Chorioamnionitis (CHORIO), placental insufficiency, and preterm birth are well-known antecedents of perinatal brain injury (PBI). Heme-oxygenase-1 (HO-1) is an important inducible enzyme in oxidative and inflammatory conditions. In the brain, HO-1 and the iron regulatory receptor, transferrin receptor-1 (TfR1), are known to be involved in iron homeostasis, oxidative stress, and cellular adaptive mechanisms. However, the role of HO pathway in the pathophysiology of PBI has not been previously studied. In this study, we set out to define the ontogeny of the HO pathway in the brain and determine if CHORIO changed its normal developmental regulation. We also aimed to determine the role of HO-1/TfR1 in CHORIO-induced neuroinflammation and peripheral inflammation in a clinically relevant rat model of PBI. We show that HO-1, HO-2, and TfR1 expression are developmentally regulated in the brain during the perinatal period. CHORIO elevates HO-1 and TfR1 mRNA expression in utero and in the early postnatal period and results in sustained increase in HO-1/TfR1 ratios in the brain. This is associated with neuroinflammatory and peripheral immune phenotype supported by a significant increase in brain mononuclear cells and peripheral blood double negative T cells suggesting a role of HO-1/TfR1 pathway dysregulation in CHORIO-induced neuroinflammation.

Introduction: Inflammatory oxidative microenvironments can alter heme oxygenase-1 (HO-1) homeostasis. Dysregulation of HO-1 favors proinflammatory signals, while transferrin receptor 1 (TfR1) regulates anti-inflammatory signal transduction. Previously, we have shown that chorioamnionitis (CHORIO) induces sustained elevations in HO-1/TfR1 at postnatal day (P)2 and mononuclear cell-driven inflammation at term age equivalent P7. Here, we hypothesized that an altered HO-1/TfR1 developmental time course would coincide with inflammatory/immune signal changes in the brain. Methods: To induce CHORIO in rats, we performed a laparotomy followed by bilateral transient uterine artery occlusion and intra-amniotic injection of lipopolysaccharide (LPS) at E18. The control group received laparotomy only with equivalent duration of anesthesia. We used real-time polymerase chain reaction (RT-PCR), multiplex electrochemiluminescent immunoassay (MECI), and flow cytometry (FC) to study changes in pro- and anti-inflammatory gene expression, immune cell secretome, and immune cells at critical and clinically relevant timepoints following CHORIO. Results: We found an acute reduction in anti-inflammatory signals in the cortex on embryonic day (E)19. This was followed by an increased proinflammatory signature on postnatal day (P)2. There were also significant alterations in the phenotypic distribution of splenic T cells on P7, a key organ in immune function. Furthermore, we showed that the microenvironment of the cortex at P21 was skewed toward a proinflammatory state by significant increases in IL-6. The prominence of T-helper cells (Th) in brain concomitant with a proinflammatory state at P21 suggests emerging inflammation and potential for neural injury. Conclusions: Defining how these and other inflammatory/immune signatures contribute to perinatal brain injury and investigating distinct immune signatures at more developmental time courses will be beneficial for targeting emerging therapies.

Background: Cutting-edge neonatal programs diagnose cerebral palsy (CP) or “high risk of CP” using validated neurobehavioral exams in combination with risk history and neuroimaging. In rat models, digital gait analyses are the gold standard adult assessment, but tools in infant rats are limited. Refinement of infant rat neurobehavioral correlates of CP will establish translational behavioral biomarkers to delineate early mechanisms of CP in both humans and rodent models of CP. Objective: To facilitate precision medicine approaches of neurodevelopmental health and integrate basic and clinical research approaches for CP, we developed and piloted a new assay of neonatal rat neurobehavior to mimic human neonate exams. Methods: Our established rat model of CP secondary to chorioamnionitis (CHORIO) that induces bilateral motor impairment reminiscent of spastic CP was used. On postnatal day 10 (P10), 5 min videos were recorded of 26 (6 sham and 20 CHORIO) animals moving freely in a cage were reviewed by an evaluator trained in the human General Movements Assessment (GMA). Non-blinded observation revealed two behaviors that differed between rat pups in each group (time spent rearing; multi-dimensional nose sweeping; and sniffing). Each video was re-coded for these criteria by an evaluator blind to group status. Differences between sham and CP groups were analyzed using a Mann–Whitney U-test or Student’s t-test (p < 0.05 level of significance). Results: Neonatal rats with CP exhibited sensorimotor impairment and decreased spatial exploration. CP rats spent significantly less time rearing (17.85 ± 1.60 s vs. 34.8 ± 2.89 s, p = 0.007) and engaged in multi-dimensional nose sweeping and sniffing (2.2 ± 0.58 episodes vs. 5.5 ± 0.96 episodes, p = 0.03) than sham controls. Conclusions: These pilot findings of harmonized translational and precision biobehavioral assays provide an opportunity for increased expediency of clinical trials at the earliest stages of brain development.