Co‐localization of Isolectin B4 (IB4), Ionized Calcium‐Binding Adapter Molecule 1 (Iba1), and Von Willebrand Factor (vWF) Immunostaining in the Mid‐Cervical Spinal Cord After Spinal Injury

Abstract

IB4 immunostaining is often used to histologically identify thin fiber non‐peptidergic afferent neurons in the spinal cord. However, IB4 can also identify both endothelial cells and microglia. In studies of adult Sprague Dawley rats with chronic (12 weeks) cervical (C2) hemisection injury (Hx; N=10), we noted robust IB4 immunostaining in mid‐cervical dorsal root ganglia cell bodies as well as spinal cord lamina II, indicating labeling of thin fiber afferents. We also observed distinct clusters of IB4+ cells in the mid‐cervical spinal cord ipsilateral to Hx in the area of the corticospinal tract (N=6 of 10 rats), and in ventrolateral (N=9 of 10) and ventromedial white matter (N=9 of 10). Incubation of tissues with primary antibodies for both IB4 and Iba1 (which can label activated microglia) revealed clear co‐localization of staining in these regions. Spinal intact rats (N=5) had robust IB4 staining in lamina II, but staining was not observed in the corticospinal tract, or ventrolateral or ventromedial white matter. The IB4‐Iba1 co‐localization after Hx likely indicates neuroinflammation and degeneration of damaged tracts. We also observed that IB4 immunostaining broadly dispersed throughout white and gray matter, in a pattern consistent with blood vessel endothelium labeling. To verify this, tissues were co‐incubated with primary antibodies against IB4 and vWF, a marker of endothelial cells. Co‐localization of vWF and IB4 immunostaining was detected throughout the mid‐cervical cord, and IB4 staining intensity was decreased in ventral gray matter caudal to Hx (P<0.05 vs. spinal intact). Reduction of IB4 intensity is consistent with vascular damage and regional tissue hypoxia. We conclude that following chronic C2Hx in adult rats, immunostaining with IB4 can identify blood vessel endothelial cells and activated microglia.Support or Funding Information1R01HL139708‐01A1 (DDF), OT2 OD023854, T32‐HD043730 (MDS), K99 HL143207‐01 (KS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.