlusion using an in situ thromboembolic model by thrombin injection into the middle cerebral artery, and tPA (10 mg/kg) was administered 20 minutes or 3 hours after ischemia. Infarct size, hemorrhages, IgG extravasation, matrix metalloproteinase 9 expression, and neutrophil infiltration were assessed 24 hours after ischemia. Results— In TLR4+/+, early reperfusion (tPA at 20 minutes) resulted infarct volume, whereas late recanalization (tPA at 3 hours) did not modify lesion size and increased the rate of the most severe hemorrhages. In TLR4−/− mice, both early and late reperfusion did not modify lesion size. Importantly, late tPA administration did not result in worse hemorrhages and in an increased bleeding area as occurred in TLR4+/+ group. In TLR4−/− animals, late reperfusion produced a lesser increase in matrix metalloproteinase 9 expression when compared with TLR4+/+ animals. Conclusions— Our results demonstrate TLR4 involvement in hemorrhagic transformation induced by delayed tPA administration, very likely by increasing matrix metalloproteinase 9 expression.

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis.

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40+rNcROP2 and rNcGRA7+rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40+rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40+rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40+rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

Specific Features of SVZ Neurogenesis After Cortical Ischemia: a Longitudinal Study

Stroke is a devastating disease with an increasing prevalence. Part of the current development in stroke therapy is focused in the chronic phase, where neurorepair mechanisms such as neurogenesis, are involved. In the adult brain, one of the regions where neurogenesis takes place is the subventricular zone (SVZ) of the lateral ventricles. Given the possibility to develop pharmacological therapies to stimulate this process, we have performed a longitudinal analysis of neurogenesis in a model of cortical ischemia in mice. Our results show an initial decrease of SVZ proliferation at 24 h, followed by a recovery leading to an increase at 14d and a second decrease 28d after stroke. Coinciding with the 24 h proliferation decrease, an increase in the eutopic neuroblast migration towards the olfactory bulb was observed. The analysis of the neuroblast ectopic migration from the SVZ toward the lesion showed an increase in this process from day 14 after the insult. Finally, our data revealed an increased number of new cortical neurons in the peri-infarct cortex 65d after the insult. In summary, we report here critical check-points about post-stroke neurogenesis after cortical infarcts, important for the pharmacological modulation of this process in stroke patients.

AhR Deletion Promotes Aberrant Morphogenesis and Synaptic Activity of Adult-Generated Granule Neurons and Impairs Hippocampus-Dependent Memory

Visual Abstract Newborn granule cells are continuously produced in the subgranular zone of dentate gyrus throughout life. Once these cells mature, they integrate into pre-existing circuits modulating hippocampus-dependent memory. Subsequently, mechanisms controlling generation and maturation of newborn cells are essential for proper hippocampal function. Therefore, we have studied the role of aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, in hippocampus-dependent memory and granule neuronal morphology and function using genetic loss-of-function approaches based on constitutive and inducible-nestin AhR–/– mice. The results presented here show that the impaired hippocampus-dependent memory in AhR absence is not due to its effects on neurogenesis but to aberrant dendritic arborization and an increased spine density, albeit with a lower number of mature mushrooms spines in newborn granule cells, a finding that is associated with an immature electrophysiological phenotype. Together, our data strongly suggest that AhR plays a pivotal role in the regulation of hippocampal function, by controlling hippocampal granule neuron morphology and synaptic maturation.