Altered Perivascular Aquaporin-4 Polarization and Laminin α1 Immunoreactivity in Canine Meningoencephalitis of Unknown Origin: A Preliminary Case–Control Brief Report

Meningoencephalitis of unknown origin (MUO) is a major neuroinflammatory disorder of dogs that is characterized by perivascular leukocyte cuffing, but methods to quantify the canine perivascular niche are lacking. This preliminary case–control report describes a dual-immunofluorescence and region-of-interest (ROI) approach to characterize the astrocyte–basement membrane interface in archival tissue. Formalin-fixed, paraffin-embedded cerebral cortex from two dogs with MUO (one granulomatous meningoencephalomyelitis [GME] and one necrotizing leukoencephalitis [NLE] pattern) and three neurologically normal controls was labeled for laminin α1 (LAMA1) and aquaporin-4 (AQP4) with 4′,6-diamidino-2-phenylindole (DAPI) counterstain, imaged by confocal microscopy, and analyzed in ImageJ/Fiji to calculate the perivascular-to-parenchymal AQP4 intensity ratio in 27 vessels. In controls, LAMA1 formed a continuous vascular outline associated with polarized perivascular AQP4. In MUO, the perivascular space was expanded by inflammatory cells, the LAMA1 outline was discontinuous or focally absent, and AQP4 was dispersed into the neuropil. Mean per-dog AQP4 ratios were lower in MUO (1.185 ± 0.129) than in controls (1.325 ± 0.138; mean ± standard deviation; exploratory Welch’s t-test, p = 0.350), a difference driven by the NLE case. Based on the observed effect size (Cohen’s d = 1.03), approximately 16 dogs per group would be needed for an adequately powered study. This approach provides a technical baseline for future studies of perivascular disruption in canine neuroinflammatory disease.