Gut Microbiome Modification as a Novel Approach for Reversing Long-Term Inflammation After Neonatal Hypoxic Ischemic Encephalopathy

Janelle Marie Korf, BS (Advisors: Louise McCullough, MD, PhD; Bhanu Priya Ganesh, PhD)

Introduction: Neonatal hypoxic-ischemic encephalopathy (HIE) arises from disrupted perinatal cerebral oxygenation and perfusion. It is a leading global cause of infant death and disability which disproportionally affects males. Like adult stroke, HIE initiates a rapid influx of inflammatory cells across the blood brain barrier and a loss of microglial homeostasis. Persistent peripheral inflammation in children with a history of HIE is recognized but its ramifications, including accelerated aging, are poorly understood. As this coincides with critical stages of brain development, interventions started months or years post-injury may still improve survivors’ quality of life. One promising intervention is to manipulate the gut microbiome, which is instrumental in immune regulation. The aim of this dissertation is to assess how post-HIE changes in gut flora and systemic inflammation intersect with aging processes, and to evaluate if microbiome-directed therapies can ameliorate long-term outcomes.

Methods: C57BL/6J mice were used under specific pathogen-free (SPF) or germ-free conditions. Neonatal HI was induced via a modified Vannucci procedure in PND 7–10 pups, while adult stroke was modeled using middle cerebral artery occlusion (MCAO). Inflammation was characterized by flow cytometry and transcriptomics (NanoString). 16S rRNA sequencing was used for bacterial analysis of fecal samples and validating successful fecal microbiota transplantation (FMT). Mass spectrometry metabolomics measured alterations in tryptophan pathways.

Results: Aged mice displayed a distinct peripheral immune cell profile, characterized by an expanded population of CD11b^high B cells that trigger pro-inflammatory responses in microglia. This phenotype was reproducible in younger animals through via microbially derived factors or co-incubation with 'aged' peripheral immune cells. Next, aged GF mice exhibited lower inflammatory markers compared to SPF mice. FMT from aged mice into young mice recapitulated age-associated inflammation and behavioral deficits. In neonatal HI models, female mice had more acute inflammation (24 hours) which resolved more efficiently by one week. HI brain injury caused shift β-diversity at 24 hours, 3 weeks, and 2–5 months post-injury. This correlated with altered tryptophan metabolism, heightened inflammation, and behavioral deficits. Finally, FMT from healthy donors into adult mice with neonatal HI injury reversed dysbiosis, reduced inflammatory markers, and rescued anxiety-like behaviors. Conversely, FMT from an injured mouse into naïve mice induced similar pro-inflammatory and depressive-like phenotypes.

Conclusion: These findings demonstrate that chronic inflammation and gut dysbiosis persists well beyond the neonatal period following HIE and may overlap with natural aging processes to accelerate the emergence of 'aged' immune phenotypes. By correcting the microbiome imbalance with FMT, we successfully mitigated chronic inflammation and improved behavioral outcomes. This work highlights the gut–brain axis as a powerful therapeutic target, offering new avenues for intervention long after the initial brain injury and paving the way for novel treatments to enhance quality of life for HIE survivors.

Advisory Committee:

• Louise McCullough, MD, PhD, Chair
• Bhanu Priya Ganesh, PhD, Co-Chair
• Tina Oak Findley, MD 
• Laura Goetzl, MD
• Fudong Liu, MD
• Pamela Wenzel, PhD