Aug
12
Mon
2013
Invited Talk: Functional MR Imaging of the brain: An Overview
Aug 12 @ 11:51 am – 12:17 pm

claudiaClaudia AM Wheeler-Kingshott, Ph.D.
University Reader in Magnetic Resonance Physics, Department of Neuroinflammation, UCL Institute of Neurology, London, UK


Abstract

Detecting neuronal activity in vivo non-invasively is possible with a number of techniques. Amongst these, in 1990 functional magnetic resonance imaging (fMRI) was proposed as a technique that has a great ability to spatially map brain activity by exploiting the blood oxygenation level dependent (BOLD) contrast mechanism [1, 2]. In fact, neuronal activation triggers a demand for oxygen and induces a localised increase in blood flow and blood volume, which actually exceeds the metabolic needs. This in turns causes an increase of oxyhaemoglobin in the venous compartment, which is a transient phenomenon and is accompanied by a transient change (decrease) in the concentration of deoxyhaemoglobin. Due to its paramagnetic properties, the amount of deoxyhaemoglobin present in the venous blood affects the local magnetic field seen by the spins (protons) and determines the local properties of the MR signal. A decrease in deoxyhaemoglobin during neuronal activity, therefore, induces local variations of this magnetic field that increases the average transverse relaxation time of tissue, measured via the T2* parameter [3]. This means that there is an increase of the MR signal (of the order of a few %, typically <5%) linked to metabolic changes happening during brain function. Activation can be inferred at different brain locations by performing tasks while acquiring the MR signal and comparing periods of rest to periods of activity.

The macroscopic changes of the BOLD signal are well characterised, while the reason for the increased blood supply, exceeding demands, needs further thoughts. Here we will discuss two approaches for explaining the BOLD phenomenon, one that links it to adenosine triphosphate production [4] and enzyme saturation, the other that relates it to the very slow diffusion of oxygen through the blood-brain-barrier with a consequent compensatory high demand of oxygen [5]. Some evidence of restricted oxygen diffusion has been shown by means of hypercapnia [6], although it is not excluded that both mechanisms may be present.

Overall, the BOLD signal changes theory and its physiological basis will be presented and discussed.

References

  1. Ogawa, S., et al., Brain magnetic resonance imaging with contrast dependent on blood oxygenation. Proc Natl Acad Sci U S A, 1990. 87(24): p. 9868-72.
  2. Kwong, K.K., et al., Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation. Proc Natl Acad Sci U S A, 1992. 89(12): p. 5675-9.
  3. Bandettini PA, et al. Spin-echo and gradient-echo EPI of human brain activation using BOLD contrast: a comparative study at 1.5 T. NMR Biomed. 1994 Mar;7(1-2):12-20
  4.  Fox, P.T., et al., Nonoxidative glucose consumption during focal physiologic neural activity. Science, 1988. 241(4864): p. 462-4.
  5. Gjedde, A., et al. Reduction of functional capillary density in human brain after stroke. J Cereb Blood Flow Metab, 1990. 10(3): p. 317-26.
  6. Hoge, R.D., et al., Linear coupling between cerebral blood flow and oxygen consumption in activated human cortex. Proc Natl Acad Sci U S A, 1999. 96(16): p. 9403-8.

Aug
13
Tue
2013
Delegate Talk: Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes
Aug 13 @ 3:53 pm – 4:06 pm
Delegate Talk: Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes

Pandiaraj Manickam, Niroj Kumar Sethy, Kalpana Bhargava, Vepa Kameswararao and Karunakaran Chandran


Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes

Release of cytochrome c (cyt c) from mitochondria into cytosol is a hallmark of apoptosis, used as a biomarker of mitochondrial dependent pathway of cell death (Kluck et al. 1997; Green et al. 1998). We have previously reported cytochrome c reductase (CcR) based biosensors for the measurement of mitochondrial cyt c release (Pandiaraj et al. 2013). Here, we describe the development of novel label-free, immunosensor for cyt c utilizing its specific monoclonal antibody. Two types of nanocomposite modified immunosensing platforms were used for the immobilization of anti-cyt c; (i) Self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in conducting polypyrrole (PPy) modified screen printed electrodes (SPE) (ii) Carbon nanotubes (CNT) incorporated PPy on SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy (SEM). Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were used for probing the electrochemical properties of the nanocomposite modified electrodes. Method for cyt c quantification is based on the direct electron transfer between Fe3+/Fe2+-heme of cyt c selectively bound to anti-cyt c modified electrode. The Faradaic current response of these nanoimmunosensor increases with increase in cyt c concentration. The procedure for cyt c detection was also optimized (pH, incubation times, and characteristics of electrodes) to improve the analytical characteristics of immunosensors. The analytical performance of anti-cyt c biofunctionalized GNP-PPy nanocomposite platform (detection limit 0.5 nM; linear range: 0.5 nM–2 μM) was better than the CNT-PPy (detection limit 2 nM; linear range: 2 nM-500nM). The detection limits were well below the normal physiological concentration range (Karunakaran et al. 2008). The proposed method does not require any signal amplification or labeled secondary antibodies contrast to widespread ELISA and Western blot. The immunosensors results in simple and rapid measurement of cyt c and has great potential to become an inexpensive and portable device for conventional clinical immunoassays.

Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall
Aug 13 @ 6:39 pm – 6:49 pm
Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India

Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi


Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.

Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.

Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.

Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.