Platelet-mediated changes to neuronal glutamate receptor expression at sites of microthrombosis following experimental subarachnoid hemorrhage

Laboratory investigation

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Object

Glutamate is important in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Notably, brain extracellular and cerebrospinal fluid as well as blood glutamate concentrations increase after experimental and clinical trauma. While neurons are one potential source of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. This study investigates the hypothesis that platelet microthrombi release glutamate that mediates excitotoxic brain injury and neuron dysfunction after subarachnoid hemorrhage (SAH).

Methods

The authors used two models, primary neuronal cultures exposed to activated platelets, as well as a whole-animal SAH preparation. Propidium iodide was used to evaluate neuronal viability, and surface glutamate receptor staining was used to evaluate the phenotype of platelet-exposed neurons.

Results

The authors demonstrate that thrombin-activated platelet-rich plasma releases glutamate, at concentrations that can exceed 300 μM. When applied to neuronal cultures, this activated plasma is neurotoxic, and the toxicity is attenuated in part by glutamate receptor antagonists. The authors also demonstrate that exposure to thrombin-activated platelets induces marked downregulation of the surface glutamate receptor glutamate receptor 2, a marker of excitotoxicity exposure and a possible mechanism of neuronal dysfunction. Linear regression demonstrated that 7 days after SAH in rats there was a strong correlation between proximity to microthrombi and reduction of surface glutamate receptors.

Conclusions

The authors conclude that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might mediate brain injury after SAH.

Abbreviations used in this paper:BSA = bovine serum albumin; CNQX = 6-cyano-7-nitroquinoxaline-2,3-dione; DAB = 3,3′-diaminobenzidine; D-AP5 = D-(-)-2-Amino-5-phosphonopentanoic acid; FAST = Fast Analytical Sensing Technology; GluR2 = glutamate receptor 2; LTP = long-term potentiation; MEA = microelectrode array; PBS = phosphate-buffered saline; SAH = subarachnoid hemorrhage; RFU = relative fluorescence unit; SEM = standard error of the mean; TA-PrP = thrombin-activated plateletrich plasma.

Article Information

Current affiliation for Elliot Lass and Hoyee Wan: Faculty of Medicine, University of Toronto.

Address correspondence to: R. Loch Macdonald, M.D., Ph.D., Division of Neurosurgery, St. Michael's Hospital, University of Toronto, 30 Bond St., Toronto, ON M5B 1W8, Canada. email: macdonaldlo@smh.ca.

Please include this information when citing this paper: published online April 18, 2014; DOI: 10.3171/2014.3.JNS132130.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Quantification of thrombin-evoked glutamate release in rat plasma. Upper: Representative in vitro recording obtained during MEA recording (black line) and a sentinel site (gray line). The arrow denotes application of thrombin (1.5 U/ml). After approximately 60 seconds, glutamate is released during thrombus formation and the concentration spikes from 175 μM to a maximum amplitude of approximately 425 μM. Lower: Quantification of data. Baseline glutamate levels in plasma diluted in Tyrode's buffer (1:1) were 216.9 ± 48.5 μM. After addition of 1.5 U of thrombin, the maximum peak response of glutamate was 336.5 ± 73.0 μM; an average 120 μM increase in glutamate concentrations. Paired 2-tailed t-test: t5 = 4.2; p = 0.009, n = 6, *p < 0.05. Bar graph shows mean ± SEM (error bars).

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    Platelet-rich plasma increases neuronal cell death. Left: Representative images of Topro-3 (nuclear) and propidium iodide (dead or dying) nucleic acid stains. Propidium iodide staining is markedly increased following exposure of cultures to TAPrP, in concentrations of both 1:4 and 1:20 (platelet-rich plasma [PrP]/extracellular fluid [ECF]). Addition of glutamate receptor antagonists D-AP5 and CNQX attenuates cell death only modestly. 1-mM glutamate was used as a positive control. Bar = 200 μm. Right: Graphical representation of cell death across all treatment conditions, normalized to control.

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    TA-PrP markedly downregulates neuronal GluR2 expression. A: Representative 20× images of Topro-3 (nuclear) and GluR2 surface staining in cultured neurons. TA-PrP reduces surface GluR2 staining, an effect attenuated by concomitant glutamate receptor antagonism. Bar = 100 μm. B: Representative 63× oil immersion lens images of all treatment conditions. There is abundant surface staining in control neurons, but staining is reduced after exposure to the activated plasma. Return of staining is noted with glutamate receptor blockade. Bar = 10 μm. C: Graphical representation of GluR2 expression across all treatment conditions. **Significant reduction from control (p < 0.01). + Significant increase from plasma alone (p < 0.05). IOD = integrated optical density.

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    Subarachnoid hemorrhage in vivo reduces GluR2 staining adjacent to microthrombi. A: Neurons are from animals 6 days post-SAH. Representative images showing autofluorescent microthrombotic vessels (white circle), GluR2 DAB reactivity, and DAB masks that were generated in ImageJ to quantify integrated optical density (IOD) of GluR2 staining per cell. Note the marked reduction of staining near the thrombus, with recovery of signal as the neurons radiate outward. Scale bar = 50 μm. B: 40× images of the vessels shown in A. Two examples are shown of thrombotic vessels with both adjacent neurons (left panels, approximately 30 μm from the vessel) and neurons farther away (approximately 100 μm). There is a paucity of staining next to the vessels. C: No such relationship exists between neurons and nonthrombosed microvessels. There is abundant GluR2 staining both near and away from these vessels. Bars = 10 μm. D: Linear regression analysis demonstrated a marked relationship between surface GluR2 expression and distance from microthrombi (upper graph, R2 = 0.6985). There is no correlation between distance from clean vessels and GluR2 expression (lower graph, R2 = 0.0997).

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