Study Design and Sampling:
This was an analytical case-control that was carried out on 142 infants born in Imam Khomeini, Chamran, and Shariati Hospitals in Tehran city, Iran. From 2020 to 2022 the samples were randomly selected and divided into two groups: the control group (preterm infants whose mothers had not any chorioamnionitis, n=78) and preterm infants whose mothers had chorioamnionitis (n= 64). The whole blood of the umbilical cord and blood from the heel were collected as soon as possible after delivery. The infants were followed to evaluate their intracranial hemorrhage in the first week after that they would evaluate by sonography to assess the extent of brain injury and also assess whether involved with epilepsy by aEEG.
The inclusion criteria were both groups were similar in relation to gestational age, premature infants (<37 weeks) with no history of genetic or metabolic and Coagulation disorders. Excluded criteria were infants with severe asphyxia with an Apgar score after 5 minutes of <4, major congenital malformations in the heart, lung, and neurologic signs, cancer, and blood disorder.
ELISA assay:
The plasma of umbilical cord YKL-40 concentration was determined in both groups by the YKL-40 ELISA kit (ab 255719) according to the manufacturer’s protocol. Protein concentrations were measured by using the Microplate Reader, MPR-H200BC.
Cell cultures and cell lines:
The Human Oligodendrocyte Progenitor Cells, Human Microglia Cells, Human Dendritic Cells, Human Astrocytes, and Human Neural Hindbrain Stem Cells (# ABC-TC3732, ABC-TC3704, ABC-TC3973, ABC-TC3969, and ABC-H0027X respectively) cell lines were obtained (accegen biotech, Fairfield, NJ, USA). All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; HyClone), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a 95% humidified air atmosphere with 5% carbon dioxide (CO2).
Transfection of miRNAs to selected cell lines:
In this study, we obtained miRNA-431, miRNA-34a, miRNA-21, and miRNA138 and the negative control (NC) miRNA (table 1) from Santa Cruz Biotechnology (California, USA). The miRNAs and 4 µl/ml of transfection medium; Lipofectamine TM2000 transfection reagent (Thermo Fisher Scientific, USA) were diluted separately in Opti-MEM I Medium (Thermo Fisher Scientific) and incubated for 20 minutes. The mixtures were added to the culture medium and cells, and the complete growth medium was then added and incubated in a CO2incubator.
Mice:
The mouse strain used was five- to six-week C57BL/6 mice from the geniran lab (Iran). All animal studies were approved by the Laboratory Animal Care and Use Committees of the Shahid Beheshti University and complied with the Institutional Animal Care and Use Committee (IAC UC) guidelines. All mice were housed under specific pathogen-free conditions and fed with regular chow and free to drink purified water. Also, mice were exposed to systemic isobaric hypoxia for 30 min, and then induced hypoxia mice were divided into 4 groups (group 1; mice under the condition of hypoxia without interference, group 2; mice with an injection of the miR-21-siRNAs-HVCN1 in GC4-targeted nanoparticles (37), group 3; mice with an injection of miR-138-siRNAs- HIF-1a in GC4-targeted nanoparticles, group 4; mice with an injection of the miR-21-siRNAs-HVCN1 and miR-138-siRNAs-HIF-1a were injected together) with 30 animals per group. In table 2 the sense of siRNAs that encoded in GC4-targeted nanoparticles with miRNAs was shown. Mice were anesthetized by isoflurane inhalation and the mixture of siRNAs 5μM in 200 μl PBS, 2G-(SNMe3I)11-FITC 15 mg/kg in 200 μl PBS, dendriplex 15 mg/kg in 200 μl PBS was injection through retro-orbital. Mice were sacrificed 72 hr post-treatment.
Evaluation of electroencephalographic (EEG):
Mice were given 5% isoflurane to induce profound anesthesia. The electrodes (Physiometrix Inc, North Billerica, MA, USA) were implanted into the prefrontal cortex (PFC) (three-shank, A4x4 recording sites, 100 μm spacing, 2.0 mm deep) and lateral entorhinal cortex (LEC) (one-shank, A1x16 recording sites, 100 μm spacing, 2 mm deep, 10° angle from the vertical plane) and olfactory bulb (OB) (one-shank, A1x16 recording sites, 50 μm spacing, 1.4–1.8 mm deep) (38).
Tissue preparation:
Mice were injected transcardially with 15 ml containing 0.2% of the vasodilator sodium nitrite, NaNO3, and 15 ml of fixative (4% paraformaldehyde, 0.2% picric acid in 0.16 M sodium phosphate buffer, pH 7.1). All this process was performed under deep anesthesia. After weighting the organs of brain, coronal and sagittal cryostat sections were collected on gelatin-coated slides and stored at −80°C.
Immunohistochemistry (IHC):
After washing tissue sections in 0.01 M phosphate-buffered saline (PBS) twice for 15 min at room temperature, submerged in 3% H2O2 in PBS for 45 min to block endogenous peroxidases. Tissue sections were incubated overnight at 4°C with the primary antibody solutions anti-rabbit HVCN1, S100b (1:200; #21008 Thermo fisher and #ab11178 Abcam), anti-mouse GFAP (1:500; #PA1-10004 Thermo fisher), anti-rabbit NeuN (1: 250; Cat #PA5-78499 Thermo fisher), rabbit anti-Caspase 3 (1: 200; #ab32351 Abcam), and anti-rabbit DAPI (1:500; #1700624 Life Technologies, CA, USA). All raised rabbits and each targeting the C-terminus of their respective antigen were used and washed in PBS for 15 min. The second antibody; the mouse glial fibrillary acidic protein (GFAP) marker was used (the second antibody was conjugated with fluorescein anti-rabbit IgG and combined with a biotin-conjugated sheep anti-mouse IgG). Fluorescence staining was observed using a Zeiss Axioplan fluorescence microscope.
Extraction of total RNA from whole blood:
The whole blood (300 μl) was added to 700 μl TriFast reagent (Roche Diagnostics GmbH, Mannheim, Germany) and stored at −80°C, and the whole blood was diluted 1:1 by RBC Lysis Buffer. The mixtures were centrifuged for 10 min in 50 ml reaction tubes at 1,000 rpm and 4°C and then were suspended in 1 ml TriFast reagent and ceramic beads, subjected to mechanical homogenization in the MagNA Lyzer instrument (Roche Diagnostics GmbH, Mannheim, Germany) at 6,000 rpm and stored at −80°C. Total RNA was extracted according to the manufacturer’s instructions kit (Roche Diagnostics GmbH, Mannheim, Germany).
Quantitative real-time PCR (qRT-PCR) assay:
Evaluation of the mRNA expression was performed by qPCR. First‐strand complementary DNA (cDNA) was synthesized using 0.8 µg of total RNA with 4 μl 5x reaction Buffer (Promega, Mannheim, Germany), 1 μl 20 U of Ribolock RNase Inhibitor (Fermentas, Vilnius, Lithuania), 1 μl Random Primers 50 mM (Invitrogen, Carlsbad, USA), 2 μl dNTPs 10 mM (Fermentas, Leon-Rot, Germany), 1 μl 200 U of MMLV H- Reverse Transcriptase (Promega), and using 0.4µM of each primer. The negative control was also added without an enzyme for prohibiting DNA contamination. Gene expression of 4 genes was quantified using the Rotor‐Gene 6000 thermal cycler (QIAGEN, Hilden, Germany).
The qRT-PCR was performed using primers specific for CHI3L1 (YKL-40), S100b, CACNA1A, Hv1 (HVCN1), and Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) as an internal control (table 3 and 4) using TaqMan real-time PCR. In short, RT-PCR was carried out using the 5 μL of Master Mix SyberGreen (10 μmol/L), 0.5 μL of each primer, and 0.8 μL of cDNA according to Real Q Plus Master Mix Green kit (Amplicon, Denmark). The melting curve analysis denaturation (95°C, 2min), cycling program (30 cycles: 95°C denaturation (20 s); 53°C annealing (30 s); 72°C elongation (30 s)) were performed using the Thermo-cycler ABI system. The threshold cycle was detected and the quantitative gene expression was calculated using the 2−△△Ct method.
Cell apoptotic analyses and flow cytometer (FCM):
For apoptosis detection, cells in the medium were collected after 48 h of treatment with miRNAs. Using an Annexin V Apoptosis Detection Kit using PE-conjugated Annexin V by Annexin V Apoptosis Detection Kit (Bio-Rad, California, USA), cells were stained with Annexin V-PE and propidium iodide (PI) according to the manufacturer’s instructions. Cells without transfecting miRNAs were used as the control for double staining. Cells were analyzed immediately using the BD FACSLyric FCM (Becton, Dickinson and Company, New Jersey, United States). For each measurement, at least 20,000 cells were counted.
Western blots and antibodies:
Blood of infants, tissue sections, and cell lines was lysed in 10 mM Tris pH 7.4 buffer containing 0.1% SDS, a protease inhibitor cocktail, and DNase (Promega). Primary antibodies used for western blot analysis were anti-rabbit YKL-40 (Abcam, ab255297), anti-rabbit HIF-1a (Abcam, ab243861), anti-rabbit HVCN1 (Sigma-Aldrich, #84329), anti-rabbit S100B (Abcam, ab52642), anti-mouse GABDH (Abcam, ab8245 ). Anti-rabbit IgG-HRP was added at room temperature and the bound proteins were visualized by Electrochemiluminescence (ECL) reagents and subsequent autoradiography.
Statistical Analysis:
An Independent sample T-test was used to compare the mean difference of YKL40, HIF, HVCN1, and S100b between preterm infants born without inflammation and preterm infants with inflammation. The One-way ANOVA and paired t-test were applied to evaluate the mean difference between intracranial hemorrhage and epilepsy with inflammation in mice and infants. Spearman correlation analysis was used to define possible correlations between YKL-40, S100B, HVCN1, and HIF-1a with epilepsy and intracranial hemorrhage. The Oligodendrocyte Progenitor, Microglia, Dendritic, Astrocytes, and Neural Hindbrain cell lines were evaluated using one-way ANOVA followed by Tukey's post hoc test. Statistical analyses were performed by using the SPSS version 22 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism v8.00 (GraphPad Software, San Diego, CA, USA).