Hapln4 is a link protein which stabilizes the binding between lecticans and hyaluronan in perineuronal nets (PNNs) in specific brain regions, including the medial nucleus of the trapezoid body (MNTB). The aim of this study was: (1) to reveal possible age-related alterations in the extracellular matrix composition in the MNTB and inferior colliculus, which was devoid of Hapln4 and served as a negative control, (2) to determine the impact of the Hapln4 deletion on the values of the ECS diffusion parameters in young and aged animals and (3) to verify that PNNs moderate age-related changes in the ECS diffusion, and that Hapln4-brevican complex is indispensable for the correct protective function of the PNNs. To achieve this, we evaluated the ECS diffusion parameters using the real-time iontophoretic method in the selected region in young adult (3 to 6-months-old) and aged (12 to 18-months-old) wild type and Hapln4 knock-out (KO) mice. The results were correlated with an immunohistochemical analysis of the ECM composition and astrocyte morphology. We report that the ECM composition is altered in the aged MNTB and aging is a critical point, revealing the effect of Hapln4 deficiency on the ECS diffusion. All of our findings support the hypothesis that the ECM changes in the MNTB of aged KO animals affect the ECS parameters indirectly, via morphological changes of astrocytes, which are in direct contact with synapses and can be influenced by the ongoing synaptic transmission altered by shifts in the ECM composition.