413 - Cytokine concentrations in brain and blood after exposure of neonatal rats to hypoxia-ischemia along with treatment with inter-alpha inhibitor proteins
Saturday, April 23, 2022
3:30 PM – 6:00 PM US MT
Poster Number: 413 Publication Number: 413.237
Liam M. Koehn, Women & Infants Hospital of Rhode Island, greensborough, Victoria, Australia; Xiaodi Chen, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Richard Tucker, Women & Infants Hospital of Rhode Island, Providence, RI, United States; Yow-Pin Lim, ProThera Biologics, Inc., Providence, RI, United States; Barbara Stonestreet, Women & Infants Hospital of Rhode Island, Providence, RI, United States
Postdoctoral Fellow Women & Infants Hospital of Rhode Island greensborough, Victoria, Australia
Background: Neonatal hypoxia-ischemia (HI) is a major cause of infant mortality and long-term cognitive impairment. Inter-alpha inhibitor proteins (IAIPs) are novel neuroprotective agents that attenuate brain injury and improve behavioral outcomes in neonatal rats exposed to HI. A potential mechanism of action for treatment with IAIPs could be the attenuation of increases in cytokine concentrations in the systemic circulation and central nervous system, thereby limiting the progression of inflammatory brain injury after HI.
Objective: To study: 1) the time course of changes in cytokine concentrations in serum and cortical brain homogenates after HI-related brain injury and 2) determine whether treatment with IAIPs (30 or 60 mg/kg) attenuates HI-related changes in serum and brain cytokines.
Design/Methods: Postnatal day-7 rats were randomly assigned to 4 groups: sham-placebo (PL), hypoxic/ischemic-PL (HI; right carotid artery ligation, 90 min 8% oxygen), HI-30 mg/kg or 60 mg/kg of IAIPs. IAIPs or PL were injected IP immediately after HI or sham-normoxia. Rats were culled at 6, 12, 24 or 36 h after HI or normoxia. Blood was centrifuged to produce serum and protein was extracted from right brain cortical homogenates. Cytokine measurements were performed using Bio-Plex Rat Cytokine 23-plex Assay (Biorad) on a Bio-Plex 200 system. Cytokines analyzed included interleukins (IL), interferons (IFN), chemokine C-C motif ligands (CCL), chemokine C-X-C motif ligands (CXCL), granulocyte colony stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF).
Results: IAIP treatment (30 and 60 mg/kg) attenuated (p < 0.05) increases in cytokine concentrations after HI in blood (6 h IL-5; 12 h IL-4, IL-5, IL-10; 24 h IL-5, G-CSF; Table 1) and brain (36 h IL-1α, IL-12, IFN-γ, M-CSF, VEGF; Table 2). Treatment did not attenuate changes to blood IL-1β (6-12 h) or brain CXCL1 (6-12 h), CCL3 (6-36 h), CCL20 (12 h) or CCL5 (36 h).Conclusion(s): Neonatal HI is associated with increases in cytokine concentrations that are tissue specific (blood or brain) and change over time after injury (6-36 h). A single injection of IAIPs attenuated the majority of cytokine related increases occurring in blood within the initial 24 h after HI and in brain at 36 h after HI. Dose-dependent effects were not observed. Therefore, treatment with IAIPs have specific immunomodulatory effects on cytokines in blood and brain after neonatal HI that could represent a component of the neuroprotective effects of treatment with IAIPs. Liam Koehn (7).pdf Mean right brain cortex cytokine fold changes 6, 12, 24 and 36 h after hypoxia-ischemia (HI).brainfigure2.jpegComparisons between sham-placebo and HI-placebo (HIPL), HI-30 mg/kg IAIP (30) or HI-60 mg/kg IAIP (60) are shown. Values are mean fold changes compared to sham-placebo (n=6-15). Measurements were made on a Bioplex200 (Biorad) using the Bio-Plex Pro Rat Cytokine 23-Plex Assay kit (Biorad). Cytokines listed are: chemokine C-X-C motif ligand 1 (CXCL1), chemokine C-C motif ligand 2 (CCL2), chemokine C-C motif ligand 3 (CCL3), chemokine C-C motif ligand 5 (CCL5), interferon-γ (IFN-γ), interleukin-1α (IL-1α), macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Bold red numbers indicate significant increases compared with sham-placebo, * p < 0.05. Bold blue numbers indicate significant decreases compared with HI-PL, ^ p < 0.05. Bold black numbers indicate both significant increases compared to sham but decreases compared to HI-PL. Statistical analysis was completed using generalized linear model least squares mean, adjusted for multiple comparisons using Tukey-Kramer.