Identification of microRNAs Involved in Cerebral Collateral Regulation
Identification of microRNAs Involved in Cerebral Collateral Regulation
Patients >18 years of age presenting to Ohio State Wexner Medical Center Emergency department
 with stroke symptoms, within 6 hours of last know well and found to have acute anterior
 circulation large vessel occlusion (LVO) will be included in this study. The purpose of this
 study is to evaluate the differential expression of exosomal microRNAs in patients with
 stroke due to acute LVO as compared to healthy controls. In addition, the investigators will
 also evaluate the differential expression of exosomal microRNA in patients with good vs poor
 collateral grade.
Patients aged >18 years presenting to the Emergency Department at the Ohio State University
 (OSU) Wexner Medical Center with acute ischemic stroke will be screened. The investigators
 will include patients with anterior circulation acute ischemic stroke secondary to sudden
 acute large vessel occlusion (LVO). Healthy subjects will be used as controls to assess
 miRNAs specific to stroke population. Since hemodynamically significant preexisting stenosis
 may result in alteration of miRNA expression, the investigators will exclude patients with
 >50% stenosis of internal carotid artery (ICA), >50% stenosis of bilateral vertebral arteries
 as well as patients with moderate to severe intracranial atherosclerotic disease as seen in
 CT Angiogram (CTA) of the head and neck. To minimize confounders, patients with known
 moderate to severe peripheral vascular disease or symptomatic coronary artery disease with
 history of stent or coronary artery bypass graft will also be excluded. Prisoners and
 Pregnant women will be excluded from this study as well.Differential expression of exosomal
 microRNAs in patients with acute LVO compared to healthy controls will be evaluated. In
 addition, a Neuroradiologist will grade the cerebral collateral circulation as (i) good or
 (ii) poor, after carefully reviewing CTA and CT perfusion (CTP) studies in each patient using
 validated collateral scoring method. Thirty patients with large vessel occlusion will be
 enrolled in this study in addition to 15 healthy controls (n=45). Blood samples will be drawn
 at 4 different time points from symptom onset for each patient: (i) 0-6 hours, (ii) 6-12
 hours, (iii) 12-24 hours and (iv) 5-7 days. Differential MiRNA expression will also be
 evaluated between the patients with varying collateral grades

 As the process of collateral maturation likely begins shortly after hemodynamic changes
 induced by LVO, the investigators plan to collect the first sample within 0-6 hours. Three
 blood samples will be collected during the first 24 hours to increase the chances of
 capturing the miRNA expression profile at the beginning of the collateral remodeling process.
 miRNA profiles of these patients will then be analyzed and compared to healthy volunteers. In
 addition, comparison will be done between patients with good and poor cerebral collateral
 grade.We will analyze the miRNA profiles of these patients using methods described below.

 Exosome Isolation and Characterization: Quantification of exosome number and size will be
 carried out using a NanoSight NS300 (Nanosight, UK) at the OSU CCC Analytical Cytometry Core
 as previously described. Briefly, exosomes will be isolated from cell-free plasma using Total
 Exosome Isolation Kit (Invitrogen) according to the manufacturer's instructions. Exosomes
 will be suspended in PBS buffer and stored at 4° C for up to 7 days. The exosome suspension
 will then be diluted to achieve a working concentration between 2 x 108 - 8 x 108 for use in
 the NanoSight NS300, after which exosomes will be counted and sized11.

 Total RNA Isolation: Total RNA will be isolated from exosomes suspended in PBS using the
 Circulating Nucleic Acid Extraction Kit (Qiagen) as previously described12. DNA contamination
 will be mitigated through use of Qiagen's RNase-free DNase set. RNA will be concentrated and
 purified using the RNeasy MinElute Cleanup Kit (Qiagen) as described.

 NanoString Assay: The multiplexed nanoString nCounter miRNA system (nanoString Technologies)
 at The Ohio State University CCC Genomics Shared Resource will be used for miRNA expression
 profiling as described12. Total RNA (100ng) isolated from exosomes will be used as input
 material. Small RNA samples will be prepared by ligating a specific DNA tag onto the 3'-end
 of mature miRNAs according to manufacturer's instructions (nanoString Technologies). These
 tags will normalize the melting temperatures of the miRNAs and provide identification for
 each miRNA species in the sample. Excess tags will be washed away, and the resulting material
 hybridized with miRNA:tag-specific nCounter reporter probes. Hybridized probes will then be
 purified and immobilized on a streptavidin-coated cartridge using the nCounter Prep Station
 (nanoString Technologies). The nCounter Digital Analyzer will be used to count individual
 fluorescent barcodes and quantify miRNA molecules present in each sample.

 miRNA Expression Profile Normalization and Analyses: Quality control, normalization, and data
 analyses will be performed using nSolver 2.0 Analysis Software (nanoString Technologies) as
 previously described. Hierarchical clustering of miRNAs identified through the nanoString
 query will be conducted using dChip (v 1.3) software. Differentially expressed miRNAs in
 patients with good or poor collateral scores (as described above) will be defined by a
 greater than or less than 2-fold change using one-way analysis of variance (ANOVA) assay with
 a significance level of p<0.05 and with correction for false discover rate.

 RT-PCR Validation of Exosomal miRNAs: Select miRNAs isolated from exosomes will be reverse
 transcribed using the miRCURY LNA Universal cDNA synthesis kit (Exiqon) according to the
 manufacturer's protocol as described previously. miRNAs will be quantified by real-time PCR
 using ExiLENT SYBR Green (Exiqon) on a Mx3000P qPCR platform, with expression normalized to a
 housekeeping reference (i.e. SNORD44).
Inclusion criteria:

 1. Age 18 years or older.

 2. Diagnosis of Acute Ischemic Stroke secondary to LVO

 3. LVO from presumed embolic source (<50% ICA)

 4. CT perfusion/RAPID images showing salvageable penumbra (evaluated by PI)

 Exclusion:

 1. >50% stenosis of internal carotid artery

 2. >50% stenosis of bilateral vertebral arteries.

 3. Patients with moderate to severe intracranial atherosclerotic disease as seen in CTA
 of head and neck.

 4. Known moderate to severe PVD or symptomatic CAD

 5. Past medical history of stent or coronary artery bypass surgery

 6. Prisoners

 7. Pregnant women

 8. Previous stroke within 30 days

 9. Intracranial vascular malformation or evidence of moya-moya disease

 10. Serious advanced or terminal illness per judgment of the investigator with life
 expectancy <1yr

 11. Known allergy to iodine that precludes CTA or CTP studies

 12. Presumed septic embolus or suspected bacterial endocarditis