50 - Hepatic Apoptotic and Inflammatory Responses Following Hypoxic–Ischemic Brain Injury in Neonatal Rats

Publication Number: 1043.50

Bingqing Wang, Women & Infants Hospital of Rhode Island, PROVIDENCE, RI, United States; Zexi Zang, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Benjamin Lee, Women & Infants Hospital of Rhode Island, White Plains, NY, United States; Jane Hwang, Women & Infants Hospital of Rhode Island, Providence, RI, United States; Darren Chen, Brown University, PROVIDENCE, RI, United States; Hang Xing, Women & Infants Hospital of Rhode Island, Providence, RI, United States; Shaolei Lu, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Xiaodi F.. Chen, Women & Infants Hospital of Rhode Island, Providence, RI, United States

Background: Perinatal hypoxic-ischemic (HI) injury not only damages the brain but also induces systemic metabolic and inflammatory disturbances. During HI encephalopathy (HIE), impaired perfusion and inflammatory activation can affect peripheral organs such as the liver. The liver plays a central role in metabolic regulation and immune homeostasis; thus, hepatic responses may contribute to the overall pathophysiology of HIE. Our previous bioinformatic analysis of liver-brain interactions following HI identified enrichment in inflammation- and metabolism-related pathways, such as SOCS3 signaling, prompting further evaluation of hepatic injury and key molecular markers after HI.

Objective: To examine hepatic histopathological changes and apoptotic and inflammatory markers following HI injury in neonatal rats.

Design/Methods: P7 rats were randomly assigned to Sham, HI-0H, HI-3H, or HI-24H groups. HI was induced by right carotid artery ligation and 8% O₂ for 120 min. Livers were collected 0 h, 3 h, and 24 h after HI. Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 10 µm. Sections were stained with hematoxylin & eosin (H&E) and immunolabeled for cleaved caspase-3, phosphorylated AMPKα (pAMPKα), and SOCS3. Quantification was performed using QuPath. Positive signals were normalized to total cell count. Statistical analysis was performed with one-way ANOVA following with Tukey's post hoc test.

Results: HE staining showed progressive hepatic degeneration and sinusoidal dilation after HI. Suzuki's scores increased from 4.8 ± 0.75 at 0 h to 6.1 ± 0.82 at 3 h and 10.0 ± 0.79 at 24 h (P < 0.001), indicating worsening hepatic injury over time. Cleaved caspase-3-positive cells increased at 3 h compared with Sham, HI-0H and HI-24H (P < 0.001). pAMPKα showed a downward trend across all HI groups, although the change was not statistically significant. SOCS3 expression decreased after HI (P < 0.0001 vs Sham) and remained suppressed at 24 h.

Conclusion(s): Hepatic injury severity increased progressively within 24 h after HI, while apoptotic activity was highest at 3 h during the 0-24 h observation window. SOCS3 expression decreased markedly after HI and remained suppressed at 24 h, indicating a persistent reduction in anti-inflammatory signaling. The inverse changes of SOCS3 and cleaved caspase-3 indicate a potential association between reduced SOCS3 signaling and increased hepatic apoptosis following neonatal HI injury, warranting further investigation.

Figure A. Hepatic histopathology and quantitative analysis following hypoxic-ischemic (HI) injury in neonatal rats. Figure B. Quantitative immunohistochemistry analysis of cleaved caspase-3 (C-CAS3), phosphorylated AMPKα (pAMPKα), and SOCS3.
Figure A Representative hematoxylin & eosin (H&E)-stained liver sections showing progressive hepatic degeneration and sinusoidal dilation in the HI-0H, HI-3H, and HI-24H groups (n=5) compared with Sham (Suzuki's score = 0, not shown). Histopathological severity was assessed using Suzuki's scoring system. Scores increased from 4.8 ± 0.75 at 0 h (HI-0H) to 6.1 ± 0.82 at 3 h (HI-3H) and 10.0 ± 0.79 at 24 h (HI-24H) (P < 0.001).
Figure B Representative liver sections (n =1-3) showing cleaved caspase-3-positive cells that increased at 3 h compared with Sham, HI-0H, and HI-24H (P < 0.001). SOCS3 expression decreased after HI (P < 0.0001 vs. Sham) and remained suppressed at 24 h. pAMPKα showed a downward trend across all HI groups without significant differences.