Time Dependent Changes in the Ovine Neurovascular Unit; A Potential Neuroprotective Role of Annexin A1 in Neonatal Hypoxic-Ischemic Encephalopathy

Abstract

Perinatal brain injury following hypoxia-ischemia (HI) is characterized by high mortality rates and long-term disabilities. Previously, we demonstrated that depletion of Annexin A1, an essential mediator in BBB integrity, was associated with a temporal loss of blood-brain barrier (BBB) integrity after HI. Since the molecular and cellular mechanisms mediating the impact of HI are not fully scrutinized, we aimed to gain mechanistic insight into the dynamics of essential BBB structures following global HI in relation to ANXA1 expression. Global HI was induced in instrumented preterm ovine fetuses by transient umbilical cord occlusion (UCO) or sham occlusion (control). BBB structures were assessed at 1, 3, or 7 days post-UCO by immunohistochemical analyses of ANXA1, laminin, collagen type IV, and PDGFRβ for pericytes. Our study revealed that within 24 h after HI, cerebrovascular ANXA1 was depleted, which was followed by depletion of laminin and collagen type IV 3 days after HI. Seven days post-HI, increased pericyte coverage, laminin and collagen type IV expression were detected, indicating vascular remodeling. Our data demonstrate novel mechanistic insights into the loss of BBB integrity after HI, and effective strategies to restore BBB integrity should potentially be applied within 48 h after HI. ANXA1 has great therapeutic potential to target HI-driven brain injury.

Keywords: Annexin A1; blood-brain barrier (BBB) integrity; hypoxic-ischemic encephalopathy; neurovascular unit (NVU); therapeutic window.

Figure 1

(a) Schematic representation of the NVU including vascular cells (endothelial cells, pericytes), basement membrane, and astrocyte end-foot in the CNS; (b) structural illustration of the basement membrane components including laminin and collagen type IV, endothelial cells and the pro-resolving signature of Annexin A1 binding to the formyl peptide receptor 2 (FPR2). Created with Biorender.com.

Figure 2

ANXA1 and laminin qualitative scoring (a,c), and integrated density measurements (b,d) on 1, 3 and 7 days after HI insult in the cerebral microvasculature; (e) representative images of consecutive sections of cerebral blood vessels stained with ANXA1 and laminin. An average of 40 fields of view per animal were analyzed. Graphs show the average integrated density per field of view (mean grey value of stained area × percentage of stained area) for ANXA1 and laminin expression in coronal brain sections. Statistical analysis was performed with a Kruskal-Wallis test followed by Dunn’s post hoc test. Bars represent mean ± SEM. Scale bar 50 μm. 400× magnification * p < 0.05, ** p < 0.01, # p = 0.06.

Figure 3

Collagen type IV (a) qualitative scoring; (b) integrated density measurement on 1, 3 and 7 days after HI; (c) representative images of consecutive brain sections of cerebral blood vessels stained with collagen type IV. An average of 40 fields of view per animal were analyzed. Graphs show the average integrated density (mean grey value of stained area × percentage of stained area) of collagen type IV expression in coronal brain sections. Statistical analysis was performed using a Kruskal-Wallis test followed by Dunn’s post hoc test. Bars represent mean ± SEM. Scale bar 50 μm. 400× magnification * p < 0.05, # p =0.06.

Figure 4

Pericyte coverage surrounding brain microvasculature. (a) Qualitative scoring of PDGFRβ at 1, 3 and 7 days post HI or sham; (b) Representative microscopic images of PDGFRβ in the microvasculature at 3 and 7 days sham and HI treatment. Arrows indicate pericytes surrounding capillaries at 7d HI. Statistical analysis was performed using a Kruskal-Wallis test followed by Dunn’s post hoc test. Bars represent mean ± SEM. Scale bar 50 μm. 400× magnification * p < 0.05, # p = 0.06.

Figure 5

(a) Fetal sheep model with placental cotyledons (red dots) and umbilical cord (occluder) depicted; (b) Experimental design. Fetuses were instrumented at GA 102 (d-4) followed by 25 min of umbilical cord occlusion (UCO) or sham occlusion (d0) after four days of recovery. On d1, d3 and d7 fetuses were sacrificed and brain tissue was collected. Abbreviations: END—end of experiment; UCO—umbilical cord occlusion; GA—gestational age; HI—Hypoxia-Ischemia; SAL– saline; IN—instrumentation. Created with Biorender.com.

Figure 6

Scoring systems of ANXA1, laminin, collagen type IV, and PDGFRβ in cerebral blood vessels (1 = minor, 2 = moderate, 3 = intense immunoreactivity (IR), magnification 400×, scale bar 50 μm).