276 - Short-term Neuropathological Effects of Azithromycin Treatment in a Ferret Model of Extremely Preterm Brain Injury

Publication Number: 4272.276

Shivani Jayaprakasam, University of Washington School of Medicine, Redmond, WA, United States; Olivia C. Brandon, University of Washington School of Medicine, Seattle, WA, United States; Kylie Corry, University of Washington School of Medicine, SEATTLE, WA, United States; Olivia Mohn, University of Washington School of Medicine, Seattle, WA, United States; Daniel Moralejo, University of Washington School of Medicine, Seattle, WA, United States; Sarah Kolnik, University of Washington - Seattle Children's Hospital, Seattle, WA, United States; Janessa Law, University of Washington School of Medicine, Seattle, WA, United States; Sandra E. Juul, University of Washington, Seattle, WA, United States; Thomas Wood, University of Washington, Seattle, WA, United States

Background: Perinatal hypoxia-ischemia (HI) in preterm infants alters cortical gyrification and white matter maturation, resulting in long-term neurodevelopmental disability. Ferrets have gyrified brains with white matter structure more comparable to humans than rodent models, providing a promising platform to model preterm HI-a condition for which no targeted therapies are currently available. Azithromycin (Az), an FDA-approved antibiotic with anti-inflammatory properties, has shown promise as a neuroprotective agent due to its ability to modulate microglial activation.

Objective: We hypothesized that Az would reduce microglial activation and preserve oligodendrocytes in a postnatal day (P)14 ferret model of HI, equivalent to extremely preterm neonates.

Design/Methods: Ferret kits received lipopolysaccharide (LPS), bilateral carotid artery ligation, and hypoxia-hyperoxia-hypoxia (hypoxia-ischemia/hyperoxia, HIH; O₂ at 8% and 80%) before being randomized to saline vehicle (n=20) or Az (n=23, 25 mg/kg IP daily for 5 days). At P21, morphometric analyses and quantitative immunohistochemistry for microglia (Iba-1) and oligodendrocyte lineage cells (Olig2) in cortex, subcortical white matter and corona radiata, corpus callosum, hippocampus, and thalamus were performed (Fig.1). Groups were compared using Kruskal-Wallis tests with Dunn's correction.

Results: Compared to heathy littermate controls, HIH increased composite global injury scores (comprising body weight gain, neuropathology scoring, brain morphometry, and Iba-1 staining intensity), with no detectable effect of Az treatment (Fig.2A). Pathology scores were significantly higher, and gyral widths, sulcal lengths, and body weight significantly lower, in both vehicle and Az-treated groups compared to controls (Fig.2B-F). Oligodendrocyte density was not significantly altered between groups (Fig.3A-D). When summed across white matter and cortical regions, Iba-1 staining was increased after HIH and lower in the Az group than in the vehicle group. This difference was not significant (Fig. 3E). Within individual cortical and white matter regions, Az treatment did not significantly reduce Iba-1 staining intensity (Fig. 3F-H).

Conclusion(s): Az treatment resulted in non-significant reductions in global Iba-1 staining intensity after an inflammation-sensitized HIH model in the extremely preterm-equivalent ferret, but there was no evidence of short-term global neuroprotection. Long-term studies including neurobehavioral testing, advanced imaging, and neuropathological assessments are required to determine whether Az may be neuroprotective in the gyrified preterm-equivalent brain.

Figure 1: Morphometric and Quantitative Immunohistochemistry (qIHC) Methodology.
(A) Top and side views of ferret brains for sulcal and gyral brain morphometry measurements. The brains shown are from control (left), vehicle-treated (middle) and Az-treated (right). Brains were then sliced coronally for qIHC staining. (B) Oligodendrocyte (Olig2, top row) and microglia (Iba-1, bottom row) staining in the left posterior gyrus of P21 ferret brain slices representing the median value in Control (I, IV), Vehicle (II, V), and Az-treated (III, VI) groups. All images are at 20× magnification. (C) Coronal Olig2 immunohistochemistry example from an animal in the Az group. Regions of interest were traced in VisioPharm to calculate the percentage of positive staining in each region.

Figure 2: Global Injury Indicators.
(A) A global injury score was derived from average ranks across whole-slice Iba-1 staining intensity, body weight, overall neuropathology score, summed gyral width, and summed sulcal length. Iba-1 (microglia) staining values were normalized by calculating fold differences for each experimental round and sex relative to the median of the corresponding control group. The global rank (0-4) reflects the extent of injury, with zero indicating values closest to the control median. Control animals had significantly lower global scores than vehicle and Az-treatment groups (p = 0.002, 0.001). (B) Global neuropathology averaging cortical lesions, mineralization, thalamus/midbrain ventricle enlargement, and hippocampal lesions, each ranked for severity (0-4 or 0-5). Control animals had significantly lower average pathology scores than the vehicle and the Az group (p = 0.003, 0.02). (C-D) Brain morphology measured as summed gyral widths (C) and summed sulcal lengths (D). HI resulted in significantly smaller gyri and sulci compared to controls (p < 0.001, 0.05). Az-treated animals also had lower measurements than controls (p = 0.002, 0.005). (E-F) Weight trajectories in female (E) and male (F) ferret kits. HI exposure resulted in weight loss and delayed weight increases, with vehicle and Az-treated groups consistently below uninjured controls. For all analyses, Kruskal-Wallis tests with Dunn's correction were used. Any p<0.05 was considered significant. Error bars represent median±IQR.

Figure 3: Regional quantification of oligodendrocytes (Olig2) and microglia (Iba-1) positive staining.
(A) Olig2 staining in white matter regions (corona radiata, subcortical white matter (SWM), and corpus callosum) showed no difference across groups. (B-D) No Olig2 staining differences were detected between control, vehicle, and Az-treated groups in the posterior gyri across the cortex (B), SWM (C), or corona radiata (D). (E) Iba-1 staining in white matter and cortical regions (corona radiata, SWM, corpus callosum, and cortex) showed that control animals had significantly lower gyral Iba-1 ranking than the vehicle and Az-treated groups (p < 0.001, 0.03). (F-H) In the posterior gyri, Az-treated and Vehicle animals were similar across the cortex (F), SWM (G), and corona radiata (H). Control animals had significantly lower Iba-1 staining than vehicle and Az groups (p < 0.001, 0.005). These statistical trends were similar across all other gyri (not shown). For all analyses, values were normalized as fold differences from the sex- and round-specific control median. Statistical testing was performed using Kruskal-Wallis tests with Dunn's correction. Any p<0.05 was considered significant. Error bars represent median±IQR.