Extracellular matrix (ECM) is a network of macromolecules which has two forms-perineuronal nets (PNNs) and a diffuse ECM (dECM)-both influence brain development, synapse formation, neuroplasticity, CNS injury and progression of neurodegenerative diseases. ECM remodeling can influence extrasynaptic transmission, mediated by diffusion of neuroactive substances in the extracellular space (ECS). In this study we analyzed how disrupted PNNs and dECM influence brain diffusibility. Two months after oral treatment of rats with 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan (HA) synthesis, we found downregulated staining for PNNs, HA, chondroitin sulfate proteoglycans, and glial fibrillary acidic protein. These changes were enhanced after 4 and 6 months and were reversible after a normal diet. Morphometric analysis further indicated atrophy of astrocytes. Using real-time iontophoretic method dysregulation of ECM resulted in increased ECS volume fraction α in the somatosensory cortex by 35%, from α = 0.20 in control rats to α = 0.27 after the 4-MU diet. Diffusion-weighted magnetic resonance imaging revealed a decrease of mean diffusivity and fractional anisotropy (FA) in the cortex, hippocampus, thalamus, pallidum, and spinal cord. This study shows the increase in ECS volume, a loss of FA, and changes in astrocytes due to modulation of PNNs and dECM that could affect extrasynaptic transmission, cell-to-cell communication, and neural plasticity.

• Keywords
• extracellular diffusion, extracellular matrix, extracellular transmission, hyaluronan synthase, perineuronal nets, plasticity,
• MeSH
• Astrocytes drug effects   MeSH
• Extracellular Matrix * drug effects   metabolism   pathology   MeSH
• Extracellular Space * drug effects   metabolism   MeSH
• Glial Fibrillary Acidic Protein metabolism   MeSH
• Hymecromone pharmacology   MeSH
• Rats MeSH
• Hyaluronic Acid metabolism   MeSH
• Brain * drug effects   metabolism   MeSH
• Nerve Net * drug effects   pathology   MeSH
• Rats, Sprague-Dawley MeSH
• Rats, Wistar MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glial Fibrillary Acidic Protein MeSH
• Hymecromone MeSH
• Hyaluronic Acid MeSH

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.

• Keywords
• Aging, Diffusion, Extracellular matrix, Extracellular space, Hapln4,
• MeSH
• Trapezoid Body metabolism   pathology   MeSH
• Diffusion * MeSH
• Extracellular Matrix Proteins deficiency   MeSH
• Extracellular Matrix metabolism   pathology   MeSH
• Extracellular Space metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Protein Deficiency metabolism   pathology   MeSH
• Organ Culture Techniques MeSH
• Peripheral Nerves metabolism   pathology   MeSH
• Nerve Tissue Proteins deficiency   MeSH
• Auditory Pathways metabolism   pathology   MeSH
• Aging metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Extracellular Matrix Proteins MeSH
• Hapln4 protein, mouse MeSH Browser
• Nerve Tissue Proteins MeSH

Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron-glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment.

• Keywords
• astrocytes, diffusion, extracellular matrix, extracellular space, tortuosity, volume fraction,
• MeSH
• Anisotropy MeSH
• Astrocytes pathology   MeSH
• Diffusion MeSH
• Extracellular Matrix physiology   MeSH
• Humans MeSH
• Cell Communication physiology   MeSH
• Synaptic Transmission physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

To understand the structural alterations that underlie early and late changes in hippocampal diffusivity after hypoxia/ischemia (H/I), the changes in apparent diffusion coefficient of water (ADC(W)) were studied in 8-week-old rats after H/I using diffusion-weighted magnetic resonance imaging (DW-MRI). In the hippocampal CA1 region, ADC(W) analyses were performed during 6 months of reperfusion and compared with alterations in cell number/cell-type composition, glial morphology, and extracellular space (ECS) diffusion parameters obtained by the real-time iontophoretic method. In the early phases of reperfusion (1 to 3 days) neuronal cell death, glial proliferation, and developing gliosis were accompanied by an ADC(W) decrease and tortuosity increase. Interestingly, ECS volume fraction was decreased only first day after H/I. In the late phases of reperfusion (starting 1 month after H/I), when the CA1 region consisted mainly of microglia, astrocytes, and NG2-glia with markedly altered morphology, ADC(W), ECS volume fraction and tortuosity were increased. Three-dimensional confocal morphometry revealed enlarged astrocytes and shrunken NG2-glia, and in both the contribution of cell soma/processes to total cell volume was markedly increased/decreased. In summary, the ADC(W) increase in the CA1 region underlain by altered cellular composition and glial morphology suggests that considerable changes in extracellular signal transmission might occur in the late phases of reperfusion after H/I.

• MeSH
• Astrocytes pathology   MeSH
• Cell Death MeSH
• Time Factors MeSH
• Diffusion MeSH
• Diffusion Magnetic Resonance Imaging MeSH
• Extracellular Space metabolism   MeSH
• Gliosis etiology   pathology   MeSH
• CA1 Region, Hippocampal pathology   physiopathology   MeSH
• Hypoxia complications   pathology   physiopathology   MeSH
• Immunohistochemistry MeSH
• Brain Ischemia complications   pathology   physiopathology   MeSH
• Microscopy, Confocal MeSH
• Rats MeSH
• Neuroglia pathology   MeSH
• Cell Count MeSH
• Rats, Wistar MeSH
• Cell Proliferation * MeSH
• Reperfusion MeSH
• Body Water metabolism   MeSH
• Imaging, Three-Dimensional MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is approximately 20% and the tortuosity is approximately 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain.

• MeSH
• Diffusion MeSH
• Extracellular Space chemistry   diagnostic imaging   physiology   MeSH
• Quaternary Ammonium Compounds MeSH
• Humans MeSH
• Brain Chemistry physiology   MeSH
• Brain cytology   physiology   MeSH
• Neuroglia physiology   MeSH
• Neurons physiology   MeSH
• Radionuclide Imaging MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Quaternary Ammonium Compounds MeSH
• tetramethylammonium MeSH Browser