e.g. compression of neural element after vascular damage (double the size of the primary) Secondary injury is initiated by primary injury s
Spinal cord lesion in rats. Cytokines are also involved – they trigger apoptosis Cellular components such as neutrophils, once at the site of injury, secrete cytokines and lytic enzymes
Mention Ally started all this work First: make gels. Then: stimuli... Glutamate is the main agonist Glycine: co-agonist
Microscope: 2 fluorescent filters
How quickly cells die – apoptosis/necrosis? compare cell death with other stimuli see PRARIY in cells protective activity? optimal dose of PRARIY Control
w/o FCS gels had better response to glutamate Longer incubation times – literature search Glutamate treated gels had less cells – response may be greater than seen
Say what things are e.g. FITC PRARIY is green... In figure 4 (A), peptide around the nucleus, bright green disc caveolae? Further investigation The CM DiI (red) shows the cytoplasm; green PRARIY in the cytosol Peptide is cytoplasmic, proving it enters the cell
reducing cell death by 27% This is still a work in progress
Glutamate vs 20 ug *; Glutamate vs 200 ng **; Glutamate vs 20 ng **;
At most doses... Not 20 ug
Others have used PRARI as the negative control
PRARIY results – rough estimates at the moment Time limitation... Ally also saw this neuroprotection
Some time between 6 and 24? Monitor cell viability after gels are set and throughout experiments. Investigate different cell culture media Then repeat the experiments outlined in the refined model and investigate the mechanism of PRARIY Question – delivery in human? Peptide implants that release the peptide. Investigate the mechanism and find the target... Question – concentration of glutamate in vivo? I used higher concentrations, there weren’t all the other stimuli etc Question – current treatments of SCI? Anti-inflammatory drugs + surgery to stabilise the patient
Aim : To investigate the activity of PRARIY in an in vitro model of spinal cord injury
Set up a culture system to investigate neuronal cell death.
Characterise the uptake and localisation of fluorescently labelled peptide PRARIY in neuronal cells.
Is this peptide protective?
Where is the peptide localised?
Compare neuroprotection seen with PRARIY to a scrambled control peptide YRPIRA, to investigate whether it is this particular sequence of amino acids in the peptide which confers the neuroprotective characteristics.
Approaches to creating a model of cell death in vitro
3D cell culture of 2 neuronal cell lines
Pure neuronal cultures
More reproducible than primary cultures
Traps the cells in collagen matrix/scaffold
Viability was assessed before setting in gels
10 mM Glutamate & 50 µM Glycine
Act on NMDA receptors and induce excitotoxicity
Glycine is a co-agonist of the NMDA receptor
Approaches to creating a model of cell death in vitro (2)
Other stimuli – induce cell death in the CNS
1 μg/mL TNF α – inflammatory stimulus
100 mM NMDA – excitotoxic stimulus
200 μg/mL Propidium iodide
Red stain for dead cells
1 μg/mL Hoechst
Blue stain for all cells regardless of viability
Approaches to creating a model of cell death in vitro
Figure 1 . Examples of staining with 200 μg/mL Propidium iodide (A) and 1 μg/mL Hoechst (B) in live/dead staining of collagen gels.
Time course death of B104 cells Differences in death were seen between control and treated gels, shown in figure 2 with the optimised protocol. At 24 and 48 hours, there were significant differences between control and treated. *** ***
Excitotoxic and inflammatory stimuli comparison – B104 Similar amounts of death were elicited by each of the stimuli. Cell death was seen with and without the presence of FCS. More death was seen without, so this was chosen as the treatment vehicle.
PRARIY localisation in B104 2D culture FITC-PRARIY entered the B104 cells. The peptide is largely cytoplasmic (figure 4 A and C), creating an orange colour with the co-localisation of the CM DiI. D Figure 4. B104 24hr Fluorescent peptide uptake. Hoechst with FITC-PRARIY (A), FITC-PRARIY only (B), an overlay of 3 images (C) and FITC-PRARIY with CM DiI (D). A B C D
PRARIY rescue of B104 cells PRARIY with glutamate had a significant effect on the amount of cell death by glutamate at 24 hours, but the level of death in the control was very high. *** ***
Dose response of B104 cells to PRARIY There was no dose-dependent effect seen with B104 cells but there were significant differences between glutamate treated gels and the different doses of the peptide. There could however be a dose-dependent effect with lower doses and further investigation is needed. * ** **
Dose response of NG108 cells to PRARIY Again there was no dose-dependent effect seen in NG108 cells, but PRARIY brought the levels of death to that seen in the control. This is the only NG108 experiment where the control levels of death has been significantly lower than that seen with glutamate. ** *** *** *
Initial comparison of PRARIY and YRPIRA No difference between the two peptides but both reduced glutamate levels of death however glutamate levels of death were not high. More investigation is needed but there was limited time to repeat these experiments etc. *** ***
Scrambled peptide YRPIRA elicits protection in NG108 cells There was a significant difference between the two peptides (n=4, P<0.05). Also PRARIY and YRPIRA significantly reduced cell death. And again control levels of death were lower than previously seen. ** *** *