Brain research. Brain research protocols最新文献

{"title":"Comparison of contrast, sensitivity and efficiency of signal amplified and nonamplified immunohistochemical reactions suitable for videomicroscopy-based quantification and neuroimaging","authors":"Oliver Schmitt,&nbsp;Stefan Preuße,&nbsp;Stefan Jean-Pierre Haas","doi":"10.1016/j.brainresprot.2003.10.003","DOIUrl":"10.1016/j.brainresprot.2003.10.003","url":null,"abstract":"<div><p>In recent years, many different technical modifications of immunohistochemical methods have been developed. The selection of a suitable technique for quantitative purposes such as mapping studies can be quite difficult. Various features of a certain method must be considered such as the sensitivity, costs, duration and practicability with respect to serial sectioned specimens. Background and foreground difference or contrast and the influence of artifacts are major problems of quantitative immunohistochemistry<span>. It is not known which of the different modifications of immunohistochemical signal amplifications and non-amplifications gives optimal results in respect to image analytical-based quantification. However, for image analysis, it is important to analyze sections which offer a sufficient contrast between foreground and background. The sensitivity of a system is crucial when quantitative immunohistochemistry should be applied to scarce material with longer postmortem and storage times which occur often by processing human brains. In addition, the enzyme–substrate reactions have an obvious influence on this criterion; therefore, different substrates were also tested. The contrast may be as well effected by the quality and specificity of the primary antibody, the type of tissue and naturally by preparative (fixation, postmortem delay, storage) and individual factors (age, circadian effects, diseases, sex). Because all of these factors may yield to different results by combining them with different neuronal structures, we used three different antigen expressions for a specific analysis: fibrillary, granulary and perikaryal antigen distributions in brains from Wistar rats.</span></p><p>Principally, the sensitivity of the modifications of immunohistochemical amplifications is revealed more strongly than without enhancement steps; however, the contrast between foreground and background structures does not necessary increase by applying a certain amplification technique. The lowest contrast (15%) was detected after applying the labelled streptavidin–biotin technique. All other methods offer comparable contrasts in between 30% and 40%. The <em>catalyzed signal amplification</em><span> reaction has been found to give optimal results (40% contrast) for image analysis. However, from the technical point of view and variability of protein expression, storage and postmortem delay, it was necessary to adapt the commercial CSA Kit from Dako (K1500). The modified technique, called C2 method, offers better results with respect to sensitivity, total costs, duration and contrast (60%) and variability of contrast.</span></p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 3","pages":"Pages 157-171"},"PeriodicalIF":0.0,"publicationDate":"2004-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.10.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40835327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and culture of motor neurons from the newborn mouse spinal cord","authors":"Kirstie N. Anderson ,&nbsp;Allyson C. Potter ,&nbsp;Loretta G. Piccenna ,&nbsp;Alvin K.J. Quah ,&nbsp;Kay E. Davies ,&nbsp;Surindar S. Cheema","doi":"10.1016/j.brainresprot.2003.10.001","DOIUrl":"10.1016/j.brainresprot.2003.10.001","url":null,"abstract":"<div><p><span>A protocol for the isolation and culture of motor neurons from postnatal day 1 mouse spinal cord is described. After 72 h in culture, phase contrast microscopy<span> reveals healthy cells with motor neuronal morphology and extensive neuritic processes. These neurons express the 75-kDa low-affinity neurotrophin receptor (p75NTR) and choline acetyltransferase (ChAT), both </span></span>proteins are specifically expressed by neonatal and embryonic motor neurons in vivo. This protocol can be adapted for various postnatal motor neuron assays.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 3","pages":"Pages 132-136"},"PeriodicalIF":0.0,"publicationDate":"2004-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.10.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40835323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A model for implanting neuronal tissue into the cochlea","authors":"Petri Olivius ,&nbsp;Leonid Alexandrov ,&nbsp;Josef M. Miller ,&nbsp;Mats Ulfendahl ,&nbsp;Dan Bagger-Sjöbäck ,&nbsp;Elena N. Kozlova","doi":"10.1016/j.brainresprot.2003.11.002","DOIUrl":"10.1016/j.brainresprot.2003.11.002","url":null,"abstract":"<div><p>Immature dorsal root ganglion (DRG) neurons have previously been shown to survive implantation to the cavity of extirpated adult native DRG, send axons via the dorsal root into the host spinal cord and make functional sypnatic connections. Regeneration or replacement of the auditory nerve would provide a major intervention in the clinical treatment of severe hearing impairment. In this study we have exploited the potential of fetal DRG neurons to survive allografting into the cochlea of adult guinea pigs. In some animals implantation of fetal DRGs was combined with infusion of neurotropic substances into the cochlea. Survival of the implanted DRG neurons was found in the majority of grafted animals. Treatment with neurotrophic factors significantly increased the number of surviving implanted DRG neurons. However, even in the absence of neurotrophic substances survival of DRG neurons was found in a majority of the animals, indicating the presence of endogenous growth promoting factors within the cochlea and/or an intrinsic capacity of fetal DRG neurons themselves to survive in this heterotropic location.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 3","pages":"Pages 152-156"},"PeriodicalIF":0.0,"publicationDate":"2004-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.11.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40835326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Free colour illustrations in the online version of articles","authors":"","doi":"10.1016/S1385-299X(03)00100-4","DOIUrl":"https://doi.org/10.1016/S1385-299X(03)00100-4","url":null,"abstract":"","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Page iii"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1385-299X(03)00100-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"137275962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A fluorescence-based method to assess plasma protein extravasation in rat dura mater using confocal laser scanning microscopy","authors":"Sigrid Schuh-Hofer,&nbsp;Carsten Boehnke,&nbsp;Uwe Reuter,&nbsp;Wiebke Siekmann,&nbsp;Ute Lindauer,&nbsp;Guy Arnold,&nbsp;Ulrich Dirnagl","doi":"10.1016/j.brainresprot.2003.08.003","DOIUrl":"10.1016/j.brainresprot.2003.08.003","url":null,"abstract":"<div><p><span><span>We describe a nonradioactive, fluorescence-based method to assess plasma protein extravasation (PPE) in rat dura mater using </span>confocal laser scanning microscopy<span> (CLSM). Unilateral PPE can be induced by electrical stimulation of the ipsilateral trigeminal ganglion<span> (TG) and is widely used as an experimental migraine model. The gold standard to determine PPE in the meninges is based on the detection of radiolabeled albumin (</span></span></span>^[125]<span>I-BSA). The aim of this study was to develop a nonradioactive, histological method to quantify PPE in the meninges. The fluorescent dye Evans Blue (50 mg/kg) was injected intravenously to the rat 7 min prior to TG stimulation. PPE in dura mater was detected by a CLSM. The amount of extravasated Evans Blue in the dura mater was measured at six to eight regions of interest (ROIs) in the vicinity of large meningeal vessels. The ratio of the average fluorescence intensity within dura mater of the “stimulus side”, compared to the contralateral “control side”, was calculated for each animal. By using this method, The PPE ratio was 1.67±0.12 (</span><em>n</em>=5). Intravenous injection of three different dosages of the 5HT_1B/1D<span>-receptor agonist sumatriptan (25, 50, and 100 μg/kg) 15 min prior to stimulation attenuated PPE by 42±12%, 49±9%, and 86±15%, respectively (</span><em>p</em>&lt;0.01). The approximated ED₅₀ value was 48 μg/kg. Our results are in accordance with previous reports in the literature using the radioactive approach. We conclude that CLSM is a safe, sensitive, and reliable method to assess PPE in rat meninges in an experimental migaine model.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 77-82"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.08.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of the effect of chronic administration of drugs on rat behavior in the water maze task","authors":"Leticia F Pettenuzzo,&nbsp;Angela T.S Wyse,&nbsp;Clóvis M.D Wannmacher,&nbsp;Carlos S Dutra-Filho,&nbsp;Carlos Alexandre Netto,&nbsp;Moacir Wajner","doi":"10.1016/j.brainresprot.2003.09.002","DOIUrl":"10.1016/j.brainresprot.2003.09.002","url":null,"abstract":"<div><p>Tissue accumulation of intermediates of the metabolism occurs in various inherited neurodegenerative disorders, including methylmalonic acidemia (MA). Animal cognition is usually tested by measuring learning/memory of rats in behavioral tasks. A procedure in which rats are chronically injected with the metabolites accumulating in the neurometabolic disorder methylmalonic acidemia from the 5th to the 28th day of life is described. The animals were allowed to recover for approximately 30 days, after which they were submitted to the Morris water maze task. This behavioral task consisted of two steps. The first one is called the acquisition phase, where rats were trained for 5 consecutive days performing four trials per day to find the submerged platform. On each trial, the rat was placed in the water in one of four start locations (N, S, W and E). The animal was then allowed to search for the platform for 60 s. Once the rat located the platform, it was permitted to remain on it for 10 s. The acquisition phase was followed by the probe trial 24 h later, in which the platform is not present. The time spent in the quadrant of the former platform position and the correct annulus crossings were obtained as a measure for spatial memory. The next step was the reversal learning (reversal phase) performed 2 weeks later. Animals were trained for 4 days (four trials per day) to find the hidden platform, which had now been moved to a position diagonally opposite (reversed) from its location in the acquisition phase. On the next day, all animals were submitted to a second probe trial, similar to the first one. We observed that rats chronically injected with methylmalonic acid (MA), although presenting no alterations in the acquisition phase, showed a long lasting reversal learning impairment. Moreover, motor activity, evaluated by the swim speed in the maze, was not altered by MA administration. These results are consistent with perseverative behavior.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 109-115"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.09.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of astrocytes from adult human optic nerve heads by immunopanning","authors":"Ping Yang ,&nbsp;M.Rosario Hernandez","doi":"10.1016/S1385-299X(03)00073-4","DOIUrl":"10.1016/S1385-299X(03)00073-4","url":null,"abstract":"<div><p><span>Most in vitro studies in the CNS require pure cultures of astrocytes. Astrocytes from the human optic nerve head (ONH, type 1B) represent a specialized population of astrocytes. Primary cells grown from human optic nerve head explants were cultured for 3–4 weeks. To select astrocytes by immunopanning, cell suspensions were placed on a P100 panning dish coated with C5 anti-neuroepithelial antibody and allowed to attach for 30 min. Nonadherent cells were plated on a second dish coated with anti-Thy1.1 antibody to deplete microglia and meningeal cells. Finally, remnant nonadherent cells were plated on a noncoated dish. Purified cells were immunostained with astrocyte markers: </span>GFAP<span><span>, vimentin<span>, Pax2, A2B5, nestin and </span></span>NCAM<span>. Other cell types were characterized by HLA-DR for microglia and smooth muscle actin<span> for vascular smooth muscle. The proportion of GFAP+ astrocytes in the cultures was determined by flow cytometry. About 95% of the cells that adhered to the C5 dish were GFAP+ astrocytes. GFAP+ astrocytes expressed vimentin, Pax2, nestin and NCAM, but not A2B5. From the Thy1.1 dish, 60–75% cells were GFAP+ astrocytes and the remainder cells were GFAP− cells. Using cloning rings, we eliminated fibroblast-like cells, smooth muscle and meningeal cells from astrocyte cultures. Smooth muscle cells and fibroblasts grew on the noncoated dish. In conclusion, immunopanning is an efficient method to get high yields of viable type 1B astrocytes from adult human ONH. The current described culture system may provide a valuable tool in studying human optic nerve head biology and disease.</span></span></span></p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 67-76"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1385-299X(03)00073-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography","authors":"Yvette P. Conley,&nbsp;Heather Brockway,&nbsp;Megan Beatty,&nbsp;Mary E. Kerr","doi":"10.1016/j.brainresprot.2003.08.005","DOIUrl":"10.1016/j.brainresprot.2003.08.005","url":null,"abstract":"<div><p>Detecting and quantifying generalized mitochondrial heteroplasmy is essential if the field of mitochondrial genetics is to advance in the arena of complex genetic disorders. The majority of techniques used to detect and quantify mitochondrial heteroplasmy focus on a known mutation or polymorphism. The necessity of knowing the mitochondrial DNA (mtDNA) change beforehand means that non-specific heteroplasmy in general cannot be assessed. In this study, we assessed the extent that denaturing high-performance liquid chromatography (dHPLC) could detect and quantify mitochondrial heteroplasmy from cerebrospinal fluid (CSF). Although we used a known polymorphism to assess reliability and sensitivity of this technique, a distinct advantage to using dHPLC for heteroplasmy detection is that the entire fragment is screened for variability and any unique fragments will be detected regardless of the placement or type of change. Our results demonstrate that dHPLC can consistently and reliably detect mitochondrial heteroplasmy in a CSF sample down to 0.01%. In addition, the level of heteroplasmy was consistent with peak height for each homoduplex, giving a reliable method to quantify level of heteroplasmy.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 99-103"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.08.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amyloid β protein deposition and neuron loss in osteopetrotic (op/op) mice","authors":"Masato Kaku,&nbsp;Keisuke Tsutsui,&nbsp;Masahide Motokawa,&nbsp;Toshitsugu Kawata,&nbsp;Tadashi Fujita,&nbsp;Shinya Kohno,&nbsp;Yuiko Tohma,&nbsp;Junji Ohtani,&nbsp;Kaoru Tenjoh,&nbsp;Kazuo Tanne","doi":"10.1016/j.brainresprot.2003.09.001","DOIUrl":"10.1016/j.brainresprot.2003.09.001","url":null,"abstract":"<div><p><span><span>Formation of senile plaques<span> (SPs) by amyloid β (Aβ) protein is a neuropathological change which characterizes Alzheimer's disease (AD), and Aβ<span> deposition and neuron loss are essential for the pathological cascade of the disease. Although the mechanism of Aβ deposition remains unclear, it has been suggested that clearance of Aβ protein may be impaired in the AD brain. Previous studies demonstrated that microglia were able to remove Aβ by releasing a </span></span></span>metalloprotease<span> or by phagocytosis, suggesting that microglia may play an important role in preventing Aβ deposition in the central nervous system (CNS). On the other hand, it was reported that the number of microglia was reduced in osteopetrotic (</span></span><em>op</em>/<em>op</em>) toothless mice resulting from the lack of functional macrophage colony-stimulating factor (M-CSF). The present study was thus designed to examine the Aβ deposition and the number of hippocampal neurons in the brain of <em>op</em>/<em>op</em> mice. A number of fibrillar plaques were detected in the cerebral cortex, hippocampus, amygdala and hypothalamus in <em>op</em>/<em>op</em> mice, however, no quantitative evidence of Aβ deposition was observed in normal mice. Moreover, the total number of pyramidal cells in the hippocampal CA1, and CA3 regions was significantly reduced in <em>op</em>/<em>op</em> mice when compared to the controls. These results demonstrate that Aβ deposition influence neuron loss and it may be suspected that expression of SPs may be in part regulated by microglia under physiological conditions.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 104-108"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.09.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A new paradigm to analyze observational learning in rats","authors":"Maria G Leggio ,&nbsp;Alessandro Graziano ,&nbsp;Laura Mandolesi ,&nbsp;Marco Molinari ,&nbsp;Paola Neri ,&nbsp;Laura Petrosini","doi":"10.1016/j.brainresprot.2003.08.001","DOIUrl":"10.1016/j.brainresprot.2003.08.001","url":null,"abstract":"<div><p>A new paradigm of learning was developed through observational training in which rats repeatedly observed companion rats performing different spatial tasks. Observer animals were separately housed in small cages suspended over a water maze tank. They repeatedly observed companion actor rats performing spatial tasks differing according to the experimental requirements. After the observational training, observer animals were or not surgically hemicerebellectomized. This surgical ablation was performed to block any further acquisition of new behavioral strategies during actual performance of swimming task. When cerebellar symptomatology stabilized, observer animals were actually tested in the Morris Water Maze (MWM) task they had previously only observed. The observer rats displayed exploration abilities that closely matched the previously observed behaviors. The results obtained indicate that it is possible to learn complex behavioral strategies by observation using this new protocol. Furthermore, acquisition of the single facets that form the behavioral repertoire can be separately studied.</p></div>","PeriodicalId":79477,"journal":{"name":"Brain research. Brain research protocols","volume":"12 2","pages":"Pages 83-90"},"PeriodicalIF":0.0,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.brainresprot.2003.08.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24072656","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}