Point Counting Stereology Research Articles

Stereological study of organelle distribution in human mature oocytes

The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques.

Estimate of the crystallization volume fraction in lithium disilicate glass-ceramics using Fourier transform infrared reflectance spectroscopy

Fourier transform infrared reflectance spectroscopy (FTIR-RS) was investigated as a potential tool for determining the volume fraction of crystals in partially-crystallized lithium disilicate (LS2) glasses. The LS2 glass samples were partially-crystallized at two temperatures, 583 °C and 595 °C, as a function of time. These temperatures were selected based on thermal analysis of LS2 glass. The FTIR spectra of the polished sample surfaces were obtained in specular reflectance mode. The volume fraction of crystals in the samples was obtained via a point-count stereology technique. A relation between FTIR-RS peak intensities and the volume fraction of crystals in the partially-crystallized LS2 glasses was then determined. This relation can provide a convenient quality control process for glass-ceramic industry.

CONTROL OF LOW-RESOLUTION SCANNING OF OVARIAN TUMOR STROMAL COMPARTMENT

The active search for markers of prognostic impact and therapeutic response is of crucial importance in oncology. The amount of stroma is a potential indicator with a significant impact on tumor growth. To solve the problems linked to tumor heterogeneity and, hence, of quantization at the microscopic level, we developed an image-analysis strategy dedicated to estimating the proportion of tumor connective tissue in whole histological sections at low resolution. 2,700-dot-per-inch numerical images were acquired with a calibrated slide scanner and automatically analyzed. We performed a quality control in order to evaluate the efficiency of the proposed method of analysis at the selected resolution. The point-counting method and the interactive drawing of stroma limits on equivalent mosaic microscopic images were "the gold standards." The proportions of stroma estimated on microscopic images (magnification with a 4× objective corresponding to 16,200 dots per inch) and on slide scanner images (2,700 dots per inch) through point counting stereology or manual delineation were equivalent (p < 0.0001). The results obtained through automatic image processing of scanner images were also close to the gold standard (95% correlation, p < 0.0001). The proposed method makes it possible to estimate the stroma amount of ovarian carcinomas in whole histological sections with a simple, fast, and low-cost acquisition device.

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.

Skeletal Muscle Ultrastructure in Normal Pregnancy and Preeclampsia

The aim of this study was to assess by quantitative methods whether the assumed metabolic disturbance underlying preeclampsia would be reflected in muscle cell composition of lipid, mitochondria, or glycogen. We have reported mitochondrial dysfunction in preeclampsia, and since accumulation of lipid in skeletal muscle is a feature in mitochondrial disorders, our hypothesis was that preeclamptic women would have an increased content of triglyceride droplets. Quantitative investigation of the skeletal muscle ultrastructure was performed in 10 women with severe preeclampsia and in 6 normotensive pregnant women. Biopsy specimens from musculus rectus abdominis were taken during cesarean section and prepared for electron microscopy. Random pictures were taken by transmission electron microscopy, and point-counting stereology was performed. Preeclamptic women did not have a higher lipid volume fraction than normotensive pregnant women, and we had to reject our hypothesis. On the contrary, there was a tendency towards a lower triglyceride volume fraction in preeclampsia. We did not detect differences in relative volumes of mitochondria or glycogen in skeletal muscle between the two groups.

Effects of Increased Pulse Pressure on Cerebral Arterioles

The goal of this study was to examine the hypothesis that increases in pulse pressure produce hypertrophy of cerebral arterioles, even in the absence of increases in mean pressure. Sprague-Dawley rats underwent creation of an arteriovenous fistula and clipping of one carotid artery at 1 month of age. Rats that underwent exposure of the abdominal aorta without fistula production and unilateral carotid clipping served as controls. At about 6 months of age, the mechanics of sham and clipped pial arterioles were examined in vivo in anesthetized rats. Stress-strain relations were calculated from measurements of pial arteriolar pressure (servo null) and diameter and cross-sectional area of the arteriolar wall. Point counting stereology was used to quantify individual components in the arteriolar wall. Before deactivation of smooth muscle with EDTA, cross-sectional areas of the vessel wall and pulse pressures in sham pial arterioles were significantly greater (P < .05) in arteriovenous fistula rats than in control rats (cross-sectional area, 1468 +/- 100 versus 1129 +/- 104 microns 2; pulse pressure, 26 +/- 1 versus 14 +/- 1 mm Hg). In contrast, systolic and mean pressures in sham arterioles were not significantly different and diastolic pressure was significantly less in arteriovenous fistula rats (systolic pressure, 69 +/- 1 versus 67 +/- 4 mm Hg; mean pressure, 52 +/- 2 versus 57 +/- 3 mm Hg; diastolic pressure, 43 +/- 2 versus 53 +/- 3 mm Hg). Carotid clipping normalized cross-sectional area of the vessel wall (1083 +/- 86 microns 2) and pulse pressure (12 +/- 1 mm Hg) in pial arterioles of arteriovenous fistula rats. During maximal dilatation, the stress-strain curve in sham arterioles of arteriovenous fistula rats was shifted to the right of the curve in control rats, which indicates that arteriovenous fistulae increase passive distensibility of cerebral arterioles. The proportion of distensible components in the vessel wall (smooth muscle, elastin, and endothelium) was increased in sham arterioles of arteriovenous fistula rats. These findings (1) suggest that increases in pulse pressure, even in the absence of increases in mean pressure, are sufficient to produce hypertrophy of cerebral arterioles and (2) provide support for the concept that increases in distensibility of cerebral arterioles in association with hypertrophy of the vessel wall may be related to alterations in wall composition.

Comparison of ultrastructural characteristics of gonadotropin-releasing hormone neurons in prepubertal and adult male rats

Gonadotropin-releasing hormone neurons from prepubertal (29-day-old) and adult (three-month-old) male rats were demonstrated immunocytochemically using the LR1 antibody, and prepared for electron microscopic examination. Gonadotropin-releasing hormone neurons were equally immunoreactive in the two age groups, but there were heavy deposits of reaction product in the outer nuclear envelope of these neurons in prepubertal animals. Point count stereology on electron micrographic montages of gonadotropin-releasing hormone neurons at x25,000 was used to compare the relative proportion of cytoplasm containing various subcellular organelles. More of the cytoplasm was occupied by Golgi apparatus and secretory vesicles in the prepubertal animals. The representation of mitochondria was equal in the two age groups, while there were more lysosomes in the gonadotropin-releasing hormone neurons from adult animals. The density of synaptic input to the neurons was estimated using quantitative morphometrics on electron micrographs of three levels of section through the neuron, magnified x25,000. The percentage of the perikaryal membrane with synaptic contacts was greater in the gonadotropin-releasing hormone neurons from adults. Most strikingly, there were gonadotropin-releasing hormone terminals on gonadotropin-releasing hormone soma of these neurons in prepubertal animals, but not in the adults. The highly immunoreactive outer nuclear envelope and relative larger representation of Golgi and secretory vesicles in gonadotropin-releasing hormone neurons in prepubertal animals suggest that these cells are actively synthesizing peptides, including gonadotropin-releasing hormone. The large representation of Golgi apparatus may also reflect the active biosynthesis of membrane in association with the elaboration of neuronal processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural changes in gonadotropin-releasing hormone neurons as a function of age and ovariectomy in rats

In this study we examined the effects of aging on various aspects of the ultrastructure of gonadotropin-releasing hormone neurons in female rats, including the density of synaptic input and the volume fraction of various subcellular organelles. In addition, we explored the possibility that removal of estrogen might provide a protective effect on the aging of the gonadotropin-releasing hormone neuron as exposure to gonadal steroids alters the time course of reproductive aging. Our experimental groups included four- and 18–20-month-old virgin female rats divided as follows: young intact, young short-term ovariectomized, old intact, old short-term ovariectomized and old long-term ovariectomized. Brain tissue was processed for immunocytochemical detection of gonadotropinreleasing hormone neurons and selected cells from the preoptic area were chosen for electron microscopic examination. The percentage of plasma membrane containing synaptic modification was quantified using a morphometrics program, and the volume fraction of lysosomes/lipofuscin, rough endoplasmic reticulum and Golgi apparatus were estimated using point count stereology. Whereas we had previously found a significant increase in the density of synaptic input to gonadotropinreleasing hormone neurons in aged virgin male rats, the density of synaptic input to gonadotropin-releasing hormone cells in the virgin female was not affected by age. The volume fraction of lysosomes/lipofuscin was increased in all age groups. Aging produced a dramatic decrease in the volume fraction of rough endoplasmic reticulum as well as a decrease in Golgi, suggesting a general decrease in biosynthetic activity of the cells. There was no apparent protective effect of long-term reduction in exposure to estrogen over the life span on these aging changes. Ovariectomy had paradoxical effects depending on the age of the animal: there was a decrease of the rough endoplasmic reticulum in the young rat and an increase in the old animal. We also noted that ovariectomy did not result in hypertrophied glial ensheathment of gonadotropin-releasing hormone neurons in female rats as we had observed in the Rhesus macaque. We conclude that aging changes in the gonadotropin-releasing hormone system are affected by factors related to the sex and hormonal condition of the animal.

Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.

The purpose of this study was to examine effects of hypertension on mechanics of cerebral arterioles in nongenetic and genetic models of chronic hypertension. Pressure (servo null) and diameter were measured in pial arterioles of anesthetized renal hypertensive rats (one-kidney, one clip), uninephrectomized normotensive rats, spontaneously hypertensive rats, and normotensive Wistar-Kyoto rats. During maximal dilatation with EDTA, external diameter of pial arterioles at 70 mm Hg pial arteriolar pressure was not significantly different in renal hypertensive and normotensive rats (86 +/- 5 [mean +/- SEM] versus 84 +/- 4 microns) but was less in spontaneously hypertensive rats than in Wistar-Kyoto rats (81 +/- 3 versus 92 +/- 3 microns; p < 0.05). Cross-sectional area of the arteriolar wall (histological) was greater in renal hypertensive than in normotensive rats (1,360 +/- 131 versus 952 +/- 89 microns 2; p < 0.05) and in spontaneously hypertensive rats than in Wistar-Kyoto rats (1,294 +/- 97 versus 817 +/- 86 microns 2; p < 0.05). The stress-strain relation obtained from pressure-diameter data during maximal dilatation with EDTA indicated that distensibility of pial arterioles, when fully relaxed, was greater in renal hypertensive and spontaneously hypertensive rats than in normotensive and Wistar-Kyoto rats. We used point-counting stereology to quantitate composition of pial arterioles in renal hypertensive rats. Cross-sectional area of smooth muscle and elastin was significantly greater in renal hypertensive than in normotensive rats (smooth muscle, 947 +/- 108 versus 620 +/- 62 microns 2; elastin, 101 +/- 11 versus 55 +/- 6 microns 2; p < 0.05), whereas cross-sectional area of collagen and basement membrane was not significantly different in the two groups (collagen, 6 +/- 1 versus 5 +/- 1 microns 2; basement membrane, 120 +/- 12 versus 104 +/- 8 microns 2). Thus, we conclude that 1) cerebral arterioles undergo hypertrophy in both renal hypertensive and spontaneously hypertensive rats; 2) cerebral arterioles in renal hypertensive rats do not undergo "remodeling" with a reduction in external diameter, whereas external diameter is smaller in spontaneously hypertensive than in Wistar-Kyoto rats; 3) distensibility of cerebral arterioles, when fully relaxed, is increased in renal hypertensive rats and is greater in spontaneously hypertensive than in Wistar-Kyoto rats; and 4) the distensible components of the arteriolar wall are increased disproportionately in cerebral arterioles of renal hypertensive rats, which may contribute to increases in arteriolar distensibility.

Computer assisted data collection for stereology: rationale and description of point counting stereology (PCS) software.

The paper describes microcomputer software for point counting stereology. Stereology includes a collection of statistical methods that quantify the images of light and transmission electron microscopy. The methods use test grids placed over images to collect raw data, which includes counts of points, intersections, transections, and profiles. In turn, the counts are included in stereological equations that give estimates of compartmental volumes, surfaces, lengths, or numbers. These parameters describe the composition of a structure in three-dimensional space. The PCS (point counting stereology) System Software III serves as a data collection, storage, and management tool. Users set up point counting protocols without programming, enter data by pressing predefined function (MS-DOS) or alphabetic keys (UNIX), store data in files, select files for analysis, and calculate results as stereological densities. The latest version of the PCS software includes a new user interface and is designed as a research "front end" that can feed data either into the calculation tools of a stereology tutorial (Bolender, 1992, this issue) or into the analysis routines of quantitative morphology databases (Bolender and Bluhm, 1992).

Structural transformations indistinquishable by point-count stereology

Confidence in 3D multi-segment foot models has been limited by a lack of repeatability data, particularly in older populations that may display unique functional foot characteristics. This study aimed to determine the intra and inter-observer repeatability of stance phase kinematic data from a multi-segment foot model described by Leardini et al. [2] in people aged 50 years or older. Twenty healthy adults participated (mean age 65.4 years SD 8.4). A repeated measures study design was used with data collected from four testing sessions on two days from two observers. Intra (within-day and between-day) and inter-observer coefficient of multiple correlations revealed moderate to excellent similarity of stance phase joint range of motion (0.621–0.975). Relative to the joint range of motion (ROM), mean differences (MD) between sessions were highest for the within-day comparison for all planar ROM at the metatarsus-midfoot articulation (sagittal plane ROM 5.2° vs. 3.9°, MD 3.1°; coronal plane ROM 3.9 vs. 3.1°, MD 2.3°; transverse plane ROM 6.8° vs. 5.16°, MD 3.5°). Consequently, data from the metatarsus-midfoot articulation in the Istituto Ortopedico Rizzoli (IOR) foot model in adults aged over 50 years needs to be considered with respect to the findings of this study.

Effects of antihypertensive treatment on composition of cerebral arterioles.

Treatment of chronic hypertension with cilazapril, but not hydralazine, attenuates changes in distensibility of cerebral arterioles that occur in stroke-prone spontaneously hypertensive rats (SHRSPs). In this study, effects of antihypertensive treatment on composition of cerebral arterioles was determined in SHRSPs. Cilazapril (45 mg/kg/day), an angiotensin converting enzyme (ACE) inhibitor, or hydralazine (18 mg/kg/day) was begun when rats were 3 months of age. Both cilazapril and hydralazine reduced systolic arterial pressure in SHRSPs (from 199 +/- 6 to 122 +/- 7 mm Hg for cilazapril versus 143 +/- 5 mm Hg for hydralazine [mean +/- SEM]; p less than 0.05). Cerebral arterioles were fixed in vivo, and the cross-sectional area of the vessel wall was measured histologically. In SHRSPs, both cilazapril and hydralazine reduced cross-sectional area of the vessel wall to values obtained in Wistar-Kyoto (WKY) rats. Thus, both cilazapril and hydralazine prevented hypertrophy of cerebral arterioles in SHRSPs. Composition of the arteriolar wall was determined with point counting stereology. Cerebral arterioles in SHRSPs contained significantly more smooth muscle and elastin than in WKY rats (1,294 +/- 157 versus 853 +/- 88 microns2, respectively, for smooth muscle and 148 +/- 13 versus 108 +/- 7 microns2, respectively, for elastin (120 +/- 8 microns2) in cerebral arterioles in SHRSPs was similar to that in WKY rats. Treatment with hydralazine was effective in preventing increases in elastin (128 +/- 14 microns2) and in attenuating increases in smooth muscle (1,008 +/- 18 microns2). The ratio of nondistensible (collagen, basement membrane) to distensible (smooth muscle, elastin, endothelium) components was greater in SHRSPs than in WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of local reduction in pressure on distensibility and composition of cerebral arterioles.

This study examined effects of local reductions in mean and pulse pressures on cerebral arterioles in normotensive Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). WKY and SHRSP underwent clipping of one carotid artery at 1 month of age. At 10-12 months of age, mechanics of pial arterioles were examined in vivo in anesthetized rats. Bilateral craniotomies were performed to expose pial arterioles in the sham and clipped cerebral hemispheres. Stress-strain relations were calculated from measurements of pial arteriolar pressure (servo null), diameter, and cross-sectional area of the arteriolar wall. Point counting stereology was used to quantitate individual components in the arteriolar wall. Before deactivation of smooth muscle with EDTA, mean (Pm) and pulse (Pp) pressures were significantly less (p less than 0.05) in clipped than in sham arterioles in WKY (Pm, 63 +/- 2 versus 73 +/- 2 mm Hg; Pp, 23 +/- 3 versus 30 +/- 3 mm Hg) and SHRSP (Pm, 94 +/- 4 versus 110 +/- 4 mm Hg; Pp, 27 +/- 2 versus 38 +/- 3 mm Hg). Cross-sectional area of the arteriolar wall was less (p less than 0.05) in clipped than in sham arterioles in both groups of rats (1,403 +/- 125 versus 1,683 +/- 125 microns2 in WKY; 1,436 +/- 72 versus 1,926 +/- 134 microns2 in SHRSP). There was a correlation between cross-sectional area of the vessel wall and pulse pressure (r2 = 0.66), but not mean pressure (r2 = 0.09). During maximal dilatation with EDTA, the stress-strain curve was shifted to the left in clipped arterioles of SHRSP, but not of WKY, which indicates that carotid clipping in SHRSP reduces passive distensibility of cerebral arterioles. The proportion of distensible components in the vessel wall (smooth muscle, elastin, and endothelium) was reduced in clipped arterioles in SHRSP, but not in WKY. These findings suggest that 1) vascular hypertrophy of cerebral arterioles is related more closely to pulse pressure than to mean pressure, and 2) reduction of pial arteriolar pressure completely prevents cerebral vascular hypertrophy and attenuates increases in passive distensibility of cerebral arterioles in SHRSP.

Effects of aging on mechanics and composition of cerebral arterioles in rats.

The purpose of this study was to examine effects of aging on the mechanics and composition of cerebral arterioles. We measured pressure (servo-null) and diameter in pial arterioles in anesthetized adult (9-12 months old) and aged (24-27 months old) Fischer 344 rats. After deactivation of smooth muscle with EDTA, diameter of pial arterioles at 70 mm Hg pial arteriolar pressure was less in aged than in adult rats (67 +/- 4 vs. 81 +/- 4 microns [mean +/- SEM], p less than 0.05). The stress-strain relation and the slope of tangential elastic modulus versus stress (6.8 +/- 0.6 vs. 5.3 +/- 0.3, p less than 0.05) indicated that distensibility of pial arterioles was reduced in aged rats. Cross-sectional area of the vessel wall, measured histologically, was less in aged than adult rats (1,239 +/- 91 vs. 1,832 +/- 180 microns2, p less than 0.05). Point counting stereology was used to quantitate smooth muscle, elastin, collagen, and basement membrane in the arteriolar wall. Cross-sectional areas of smooth muscle and elastin were significantly less in aged than adult rats (744 +/- 57 vs. 1,291 +/- 119 microns2 for smooth muscle, 52 +/- 6 vs. 113 +/- 15 microns2 for elastin; p less than 0.05), whereas cross-sectional areas of collagen and basement membrane were not significantly different in aged and adult rats (4 +/- 1 vs. 3 +/- 1 microns2 for collagen, 236 +/- 17 vs. 258 +/- 31 microns2 for basement membrane). The ratio of nondistensible (collagen and basement membrane) to distensible (smooth muscle and elastin) components was greater in aged than adult rats (0.30 +/- 0.01 vs. 0.18 +/- 0.01, p less than 0.05). Thus, we conclude that, during aging, cerebral arterioles undergo atrophy, distensibility of cerebral arterioles is reduced, and the relative proportion of distensible elements, elastin and smooth muscle, is reduced in the arteriolar wall.

Ultrastructural differences between smooth and thorny gonadotropin-releasing hormone neurons

It has been suggested that gonadotropin-releasing hormone neurons with irregular contours receive more synaptic input than do those with smooth contours. To test this hypothesis, a morphometric analysis of gonadotropin-releasing hormone neurons of differing shapes was made. Male Sprague-Dawley rats were perfused and 40-μm tissue sections from the preoptic area were treated for the demonstration of gonadotropin-releasing hormone immunoreactivity, using the avidin-biotin horseradish peroxidase method with 3,3'-diaminobenzidine as the chromogen. Gonadotropin-releasing hormone neurons with either a smooth or a highly irregular outline, sampled at three depths ultrastructurally, were compared for density of synaptic innervation and for relative content of various subcellular organelles. Point counting stereology comparisons of five gonadotropin-releasing hormone neurons of the two shapes in four rats revealed that smooth neurons contained less cytoplasm than “thorny” neurons, but the size of their nuclei was the same. There were more and larger nucleoli in smooth cells. Thorny neurons contained more Golgi apparatus and more mitochondria, but the amount of rough endoplasmic reticulum was the same in neurons of the two morphological types. There was no difference in the incidence of synapses or in the density of synaptic input as measured per unit of neuronal membrane in smooth and thorny gonadotropin-releasing hormone neurons. These results suggest that smooth-contoured gonadotropin-releasing hormone neurons may be more actively transcribing a message while thorny neurons are more actively engaged in peptide processing and packaging. In that the density of synaptic input was the same, these apparent functional differences among gonadotropin-releasing hormone neurons of differing shapes may be independent of synaptic influences; but this must be tested by chemical characterization of the synapses.

Effect of sympathetic nerves on composition and distensibility of cerebral arterioles in rats.

1. The goals of this study were to examine the effects of chronic sympathetic denervation on the mechanics and composition of cerebral arterioles in normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). 2. We used an in vivo method to examine the mechanics of pial arterioles in 10- to 12-month-old, anaesthetized WKY and SHRSP that had undergone unilateral removal of the superior cervical ganglion at 1 month of age. Bilateral craniotomies were performed in each rat to expose pial arterioles in the innervated and denervated cerebral hemispheres. Arterioles were deactivated with EDTA. Incremental distensibility and stress-strain relationships were calculated from measurements of pial arteriolar pressure (servo null), diameter and cross-sectional area of the arteriolar wall. Point counting stereology was used to quantify volume density and cross-sectional area of individual components in the arteriolar wall. 3. Chronic sympathetic denervation reduced cross-sectional area of the arteriolar wall by 16 +/- 2% (mean +/- S.E. of mean; P less than 0.05) in WKY and 44 +/- 3% in SHRSP. During maximal dilatation with EDTA, incremental distensibility was reduced and the stress-strain curve was shifted to the left in denervated arterioles of SHRSP, but not WKY. These findings indicate that sympathetic denervation in SHRSP attenuates the development of hypertrophy in pial arterioles and reduces arteriolar distensibility. The ratio of non-distensible (collagen and basement membrane) to distensible (smooth muscle, elastin and endothelium) components was reduced in denervated arterioles in SHRSP, but not WKY. 4. Thus, sympathetic nerves have trophic effects on cerebral arterioles in WKY and, to a greater degree, in SHRSP. Sympathetic nerves also contribute to increases in distensibility of cerebral arterioles in SHRSP, but not WKY. The increase in arteriolar distensibility is accompanied by a disproportionate increase in the more compliant elements of cerebral arterioles.

Mechanics and composition of arterioles in brain stem and cerebrum.

The goal of this study was to compare mechanics and composition of arterioles in brain stem and cerebrum. We calculated stress and strain of pial arterioles in anesthetized rats from measurements of pial arteriolar pressure (servo-null), diameter, and cross-sectional area of the vessel wall. Composition of pial arterioles was quantitated using point-counting stereology. Before deactivation of smooth muscle with ethylenediaminetetraacetic acid (EDTA), pial arteriolar pressure and diameter were 28 and 30% greater (P less than 0.05) in brain stem than cerebrum. After EDTA, diameter of arterioles was similar in brain stem and cerebrum. Cross-sectional area of the arteriolar wall was 32% greater (P less than 0.05) in brain stem than cerebrum. Stress-strain curves indicated that distensibility of pial arterioles is greater in brain stem than cerebrum. The proportion of nondistensible (collagen and basement membrane) to distensible (elastin, smooth muscle, and endothelium) components was 20% less (P less than 0.05) in brain stem than cerebral arterioles. We conclude that 1) cross-sectional area of the vessel wall in arterioles of comparable size is greater in brain stem than cerebrum, 2) distensibility of arterioles is greater in brain stem than cerebrum, despite greater cross-sectional area of the arteriolar wall in brain stem, and 3) the proportion of elastic components is greater in brain stem than cerebral arterioles, which may contribute to greater arteriolar distensibility in brain stem.

Coronary vasodilator reserve, capillarity, and mitochondria in trained hypertensive rats

To test the hypothesis that exercise training can reverse the decrements in coronary reserve, capillary density, and mitochondrial volume density evident during established hypertension, we trained spontaneously hypertensive (SHR) and normotensive (WKY) rats on a treadmill over a 3-mo period. At 7 mo of age we used microspheres to evaluate myocardial perfusion in conscious rats. Exercise training did not alter hypertension or left ventricular hypertrophy but did increase maximal O2 consumption in both SHR and WKY. A decrement in left and right ventricular coronary reserve in SHR, compared with WKY, was indicated by 1) a smaller increment in myocardial perfusion during maximal vasodilation with dipyridamole and 2) a higher minimal coronary vascular resistance per unit mass. Exercise training had no significant effect on any index of myocardial perfusion in SHR or WKY. A 12% decrement in capillary numerical density in the endomyocardium of SHR was not reversed by exercise training. We estimated the volume densities of mitochondria, myofibrils, and sarcoplasm using electron microscopy and point-counting stereology on perfusion-fixed hearts. None of the parameters in either SHR or WKY was changed by exercise training. It is concluded that exercise training does not reverse the decrements in coronary reserve and capillary numerical density associated with hypertension in adult rats. Moreover the previously observed enhancement of mitochondrial volume density due to exercise in young hypertensive rats was not observed in adult SHR.

Computer-assisted morphometry using video-mixed microscopic images and computer graphics.

A microcomputer system has been developed for collection and analysis of morphometric data from video images of histological sections. Microscopic fields of view are visualized on a video monitor and are overlaid with computer-generated graphics by means of a video mixer. Planimetric data are entered by drawing on a digitizing tablet while observing the drawn image superimposed on the tissue. Programs are available to calculate various geometric parameters, count objects in a field, perform descriptive statistics, and measure volume and surface area of a solid from a series of cross sections. A program for point-counting stereology presents single points for scoring within a user-defined reference area. The potential applications of interactive graphics in morphometry are discussed.

Timing of the quantitative recovery in the regenerating rat lung.

Growing rats 23 days of age were subjected to the resection of the upper and middle lobes of the right lung (25% of total lung volume). On postoperative days 1, 4, 6, 9, 12, 18, and 30, the lungs of 5 animals were fixed by intratracheal instillation of glutaraldehyde, their volumes measured by water displacement, and the lung tissue processed for quantitative light (LM) and electron microscopic (EM) investigations. For each group, 3 age-matched sham-operated, and 4 normal rats served as controls. Sham operation consisted in entering the pleural space, and collapsing and ventilating the lungs. In all animals, the following parameters were measured by means of point and intersection counting stereology at either LM or EM-levels: volume densities of lung parenchyma and of its components (airspaces, tissue, capillary blood) and surface densities of airspaces and of capillaries. From these data absolute parameter values were calculated for each lung. The results showed that after bilobectomy the remaining lung re-expanded rapidly first by an overinflation of the airspaces and after Day 4 by an increase in tissue mass and capillary volume. On Days 9 and 12 the operated lungs did not differ quantitatively from control lungs. Later, however, further sequels of the bilobectomy were detected: On Day 18, lobectomy-lungs were smaller than controls, and on Day 30 lungs, the left lung was significantly different in structure from the right lung and from control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)