Glutamate Receptor Peptides as Potential Neurovascular Biomarkers of Acute Stroke

Abstract

In this chapter different scenarios of biomarkers evaluating ischemic stroke caused by small vessel occlusions and leading to cerebral infarction are considered. Results of assays detecting glutamate receptor (GluR) peptides alone or combined into biomarkers panel to assess microvessel and small vessel strokes are explored in case report studies. A clinical protocol of neurovascular biomarkers implying GluR peptides is suggested for translational research assessing the severity of acute ischemic events based on structural location of cerebral infarction. It is proposed that the combination of clinical, biochemical, and radiological data might increase the diagnostic certainty of suspected acute ischemic stroke due to small occlusions in selected patients for timely and personalized therapy.

Appendices

Appendix 1: Case-Report Studies of NR2 Peptide Contents in Patients with Preexisting Conditions

Cardioembolic stroke. A 67-year-old female with prior aortic valve replacement surgery and diabetes mellitus (DM, 227 mg/dL glucose) was admitted to ER within 2 h of stroke onset (NIH stroke score 10) with ischemic lesions in right middle cerebral artery area (cardioembolic subtype) seen on CT (Fig. 3a). The measurement of NR2 peptide assay yielded in 4.71 ng/mL (preliminary cutoff of 0.5 ng/mL) depicted the acute cerebral ischemia.

Atherothrombotic stroke. A 82-year-old male presented with a history of stenosis of right (40%) and occlusion in left carotid artery, motor aphasia, dysphagia, and right hemiparesis (NIH stroke scale 10). He presented within 10 h post-symptom onset and had head CT that showed acute ischemic changes in territory of left middle cerebral artery (thrombotic subtype, Fig. 3b). As a part of an ongoing clinical study a blood sample was taken while he had the neurological deficit and showed elevated NR2 peptide of 2.34 ng/mL.

Stroke of unknown origin in vertebral region. A 52-year-old female patient presented after series of TIA within 2 weeks with last resolved with symptoms of dysarthria and hemihypesthesia within 12 h since onset. The CT was normal, and MRI showed acute lacunar infarct in the posterior-left sections of the Varoliev Bridge of hindbrain with hyperintensities on DWI and T2 (Fig. 3c, d). The major function of Varoliev Bridge is to pass nerve impulses to the cortex and to the spinal cord. NR2 peptide was 1.68 ng/mL and about three times higher than the normal levels in healthy controls.

Stroke due to neurovasculitis. Neurovasculitis and renal disorders may show elevated NR2 biomarker in certain cases due to NMDA receptors response to microvessel inflammation [43]. A 21-year-old woman “crystal” methamphetamine user presented with headaches and visual complaints and a T2-weighted MRI scan showed multiple bilateral subcortical areas of white matter abnormality. This was followed by a worsening in condition that resulted in admission to the hospital with left hemiparesis and delirium. MRI scans after admission showed acute right hemispheric infarction and diffuse vasospasm of cerebral vessels without the beading associated with typical vasculitis on magnetic resonance angiography (Fig. 4a). Initial ischemic lesion in left hemisphere revealed by routine CT (Fig. 4b) progressed further (Fig. 4c). Time course of NR2 peptide obtained in this case showed significant and prolonged elevation within a week suggesting an ongoing ischemic process (Fig. 4d).

Fig. 4

Acute stroke due to neurovasculitis. (a) MRA brain on admission depicts vasospasm that creates large areas of the brain with no visible vessels (arrows), (b) routine CT scan on admission, (c) FLAIR shows region of stroke, (d) NR2 peptide monitoring (cutoff of 0.5 ng/mL)

Appendix 2: Case-Report Studies of AMPAR Peptide Contents in Patients with Subcortical Strokes Including Lacunar Lesions

Lacunar strokes. A 43-year-old hypertensive Afro-American woman presented with sudden symptoms of left-sided weakness, incoordination, unsteadiness, cerebellar ataxic dysarthria, and dysphonia. The apparent diffusion coefficient (ADC) sequence shows an acute bilateral pontine infarct (Fig. 5a). Drastically increased NR2 (9.32 ng/mL) and AMPAR (7.03 ng/mL) peptides were detected in the patient’s plasma on admission.

Fig. 5

Radiological findings for case reports (AMPAR peptide). (a) ADC sequence of lacunar stroke, (b) CT of acute IS in vertebral basilar area, (c) T2-weighted FLAIR of acute IS after snowboarding accident, (d) FLAIR and T2-weighted images for the same patient with hemorrhagic transformation

Stroke in vertebral basilar area. A 59-year-old Caucasian male patient presented with a history of hypertension and symptoms of left hemiparesis, imbalance during walking, facial asymmetry, and double vision within 15 h after the onset. The CT showed acute infarct in vertebral basilar area with multiple arachnoid cysts in basal nuclei in deep frontal lobe and brainstem (Fig. 5b). The elevated NR2 (1.8 ng/mL) and AMPAR (3.1 ng/mL) peptides were measured compared to that for controls (preliminary cutoffs are 0.5 and 0.4 ng/mL, respectively).

Ischemic stroke after snowboarding accident. The case of multiple concussions resolved in stroke after snowboarding accident with white matter lesions depicted on Fig. 5c. A 22-year-old Caucasian male had a seizure on the day of the accident with following admission to hospital. The transformation from initial normal CT image to subcortical lesions in left hemisphere was observed within the next 48 h and accompanied increased AMPAR peptide (1.6 ng/mL at preliminary cutoff 0.4 ng/mL) and control level of NR2 peptide (0.5 ng/mL) that could be associated with cytotoxic edema formation in subcortical structures with presumably AMPAR localization (Figs. 1b and 5c).

Hemorrhagic transformation. The contusion acquired by a 31-year-old Caucasian woman occurred after a car accident and an impact with a tree. She had acute symptoms of incoordination, low alertness and was hyporeflexic. The subcortical areas of hemorrhage are seen on axial Flair and T2-weighed scans registered on seventh day after the injury (Fig. 5d). AMPAR peptide concentration of 7.2 ng/mL and NR2 peptide of 2.0 ng/mL are measured compared to non-injured controls (0.5–1.0 ng/mL range).

Appendix 3

1.1 Preliminary Critical Values of NR2 Peptide

Samples from apparently healthy males/females (52 M/102 F) and persons (42 M/35 F) with preexisting conditions (Table 2) in the clinically relevant age range of 30–70 years were evaluated using the NR2 peptide assay.

Table 2 The distribution of persons with preexisting conditions for NR2 peptide assay

Table 3 The distribution of NR2 peptide across enrolled population

1.2 AMPAR Peptides Preliminary Reference Values

Total of 128 apparently healthy males/females (75 M/52 F) including 53 persons (28 M/25 F) with preexisting conditions (Table 4) in the clinically relevant age range of 30–70 years included to evaluate reference interval for AMPAR peptide assays.

Table 4 The distribution of persons with preexisting conditions for AMPAR peptide assay

Table 5 The distribution of AMPAR peptides across population investigated

Appendix 4: Sample Size Calculations

The sample size calculation for the first phase is based on the following assumptions:

• 1. The prevalence rate of ischemic events in the enrolled cohort is 80%.

• 2. A transformation of each peptide measure is normally distributed for trial participants with and without ischemic events.

It is assumed that the standard deviation for the non-ischemic participant is one and the counterpart for the ischemic participants is d₀ without loss of generality. With n participants per time window in the first phase, the following table provides the standard error for the ${\mathrm{pAUC}}_{0.75}^{0.95}$ estimate for each time window and the standard error of the average ${\mathrm{pAUC}}_{0.75}^{0.95}$ (displayed in the parenthesis) when the true ${\mathrm{spAUC}}_{0.75}^{0.95}$ is 0.90.

Results summarized in the Table 6 suggest that a sample size n = 100 per time window is adequate for the first phase of the trial with a power of no less than 86% for the above settings. With n = 100 per time window, the 95% confidence interval for the average ${\mathrm{spAUC}}_{0.75}^{0.95}$ across the 3-time windows is expected to be (0.84, 0.96), which is sufficiently tight clinically.

Table 6 Power of study calculations