Jinmin Zhu

Prolonged Therapeutic Window for Ischemic Brain Damage Caused by Delayed Caspase Activation

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimers disease and Huntingtons disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.

Neuroprotective effects of gelsolin during murine stroke

Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.

A Novel Computational Approach for Automatic Dendrite Spines Detection in Two-Photon Laser Scan Microscopy

BACKGROUND Recent research has shown that there is a strong correlation between the functional properties of a neuron and its morphologic structure. Current morphologic analyses typically involve a significant component of computer-assisted manual labor, which is very time-consuming and is susceptible to operator bias. The existing semi-automatic approaches largely reduce user efforts. However, some manual interventions, such as setting a global threshold for segmentation, are still needed during image processing. METHODS We present an automated approach, which can greatly help neurobiologists obtain quantitative morphological information about a neuron and its spines. The automation includes an adaptive thresholding method, which can yield better segment results than the prevalent global thresholding method. It also introduces an efficient backbone extraction method, a SNR based, detached spine component detection method, and an attached spine component detection method based on the estimation of local dendrite morphology. RESULTS The morphology information obtained both manually and automatically are compared in detail. Using the Kolmogov-Smirnov test, we find a 99.13% probability that the dendrite length distributions are the same for the automatic and manual processing methods. The spine detection results are also compared with other existing semi-automatic approaches. The comparison results show that our approach has 33% fewer false positives and 77% fewer false negatives on average. CONCLUSIONS Because the proposed detection algorithm requires less user input and performs better than existing algorithms, our approach can quickly and accurately process neuron images without user intervention.

Sequence of κ-opioid receptor cDNA in the R1.1 thymoma cell line

Abstract To our knowledge, this is the first demonstration of κ-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the κ-opioid receptor is expressed by the immature T cell line R1.1 We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse κ-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5′ region upstream of the ATG start codon. Comparison of these sequences to the previously published κ-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5′ non-coding region.

Segmentation of touching cell nuclei using gradient flow tracking

Reliable cell nuclei segmentation is an important yet unresolved problem in biological imaging studies. This paper presents a novel computerized method for robust cell nuclei segmentation based on gradient flow tracking. This method is composed of three key steps: (1) generate a diffused gradient vector flow field; (2) perform a gradient flow tracking procedure to attract points to the basin of a sink; and (3) separate the image into small regions, each containing one nucleus and nearby peripheral background, and perform local adaptive thresholding in each small region to extract the cell nucleus from the background. To show the generality of the proposed method, we report the validation and experimental results using microscopic image data sets from three research labs, with both over‐segmentation and under‐segmentation rates below 3%. In particular, this method is able to segment closely juxtaposed or clustered cell nuclei, with high sensitivity and specificity in different situations.

High Content Image Analysis for Human H4 Neuroglioma Cells Exposed to CuO Nanoparticles

BackgroundHigh content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study.ResultsThe cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) a novel local intensity maxima detection method based on the gradient vector field has been established; and (3) a statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the proposed image analysis system.ConclusionThe proposed automated image analysis system can effectively segment the images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results confirmed our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles.

Characterization of κ-opioid receptor transcripts expressed by T cells and macrophages

We have found that the immature T cell lines R1.1 and DPK and the macrophage lines P388D1 and WEHI-3 also express κ-opioid receptor (KOR) mRNA. Characterization of the KOR transcripts in both brain tissue and these T cells has revealed both the normal full-length as well as a truncated form of the mRNA. Our results show that the truncated transcript lacks the second exon. Primary macrophages express this truncated form of the transcript in the absence of detectable levels of the full-length form. These results suggest a degree of heterogeneity in the expression of the opioid receptors which has not previously been reported.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimers disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Methylphenidate and μ opioid receptor interactions: A pharmacological target for prevention of stimulant abuse

Methylphenidate (MPH) is one of the most commonly used and highly effective treatments for attention deficit hyperactivity disorder (ADHD) in children and adults. As the therapeutic use of MPH has increased, so has its abuse and illicit street-use. Yet, the mechanisms associated with development of MPH-associated abuse and dependence are not well understood making it difficult to develop methods to help its mitigation. As a result, many ADHD patients especially children and youth, that could benefit from MPH treatment do not receive it and risk lifelong disabilities associated with untreated ADHD. Therefore, understanding the mechanisms associated with development of MPH addiction and designing methods to prevent it assume high public health significance. Using a mouse model we show that supra-therapeutic doses of MPH produce rewarding effects (surrogate measure for addiction in humans) in a conditioned place preference paradigm and upregulate μ opioid receptor (MOPR) activity in the striatum and nucleus accumbens, brain regions associated with reward circuitry. Co-administration of naltrexone, a non-selective opioid receptor antagonist, prevents MPH-induced MOPR activation and the rewarding effects. The MPH-induced MOPR activation and rewarding effect require activation of the dopamine D1 but not the D2-receptor. These findings identify the MOPR as a potential target for attenuating rewarding effects of MPH and suggest that a formulation that combines naltrexone with MPH could be a useful pharmaceutical approach to alleviate abuse potential of MPH and other stimulants.

Detection of К-Opioid Receptor mRNA in Immature T Cells

Opioid receptors were first detected on the cells of the nervous system (1,2). Using radiolabeled ligands, cell lines developed from cells of the immune system have been shown to express the kappa opiate receptor (3,4). One example is the R1.1 T-thymoma developed from a C58/J mouse. This cell line has been shown to express the surface markers Thy 1.2 and H-2 k(5). This cell line has also been shown to express the kappa receptor by receptor binding assays (4). Little is known about the differentiation stage or functional capacity of this T cell line.