Animals. Wild type C57BL/6J mice (WT) were purchased from The Jackson Laboratory (#000664, Bar Harbor, ME). Mice deficient for the SP-A gene (SP-A−/−) were obtained from Dr. Carole Mendelson [38] and were originally generated in the lab of Dr. Samuel Hawgood [39]. To reduce genetic differences in our wild type and SP-A−/− lines used in this study, backcrossing protocols were used as previously described [17]. All three neuroinflammatory models were performed in 7- to 10-day-old (P7-P10) mice as previous research has indicated that the brains of 7- to 10-day-old mice and rats are similar to third-trimester human fetuses (and thus premature human newborns) in regards to cellular proliferation and myelination, neuroanatomy, neurochemical indices, and neuroinformatics [40–43]. Pups were kept with their dams at all times, with the exception of the brief period during surgery. SP-A expression was evaluated in 6-week-old mice as previous research has indicated lower levels of SP-A in neonatal mice compared with older animals. Animals of both sexes were included in each experimental group in this study. A total of 145 pups were used in the study.
Sepsis model. P7 mice were injected intraperitoneally with lipopolysaccharide (LPS-EB Ultrapure; Escherichia coli; strain: 0111:B4; 100 µg/kg; InvivoGen, San Diego, CA) diluted to 0.1 µL/g. The dose of LPS (100 µg/kg) used in this study has been previously shown in Sprague-Dawley rats to produce mild fever [44], transient activation of cerebral microglia, and a long-lasting increase in hippocampal excitability [45]. Furthermore, peripheral LPS administration at doses of 20 µg/kg up to 100 µg/animal in mice have been shown to increase pro-inflammatory cytokine expression in the brain [46, 47]. Pups in the control group received the same volume of sterile phosphate-buffered saline (PBS). Pups were returned to their dams, then at 24 hours post-injection, pups were deeply sedated with isoflurane, cervically dislocated, and decapitated to harvest brain tissue.
IVH model. P7 mice received unilateral intraventricular hemoglobin injection using a modification of a previously described rat model of neonatal IVH [48]. Prior to IVH induction, pups were sedated throughout the procedure with isoflurane (5% for induction and 2–3% for maintenance). Pups were secured in a warmed stereotaxic frame using nonrupture ear bars (Stoelting, Wood Dale, IL). The scalp was prepped with 10% povidone-iodine, and a midline skin incision was made to expose bregma. Using a stereotaxic injector (Stoelting, Wood Dale, IL) equipped with a Hamilton syringe (model 701 RN, 30G, point style 4, removable needle), the right lateral ventricle was accessed at coordinates 1 mm lateral, 3 mm posterior, and 2 mm deep from bregma. Injections of 150 mg/ml of hemoglobin (MP Biomedicals, Irvine, CA) prepared in phosphate-buffered saline (PBS) or PBS alone (control group) were delivered at a rate of 6.67 µl per minute, until a total of 10 µl was injected. The syringe was left in place for an additional 1 minute to reduce retrograde flow upon removal. Incisions were closed with Vetbond tissue adhesive (3M Corp, St. Paul, MN), and animals were returned to their dams. All mice were then deeply sedated with isoflurane, cervically dislocated, and decapitated at 24 hours post-surgery to harvest brain tissue.
HIE model. Unilateral HIE was induced in P8-10 mice (weight 5–6 grams) using the modified Rice-Vannucci model [49, 50]. After sedation with isoflurane (4% for induction and 1.5-2% for maintenance) and local anesthesia with bupivacaine infiltration to minimize pain and distress, the surgical site was cleaned with 10% povidone-iodine and a midline cervical incision was made. The right common carotid artery was isolated and occluded through 8-Watt electrocoagulation. For mice in the sham groups, the carotid artery was visualized and isolated but not cauterized. The skin incision was closed with Vetbond tissue adhesive and infiltrated with additional local anesthesia. All mice were subjected to ischemic surgery within 5 minutes. Mice then received a subcutaneous injection of 0.3 mL normal saline to prevent dehydration during recovery. After surgery, the pups were kept warm using a temperature-controlled blanket and allowed to recover for 2 hours. Two control groups (True Sham and Sham + Hypoxia) were used in order to control for the effects of hypoxia alone. To induce hypoxia, the HIE and Sham + Hypoxia groups were placed in a chamber containing 10% oxygen and 90% nitrogen at 36°C for 45 minutes. After that, the animals were replaced on a temperature-controlled blanket for 20 min and then returned to their dams. The True Sham group remained in normoxia. All mice were then deeply sedated with isoflurane, cervically dislocated, and decapitated at 24 hours post-surgery to harvest brain tissue.
Assessment of SP-A and cytokine expression. Expression of SP-A was determined via standard reverse transcription PCR analysis, while expression of cytokines IL-1β, IL-6, CXCL1, TNF-α, and IL-10 was determined via real-time quantitative reverse transcription PCR (qRT-PCR) as described previously [17]. Brains were harvested, flash frozen in liquid nitrogen, then transferred to -80°C freezer until ready for RNA isolation. Brain tissue was mechanically homogenized then processed to isolate RNA using RNeasy Mini Kit (Qiagen, Germantown, MD) per manufacturer’s instructions. RNA concentration was quantified using TECAN (Infinite 200 Pro, Männedorf, Switzerland), then reverse transcription was performed for complimentary DNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). For cytokine analysis, qRT-PCR was performed using a LightCycler 480II System (Roche Diagnostics, Indianapolis, IN) and the iTaq Universal SYBR Green Supermix (Bio-Rad) per manufacturer’s instructions using primers specific for target mRNAs (see Table 1). Data was calculated by the comparative CT method (CT, threshold cycle) and each sample was duplicated to ensure accuracy. Cytokine expression was determined via replicated 2−ΔΔC(t) values and normalized to WT control values = 1 [51, 52]. For analysis of SP-A expression in tissue, standard RT-PCR was performed using primers specific for mouse SP-A and β-actin (see Table 1). Briefly, mRNA isolated from lung and brain tissue was subjected to reverse transcription to produce cDNA which was then subjected to 50 cycles of PCR. Negative controls were included in the analysis in which the reverse transcription step was eliminated.
Table 1
Sequence of primers used in RT-PCR analysis.
Mouse Target Gene | Forward Primer | Reverse Primer | Amplicon |
IL-1β | 5’-GCCACCTTTTGACAGTGATGAG | 5’-AAGGTCCACGGGAAAGACAC | 218 bp |
IL-6 | 5’-TAGTCCTTCCTACCCCAATTTCC | 5’-TTGGTCCTTAGCCACTCCTTC | 75 bp |
CXCL1 | 5’-CTGCACCCAAACCGAAGTC | 5’-AGCTTCAGGGTCAAGGCAAG | 66 bp |
TNF-α | 5’-CAGCCTCTTCTCATTCCTGC | 5’-GGTCTGGGCCATAGAACTGA | 132 bp |
IL-10 | 5’-GCTCTTACTGACTGGCATGAG | 5’-CGCAGCTCTAGGAA-GCATGTG | 104 bp |
SP-A | 5’-GTGCACCTGGAGAACATGGA | 5’-TGACTGCCCATTGGTGGAAA | 177 bp |
β-actin | 5’-CATGTACGTTGCTATCCA | 5’-CTCCTTAATGTCACGCAC | 249 bp |
18S | 5’-GTAACCCGTTGAACCCCATT | 5’-CCATCCAATCGGTAGTAGCG | 150 bp |
Statistical analysis. Cytokine mRNA expression was determined via one-way analysis of variance (ANOVA). Statistical significance was considered at p ≤ 0.05 for all statistical analyses. ANOVA was performed in Jamovi (Version 1.6.15, Sydney, Australia). Values are graphed as average mean ± standard deviation (SD).