Brain extracellular space (ECS) is a narrow, irregular space, which provides immediate living environment for neural cells and accounts for approximately 15%-20% of the total volume of living brain. Twenty-five years ago, as an interventional radiologist, the author was engaged in investigating early diagnosis and treatment of cerebral ischemic stroke, and the parameters of brain ECS was firstly derived and demonstrated during the study of the permeability of blood-brain barrier (BBB) and its diffusion changes in the cerebral ischemic tissue. Since then, the author and his team had been working on developing a novel measuring method of ECS: tracer-based magnetic resonance imaging (MRI), which could measure brain ECS parameters in the whole brain scale and make the dynamic drainage process of the labelled brain interstitial fluid (ISF) visualized. By using the new method, the team made a series of new findings about the brain ECS and ISF, including the discovery of a new division system in the brain, named regionalized ISF drainage system. We found that the ISF drainage in the deep brain was regionalized and the structural and functional parameters in different interstitial system (ISS) divisions were disparate. The ISF in the caudate nucleus could be drained to ipsilateral cortex and finally into the subarachnoid space, which maintained the pathway of ISF-cerebrospinal fluid (CSF) exchange. However, the ISF in the thalamus was eliminated locally in its anatomical division. After verifying the nature of the barrier structure between different drainage divisions, the author proposed the hypothesis of "regionalized brain homeostasis". Thus, we demonstrated that the brain was protected not only by the BBB, which avoided potential exogenous damage through the vascular system, but was also protected by an internal ISF drainage barrier to avoid potentially harmful interference from other ECS divisions in the deep brain. With the new findings and the proposed hypothesis, an innovative therapeutic method for the treatment of encephalopathy with local drug delivery via the brain ECS pathway was established. By using this new administration method, the drug was achieved directly to the space around neurons or target regions, overwhelming the impendence from the blood-brain barrier, thus solved the obstacles of low efficiency in traditional drug investigation. At present, new methods and discoveries developed by the author and his team have been widely applied in several frontier fields including neuroscience, new drug research and development, neurodevelopment aerospace medicine, clinical encephalopathy treatment,new neural network modeling and so on.
Brain ; Caudate Nucleus ; Extracellular Fluid ; Extracellular Space ; Magnetic Resonance Imaging

OBJECTIVETo analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy adults.

METHODSThe 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking analysis (NTA).

RESULTSuEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed that the number of 24-hour uEVs amount ranged from 3.56 × 10¹² particles to 5.12 × 10¹² particles, with a CV of 14.23%. The proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49)× 10⁹ particles; the proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a number range of (1.00-1.77)× 10¹² particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86)× 10¹² particles.

CONCLUSIONThe established HFD method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies.

Extracellular vesicles (EVs) are tiny biological nanovesicles ranging from approximately 30-1000 nm in diameter that are released into the extracellular matrix of most cell types and in biofluids. The classification of EVs includes exosomes, microvesicles, and apoptotic bodies, dependent on various factors such as size, markers, and biogenesis pathways. The transition of EV relevance from that of being assumed as a trash bag to be a key player in critical physiological and pathological conditions has been revolutionary in many ways. EVs have been recently revealed to play a crucial role in stem cell biology and cancer progression via intercellular communication, contributing to organ development and the progression of cancer. This review focuses on the significant research progress made so far in the role of the crosstalk between EVs and stem cells and their niche, and cellular communication among different germ layers in developmental biology. In addition, it discusses the role of EVs in cancer progression and their application as therapeutic agents or drug delivery vehicles. All such discoveries have been facilitated by tremendous technological advancements in EV-associated research, especially the microfluidics systems. Their pros and cons in the context of characterization of EVs are also extensively discussed in this review. This review also deliberates the role of EVs in normal cell processes and disease conditions, and their application as a diagnostic and therapeutic tool. Finally, we propose future perspectives for EV-related research in stem cell and cancer biology.
Biomarkers/metabolism* ; Cell-Derived Microparticles/metabolism* ; Exosomes ; Extracellular Vesicles/metabolism* ; Humans ; Neoplasms/metabolism*

Qi, blood and the meridians are fundamental concepts in Chinese medicine (CM), which are components of the human body and maintain physiological function. Pathological changes of qi, blood and meridians may lead to discomfort and disease. Treatment with acupuncture or herbal medicine aims to regulate qi and blood so as to recover normal function of the meridians. This paper explores the nature of qi as well as compares and correlates them with the structures of the human body. We propose a conceptualization of qi as being similar to the interstitial fluid, and the meridians as being similar to interstitial space of low hydraulic resistance in the body. Hence, qi running in the meridians can be understood as interstitial fluid flowing via interstitial space of low hydraulic resistance.
Acupuncture Points ; Connective Tissue ; physiology ; Extracellular Fluid ; physiology ; Extracellular Space ; physiology ; Humans ; Meridians ; Qi ; Water

Dialysis unphysiology was first discussed by Carl Kjellstrand in 1975 for the possible negative effects of the unphysiology of intermittent dialysis treatment. Current hemodialysis practices are still unphysiologic because they cannot keep blood chemistries within normal limits, both before and after dialysis. In addition, the discontinuous nature of hemodialysis causes saw-tooth volume fluctuations, and the extracellular fluid volume expansion during the interdialytic period may lead to hypertension and adverse cardiovascular consequences. Sodium, which is accumulated over the interdialytic period, may be divided into two fractions. The one is the fraction of osmotically active sodium which is mainly confined to the extracellular space, and the other is that of water-free (osmotically inactive) sodium which diffuses into the intracellular space. Both contribute to the pathogenesis of hypertension because the former may act to expand extracellular fluid volume and the latter may cause vasoconstriction in the long run by increasing cytosolic concentration of calcium in the vascular smooth muscle cells. Even in intensive hemodialysis, it may take several weeks to months for water-free sodium storage in the vascular smooth muscle cells to be relieved. This may be an explanation for the lag phenomenon, i.e., the delay of blood pressure decrease after normalization of extracellular fluid volume shown in the Tassin experience. Modest restriction of dietary sodium intake, the dialytic session length long enough to maintain a high ultrafiltration volume, and the reasonably low dialysate sodium concentration are required to avoid unphysiology of positive sodium balance in current hemodialysis practice.
Blood Pressure ; Calcium ; Cytosol ; Dialysis ; Extracellular Fluid ; Extracellular Space ; Hypertension ; Intracellular Space ; Muscle, Smooth, Vascular ; Renal Dialysis ; Sodium ; Sodium, Dietary ; Ultrafiltration ; Vasoconstriction

No abstract available.
Extracellular Space* ; Ischemia* ; Myocytes, Cardiac*

The authors observed the histological and ultrastructural findings in a study of the fibrosis of the extraocular muscles following a posterior fixation suture in rabbits. the early findings demonstrated hyalinosis, clumping of the nuclei of muscle cells and collagenization of the muscle tissue. However, there was no evidence of collagen tissue in the degenerated muscle fiber, reserving cell membrane and basement membrane ultrastructurally. The late findings showed splitting and irregular stain of the muscle fibers, many nuclei of presumed muscle cell and filamentous structure at the collagen tissue in the extracellular space. Electron microscopic study showed atrophy and angulation with distortion of the myofibrillar matrix, along with other cytoplasmic degenerative phe nomena in the muscle fibers. Clumps of the well-arranged microfibrils(Mf) and irregularly arranged Mf with fine granular materials(FGM) were adjacent to the immature collagen fibrils. Many of the Mf and FGM were closely associated, and the number of the Mf and FGM decreased with the degree of maturity of the collagen fibrils. The anatomical structure of the myofibrill was identified as that of the Mf. From these findings, it would appear that formation of collagen fiber occurs in the extracellular space and that Mf and FGM are primarily responsible for the formation of the collagen fibils.
Atrophy ; Basement Membrane ; Cell Membrane ; Collagen ; Cytoplasm ; Extracellular Space ; Fibrosis* ; Microfibrils ; Muscle Cells ; Muscles ; Myofibrils ; Rabbits* ; Sutures*

This study was carried out to investigate the ultrastructural changes of lens fiber cells, length of gap junctions, and distribution of proteoplycans in cataractous lens. The cataract was induced by stabbing the posterior surface of the lens with 25 gauge needle. The lens were obseved by electron microscopy with estimation of the gap junction. Alcian blue stain was performed for examination of proteoglyscas. The results were as follows. Characteristic changes of the cataractous lens fiber cells were swelling and condensation of the cytoplasm, alterations of cytoplasmic granules including changes of density and size of cytoplasmic granules, and formation of microfibrills, high electron-dense amorphous substance, electron-dense particles, variably sized globular bodies, and multilamellar bodies. The lengths of gap junctions were 2.51+/-1.09um in control and 1.06+/-0.33um in cataractous lens fiber cells(P<0.001). Proteoglycans showing high electron-dense amophorous substance were distributed in the intercellular spaces along the cytoplasmic membrane in control group, but disapperaed in cataractous lens fiber cells. According to these results, it is conceivalble that the changes of the gap junctions and proteoglycans were closely related to the pathogenesis of cataract.
Alcian Blue ; Cataract* ; Cell Membrane ; Cytoplasm ; Cytoplasmic Granules ; Extracellular Space ; Gap Junctions ; Microfibrils ; Microscopy, Electron ; Needles ; Proteoglycans

Melatonin could be used as an anticancer agent to suppress the proliferation of tumor cells and induce the differentiation of cancer cells. HeLa, HepG2, A549, L929, and NIH/3T3 cell lines were cultivated in alpha-MEM with 0.2 mM/2 mM melatonin. The influences of melatonin on quantitative changes of glycoprotein, fibronectin, laminin and actin related to the metastasis of tumor cells investigated with PAS or PAP at light microscopic level. To elucidate the possibility of antitumor actions of melatonin, the changes of cell organelles were observed under transmission electron microscope. Cell proliferation was suppressed after treatment with 2 mM melatonin for 2 or 3 days. Compared with control groups, the amounts of glycoprotein, fibronectin, laminin and actin in all cell lines at 1st, 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin were generally increased. Heterochromatin in the nucleus formed clumps in all cell lines at 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin. The numerical increase of rough endoplasmic reticulum and golgi complex observed in HeLa and L929 cells treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. The number of lysosomes increased in HeLa, A549, and L929 cells treated with 0.2 mM and 2 mM melatonin at 3rd day. The number of vesicles increased in all cell lines treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. Taken together, antimitotic effect of melatonin can be expected at least 2day after treatment with 2 mM melatonin. The production of fibronectin and laminin in all cell lines treated with 0.2 mM or 2 mM melatonin increased. Therefore, the increase of amounts of extracellular matrix proteins in the extracellular space can be expected. And the increase of amounts of actin connected to the extracellular matrix proteins through the integrin of plasma membrane seemed to strengthen cell attachment. In order to metastasize of cancer cells, it is important for them to secrete various enzymes to pass through the extracellular matrix proteins. Hence, it will be more difficult for the cells to metastasize into other regions due to the increase of the extracellular matrix proteins. It was postulated that the clumps of heterochromatin and the numerical increase of vesicles induced by treatment with 0.2 mM and 2 mM melatonin could be represented for the actions of melatonin as morpholgical criteria.
Actins ; Cell Line* ; Cell Membrane ; Cell Proliferation ; Endoplasmic Reticulum, Rough ; Extracellular Matrix Proteins* ; Extracellular Matrix* ; Extracellular Space ; Fibronectins ; Glycoproteins ; Golgi Apparatus ; Heterochromatin ; Laminin ; Lysosomes ; Melatonin* ; Neoplasm Metastasis ; Organelles

OBJECTIVE: Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the complex biochemical mechanism between the ovum and the oviduct. METHODS: Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue. RESULTS: The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells. CONCLUSION: Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.
Actin Cytoskeleton ; Adhesives ; Animals ; Antibodies ; Antibodies, Monoclonal ; Basement Membrane ; Cell Membrane ; Cumulus Cells ; Cytoplasm ; Embryonic Structures ; Endoplasmic Reticulum, Smooth ; Epitopes ; Extracellular Matrix ; Extracellular Matrix Proteins ; Extracellular Space ; Female ; Fertilization ; Fibronectins* ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique ; Formaldehyde ; Gap Junctions ; Immunohistochemistry ; Laminin* ; Microscopy, Confocal ; Microscopy, Electron* ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Microtubules ; Microvilli ; Mitochondria ; Oocytes ; Organelles ; Oviducts ; Ovulation ; Ovum ; Sperm-Ovum Interactions ; Tenascin* ; Zona Pellucida