Investigating biochemical aspects of stroke

There is a long-standing interest in understanding the biochemistry of stroke, including the effect of neuroprotective peptides and the ectopeptidases that regulate their activity. The problem is complex, as the transient decrease in blood flow to the brain, or an area of the brain, causes short-term changes that can lead to excess production of reactive oxygen species (ROS) which may be injurious, to alterations in the expression of various genes with concomitant changes in protein synthesis happening in hours, to longer term changes measured in days. We are pursuing two independent directions. One relates to the short-term production of ROS, and the long-noted differences in the susceptibility of certain neurons to stroke. Here, we are investigating the hippocampal formation's response to stroke-like conditions, oxygen-glucose deprivation and reperfusion (OGD-RP) using organotypic hippocampal slice cultures and GFP-based sensors for peroxide and the glutathione redox status (first developed by Remington and Tsien and improved by Meyer and Dick). The second area relates to longer-term changes. In particular we are focused on neuropeptides and how their influence on the well-being of neurons following stroke or stroke models may be controlled or altered. There is very little understanding of the role of ectopeptidases, membrane-bound peptidases that hydrolyze peptides in the extracellular space, in controlling the influence of neuropeptides in promoting or inhibiting neuronal recovery from an insult. Primarily, this is due to the lack of an appropriate method to determine the activity of these enzymes in functioning tissue.

The determination of ectopeptidase activity in organotypic hippocampal slice cultures using electroosmotic perfusion (Ou 2014).

Brain tissue has a significant zeta potential (Guy 2008; Guy 2009). Thus, electroosmotic flow can be induced in organotypic hippocampal slice cultures (Xu 2010). We use electroomosis to perfuse brain tissue with neuroprotective peptides – ex vivo in organotypic hippocampal slice cultures via electroosmotic push-pull perfusion, EOPPP, Hamsher 2013). Ectopeptidases hydrolyze the peptides into smaller peptides that may be active or inactive. Peptides are quantitated using capillary liquid chromatography (cLC). Peptidase activity is inferred from peptide quantitation and a computed peptide residence time in the tissue.

Left: apparatus. Source and sampling capillaries are positioned with one end in a buffer with an electrode to a voltage or current source and the other end positioned in the culture as shown on the right. Right: Peptide substrate, a D-amino acid analog as an internal standard, and a Texas Red-labeled 70 kDa dextran to visualize flow during each experiment are in the source probe. Upon initiating current flow, electrophoresis and electroosmosis proceed to carry the solutes through the tissue. A fraction of the solutes is collected (a higher fraction is collected if the diffusion process is slower, the current is higher, and the collection capillary is larger).

Left: apparatus. Source and sampling capillaries are positioned with one end in a buffer with an electrode to a voltage or current source and the other end positioned in the culture as shown on the right. Right: Peptide substrate, a D-amino acid analog as an internal standard, and a Texas Red-labeled 70 kDa dextran to visualize flow during each experiment are in the source probe. Upon initiating current flow, electrophoresis and electroosmosis proceed to carry the solutes through the tissue. A fraction of the solutes is collected (a higher fraction is collected if the diffusion process is slower, the current is higher, and the collection capillary is larger).

Redox sensors and fluorescence imaging

Goals: Understand the management of reactive oxygen species (ROS) in the neurons of rat OHSCs.

Grx1-roGFP2 sensor response under normal conditions, oxidizing conditions, and reducing conditions. Single pyramidal cells in organotypic hippocampal slice cultures.

Grx1-roGFP2 sensor response under normal conditions, oxidizing conditions, and reducing conditions. Single pyramidal cells in organotypic hippocampal slice cultures.

Approaches: Fluorescence imaging of NAD(P)H, and roGFP2-based sensors for hydrogen peroxide and glutathione (GSH) are used to monitor the redox status of single rat hippocampal neurons in an OHSC (Yin 2015). Pharmacological treatments are used in conjunction with these sensors to understand the pathway of ROS generation and removal.

Results: NAD(P)H/NAD(P)+ works in concert with GSH/GSSG and thioredoxin redox couples to combat increases in ROS levels.

 

 


Fast, online microdialysis-capillary LC of neurotransmitters

Microdialysis is a widely used method for assessement of changes in concentrations of chemical species in the extracellular space of tissue. Early work by Justices' group at Emory demonstrated that following changes in extracellular concentrations on the one-minute timescale was possible, and further that monitoring changes on the five-minute timescale online was also possible. However, until recently this was not pursued. Today, typical microdialysis sampling times are 20 minutes-per-data point (Yang 2013). We (Liu 2010; Zhang 2012; Zhang 2013; Gu 2015) and the Andrews group (Liu 2010; Yang 2013; Yang 2015). Sampling and analysis online and continuously at one minute-per-measurement provides abundantly more information than experiments that sample less frequently. We see oscillations in concentrations of serotonin when release is stimulated by retrodialysis of "high potassium" as shown in the figure. The figure represents a portion of a 16-hour continuous measurement.

Dialysis concentration vs time for one-minute (500 nL) samples from rat striatum. A sample chromatogram is shown in the inset.

Dialysis concentration vs time for one-minute (500 nL) samples from rat striatum. A sample chromatogram is shown in the inset.