Analysis Of Histological Sections Research Articles

Improved neoangiogenesis in transmyocardial laser revascularization combined with angiogenic adjunct in a pig model

Recent research has revealed neoangiogenesis as a basic phenomenon in transmyocardial laser revascularization (TMLR). Theoretically, myocardial neoangiogenesis could be further enhanced by the addition of angiogenic growth factors. Triads of TMLR channels were created in the lateral wall of the left ventricles of 12 pigs (mean body weight 73+/-5 kg), using a holmium:yttrium-aluminium garnet (YAG) laser. The animals were allocated randomly either to receive an injection of 100 microg of a bovine bone-derived growth factor mixture within the triads (n=6), or to a control group (n=6). Animals were killed 1 month later. Capillary and arteriolar densities were determined by computed morphometric analysis of histological sections of the triads. The capillary density of myocardial areas within the triads was significantly greater in the group receiving the bovine bone-derived growth factor mixture than in the control group (14.3+/-3.5/mm(2) and 5.7+/-1.4/mm(2) respectively; P<0.001). The difference was also significant when considering arteriolar density (0.7+/-0.4/mm(2) and 0.2+/-0.1/mm(2) respectively; P<0.001). For comparison, capillary and arteriolar densities of the TMLR channel scars were 48.7+/-9.7 and 1.9+/-0. 5/mm(2) respectively in the angiogenic group, and 46.3+/-13.7 and 2. 3+/-1.3/mm(2) respectively in the control group (no significant differences). These results demonstrate that the addition of angiogenic factors to TMLR stimulates neoangiogenesis significantly in the areas adjacent to the channels, but not within the channel scars. The latter are themselves strongly vascularized. Hence this combined approach, potentiating the effect of TMLR by establishing vascular connections between the neovessels of the channel scars, has the potential for improved clinical outcome.

Biocompatibility of HEMA copolymers designed for treatment of CNS diseases with polymer-encapsulated cells.

Surrounding the cells with a semipermeable polymeric membrane allows transplanting unmatched xenogeneic cells without a risk of their rejection. We prepared and tested several 2-hydroxyethyl methacrylate (HEMA) copolymers with alkyl methacrylates or acrylates to find out which was the most valuable for cell encapsulation. On the basis of optimum physical properties and good results of cytotoxicity tests, HEMA-EMA copolymer was chosen as a suitable candidate for encapsulation and immunoprotection of xenogeneic cells before their grafting into the central nervous system (CNS). To characterize the biocompatibility of p(HEMA-co-EMA) copolymer in the CNS, we implanted microcapsules made of this hydrogel into the brains of adult rats that were allowed to survive for 0.5, 1, 3, 6, and 9 months. Analysis of histological sections containing the implantation site was aimed at assessment of the cellular density at the implant-brain interface and identification of cell types participating in a tissue reaction. Our results indicated that the tissue reaction that was observed was caused largely by the implantation procedure because HLA-DR- and GSI-B4-positive macrophages/microglia infiltrated mainly the implantation channel. The number of these cells declined with time, which was true also for GFAP-positive reactive astrocytes, as well as for foreign body giant cells. The amount of connective tissue components surrounding the implanted microcapsules increased only slightly. These findings indicated that p(HEMA-co-EMA) hydrogel was well tolerated after implantation in the brain.

Macromolecule uptake in human melanoma xenografts: relationships to blood supply, vascular density, microvessel permeability and extracellular volume fraction

The uptake of albumin–Evans blue in human melanoma xenografts was studied and related to blood supply, vascular density, microvessel permeability and extracellular volume fraction in an attempt to identify transport barriers limiting the delivery of macromolecular therapeutic agents to tumours. Three melanoma lines (A-07, R-18, U-25) were included in the study. Tissue concentrations of albumin–Evans blue were determined by spectrophotometry. The [ 86Rb] uptake method was used to measure tumour blood supply. Vascular density was determined by stereological analysis of histological sections. Microvessel permeability was measured by using the indicator diffusion method. Contrast-enhanced magnetic resonance imaging was used to measure tumour extracellular volume fraction. The fractional volume of the extracellular space governed the uptake of albumin–Evans blue in the tumours. The uptake of albumin–Evans blue in the extracellular space was primarily limited by transport in the vasculature and not by transport across the microvascular wall or the transport through the interstitium. Our study thus suggests that novel strategies for improving the delivery of macromolecular therapeutic agents to tumours should focus on enhancing the tumour blood supply, increasing the half-life of the therapeutic agent in the blood plasma and/or enhancing the volume of the extracellular space available to macromolecules rather than on increasing the permeability of the microvascular wall or improving diffusion conditions in the tumour interstitium.

Methodological principles for fractal analysis of trabecular bone.

A standardized methodology for the fractal analysis of histological sections of trabecular bone has been established. A modified box counting method has been developed for use on a PC-based image analyser. The effect of image analyser settings, magnification, image orientation and threshold levels was determined. Also, the range of scale over which trabecular bone is effectively fractal was determined and a method formulated to calculate objectively more than one fractal dimension from the modified Richardson plot. The results show that magnification, image orientation and threshold settings have little effect on the estimate of fractal dimension. Trabecular bone has a lower limit below which it is not fractal (lambda < 25 microm) and the upper limit is 4250 microm. There are three distinct fractal dimensions for trabecular bone (sectional fractals), with magnitudes greater than 1.0 and less than 2.0. It has been shown that trabecular bone is effectively fractal over a defined range of scale. Also, within this range, there is more than one fractal dimension, describing spatial structural entities. Fractal analysis is a model-independent method for describing a complex multifaceted structure, which can be adapted for the study of other biological systems. This may be at the cell, tissue or organ level and complements conventional histomorphometric and stereological techniques.

De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model: a novel concept and capability.

This study was undertaken to characterize the duration of long-term growth factor delivery by poly(lactic-co-glycolic-acid)-polyethylene glycol (PLGA/PEG) microspheres and to evaluate the potential of long-term delivery of insulin and insulin-like growth factor-1 (IGF-1) for the de novo generation of adipose tissue in vivo. PLGA/PEG microspheres containing insulin and IGF-1, separately, were produced by a double-emulsion solvent-extraction technique. In the first phase of the experiment, the in vitro release kinetics of the microspheres were evaluated for the optical density and polyacrylamide gel electrophoresis of solutions incubated with insulin-containing microspheres for four different periods of time (n = 1). The finding of increased concentrations of soluble insulin with increased incubation time confirmed continual protein release. In the second stage of the experiment, 16 rats were divided equally into four study groups (insulin, IGF-1, insulin + IGF-1, and blank microspheres) (n = 4). Insulin and IGF-1 containing microspheres were administered directly to the deep muscular fascia of the rat abdominal wall to evaluate the potential for de novo adipose tissue generation via adipogenic differentiation from native nonadipocyte cell pools in vivo. Animals treated with blank microspheres served as an external control group. At the 4-week harvest period, multiple ectopic islands of adipose tissue were observed on the abdominal wall of the animals treated with insulin, IGF-1, and insulin + IGF-1 microspheres. Such islands were not seen in the blank microsphere group. Hematoxylin and eosin-stained sections of the growth factor groups demonstrated mature adipocytes interspersed with fibrous tissue superficial to the abdominal wall musculature and continuous with the fascia. Oil-Red-O stained sections demonstrated that these cells contained lipid. Computer-aided image analysis of histologic sections confirmed that there were statistically significant increases in the amount of "ectopic" adipose neotissue developed on the abdominal wall of animals treated with growth factor microspheres. In conclusion, this study confirms the long-term release of proteins from PLGA/PEG microspheres up to 4 weeks and demonstrates the potential of long-term local insulin and IGF-1 to induce adipogenic differentiation to mature lipid-containing adipocytes from nonadipocyte cell pools in vivo at 4 weeks.

Influence of different staining techniques on the DNA analysis of histological sections.

The use of stained histological sections as a source for DNA may be necessary in forensic case work if confusion of tissue is suspected, for identification or paternity cases. To elucidate the influence of different staining techniques on the PCR amplification of DNA, histological sections of liver tissue were prepared using eight different staining techniques and two histochemical methods. The DNA of the sections was extracted by a modified Chelex extraction and amplified using a commercial triplex kit. Staining with hematoxylin-eosin, hemalum-eosin, azan, periodic acid-schiff and prussian blue showed no adverse effect on the amplification of DNA while the extracts of tissue stained by Masson-Goldner, Ladewig and elastica-van Gieson methods had to be purified before amplification was possible. Staining with phosphoric tungsten acid hematoxylin and Gomori led to a degradation of DNA probably due to the use of potassium permanganate solution.

Hypoxia-Dependent Retinal Toxicity of Bioreductive Anticancer Prodrugs in Mice

The bioreductive anticancer prodrug CI-1010 ((2R)-1-[(2-bromoethyl)amino]-3-(2-nitro-1H-imidazol-1-yl)-2-propanol hydrobromide) is an alkylating nitroimidazole which shows selective toxicity against hypoxic cells in murine tumors, but causes extensive apoptosis in the outer retina in rodents and monkeys. This irreversible retinal toxicity has terminated preclinical development of CI-1010. We have investigated whether such toxicity is due to physiological hypoxia in the retina, and whether it is a general feature of hypoxia-selective bioreductive drugs. Retinal damage was quantified by morphometric analysis of histological sections following treatment of female C57Bl6 mice. Both CI-1010 and tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide), a bioreductive drug in Phase III clinical trial, caused a time and dose-dependent loss of photoreceptor cells of the outer retina following administration of single intraperitoneal doses. The lesion caused by TPZ was qualitatively similar to that with CI-1010, but was less severe at equivalent fractions of the maximum tolerated dose (as defined by lethality). With both bioreductive drugs, lesion severity was increased if animals breathed 10% O2 for 3 h after drug administration, while breathing 95% O2/5% CO2 was protective. Other hypoxia-selective bioreductive drugs tested (the quinone porfiromycin, the anthraquinone N-oxide AQ4N and the nitrogen mustard prodrugs SN 23816 and SN 25341) did not cause retinal damage at their maximum tolerated doses. This study suggests that the retinal toxicity of bioreductive drugs might be avoided by manipulation of tissue hypoxia using 95% O2/5% CO2, although this intervention could suppress antitumor activity. The finding that not all bioreductive drugs cause retinal toxicity suggests this toxicity can be avoided through appropriate drug design.

Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome

During embryonal development primitive hematopoiesis can be observed first in the yolk sac, in which both hematopoietic and endothelial cells are derived from a common precursor, the hemangioblast. Whether cells with this dual differentiation potential persist during postnatal life is unknown. A cell line was derived from a patient with secondary acute leukemia. Because of its ability to grow in soft agar and in SCID mice, this cell line was analyzed for expression of differentiation antigens by fluorescence-activated cell sorter analysis, immunocytochemistry, fluorescent in situ hybridization (FISH) analysis with simultaneous cell surface staining, and polymerase chain reaction (PCR). A new cell line was established from a patient with essential thrombocytosis that transformed into acute leukemia. The patient's initial clinical presentation included skin and lymph node infiltrations that were taken for an angiosarcoma due to positivity for CD34, CD31, and von Willebrand factor on immunohistology. In addition to hematopoietic markers, leukemic cells expressed endothelial antigens such as CD62E, CD105, and bound Ulex europäeus lectin-1. Immunocytochemistry revealed positive staining for vascular endothelial growth factor receptor type 2 (KDR), Tie-2/Tek, the angiopoietin receptor, and vascular endothelial cadherin. These results were confirmed by PCR analysis. Simultaneous staining for CD62E and FISH analysis showed that cells with endothelial characteristics belonged to the leukemia. FISH analysis of histologic sections of the lymph node infiltration confirmed this manifestation as part of the leukemic process. The derived cell line, UKE-1, forms colonies in soft agar and is tumorigenic in SCID mice. This new cell line, UKE-1, appears to combine hematopoietic and endothelial features, indicating the close ontogenic relation of both lineages.

A new constitutive framework for arterial wall mechanics and a comparative study of material models

In this paper we develop a new constitutive law for the description of the (passive) mechanical response of arterial tissue. The artery is modeled as a thick-walled nonlinearly elastic circular cylindrical tube consisting of two layers corresponding to the media and adventitia (the solid mechanically relevant layers in healthy tissue). Each layer is treated as a fiber-reinforced material with the fibers corresponding to the collagenous component of the material and symmetrically disposed with respect to the cylinder axis. The resulting constitutive law is orthotropic in each layer. Fiber orientations obtained from a statistical analysis of histological sections from each arterial layer are used. A specific form of the law, which requires only three material parameters for each layer, is used to study the response of an artery under combined axial extension, inflation and torsion. The characteristic and very important residual stress in an artery in vitro is accounted for by assuming that the natural (unstressed and unstrained) configuration of the material corresponds to an open sector of a tube, which is then closed by an initial bending to form a load-free, but stressed, circular cylindrical configuration prior to application of the extension, inflation and torsion. The effect of residual stress on the stress distribution through the deformed arterial wall in the physiological state is examined. The model is fitted to available data on arteries and its predictions are assessed for the considered combined loadings. It is explained how the new model is designed to avoid certain mechanical, mathematical and computational deficiencies evident in currently available phenomenological models. A critical review of these models is provided by way of background to the development of the new model.

Interstitial Fluid Pressure and Capillary Diameter Distribution in Human Melanoma Xenografts

Tumors have been shown to differ substantially in interstitial fluid pressure (IFP), but the biological properties of tumors governing the intertumor heterogeneity in IFP have not been identified conclusively. The purpose of the work reported here was to investigate whether the IFP of tumors is influenced significantly by the diameter distribution of the capillaries and hence by the geometric resistance of the capillary network to blood flow. Tumors of three human melanoma xenograft lines (D-12, R-18, U-25) showing similar capillary densities were included in the study. IFP was measured using the wick-in-needle technique. Capillary diameter distribution was determined by stereological analysis of histological sections. The lines differed significantly in tumor IFP (P < 0.05) and capillary diameter distribution (P < 0.05). Mean IFP was 6 mm Hg (D-12), 17 mm Hg (R-18), and 11 mm Hg (U-25). The mean of the mean capillary diameter was 13.1 μm (D-12), 10.9 μm (R-18), and 12.0 μm (U-25). The sequence of the lines from low to high IFP was the same as the sequence of the lines from large to small mean capillary diameter: D-12, U-25, R-18. Also, individual tumors of the same line differed substantially in IFP and in mean capillary diameter. IFP ranged from 2 to 15 mm Hg (D-12), from 2 to 36 mm Hg (R-18), and from 4 to 30 mm Hg (U-25). Mean capillary diameter ranged from 11.0 to 14.6 μm (D-12), from 9.5 to 11.7 μm (R-18), and from 10.4 to 13.0 μm (U-25). Inverse linear correlations between tumor IFP and mean capillary diameter were found for each of the melanoma lines [P < 0.05, R2 = 0.85 (D-12); P < 0.05, R2 = 0.86 (R-18); P < 0.01, R2 = 0.93 (U-25)]. Moreover, the IFP and mean capillary diameter of individual tumors varied with tumor size in all lines. IFP decreased during tumor growth whereas mean capillary diameter increased with increasing tumor volume (P < 0.001). Taken together, these data suggest that the diameter distribution and hence the geometric resistance of the capillary network exerts significant influence on the IFP of tumors.

The Angular Branch of the Thoracodorsal Artery and Its Blood Supply to the Inferior Angle of the Scapula: An Anatomical Study

An anatomical study of the thoracodorsal arterial system was performed; it focused on the angular branch. The aim of the study was to document the anatomical variations of this pedicle and to delineate the area of supply to the inferior angle of the scapula with a view to free bone transfer. A total of 81 cadaver dissections were performed; they revealed the constant presence of the thoracodorsal artery and four vascular patterns of origin of the angular branch. Selective India ink perfusion studies performed on 11 sides in six fresh cadavers demonstrated a reliable supply to the inferior angle of the scapula to the extent of 6 cm of the vertebral margin and 3 cm of the lateral margin of the scapula. Histologic analysis of sections of this region of the scapula confirmed the presence of ink within the periosteal, cortical, and medullary vascular channels, implying the viability of this area of bone if transferred based on the angular branch.

The effect of prolonged iron loading on the chemical form of iron oxide deposits in rat liver and spleen

Female Porton rats were loaded with iron either by supplementing the diet with 2.5% carbonyl iron for up to 22 months (18 rats) or by regularly injecting rat blood cells intraperitoneally for up to 10 months (eight rats). 57Fe Mössbauer spectroscopy of freeze-dried samples of liver and spleen was used to analyse the chemical forms of iron deposited in these tissues over the period of iron loading. A sextet signal in the Mössbauer spectra was identified as being due to a form of haemosiderin based on the structure of the mineral goethite. The spectral parameters of the sextet signal in the rat tissues indicate that the goethite-like haemosiderin particles are less crystalline than those found in iron-loaded human tissues. For the dietary-iron-loaded rat livers, the fraction ( F s) of the Mössbauer signal in the form of this sextet was found to increase significantly (from approx 0.04 to 0.09) with the age of the rats ( r=0.77, P<0.0005). This indicates that the fraction of liver iron in the form of the goethite-like haemosiderin increases with age of the rat and hence with the duration of iron loading. In addition, F s for these livers was found to increase significantly with the fraction of iron in non-parenchymal cells as measured by computer-assisted morphometric analysis of histological sections ( r=0.71, P<0.005).

Polymetallism and osseointegration in oral implantology: pilot study on primate.

In oral implantology, successful results in osseointegration are obtained in the medium term (6-12 months) with commercially pure titanium implants. However, current superstructures can be of a different nature (precious metal or titanium) and of different manufacture (cast or machine-produced). Polymetallism between the implant and the superstructure may lead to conditions of galvanic corrosion, and influence osseointegration. The study described establishes, on the one hand, the procedures of animal experimentation in primates and on the other, the techniques of analysis of histological sections. The first technique of analysis is based on histomorphometry and leads to the definition of an osseointegration index. The second technique of analysis consists of X spectrometry by dispersion of energy which enables a spectral analysis of selected points below the crestal neck of the implant (vulnerable area in the case of corrosive attack) to be obtained. It is noted that after 6months, two of which were of activation, osseointegration did not vary according to the nature of the superstructure (precious alloy or titanium). After 2 months, the presence of a precious alloy superstructure lead to titanium migration towards the area around the cervical region of the implant (10-50 microm). This phenomenon did not occur with a titanium implant. It can therefore be presumed that polymetallism leads to detectable corrosion after 2 months but without apparent modification of osseointegration.

Thermographic and histological evaluation of laser skin resurfacing scans

In general, ablative laser skin resurfacing procedures have shown good short-term efficacy. However, the mechanisms underlying laser skin resurfacing remain poorly understood. We performed a quantitative study to investigate the thermal response of skin to CO/sub 2/ laser irradiation. Raster scans were performed on an in vivo rat model and radiometric surface temperatures measured using a thermal camera. Temperatures approaching 400/spl deg/C were measured during the scans and remained above the initial skin temperature for durations longer than ten seconds. Analysis of histology sections showed that the epidermis remained partially intact after three passes. To explain the observed trends in the temperature response and histology, the dynamics of optical and thermal parameters were investigated. Water vaporization played a key role in governing the response of the skin to subsequent laser passes. Char formation and pulse stacking altered the thermal effects.

Amygdala damage in experimental and human temporal lobe epilepsy.

The amygdala complex is one component of the temporal lobe that may be damaged unilaterally or bilaterally in children and adults with temporal lobe epilepsy (TLE) or following status epilepticus. Most MR (magnetic resonance) imaging studies of epileptic patients have shown that volume reduction of the amygdala ranges from 10-30%. In the human amygdala, neuronal loss and gliosis have been reported in the lateral and basal nuclei. Studies in rats have more specifically identified the amygdaloid regions that are sensitive to status epilepticus-induced neuronal damage. These areas include the medial division of the lateral nucleus, the parvicellular division of the basal nucleus, the accessory basal nucleus, the posterior cortical nucleus, and portions of the anterior cortical and medial nuclei. Otherwise, other amygdala nuclei, such as the magnocellular and intermediate divisions of the basal nucleus and the central nucleus, remain relatively well preserved. Amygdala kindling studies in rats have shown that the density of a subpopulation of GABAergic inhibitory neurons that also contain somatostatin may be reduced even after a low number of generalized seizures. While analyses of histological sections and MR images indicate that in approximately 10% of TLE patients, seizure-induced damage is isolated to the amygdala, more often amygdala damage is combined with damage to the hippocampus and/or other brain areas. Moreover, recent data from rodents and nonhuman primates suggest that structural and functional alterations caused by seizure activity originating in the amygdala are not limited to the amygdala itself, but may also affect other temporal lobe structures. The information gathered so far on damage to the amygdala in epilepsy or after status epilepticus suggests that local alterations in inhibitory circuitries may contribute to a lowered seizure threshold and greater excitability within the amygdala. Furthermore, damage to select nuclei in the amygdala may predict impairment of performance in behavioral tasks that depend on the integrity of the amygdaloid circuits.

Estimating age and growth in long‐lived temperate freshwater crayfish using lipofuscin

1. In ecological studies on freshwater crayfish, determination of basic population parameters is often complicated by the lack of a suitable age estimation method.2. Previously, lipofuscin age pigment in the olfactory lobe cell masses (OLCM) of short‐lived tropical crayfish has been used for accurate age determination. Here we present the first test of this method on a longer‐lived, temperate species, the signal crayfish, Pacifastacus leniusculus.3. Confocal fluorescence microscopy and image analysis of histological sections were used to quantify OLCM lipofuscin in a reference sample of Swedish P. leniusculus from several known year‐classes, reared under naturally variable temperature conditions. Lipofuscin concentration was linearly associated with age (r2 = 92.4%) and produced much more accurate age estimates than conventional body size‐based procedures.4. A model derived from the crayfish of known‐age was used to estimate the ages of wild P. leniusculus from an English stream. The relationship between lipofuscin‐estimated age and carapace length suggested relatively slow growth in this wild population, consistent with a high population density and severe competition. The analysis also extended the known longevity of P. leniusculus to approximately 16 years.5. The lipofuscin method for determining age and growth may be widely applicable to freshwater crayfish, with probable further potential both within and outside the Crustacea.

Quantitative analysis of microvascular changes in diffuse astrocytic neoplasms with increasing grade of malignancy

Because glioblastoma multiforme (GBM) frequently shows striking, glomeruloid microvascular proliferation (MVP), this tumor has become a strong candidate for anti-angiogenic therapy. However, the efficacy of anti-angiogenic treatment may rather be determined by the extent of classic angiogenesis with the formation of delicate microvascular sprouts. Therefore, this study differentially quantifies the microvascular changes in supratentorial diffuse astrocytic neoplasms by computerized image analysis of histological sections in which the microvessels were highlighted by a combined anti-collagen IV/MIB-1 staining. Four microvascular parameters (number, area, perimeter, diameter), the cellularity of the glial tissue, and the MIB-1 labeling index were assessed in biopsies of astrocytoma (A, n = 13), anaplastic astrocytoma (AA, n = 14), and GBM (n = 20), and in normal cerebral cortex (n = 7) and white matter (n = 7). In As and AAs, the microvascular parameters were not significantly different from each other, and the microvascular changes were generally limited compared with WM and CX. In contrast, in GBMs the microvascular parameters were highly variable, and their overall mean value was significantly increased compared with As and AAs (ranging from 1.3× for vessel diameter to 3.3× for vessel area). Our study indicates that not only glomeruloid MVP, but also classic angiogenesis, occurs mainly and only locally in GBMs. Thus, this study provides evidence that As and AAs are not good candidates for anti-angiogenic therapy. The efficacy of such therapy for GBMs awaits further evaluation.

A Semiquantitative Histological Analysis of Repair of Anastomoses in the Rat Colon after Combined Preoperative Irradiation and Local Hyperthermia

Hyperthermia is a promising method for increasing the efficacy of radiation therapy of colorectal cancer. To study the histological aspects of healing of an anastomosis in the colon, after combined preoperative (sham) irradiation and (sham) hyperthermia treatment, 48 male Wistar rats were divided randomly into four groups. In each animal, a segment of the colon was treated successively by (sham) irradiation (single dose of 25 Gy X rays) and/or (sham) hyperthermia (44 degrees C, 30 min). After 5 days, a resection of the colon was performed by construction of an anastomosis: The distal limb consisted of (sham-) irradiated and/or (sham-) hyperthermia-treated bowel. Rats were killed 3 or 7 days after the surgical procedure. Evaluation of healing of the anastomosis was made by: (1) histological analysis of sections stained with hematoxylin and eosin, (2) semiquantitative measurement of collagen in the area of the anastomosis and (3) semiquantitative analysis of the number of macrophages by immunocytochemistry. Healing of the anastomoses in animals receiving irradiation or hyperthermia alone and in control animals was relatively uneventful. There were no differences between groups in formation of collagen or infiltration by macrophages in the area of the anastomosis. Animals treated with both radiation and hyperthermia showed marked necrosis, infiltration by polymorphonuclear leukocytes and rupture of the anastomosis. It is concluded that preoperative irradiation with a single dose of 25 Gy in combination with local hyperthermia at 44 degrees C for 30 min leads to disturbed repair of anastomoses.

Venous endothelial changes in therapeutic arteriovenous fistulae

Specimens of veins of therapeutic arteriovenous fistulae from five patients were examined by an en face immunohistochemical technique to investigate endothelial morphology and the presence of the vasoactive peptide endothelin-1 (ET-1). These were compared with control segments of long saphenous veins from six patients. Venous endothelium from the arteriovenous shunts was mostly intact even overlying phlebosclerotic plaques. Occasional small areas of denudation, with associated platelets, were present in the depressions of ‘jet’ lesion however. The endothelial cells were generally elongated and interspersed with foci of polyhedral cells. The control saphenous veins contained elongated endothelial cells without detectable denudation. Image analysis of histological sections of veins from the shunts indicated significantly less intact elastic tissue than control veins but greater mononucleated endothelial cell density in en face preparations. ET-1 staining was considerably stronger in endothelium from the fistulae than in the control saphenous veins and was most intense over the raised crescentic ridges of jet lesions, stenoses and phlebosclerotic plaques. Endothelial mitoses and cells with hyperchromatic nuclei stained more strongly for ET-1 than surrounding cells. These results indicate that the endothelial cells lining veins associated with arteriovenous fistulae are dynamically altered by the increased haemodynamic stresses associated with these shunts. Furthermore ET-1 may act as a localising factor associated with intimal thickening at sites of ‘jet’ lesions, stenosis and phlebosclerosis.

Interstitial Fluid Pressure, Fraction of Necrotic Tumor Tissue, and Tumor Cell Density in Human Melanoma Xenografts

Interstitial fluid pressure (IFP) has been shown to differ substantially between individual tumors, but the tumor properties governing the intertumor heterogeneity in IFP have not been identified conclusively. The purpose of the work reported here was to investigate whether the fraction of necrotic tissue and the density of tumor cells are major determinants of the intertumor heterogeneity in IFP. The study was based on the hypothesis that the resistance against fluid flow in the tumor interstitium is influenced significantly by these parameters. Xenografted tumors of four human melanoma lines (A-07, D-12, R-18, U-25) were included in the study. Tumors showing large variation in necrotic fraction but similar cell densities (D-12, U-25) were used to study the influence of necrosis on IFP, whereas tumors showing no or insignificant necrosis but large variation in cell density (A-07, R-18) were used to search for correlations between IFP and cell density. IFP was recorded using the wick-in-needle technique. Necrotic fraction and cell density were measured by stereological analysis of histological sections using an image processing system. Significant correlations between IFP and necrotic fraction were not found, implying that the IFP of tumors is not influenced significantly by the development of necrosis. The R-18 tumors, which had a high cell density, showed a significantly higher IFP than the A-07 tumors, which had a low cell density. Significant correlations between IFP and cell density were not found when individual tumors of the same line were considered. These two observations suggest that the IFP of tumors depends on the cell density, but the cell density is probably not a major determinant of the IFP.