Tissue Extracellular Space Research Articles

Nodular Cutaneous Amyloidosis of the Scalp Reconstructed With a Free Anterolateral Thigh Flap: A Case Report

i p r T r s w a c n Amyloidosis is a rare disease of unknown cause that is characterized by the extracellular deposition of an amorphous fibrillar insoluble substance (amyloid) in the extracellular spaces of various organs and tissues, resulting in the loss of normal tissue structure. This can give rise to variable functional and structural impairments, depending on the location and intensity of the amount of protein deposition, eventually leading to organ dysfunction. Depending on the biochemical structure of the precursor protein, the amyloid fibrils can be deposited and can affect every organ of the body. Clinical manifestations are nonspecific and are determined by the organ or system concerned. The definitive diagnosis is made by biopsy using Congo red staining. The authors report a unique case of nodular cutaneous amyloidosis in the temporal region of the scalp that was treated by excision and anterolateral thigh (ALT) free flap reconstruction.

Amiloidose localizada cutânea primária nodular: relato de caso

A amiloidose resulta da deposição de proteína amiloide fibrosa e insolúvel em espaços extracelulares de órgãos e tecidos. O depósito da substância amiloide pode ser localizado ou sistêmico e pode ser de natureza primária ou secundária. Relataremos um caso de amiloidose localizada cutânea primária nodular, manifesta por lesões pápulo-nodulares, eritêmato-acastanhadas, acometendo a região nasal, sem evidência de acometimento sistêmico. O estudo imunoistoquímico demonstrou presença de imunoglobulinas de cadeia leve kappa.

Physiological role of extracellular ribonucleases of higher plants

Extracellular space of plant tissues contains a complex set of proteins whose functions are poorly understood. The review represents a current view on the potential biological roles of extracellular ribonucleases. In particular, the relations of these enzymes to the molecular mechanisms of resistance to pathogens are discussed.

Microfiberoptic Measurement of Extracellular Space Volume in Brain and Tumor Slices Based on Fluorescent Dye Partitioning

The fractional volume occupied by extracellular space in tissues, termed α, is an important parameter of tissue architecture that affects cellular functions and drug delivery. We report a technically simple fluorescent dye partitioning method to measure α in tissue slices based on microfiberoptic detection of dye fluorescence in tissue versus overlying solution. Microfiberoptic tip geometry and dyes were selected for α determination from fluorescence intensity ratios, without the need to correct for illumination profile, light scattering/absorption, or dye binding. The method was validated experimentally using cell-embedded gels of specified α-values and optical properties. In mouse brain slices, α was strongly location-dependent, ranging from 0.16 in thalamus to 0.22 in brainstem, and was sensitive to cell volume changes. Aquaporin-4 water channel gene deletion caused significant extracellular space expansion, with α = 0.181 ± 0.002 in cortex in wild-type mice and 0.211 ± 0.003 in Aquaporin-4 knockout mice. In slices of LLC1 cell tumors grown in mice to ∼5 mm diameter, α decreased remarkably from ∼0.45 in superficial tumor to <0.25 in deeper (>100 μm) tumor. Fluorescent dye partitioning with microfiberoptic detection permits rapid, accurate, and anisotropy-insensitive determination of α-values in tissue slices.

Mediators Leading to Fibrosis—How to Measure and Control Them in Tissue Engineering

Fibrosis is the result of an excessive amount of fibrous connective tissue deposited into the extracellular matrix (ECM) space of damaged tissues from injury or disease. Collagens, particularly types I and III are the main constituents of the fibrotic scar tissue as well as a mixture of fibrotic cells. Severely fibrotic tissue will develop chronic healing problems resulting in tissue/organ dysfunction. More attention needs to be given to the fibrotic differentiation and related effects in bioengineered tissues. The current review provides an update on the mechanism behind fibrosis formation as well as technical measurements and preventions.

Multi-scale study of nanoparticle transport and deposition in tissues during an injection process

In magnetic nanoparticle hyperthermia for cancer treatment, controlling the nanoparticle distribution delivered in tumors is vital for achieving an optimum distribution of temperature elevations that enables a maximum damage of the tumorous cells while minimizing the heating in the surrounding healthy tissues. A multi-scale model is developed in this study to investigate the spatial distribution of nanoparticles in tissues after nanofluid injection into the extracellular space of tissues. The theoretical study consists of a particle trajectory tracking model that considers particle-surface interactions and a macroscale model for the transport of nanoparticles in the carrier solution in a porous structure. Simulations are performed to examine the effects of a variety of injection parameters and particle properties on the particle distribution in tissues. The results show that particle deposition on the cellular structure is the dominant mechanism that leads to a non-uniform particle distribution. The particle penetration depth is sensitive to the injection rate and surface properties of the particles, but relatively insensitive to the injected volume and concentration of the nanofluid.

Determination of tissue spaces of the isolated hindlimb of the rat using netilmicin as an extracellular space indicator.

The kinetic behaviour of drugs in extracellular space is of interest as it influences the drug's access to, and permanence in, those areas of the body upon which drugs exert pharmacological or toxicological actions. A series of experiments was carried out to characterize the vascular, interstitial and cellular spaces of the isolated hindlimb of the rat. Certain specific experimental conditions were met: body weight was under 230 g to avoid fat tissue in the preparations; a perfusion flow rate of 3 mL min(-1); and 3% of bovine albumin in the perfusate supplied at 25 degrees C to the tissues. The isolation of the hindlimb followed the method described by Ruderman with some modifications to restrict the perfusion to the right hindlimb. Tritiated water, netilmicin and methylene blue were injected separately and efferent fluid samples were collected for 15 min after solute injection. Analysis of the efferent curves was performed to calculate the statistical moments (AUC, area under concentration-time curve; MTT, mean transit time; VTT, variance of mean transit times) and solute distribution volumes, which were subsequently used to estimate the tissue spaces of the isolated hindlimb. The results revealed that methyl blue and netilmicin can be respectively used as alternatives to radiolabelled indicators of the vascular and extracellular spaces of tissues included in the rat isolated hindlimb.

Characterizing the Origin of the Arterial Spin Labelling Signal in MRI Using a Multiecho Acquisition Approach

Arterial spin labelling (ASL) can noninvasively isolate the MR signal from arterial blood water that has flowed into the brain. In gray matter, the labelled bolus is dispersed within three main compartments during image acquisition: the intravascular compartment; intracellular tissue space; and the extracellular tissue space. Changes in the relative volumes of the extracellular and intracellular tissue space are thought to occur in many pathologic conditions such as stroke and brain tumors. Accurate measurement of the distribution of the ASL signal within these three compartments will yield better understanding of the time course of blood delivery and exchange, and may have particular application in animal models of disease to investigate the relationship between the source of the ASL signal and pathology. In this study, we sample the transverse relaxation of the ASL perfusion weighted and control images acquired with and without vascular crusher gradients at a range of postlabelling delays and tagging durations, to estimate the tricompartmental distribution of labelled water in the rat cortex. Our results provide evidence for rapid exchange of labelled blood water into the intracellular space relative to the transit time through the vascular bed, and provide a more solid foundation for cerebral blood flow quantification using ASL techniques.

Index of Suspicion

Index of Suspicion

P0255 MEDICAL COMPLICATIONS IN PATIENTS WITH HIP FRACTURE

Introduction: Amyloidosis is a rare disease of unknown etiology, characterized by the deposition of amyloid in the extracellular spaces of various organs and tissues, and with an incidence of about 8 per million person per year. Objectives: 1.To review Systemic Amyloidosis cases (Primary amyloidosis:AL and Secondary amyloidosis:AA) admitted to our hospital over a period of twelve years.2.To determine the most frequent renal manifestations.3.To gain knowledge which is the organ’s biopsy for the diagnosis.4.To analysis the medical services implied in this disease. Materials and methods: We performed a retrospective descriptive statistical study of 67 patients admitted to the San Carlos Clinic Hospital from January 1996 to December 2007 with a diagnosis of Systemic Amyloidosis. Data were collected on renal clinical manifestation and biological data in blood and urine. The diagnosis of amyloidosis was established on positive biopsy for amyloidosis or negative biopsy, but meeting certain clinical criteria. Results: – Anthropometric data: Men: 41 (61.2%), women: 26 (38.8%). In AL: 19 (70.4%) were males and 8 (29.6%) females and in AA: 22 (55.0%) were males and 18 (45.0%) females. The median age of diagnosis was 67 years old (25-75 percentile: 54-75). The median age at diagnosis in AL was 73 years old (25-75 percentile: 61-81) and in AA was 62.5 years old (25-75 percentile: 43,2-73,7). – Clinical and biological criteria: *In AL: renal affectation: 23(85.2%): Proteinuria >1g/dl: 78.3% (in nephrotic range >3.5 g/dl: 83.3% and in non-nephrotic range: 22.2%); Bences-Jones proteinuria: 37.0%; renal failure (Cr>1.2) :82.6%; hypoalbuminemia (albumin 1g/dl: 69.7% (in nephrotic range >3.5g/dl: 60.9% and in non-nephrotic range: 39.1%); Bences-Jones proteinuria: 0.0%; renal failure (Cr>1.2): 84.8%; hypoalbuminemia (albumin <3): 51.5%; nephrotic syndrome: 33.3%; renal vein thrombosis: 6.1%; nephromegalia: 6.1%; microhematuria: 12.1%; anasarca: 9.1%. – Biopsy-Diagnosis: *In AL, positive biopsy: 16 (69.9%): renal biopsy: 50.0%, rectal: 25.0%; abdominal fat: 12.5%; bowel :6.3%; ganglio: 6.3%. *In AA, positive biopsy: 21 (63.6%): renal biopsy: 57.1%, rectal: 28.6%; bowel: 19.0%; abdominal fat: 4.8%; endomyocardial: 4.8%; liver: 4.8%. – Distribution of patients with renal affectations in hospital servicies: Nephrology: 28(50.0%); IM: 20 (35.7%); Cardiology: 2 (3.6%). Rest of them: less than 2% each one. Conclusions: 1. Systemic amyloidosis are more common in men than women aged between 60 and 70 years old. 2. More than 80% of patients with systemic amyloidosis have got renal manifestations. 3. The most frequent clinical manifestations are renal failure and nephrotic range proteinuria. 4. Renal biopsy is the gold standard for diagnosis systemic amyloidosis in patients with renal manifestations. 5. Admitance is carried out by nephrology and internal medicine.

P0254 RENAL AFFECTATION IN SYSTEMIC AMYLOIDOSIS. ANALYSIS OF 67 PATIENTS:

Introduction: Amyloidosis is a rare disease of unknown etiology, characterized by the deposition of amyloid in the extracellular spaces of various organs and tissues, and with an incidence of about 8 per million person per year. Objectives: 1.To review Systemic Amyloidosis cases (Primary amyloidosis:AL and Secondary amyloidosis:AA) admitted to our hospital over a period of twelve years.2.To determine the most frequent renal manifestations.3.To gain knowledge which is the organ’s biopsy for the diagnosis.4.To analysis the medical services implied in this disease. Materials and methods: We performed a retrospective descriptive statistical study of 67 patients admitted to the San Carlos Clinic Hospital from January 1996 to December 2007 with a diagnosis of Systemic Amyloidosis. Data were collected on renal clinical manifestation and biological data in blood and urine. The diagnosis of amyloidosis was established on positive biopsy for amyloidosis or negative biopsy, but meeting certain clinical criteria. Results: – Anthropometric data: Men: 41 (61.2%), women: 26 (38.8%). In AL: 19 (70.4%) were males and 8 (29.6%) females and in AA: 22 (55.0%) were males and 18 (45.0%) females. The median age of diagnosis was 67 years old (25-75 percentile: 54-75). The median age at diagnosis in AL was 73 years old (25-75 percentile: 61-81) and in AA was 62.5 years old (25-75 percentile: 43,2-73,7). – Clinical and biological criteria: *In AL: renal affectation: 23(85.2%): Proteinuria >1g/dl: 78.3% (in nephrotic range >3.5 g/dl: 83.3% and in non-nephrotic range: 22.2%); Bences-Jones proteinuria: 37.0%; renal failure (Cr>1.2) :82.6%; hypoalbuminemia (albumin 1g/dl: 69.7% (in nephrotic range >3.5g/dl: 60.9% and in non-nephrotic range: 39.1%); Bences-Jones proteinuria: 0.0%; renal failure (Cr>1.2): 84.8%; hypoalbuminemia (albumin <3): 51.5%; nephrotic syndrome: 33.3%; renal vein thrombosis: 6.1%; nephromegalia: 6.1%; microhematuria: 12.1%; anasarca: 9.1%. – Biopsy-Diagnosis: *In AL, positive biopsy: 16 (69.9%): renal biopsy: 50.0%, rectal: 25.0%; abdominal fat: 12.5%; bowel :6.3%; ganglio: 6.3%. *In AA, positive biopsy: 21 (63.6%): renal biopsy: 57.1%, rectal: 28.6%; bowel: 19.0%; abdominal fat: 4.8%; endomyocardial: 4.8%; liver: 4.8%. – Distribution of patients with renal affectations in hospital servicies: Nephrology: 28(50.0%); IM: 20 (35.7%); Cardiology: 2 (3.6%). Rest of them: less than 2% each one. Conclusions: 1. Systemic amyloidosis are more common in men than women aged between 60 and 70 years old. 2. More than 80% of patients with systemic amyloidosis have got renal manifestations. 3. The most frequent clinical manifestations are renal failure and nephrotic range proteinuria. 4. Renal biopsy is the gold standard for diagnosis systemic amyloidosis in patients with renal manifestations. 5. Admitance is carried out by nephrology and internal medicine.

Thermal Stability Of Oriented Aß25-35 Amyloid Fibril Nanoarray

Amyloid fibrils are filamentous protein deposits in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. It has been proposed that amyloid fibrils may be used in nanotechnology applications because of their self-assembly properties and stability. Recently we have shown that amyloid beta 25-35 (As25-35) forms a highly oriented, potassium-dependent network on mica. The mutant form of the peptide (As25-35_N27C), which forms an identically oriented nanoscale network, may be chemically addressed for functionalization in dedicated applications. In order to utilize the amyloid nanoarray in nanotechnology applications, uderstanding its physical and chemical stability is important.In the present work we investigated thermally induced changes in the morphology of the oriented As25-35 fibril network. The fibrils maintained high orientation stability in the temperature range of 30-70 degrees, suggesting that orientational rearrangement of As25-35 fibrils on mica is an unfavorable process. Above 45 degree a gradual decrease in fibril length and dissociation from the surface could be observed. In addition, at high temperatures (45-70 degrees) the average fibril thickness increased, indicating changes in the underlying structure or structural dynamics. Possibly, a thermally induced transition in the As25-35 peptide around 45 degree leads to structural changes in the fibril as well. The temperature-dependent changes described here need to be considered in the use of amyloid fibrils in nanotechnology applications.

Applications of cardiac magnetic resonance in electrophysiology.

Because of its wide availability, echocardiography is the most frequently used modality for diagnosis of the underlying substrate for cardiac arrhythmia. However, echocardiography is unable to visualize intramyocardial substrates for reentrant arrhythmia such as fat or scar fibrosis. Replacement of myocytes with nonviable tissue results in hypertrophy of the remaining viable cells and is associated with altered ion channel and gap junction expression. These changes affect myocardial mechanical function and promote arrhythmia.1 Because of its soft-tissue resolution, multiplanar imaging capabilities, and specialized techniques uniquely suited for diagnosis of various structural changes,2 cardiac magnetic resonance (CMR) offers significant advantages for identification of arrhythmic substrates. Ischemic cardiomyopathy (ICM) is ventricular dysfunction resulting from coronary artery disease and myocardial infarction and is a common substrate of ventricular arrhythmia. Techniques to visualize infarcted myocardium in the acute3–7 and chronic8–11 settings have been well described and rely primarily on steady-state free precession cine CMR for evaluation of function, and late gadolinium enhancement (LGE) imaging to assess scar burden and distribution. The steady-state free precession cine technique relies on short repetition times and electrocardiographic gating to provide dynamic visualization of the heart during the full cardiac cycle. The LGE technique uses an inversion recovery gradient echo sequence with optimization of the inversion time set to null the signal of normal myocardium. In the setting of ICM, images are acquired 10 to 15 minutes after intravenous administration of 0.2 mmol/kg gadolinium chelate. Gadolinium chelates are hydrophilic and have low molecular weights thereby concentrating into the extracellular fluid space. The extent of LGE is primarily determined by expansion of the extracellular space in fibrotic tissue, which slows washout of the gadolinium chelates.12 This “delayed” time period from contrast administration to scan acquisition allows clearance of the contrast medium from the normal myocardium, whereas …

Oriented epitaxial growth of amyloid fibrils of the N27C mutant β25–35 peptide

Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid beta 25-35 (A beta 25-35) forms a highly oriented, K(+)-dependent network on mica. Here, we analyzed the properties of A beta 25-35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that A beta 25-35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating A beta 25-35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of A beta 25-35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.

A Patient with Primary Amyloidosis Misrecognized as Thyrotoxicosis-induced Heart Failure

Amyloidosis is caused by deposition of insoluble amyloid protein in the extracellular space of organs and tissues. The causes of amyloidosis are classified as primary, secondary, and hereditary, and symptoms develop according to which organ is involved. Cardiac amyloidosis induces cardiomyopathy and is developed by deposition of amyloid proteins in cardiac tissue. We diagnosed a patient with rhabdomyolysis and thyrotoxicosis with underlying Graves' disease 5 years ago. The patient was readmitted recently complaining of general weakness and mild dyspnea, and was diagnosed as relapsed thyrotoxicosis. An echocardiogram was performed for the evaluation of dyspnea and the findings were compatible with infiltrative cardiomyopathy due to amyloidosis. A biopsy of the abdominal subcutaneous fat and rectal mucosa was performed, and diagnosis was amyloidosis with histologic findings. The cause of heart failure was therefore cardiac amyloidosis rather than thyrotoxicosis. This case indicates the importance of evaluating the cause of heart failure in patients with thyrotoxicosis. (J Korean Endocr Soc 23:332~336, 2008)

Identification of accessible human cancer biomarkers using ex vivo chemical proteomic strategies

One promising avenue towards the development of more selective, better anticancer drugs lies in the targeted delivery of bioactive compounds to the tumor environment by means of binding molecules specific for tumor-associated biomarkers. Eligibility of such markers for therapeutic ideally use three criteria: accessibility from the bloodstream; expression at sufficient level, and no (or much lower) expression in normal tissues. Most current discovery strategies (such as biomarker searching into body fluids) provide no clue as to whether proteins of interest are accessible, in human tissues, to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies. To address this limitation, our group recently developed two methodologies based on chemical proteomic modifications, enabling the discovery of proteins accessible from the bloodstream and the extracellular space in human pathological tissues. In this review, we will discuss the potential benefits of these methodologies for the fast discovery of therapeutically valuable biomarkers.

AAPS-FDA Workshop White Paper: Microdialysis Principles, Application and Regulatory Perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (microD) is the only tool available that explicitly provides data on the extracellular space. Although microD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of microD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of microD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on microD as a tool in drug research and development.

Comparison of the effects on glucose and lipid metabolism of equipotent doses of insulin detemir and NPH insulin with a 16-h euglycaemic clamp

The association of insulin detemir with non-esterified fatty acid binding sites on albumin may limit its transfer from the circulation into the extravascular extracellular space in adipose tissue and muscle, due to the capillary endothelial cell barrier. In the liver, the open sinusoids may expose hepatocytes to insulin detemir, enabling it to have a greater effect in the liver than in peripheral tissues. We investigated the effects of equipotent doses of insulin detemir and NPH insulin on hepatic glucose rate of appearance (Ra), peripheral glucose rate of disposal (Rd) and glycerol Ra (a measure of lipolysis) using stable isotope techniques. We also investigated the effects of these insulins on NEFA concentrations in seven healthy volunteers during a 16-h euglycaemic clamp. A higher dose of insulin detemir was also studied. There was no difference in the glucose infusion profile between insulin detemir and NPH. Insulin detemir had a greater effect on mean suppression of glucose Ra (mean difference 0.24 mg kg(-1) min(-1); CI 0.09-0.39; p<0.01), and minimum glucose Ra, with minimum low dose detemir -0.10+/-0.15 mg.kg(-1).min(-1) and minimum NPH 0.17+/-0.10 mg.kg(-1).min(-1) (p<0.02). However, it had a lesser effect on mean suppression of NEFA concentrations (mean difference -0.10 mmol/l; CI -0.03 to -0.17; ANOVA, p<0.02) than NPH. The effect of insulin detemir on glucose Rd and glycerol Ra was not different from NPH. Following high-dose detemir, total glucose infused and maximum glucose Rd were higher (p<0.02, p<0.03) and plasma NEFA concentrations lower (p<0.01) than with low-dose determir. This study suggests that insulin detemir, when compared to NPH insulin, has a greater effect on the liver than on peripheral tissues and thus has the potential to restore the physiological insulin gradient.

Restriction by Endocytosis

Key signaling molecules in vertebrate development, such as fibroblast growth factor (FGF), can have short- and long-range regional effects. Their activities are controlled by mechanisms that propagate or restrict their availability to target cells and tissues. Scholpp and Brand have examined how the signaling range of Fgf8 might be controlled in developing zebrafish embryos. By implanting a labeled source of Fgf8 in early embryos, they visualized spreading of the growth factor and determined expression of a target gene encoding sprouty4. Fgf8 was detected in endosomes in cells up to 12 cell diameters away from the source. Host embryos were also injected with various mRNAs to express proteins that interfere with endocytosis. When either a dominant-negative form of the FGF receptor or a Rab5-specific guanosine triphosphatase activating protein (GAP) was expressed to block receptor internalization or inhibit endocytosis, respectively, Fgf8 accumulated outside target cells at greater distances from the source compared with wild-type embryos. If endocytosis was stimulated by expressing more Rab5 protein, the range of Fgf8 spreading and sprouty4 expression was reduced in embryos. The data demonstrate that a Rab5-dependent endocytosis can restrict spreading of diffusible Fgf8, clearing FGF from the extracellular space of target tissues. Because target genes respond to different concentrations of growth factors, endocytosis may serve to shape cellular responses during development. S. Scholpp, M. Brand, Endocytosis controls spreading and effective signaling range of Fgf8 protein. Curr. Biol. 14 , 1834-1841 (2004). [Online Journal]

Extracellular matrix glycoproteins and diffusion barriers in human astrocytic tumours.

The extracellular matrix (ECM) and changes in the size and geometry of the extracellular space (ECS) in tumour tissue are thought to be of critical importance in influencing the migratory abilities of tumour cells as well as the delivery of therapeutic agents into the tumour. In 21 astrocytic neoplasms, the ECM composition was investigated in situ by the immunohistochemical detection of ECM glycoproteins (tenascin, laminin, vitronectin, fibronectin, collagen types I-VI). To explain the changes in ECS size and to detect barriers to diffusion in the tumour tissue, the ECM composition, the cellularity, the density of glial fibrillary acidic protein (GFAP)-positive tumour cell processes and the proliferative activity of the tumours were compared with the size and geometry of the ECS. The ECS volume fraction and the complex of hindrances to diffusion in the ECS (i.e. the tortuosity) were revealed by the real-time iontophoretic tetramethylammonium method. Increased proliferative activity of the tumours correlated with increased ECS volume fraction and tortuosity. The tortuosity of the tumour tissue was not significantly influenced by tumour cell density. Higher tortuosity was found in low-grade astrocytomas associated with the presence of a dense net of GFAP-positive fibrillary processes of the tumour cells. The increase in tortuosity in high-grade tumours correlated with an increased accumulation of ECM molecules, particularly of tenascin. We conclude that the increased malignancy of astrocytic tumours correlates with increases in both ECS volume and ECM deposition.