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
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
Extrasynaptic volume transmission, mediated by the diffusion of neuroactive substances in the extracellular space (ECS), plays an important role in short- and long-distance communication between nerve cells. The ability of a substance to reach extrasynaptic high-affinity receptors via diffusion depends on the ECS diffusion parameters, ECS volume fraction alpha (alpha=ECS volume/total tissue volume) and tortuosity lambda (lambda2=free/apparent diffusion coefficient), which reflects the presence of diffusion barriers represented by, e.g., fine astrocytic processes or extracellular matrix molecules. These barriers channel the migration of molecules in the ECS, so that diffusion may be facilitated in a certain direction, i.e. anisotropic. The diffusion parameters alpha and lambda differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Changes in diffusion parameters have been found in many physiological and pathological states, such as development and aging, neuronal activity, lactation, ischemia, brain injury, degenerative diseases, tumor growth and others, in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances and thus extrasynaptic transmission, neuron-glia communication, mediator "spillover" and synaptic crosstalk as well as, cell migration. The various changes occurring during pathological states can be important for diagnosis, drug delivery and treatment.
- MeSH
- Diffusion MeSH
- Extracellular Space * MeSH
- Humans MeSH
- Synaptic Transmission * MeSH
- Neuroglia 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
The diffusion of neuroactive substances in the extracellular space (ECS) plays an important role in short- and long-distance communication between nerve cells and is the underlying mechanism of extrasynaptic (volume) transmission. The diffusion properties of the ECS are described by three parameters: 1. ECS volume fraction alpha (alpha=ECS volume/total tissue volume), 2. tortuosity lambda (lambda2=free/apparent diffusion coefficient), reflecting the presence of diffusion barriers represented by, e.g., fine neuronal and glial processes or extracellular matrix molecules and 3. nonspecific uptake k'. These diffusion parameters differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Moreover, diffusion barriers may channel the migration of molecules in the ECS, so that diffusion is facilitated in a certain direction, i.e. diffusion in certain brain regions is anisotropic. Changes in the diffusion parameters have been found in many physiological and pathological states in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances in the CNS and thus extrasynaptic transmission, neuron-glia communication, transmitter "spillover" and synaptic cross-talk as well as cell migration, drug delivery and treatment.
- MeSH
- Anisotropy MeSH
- Central Nervous System physiology MeSH
- Diffusion MeSH
- Extracellular Space physiology MeSH
- Rats MeSH
- Drug Delivery Systems MeSH
- Humans MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Neuroglia physiology MeSH
- Neurons physiology MeSH
- Cell Movement physiology MeSH
- Signal Transduction physiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Tumor cell migration through the extracellular space (ECS) might be affected by its pore size and extracellular matrix molecule content. ECS volume fraction alpha (alpha = ECS volume/total tissue volume), tortuosity lambda (lambda(2) = free/apparent diffusion coefficient) and nonspecific uptake k' were studied by the real-time tetramethylammonium method in acute slices of human tissue. The diffusion parameters in temporal cortical tissue resected during surgical treatment of temporal lobe epilepsy (control) were compared with those in brain tumors. Subsequently, tumor slices were histopathologically classified according to the grading system of the World Health Organization (WHO), and proliferative activity was assessed. The average values of alpha, lambda, and k' in control cortex were 0.24, 1.55, and 3.66 x 10(-3)s(-1), respectively. Values of alpha, lambda, and k' in oligodendrogliomas did not significantly differ from controls. In pilocytic astrogliomas (WHO grade I) as well as in ependymomas (WHO grade II), alpha was significantly higher, while lambda and k' were unchanged. Higher values of alpha as well as lambda were found in low-grade diffuse astrocytomas (WHO grade II). In cellular regions of high-grade astrocytomas (WHO grade III and IV), alpha and lambda were further increased, and k' was significantly larger than in controls. Classic medulloblastomas (WHO grade IV) had an increased alpha, but not lambda or k', while in the desmoplastic type alpha and k' remained unchanged, but lambda was greatly increased. Tumor malignancy grade strongly corresponds to an increase in ECS volume, which is accompanied by a change in ECS structure manifested by an increase in diffusion barriers for small molecules.
- MeSH
- Analysis of Variance MeSH
- Child MeSH
- Adult MeSH
- Extracellular Space metabolism MeSH
- Glioma metabolism pathology MeSH
- Iontophoresis methods MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Cerebral Cortex cytology metabolism MeSH
- Brain Neoplasms metabolism pathology MeSH
- Child, Preschool MeSH
- Aged MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
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
Bral2 is a link protein stabilizing the binding between lecticans and hyaluronan in perineuronal nets and axonal coats (ACs) in specific brain regions. Using the real-time iontophoretic method and diffusion-weighted magnetic resonance, we determined the extracellular space (ECS) volume fraction (α), tortuosity (λ), and apparent diffusion coefficient of water (ADCW ) in the thalamic ventral posteromedial nucleus (VPM) and sensorimotor cortex of young adult (3-6 months) and aged (14-20 months) Bral2-deficient (Bral2-/- ) mice and age-matched wild-type (wt) controls. The results were correlated with an analysis of extracellular matrix composition. In the cortex, no changes between wt and Bral2-/- were detected, either in the young or aged mice. In the VPM of aged but not in young Bral2-/- mice, we observed a significant decrease in α and ADCW in comparison with age-matched controls. Bral2 deficiency led to a reduction of both aggrecan- and brevican-associated perineuronal nets and a complete disruption of brevican-based ACs in young as well as aged VPM. Our data suggest that aging is a critical point that reveals the effect of Bral2 deficiency on VPM diffusion. This effect is probably mediated through the enhanced age-related damage of neurons lacking protective ACs, or the exhausting of compensatory mechanisms maintaining unchanged diffusion parameters in young Bral2-/- animals. A decreased ECS volume in aged Bral2-/- mice may influence the diffusion of neuroactive substances, and thus extrasynaptic and also indirectly synaptic transmission in this important nucleus of the somatosensory pathway.
- Keywords
- Bral2, aging, diffusion, extracellular matrix, extracellular space,
- MeSH
- Aggrecans metabolism MeSH
- Analysis of Variance MeSH
- Diffusion Magnetic Resonance Imaging MeSH
- Extracellular Matrix Proteins deficiency genetics MeSH
- Extracellular Space diagnostic imaging genetics MeSH
- Ganglionic Stimulants pharmacology MeSH
- Quaternary Ammonium Compounds pharmacology MeSH
- RNA, Messenger MeSH
- Mice, Inbred C57BL MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Neurons cytology drug effects MeSH
- Animals, Newborn MeSH
- Nerve Tissue Proteins deficiency genetics MeSH
- Aging physiology MeSH
- In Vitro Techniques MeSH
- Thalamus cytology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Aggrecans MeSH
- Extracellular Matrix Proteins MeSH
- Ganglionic Stimulants MeSH
- Hapln4 protein, mouse MeSH Browser
- Quaternary Ammonium Compounds MeSH
- RNA, Messenger MeSH
- Nerve Tissue Proteins MeSH
- tetramethylammonium MeSH Browser
Tenascin-R (TN-R), a large extracellular glycoprotein, is an important component of the adult brain's extracellular matrix (ECM); tenascin-C (TN-C) is expressed mainly during early development, while human natural killer 1 (HNK-1) is a sulphated carbohydrate epitope that attaches to these molecules, modifying their adhesive properties. To assess their influence on extracellular space (ECS) volume and geometry, we used the real-time iontophoretic method to measure ECS volume fraction alpha and tortuosity lambda, and diffusion-weighted magnetic resonance imaging (MRI) to measure the apparent diffusion coefficient of water (ADC(W)). Measurements were performed in vivo in the cortex and CA1 hippocampal region of TN-R-, TN-C- and HNK-1 sulphotransferase (ST)-deficient adult mice and their wild-type littermate controls. In both cortex and hippocampus, the lack of TN-R or HNK-1 sulphotransferase resulted in a significant decrease in alpha and lambda. Compared with controls, alpha in TN-R-/- and ST-/- mice decreased by 22-26% and 9-15%, respectively. MRI measurements revealed a decreased ADC(W) in the cortex, hippocampus and thalamus. ADC(W) reflected the changes in alpha; the decrease in lambda indicated fewer diffusion obstacles in the ECS, presumably due to a decreased macromolecular content. No significant changes were found in TN-C-/- animals. We conclude that in TN-R-/- and ST-/- mice, which show morphological, electrophysiological and behavioural abnormalities, the ECS is reduced and its geometry altered. TN-R, as an important component of the ECM, appears to maintain an optimal distance between cells. The altered diffusion of neuroactive substances in the brain will inevitably affect extrasynaptic transmission, neuron-glia interactions and synaptic efficacy.
- MeSH
- Diffusion Magnetic Resonance Imaging methods MeSH
- Extracellular Space metabolism MeSH
- Iontophoresis methods MeSH
- Quaternary Ammonium Compounds metabolism MeSH
- Brain metabolism pathology MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Image Processing, Computer-Assisted methods MeSH
- Sulfotransferases deficiency MeSH
- Tenascin deficiency MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- HNK-1 sulfotransferase MeSH Browser
- Quaternary Ammonium Compounds MeSH
- Sulfotransferases MeSH
- tenascin R MeSH Browser
- Tenascin MeSH
- tetramethylammonium MeSH Browser
Changes in the ability of substances to diffuse in the intersticial space of the brain are important factors in the pathophysiology of cerebrovascular diseases. Extracellular space (ECS) volume fraction alpha (alpha = ECS volume/ total tissue volume), tortuosity lambda (lambda 2 = free diffusion coefficient/apparent diffusion coefficient), and nonspecific uptake (k')-three diffusion parameters of brain tissue were studied in cortex and subcortical white matter (WM) of the developing rat during anoxia. Changes were compared with the rise in extracellular potassium concentration ([K+]e), extracellular pH (pHe) shifts, and anoxic depolarization (AD). Diffusion parameters were determined from extracellular concentration-time profiles of tetramethylammonium (TMA+) or tetraethylammonium (TEA+), TMA+, TEA+, K+, and pH changes were measured using ion-selective microelectrodes. In the cortex and WM of animals at 4-12 postnatal days (P4-P12), the volume fraction, alpha, is larger than that of animals at > or = P21. Anoxia evoked by cardiac arrest brought about a typical rise in [K+]e to approximately 60-70 mM, AD of 25-30 mV, decrease in alpha, increase in lambda, and increase in k'. At P4-P6, alpha decreased from approximately 0.43 to 0.05 in cortical layer V and from approximately 0.45 to 0.5 in WM. Tortuosity, lambda, increased in the cortex from 1.50 to 2.12 and in WM from approximately 1.48 to 2.08. At P10-P12 and at P21-P23, when alpha in normoxic rats is lower than at P4-P6 by approximately 25 and 50%, respectively, the final changes in values of alpha and lambda evoked by anoxia were not significantly different from those in P4-P6. However, the younger the animal, the longer the time course of the changes. On P4-P6 final changes in alpha, lambda and k' in cortex and WM were reached after 37 +/- 3 min and 54 +/- 2 min; on P10-P12, after 24 +/- 2 and 27 +/- 3 min; and on P21-P23 at 15 +/- 1 and 17 +/- 3 min, respectively (mean +/- SE, n = 6). The time course of the changes was longer in WM than in gray matter (GM), particularly during the first postnatal week, i.e., in the period during which WM is largely unmyelinated. Changes in diffusion parameters occurred in three phases. The first slow and second fast changes occurred simultaneously with the rise in [K+]e and AD. Peaks in [K+]e and AD were reached simultaneously; the younger the animal, the longer the time course of the changes. The third phase outlasted the rise in [K+]e and AD by 10-15 min and correlated with the acid shift in pHe. Linear regression analysis revealed a positive correlation between the normoxic size of the ECS volume and the time course of the changes. Slower changes in ECS volume fraction and tortuosity in nervous tissue during development can contribute to slower impairment of signal transmission, e.g., due to lower accumulation of ions and neuroactive substances released from cells and their better diffusion from the hypoxic area in uncompacted ECS.
- MeSH
- Corpus Callosum growth & development metabolism pathology MeSH
- Diffusion MeSH
- Potassium metabolism MeSH
- Extracellular Space metabolism MeSH
- Brain Ischemia complications metabolism pathology MeSH
- Hydrogen-Ion Concentration MeSH
- Rats MeSH
- Quaternary Ammonium Compounds pharmacokinetics MeSH
- Hypoxia, Brain etiology metabolism MeSH
- Cerebral Cortex growth & development metabolism pathology MeSH
- Neuroglia metabolism pathology MeSH
- Neurons metabolism pathology MeSH
- Rats, Wistar MeSH
- Tetraethylammonium Compounds pharmacokinetics MeSH
- Tetraethylammonium MeSH
- Cell Size MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Names of Substances
- Potassium MeSH
- Quaternary Ammonium Compounds MeSH
- Tetraethylammonium Compounds MeSH
- Tetraethylammonium MeSH
- tetramethylammonium MeSH Browser
