Stereological Point Counting Research Articles

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy-to-apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17-day-old laboratory Wistar rat embryo.

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

The extent of vacuolation in non-heart-beating porcine donor liver grafts prior to transplantation predicts their viability

Livers exposed to prolonged warm ischemia (WI), such as those from non-heart-beating donors (NHBDs), are at higher risk of primary graft nonfunction (PNF). In a pig model of liver transplantation (LTx) from NHBDs, hepatocellular vacuolation, focal hepatocyte dropout, congestion, and sinusoidal dilatation appeared on biopsies taken after exposure to WI. In functioning grafts, vacuolation and sinusoidal dilatation were reversible after LTx, in contrast to PNF grafts. We studied whether the extent of these morphological signs and particularly vacuolation, present on pre-LTx biopsies, was associated with WI length and able to predict PNF, hepatocellular damage, and survival. Pre-LTx biopsies from pig livers exposed to incremental periods of WI were reviewed retrospectively. The extent of vacuolation was quantified blindly by a pathologist's semiquantitative score, validated by stereological point counting and digital image analysis, and then used to predict PNF and hepatocellular damage. On biopsies taken after WI, stereological point counting and digital analysis scoring contributed significantly in predicting PNF (P = 0.027 and P = 0.043, respectively) versus the pathologist's semiquantitative score (P = 0.058). Stereological point counting and digital image analysis predicted the extent of hepatocellular damage (P < 0.0001 and P = 0.001) versus the pathologist's semiquantitative score (P = 0.085). In conclusion, the extent of parenchymal vacuolation present on WI liver grafts reflects the severity of hepatocellular damage and predicts pig liver graft viability before LTx. Further studies are now warranted to evaluate whether these anoxic changes that are associated with liver graft viability in pigs also apply to human NHBD liver biopsies.

Validity of serial milling-based imaging system for microdamage quantification

Understanding the three-dimensional distribution of microdamage within trabecular bone may help provide a better understanding of the mechanisms of bone failure. Toward that end, a novel serial milling-based fluorescent imaging system was developed for quantifying microscopic damage in three dimensions throughout cores of trabecular bone. The overall goal for this study was to compare two-dimensional (2D), surface-based measures of microdamage extracted from this new imaging system against those from more conventional histological section analyses. Human vertebral trabecular cores were isolated, stained en bloc with a series of chelating fluorochromes, monotonically loaded, and underwent microdamage quantification via the two methods. Bone area fraction measured by the new system was significantly correlated to that measured by histological point counting ( p < 0.001, R 2 = 0.80). Additionally, the new system produced statistically equivalent ( p = 0.021) measures of damage fraction (mean ± SD), Dx.AF = 0.047 ± 0.021, to that obtained from stereological point counting, Dx.AF = 0.048 ± 0.017, at a 10% difference level. These results demonstrate that this serial milling-based fluorescent imaging system provides a destructive yet practical alternative to more conventional histologic section analysis in addition to its ability to provide a better understanding of the three-dimensional nature of microdamage.

Stereology: a simplified and more time-efficient method than planimetry for the quantitative analysis of vascular structures in different models of intimal thickening

Semiautomatic computer-assisted planimetry is currently the standard morphometric technique in models of intimal thickening. We evaluated stereological point counting as an alternative method for the measurement of cross-sectional vascular areas (lumen, intima, and media) by comparing precision, efficiency, and variance components. Sections from murine atherosclerotic aorta (n = 21), stented rabbit iliac arteries (n = 30), and porcine coronary arteries (n = 30) were analyzed at two institutes using both techniques. To determine reproducibility, porcine arteries were measured twice. Area measurements showed almost identical means and standard deviations for planimetry and stereology [e.g., intima (mm2): 0.10+/-0.11 vs. 0.12+/-0.12 (mouse), 0.60+/-0.16 vs. 0.60+/-0.15 (rabbit), and 1.92+/-1.52 vs. 1.97+/-1.61 (pig)]. Deming regression and Bland-Altman plots demonstrated a good agreement between both techniques that was not influenced by artery size. Both methods exhibited excellent repeatability, although planimetry (-0.18+/-0.27) was more precise than stereology (-0.17+/-0.47; variance, P < .01, Levene test). In addition, intraoperator variance (error inherent to the technique) was greater for stereology (1.6-15.8% vs. 4.8-33.5%), whereas interoperator variance (error between institutes) was very small for both methods (0.1-0.9% vs. 0.1-1.7%). Indeed, biologic variability was, by far, the most important variance component in all measurements (84-98% vs. 65-95%). Finally, stereology required 20% (mouse; P < .05) to 40% (pig; P < .001) less time to complete analysis. The quantification of vascular structures by planimetry and stereology yielded comparable results in all models of intimal thickening, but stereology proved to be less time-consuming. Therefore, study design may dictate the most appropriate choice of technique.

How Many Glomerular Profiles Must Be Measured to Obtain Reliable Estimates of Mean Glomerular Areas in Human Renal Biopsies?

The objective of this study was to investigate the number of glomerular profiles that are required for accurate estimates of mean profile area in a renal biopsy series. Slides from 384 renal biopsies from one center were reviewed. They contained a median of seven glomerular profiles or of four profiles without sclerosis. Profile areas were measured using stereologic point counting. The "true individual mean" for each biopsy was calculated and the "true population mean" for groups of biopsies derived. Individual and population "random sample means" then were calculated from a random sampling of profiles in each biopsy and were compared with true means for the same biopsies. The effect on the true population means of the entire group of biopsies was also assessed, as the minimum number of glomerular profiles that were required for inclusion was changed. In a single biopsy, random sampling of > or =10 profiles without exclusions and of eight profiles or more without sclerosis reliably estimated the true mean areas. In a group of 30 biopsies, random sampling of five or more glomeruli per biopsy reliably estimated the true population mean. In the aggregate series, inclusion of all 384 biopsies produced the most robust true population mean; the reliability of the estimates decreased as the numbers of eligible biopsies diminished with increasing requisite minimum numbers of profiles per biopsy. We conclude that, while > or =10 profiles might be needed for reliable area estimates in a single biopsy, far fewer profiles per biopsy can suffice when groups of biopsies are studied. In analyses of groups of biopsies, all available biopsies should be used without consideration of the number of glomerular profiles in each. Stipulation of a specific minimum number of glomeruli in each biopsy for inclusion reduces the power of analyses because fewer biopsies are available for evaluation.

Semiquantitative evaluation overestimates the degree of steatosis in liver biopsies: a comparison to stereological point counting

The degree of steatosis in liver biopsies is usually assessed by a morphological semiquantitative approach in which the histopathologist uses a four-graded scale: 0–3 or none, slight, moderate and severe. Scores 1–3 are considered to correspond to fat deposition in <33, 33–66 and >66% of the hepatocytes. There is a considerable inter- and intra-individual variation in such scoring methods and a more standardized and quantitative approach is preferable. In the present study, we compare the semiquantitative technique with the stereological point counting method in the assessment of hepatic steatosis. A total of 75 archived liver needle biopsies were used. They were selected according to the original routine diagnosis of slight, moderate or severe steatosis. In all, 10 randomly selected images from each biopsy were digitized into a computer, a point grid lattice was superimposed and the number of hits on fat globules was counted. A pathologist scored the specimens in a four-graded scale as described above. The mean liver biopsy area (volume) with fat in hepatocytes was 2.2% for grade 1, 9.2% for grade 2 and 23.1% for grade 3. The kappa value for the semiquantitative estimates was 0.71 for the unweigthed kappa and 0.87 for weighted kappa. The intraclass correlation coefficient (ICC) was 0.99 for images counted twice and 0.95 when two sets of images were captured from the same biopsy. These ICCs indicate excellent agreement and above that of the semiquantitative estimates. In conclusion, the area/volume of fat content of the hepatocytes is greatly overemphasized in semiquantitative estimation. Furthermore, the point counting technique has a better reproducibility than visual evaluation and should be preferred in estimates of liver steatosis in scientific studies and in clinical contexts when the amount of steatosis is important for treatment and prognosis, such as liver transplantation.

Effects of Age and Diet on Rat Skin Histology

To document age-related histologic morphometric changes of rat skin and the effects of calorie restriction on such changes. Fischer 344 rats of three age groups (young, 4 mo; adult, 1 year; old, 24+ months) were procured from ad libitum (AL) diet and calorie-restricted (CR) colonies of the National Institute of Aging and were used for histologic study. Each study group consisted of six animals. Skin samples from the dorsum (DS) and footpad (FP) of these animals were excised and processed for histology with staining techniques for general morphology (hematoxylin-eosin-phloxine) and for differentiation of collagen bundles and elastic fibers (Verhoeff-van Gieson technique). Light microscopic morphometric and stereologic point counting procedures were applied manually to tissue sections to obtain quantitative data on the depth of the epidermis, dermis, and stratum corneum, epidermal nuclear number, and percentage fraction of collagen, elastic fibers, capillaries, and pilosebaceous units. Data were analyzed with two-way of analysis of variance (ANOVA) to determine significant effects of age, diet, and age-diet interaction on these parameters in AL rats and their age-matched cohorts. Significant effects of age, diet, or age-diet interaction were observed in respect of the thickness of epidermis, dermis, stratum corneum of FP, epidermal nuclear number, collagen percentage fraction, and area fraction of capillaries. DS epidermis showed increasing thickness in AL group, but this was reduced in CR rats. A similar trend in DS dermal depth was observed. Fewer capillaries were present in aging CR rats. The DS epidermal nuclear profiles and collagen area fraction also showed effects of diet and age-diet interaction. Aging changes, especially the effect of CR, was more evident in the measured parameters of dorsal skin. No alterations were observed in the distribution of pilosebaceous units and elastic fiber profiles of the skin. The Fischer 344 rat shows many age-related changes in the skin, some of which are different from data reported in literature. The pattern of aging changes in skin parameters was different in the two groups, suggesting an in influence of CR. CR appears to modify the aging rate of some skin components, and this may be caused by metabolic changes imposed by diet.

Histologic modification of cutaneous aging by caloric restriction in rats

Problem: To investigate whether caloric restriction can modify age-induced intrinsic changes of the skin in Fischer 344 rats. Methods: Fischer 344 rats were procured from the National Institute of Aging and 3 groups of ad libitum (AL) and calorie-restricted (CR)rats were set up for study: young (4 months), adult (1 year), and old (24 months+). Samples of the ventral skin were subjected to histological processing and paraffin microtomy. Readings were obtained in blinded fashion, and standard manual methods of ocular grid morphometry and stereological point counting were applied. The parameters assessed were the number of epidermal nuclei, depth of epidermis, dermis, and adipose layer (μm), and percentage area of dermis occupied by collagen bundles, elastic fibers, pilosebaceous units, and capillaries. Using SPSS 11.0 and Excel statistical software the data were analyzed by 2-factor analysis of variance (general linear model-univariate procedure) to test the null hypotheses about the effects of age, diet, and age-diet interaction effects on the dependent variables, ie, various skin parameters. Results: Both age and diet had significant effects on the epidermal component of the skin. Diet effect was seen in the dermal thickness (F9.26,P.005) with larger values in aging AL animals. Dermal adipose tissue showed pronounced effects of calorie restriction (F5.89,P.021). Elastic and collagen fibers manifested tri-phasic age effect in both nutritional groups with higher values in adult animals. There was no noticeable change in the percentage area of pilosebaceous units. Conclusion: This model shows alterations of skin histomorphology with age, some of which can be modulated with caloric restriction. Significance: Microscopic studies are useful for investigating the effects of chemotherapeutic agents on the skin in suitable age-controlled laboratory animals. The use of CR animals may elucidate whether dietary control can modify the response to such therapies. Support: None reported.

Stereological estimation of total intracranial volume on CT images.

This study aims to investigate the possibility of generating stereological estimations of total intracranial volume (TIV) on CT scans. The study group included 16 consecutive patients referred for a cranial CT examination. The TIV was estimated using the stereological point counting technique. Volume measurements were optimized by systematically sampling CT sections and by defining an optimum spacing between test points of the grid. The intraobserver and interobserver variability of the optimized volumetric technique was determined. Stereological TIV estimations were compared with the respective planimetric measurements. The application of a test grid with a point spacing of 2.4 cm on 6-8 systematically sampled CT sections provided TIV estimations with a coefficient of error of less than 5%. The intraobserver and interobserver coefficient of variation values were found to be 2.4 and 4.0%, respectively. The 95% limits of agreement between stereological and planimetric TIV measurements were equal to -91.4 and 103.4 cm3. The mean time (+/- SD) needed to obtain stereological TIV estimations was 2.9 +/- 0.6 min. The application of the optimized stereological technique on CT scans enables the efficient estimation of TIV.

Stereologic assessment of the total cortical volume occupied by amyloid deposits and its relationship with cognitive status in aging and Alzheimer’s disease

Although the presence of amyloid deposits is required to establish the neuropathologic diagnosis of Alzheimer’s disease, from a clinical point of view, a direct contribution of these cerebral lesions to cognitive deficits is still controversial. The development and standardization of quantitative and accurate biochemical and neuropathologic methods may be critical to improve the postmortem diagnosis and clinicopathologic correlations. Here, we used a point counting method, based on the Cavalieri principle, to estimate the volume occupied by amyloid deposits in a discrete region of the prefrontal cortex and in the hippocampal formation, in brains from patients with cognitive status ranging from normal to severely demented. We demonstrate that the assessment of the total volume occupied by the amyloid deposits in the entorhinal cortex and subiculum can be considered an effective predictor of dementia severity. We also reveal the existence of a high degree of regional and interindividual heterogeneity in amyloid distribution and relative volume. Our data suggest that even though a correlation was observed between the stereologic point counting method and a non-stereologic random field thresholding approach, in most cases non-stereologic methods may not provide adequate samples of the tissue and may lead to unreliable estimates of amyloid burden due to the inhomogeneous distribution of amyloid in the cerebral cortex and the large variability among brains.

Stereological point-counting; an accurate method for assessing ovarian function in tilapia

We discuss the use of stereology in fisheries research and by presenting novel data, along with comparisons to conventional methodology, further confirm the effectiveness of a point-counting method that estimates the relative distributions of oocyte developmental stages within an asynchronous ovary. Point-counting methodology was applied in three experiments involving the ovaries of Tilapia zillii (Gervais), a teleost that exhibits highly asynchronous ovarian development. Quantitative data were first obtained from conventional digestion of ovarian tissue with mercury-based Gilson's Fluid and compared to that derived from stereology. Analysis of covariance demonstrated no significant difference ( P≥0.05) between counts of discrete oocyte developmental stages determined by either method. However, counts of post-ovulatory follicles (POFs) using Gilson analysis were not possible, due to the highly irregular size and shape of these structures. A second experiment compared an immediately post-spawned ovary with an ovary 5 days after spawning, presenting comparative data determined by stereology and Gilson digestion; on this occasion, Gilson analysis significantly underestimated the proportion of atretic ooytes ( P<0.05). A further experiment forms a preliminary investigation of the effect of intra-muscular injections of human chorionic gonadotropin (hCG) upon early ovarian recrudescence and is presented as an example to demonstrate the highly informative nature of stereological analysis; injections of hCG on 3 days after spawning resulted in a significant decrease of stage 5 (early vitellogenic) but significant increase of stage 6 (late vitellogenic/maturing) oocytes by day 5. We conclude that point-counting stereology is a powerful technique for the quantitative analysis of ovarian development. Moreover, stereology avoids the prolonged use of dangerous mercury-based solutions and classifies oocytes into discrete developmental stages using a combination of criteria (biochemical, histological and size distribution) and not simply oocyte size.

GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue

Glutathione S-transferases (GSTs), inducible enzymes that catalyze the detoxification of reactive electrophiles and oxidants, protect against neoplastic transformation. Prostatic adenocarcinoma and high-grade prostatic intraepithelial neoplasia (HGPIN) fail to express GSTP1, a major class of GST. This failure of expression is associated with methlyation of the GSTP1 promoter, a somatic alteration proposed to be a critical step in prostatic carcinogenesis. However, simple atrophy and post-atrophic hyperplasia-proliferative lesions associated with chronic inflammation, which we have termed "proliferative inflammatory atrophy" (PIA)-express elevated levels of GSTP1. We postulated that this increase in GSTP1 expression in PIA occurs in response to increased oxidative stress. We examined the expression of another major class of GST, GSTA1, in the human prostate. We performed immunohistochemistry against GSTA1 on formalin-fixed radical prostatectomies (n = 45). A stereological grid point counting method was used to estimate the percent of cells staining positive for GSTA1 in normal prostate, PIA, HGPIN, and adenocarcinoma. In contrast to GSTP1, normal peripheral zone epithelium was virtually devoid of GSTA1. Strikingly, though, epithelial cells in PIA demonstrated strong staining for GSTA1 (median of percent of cells staining positive = 44) as compared to those in normal peripheral zone (median = 3.0, P <.00001), HGPIN (median = 2.9, P <.00001), and adenocarcinoma (median = 3.8, P <.00001). Variations in GSTA1 were also detected between normal anatomic zones: the central zone showed an increase in the percentage of cells staining positive (median = 20.9) as compared to the transition (median = 0.47, P <.0002) and the peripheral (P <.0001) zones. Expression of GSTA1 is increased in PIA, supporting the concept that cells within these lesions are subject to localized increases in oxidative stress. Low levels of GSTA1 and GSTP1 in HGPIN and adenocarcinoma suggest a broad lack of detoxification activity in these cells, which may be associated with carcinogenesis in the prostate.

A computer‐assisted stereological quantification program: Openstereo

At present, the stereological assessment of histological sections is made possible by the use of manual counting techniques which estimate measurement parameters. These methods are tedious, time-consuming, and subject to operator error. This paper describes a UNIX-based computer program, OpenStereo, which was developed to facilitate the quantitative investigation of innervation and vascularity from histological sections. We designed OpenStereo to reduce operator error and increase the efficiency of stereological point counting for volume estimation and intercept counting for surface area analysis. The program was written in the C language for the Sun Workstation and uses the XView graphics user interface. Digital images, obtained by a variety of modalities, may be processed using stereological point counting, interceptions, planimetry, or thresholding techniques. The program displays selected images in a random fashion for analysis or processing and records the number of manually selected points or interceptions. Delineation of the reference space provides the computer with the data necessary to calculate volume or surface densities. The efficiency of OpenStereo was demonstrated by performing a pilot study on the quantification of innervation in the normal human colon and ileum. This stereological package benefits from the features of the X-windowing environment and has proved to be suitable for what has hitherto been a tedious and time-consuming task.

A quantitative morphological analysis of the cerebellar nuclei in normal and lurcher mutant mice. II. Volumetric changes in cytological components

In the present study the stereological point counting method has been used to determine volumetric changes in the cytological components of the lurcher cerebellar nuclei. The volume fraction of neuronal somata was estimated, using semithin toludine blue stained sections. Seven categories of profiles were analyzed in electron micrographs: 1) dendrites, 2) boutons on dendrites, 3) boutons on somata, 4) myelinated fibers, 5) glial perikarya, 6) vascular elements, and 7) unclassified components. Volume fraction data indicate that the volumetric composition of the wild-type murine cerebellar nuclei is: 38% myelinated fibers, 8% neuronal somata, 11% dendrites, 10% boutons on dendrites, less than 1% boutons on somata, 2.5% glial somata, and 6% vascular elements. Unclassified elements, consisting primarily of glial processes and intercellular space, comprised 23% of the volume of the wild-type cerebellar nuclei. In lurcher, the loss of myelinated axons and boutons accounts for 59% of the atrophy of the cerebellar nuclei. Loss of nuclear neurons accounts for 2%, and a reduction in dendritic arbors for another 8.3% of this atrophy. The remaining 30.7% of the lost nuclear volume results from reduced volume of glial processes, vascular elements, and intercellular space.

Coronary artery stenosis in an australian population

The purpose of this study was to examine the extent and severity of coronary stenosis in an Australian population. The subjects were autopsy cases among people aged 20-69 yrs dying in and out of hospital in the Newcastle area of New South Wales in 1985-86. Three groups were compared: coronary deaths, deaths from other natural causes and deaths due to external causes. Cross-sectional luminal narrowing in each major subepicardial artery was measured by stereological point counting. Stenosis was found, as expected, to be more severe in males than in females, increased with age and was greatest in those who died from coronary disease. Even in non-coronary deaths mean percentage luminal narrowing in the narrowest segments ranged from about 40% in people aged 20-29 yrs to over 60% in those aged 60-69 yrs. Narrowing was more pronounced in the left anterior descending artery and least in the left circumflex artery. To the extent that results from this autopsy study can be generalized to the whole population, these findings illustrate that, despite declining mortality from ischemic heart disease in Australia, coronary atherosclerosis remains a widespread problem.

Turn-specific and pigment-dependent differences in the stria vascularis of normal and gentamicin-treated albino and pigmented guinea pigs

The aims of the present study were to determine which structures in the stria vascularis (SV) may depend upon the presence of pigmented melanocytes both for normal morphology and for the expression of gentamicin ototoxicity in the inner ear. These pigment-dependent influences were inferred through comparisons of the SV in pigmented guinea pigs and in albinos containing nonpigmented melanocytes. Results were obtained from 6 albino and 8 pigmented guinea pigs given gentamicin, and from 3 albino and 3 pigmented control animals not receiving the drug. One-month old animals received gentamicin daily (100 mg/kg) for 14 days and recovered for an additional 14 days before being prepared for electron microscopy. The SV from each of the 4 cochlear turns was analyzed using stereological point counting procedures. In control animals, differences were found in the higher cochlear turns, where volume density for the marginal cells in albinos was abnormally large (turns 3 and 4), while the volume density for intermediate cells (melanocytes) was abnormally small (turn 3). Cell volume estimates for the intermediate cells were significantly smaller in the albino than pigmented control animals in the higher cochlear turns, indicating that functional abnormalities may be found in the albino cochlea. In animals exposed to gentamicin, marginal cell volume density was reduced significantly in turn 4 of albinos, but not in any region of the pigmented inner ears. Radial area of SV and estimates of the absolute volumes for marginal cells in albinos given gentamicin also were significantly reduced in turn 1 compared to their controls; such differences were not observed in the pigmented animals. The results indicate that marginal cell size is significantly reduced in albino but not pigmented animals 14 days after gentamicin exposure, and further suggest a role of pigmented melanocytes in ameliorating gentamicin-induced cochlear damage.

Hypertonic saline increases vascular permeability in the rat trachea by producing neurogenic inflammation.

In this study, we examined whether inhalation of hypertonic saline aerosols increases vascular permeability in the rat trachea, and we examined the role of neurogenic inflammation in this response. Stereological point counting was performed to measure the percent area occupied by Monastral blue-labeled blood vessels as a means of quantifying the increase in vascular permeability in tracheal whole mounts. Hypertonic saline aerosols (3.6-14.4% NaCl) increased vascular permeability in a dose-dependent fashion compared with 0.9% NaCl. Thus, the area density of Monastral blue-labeled vessels after inhalation of 3.6% NaCl was greater (21.2 +/- 3.5% mean +/- SEM, n = 5) than after 0.9% NaCl aerosol (3.3 +/- 0.9%, n = 5, P less than 0.5). The neutral endopeptidase inhibitor phosphoramidon (2.5 mg/kg, i.v.) significantly potentiated the increase of vascular permeability caused by 3.6% NaCl. Desensitization of sensory nerve endings by pretreatment with capsaicin markedly reduced the usual increase in vascular permeability caused by 3.6% NaCl, but the increase in vascular permeability induced by aerosolized substance P (10(-4) M) was unchanged. These findings suggest that hypertonic saline increases vascular permeability in the rat trachea by stimulating the release of neuropeptides from sensory nerves.

Quantitative ultrastructural analysis of hepatoprotective effects of (+)-cyanidanol-3 on alcoholic liver damage

Biochemical considerations suggest that bioflavonoids may be effective antihepatotoxic and hepatoprotective agents. We, therefore, designed a morphometric study to examine the effect of (+)-cyanidanol-3 on ethanol-induced hepatocellular alterations. Using a stereologic point and intersection counting method we determined the volume fraction of cytoplasmic components, the surface area of cytoplasmic membranes, some parameters of mitochondria, and numerical densities of some organelles, after chronic ethanol intoxication and hepatoprotective treatment of rats. Consistent with previous qualitative and quantitative descriptions, chronic ethanol feeding caused increases in parameters of mitochondria, microsome, and Golgi complex. The hepatoprotective (+)-cyanidanol-3 treatment restored most of the morphologic distortion caused by ethanol ingestion. The (+)-cyanidanol-3 treatment alone caused an elevation of the surface area of smooth endoplasmic reticulum and nonsignificant changes in mitochondrial parameters, possibly due to its inducing effect on microsomes and activating effect on mitochondrial enzymes. These data indicate that bioflavonoids could be of potential benefit in hepatotoxic or alcohol-related liver disease.

Sendai virus infection potentiates neurogenic inflammation in the rat trachea

The goal of this study was to determine whether Sendai (parainfluenza type I) virus infection potentiates neurogenic inflammatory responses in the trachea. Pathogen-free F344 male rats were inoculated into each nostril with Sendai virus or with sterile culture medium. Six days after the inoculation, a threshold dose of capsaicin (75 micrograms/kg) or the vehicle used to dissolve the capsaicin was injected intravenously, and Monastral blue pigment was injected to measure the increase in vascular permeability produced by the capsaicin or vehicle (n = 6 per group). Five minutes later the tracheae were fixed by vascular perfusion, removed, and treated with a histochemical reaction for myeloperoxidase to stain the neutrophils in the mucosa, and the magnitude of the Monastral blue extravasation was determined both by stereological point counting and by microspectrophotometry. Capsaicin produced a larger number of Monastral blue-labeled blood vessels in the rats infected with Sendai virus than in the uninfected rats or in any of the rats that received only the vehicle. In addition, capsaicin further increased the already large number of neutrophils in the tracheal mucosa of the infected rats. However, in the dose used, it had no detectable effect on neutrophils in the uninfected rats. We conclude that Sendai virus infection causes neutrophil chemotaxis but does not increase vascular permeability in the trachea; it does, however, make the trachea abnormally susceptible to neurogenic inflammation, as manifested by an unusually large increase in vascular permeability and an exaggerated influx of neutrophils in response to sensory nerve stimulation by capsaicin.