Quantitative Morphological Study Research Articles

Delayed loss of spinal motoneurons after peripheral nerve injury in adult rats: a quantitative morphological study

The existence of retrograde cell death in sensory dorsal root ganglion (DRG) cells after peripheral nerve injury is well established. However, with respect to retrograde motoneuron death after peripheral nerve injury, available data are conflicting. This may partly be due to the cell counting techniques used. In the present study, quantitative morphometric methods have been used to analyse retrograde motoneuron death induced by spinal nerve injury in adult rats. For comparison, DRG cells were also included in the study. The C7 spinal nerve was transected about 10 mm distal to the DRG and exposed to the fluorescent tracer fast blue in order to retrogradely label the spinal motoneurons and DRG cells of the C7 segment. At 1-16 weeks postoperatively, the nuclei of fast-blue-labelled C7 motoneurons and DRG cells were counted in consecutive 50-microm-thick serial sections. For comparison, the physical disector technique and measurements of neuronal density were also used to calculate motoneuron number. The counts of fast-blue-labelled motoneurons revealed a delayed motoneuron loss amounting to 21% and 31% after 8 and 16 weeks, respectively (P<0.001). The remaining motoneurons exhibited 20% (P<0.05) soma atrophy. Using the physical disector technique, the motoneuron loss was 23% (P<0.001) after 16 weeks. Calculations of neuronal density in Nissl-stained sections failed to reveal any motoneuron loss, although after correction for shrinkage of the ventral horn a 14% (P<0.001) motoneuron loss was found. The fast-blue-labelled DRG neurons displayed 51% (P<0.001) cell loss after 16 weeks, and the remaining cells showed 22% (P<0.001) soma atrophy. In summary, cervical spinal nerve injury induces retrograde degeneration of both motoneurons and DRG cells. However, to demonstrate the motoneuron loss adequate techniques for cell counts have to be employed.

Differential emotional experience induces elevated spine densities on basal dendrites of pyramidal neurons in the anterior cingulate cortex of Octodon degus.

It appears likely that, in analogy to the synaptic development of sensory and motor cortices, which critically depends on sensory or motor stimulation (Rosenzweig and Bennett, 1996), the synaptic development of limbic cortical regions are modulated by early postnatal cognitive and emotional experiences. The very first postnatal experience, which takes place in a confined and stable familial environment, is the interaction of the newborn individual with the parents and siblings (Gray, 1958). The aim of this quantitative morphological study was to analyze the impact of different degrees of juvenile emotional experience on the synaptic development in a limbic cortical area, the dorsal anterior cingulate cortex, a region which is involved in the perception and regulation of emotions. We study the precocious trumpet-tailed rat (Octodon degus) as the animal model, because, like human babies, this species is born with functional visual and acoustic systems and the pups are therefore capable of detecting even subtle environmental changes immediately after birth (Reynolds and Wright, 1979; Poeggel and Braun, 1996; Braun et al., 2000; Ovtscharoff and Braun, 2001). The results demonstrate that already a subtle disturbance of the familial environment such as handling induced significantly elevated spine densities on the basal dendrites of layer III cortical pyramidal neurons. More severe disturbances of the emotional environment, such as periodic parental deprivation with or without subsequent chronic social isolation, resulted in an elevation of spine densities of similar magnitude as seen after handling and in addition, altered spine densities confined to specific dendritic segments were observed in these groups. These observations unveil the remarkable sensitivity of the dorsal anterior cingulate cortex towards environmental influences and behavioral experiences during phases of postnatal development. The behavioral consequences of these experience-induced synaptic changes still need to be analyzed further to assess if they are beneficial or detrimental to the animals cognitive and emotional capacities in later life.

Stereologic evaluation of the pig gastric wall and of somatostatinergic and serotoninergic immunoreactive mucosal cells during perinatal development.

A quantitative morphological study using stereologic methods was performed on vertical paraffin sections of the stomach of fetal and neonatal pigs. The sections were processed for 5-HT- and SOM-immunohistochemistry. In the neonatal pigs, the volume density of the submucosal layer in the pyloric gland region was approximately 15% less compared to the cardiac gland region. This suggests that altered functional demands after birth are temporarily related to and perhaps could promote the morphological diversification between the gastric regions. The distribution of 5-HT-IR and SOM-IR mucosal cells corresponds with previous observations in the adult mammalian stomach. However, based upon our results an age-dependent maturity or even different role is suspected for 5-HT and SOM. This is substantiated by the 4-fold rise of the volume occupied by 5-HT-IR mucosal cells in the pyloric gland region during development. Secondly, the regional differences of the volume density of SOM-IR mucosal cells vary according to developmental stage. The developmental variations of 5-HT- and SOM-IR mucosal cells contrast with findings in the rodent stomach. However, they are comparable to observations in man.

Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: neuropathology.

Neuropathologic assessment of chemically induced developmental alterations in the nervous system for regulatory purposes is a multifactorial, complex process. This calls for careful qualitative and quantitative morphologic study of numerous brains at several developmental stages in rats. Quantitative evaluation may include such basic methods as determination of brain weight and dimensions as well as the progressively more complex approaches of linear, areal, or stereologic measurement of brain sections. Histologic evaluation employs routine stains (such as hematoxylin and eosin), which can be complemented by a variety of special and immunohistochemical procedures. These brain studies are augmented by morphologic assessment of selected peripheral nervous system structures. Studies of this nature require a high level of technical skill as well as special training on the part of the pathologist. The pathologist should have knowledge of normal microscopic neuroanatomy/neuronal circuitry and an understanding of basic principles of developmental neurobiology, such as familiarity with the patterns of physiologic or programmed cell de

Quantitative estimations of foveal and extra-foveal retinal circuitry in humans

For an understanding of the basis for psychophysical measurement of visual resolution, quantitative morphological studies of retinal neuronal architecture are needed. Here we report on cell densities and retinal ganglion cell:cone ratio (RGC:C) from the foveal border to the peripheral retina (34° eccentricity). Quantitative estimates of RGC and C densities were made using a modified disector method in three vertically sectioned human retinae and were adjusted for RGC displacement. In agreement with our previous data on humans, we found an RGC:C ratio close to 3 at 2–3° eccentricity. Outside the foveal border, the ratio declined to 1.0 at 7.5° eccentricity and to 0.5 at eccentricities larger than 19°. Center-to-center separation of C and RGC in addition to center-to-center separation of estimated ‘receptive fields’ was calculated at corresponding locations along the superior and inferior hemimeridians. The center-to-center separation of estimated ‘receptive fields’ was found to be more closely related to resolution thresholds from the fovea to 19° eccentricity than was the separation of RGC and C. On the basis of these quantitative estimates, models for neural circuitry involved in central and peripheral spatial vision can be discussed.

The Evolution of Morphological Diversity

The diversity of organismic form has evolved nonuniformly during the history of life. Quantitative morphological studies reveal profound changes in evolutionary rates corresponding with the generation of morphological disparity at low taxonomic diversity during the early radiation of many clades. These studies have also given insight into the relative importance of genomic and ecological factors in macroevolution, the selectivity of extinction, and other issues. Important progress has been made in the development of morphological spaces that can accommodate highly disparate forms, although this area still needs more attention. Other future directions include the relationship between morphological and ecological diversification, geographic patterns in morphological diversity, and the role of morphological disparity as a causal factor in macroevolution.

Use of low temperature and high K+ incubation media for in vitro tissue preparation for X-ray microanalysis.

Incubation of tissue slices in physiological buffers gives rise to significant changes in the intracellular ion concentrations, which may disturb subsequent X-ray microanalysis. In the present study it was attempted to design incubation conditions that retain the in vivo conditions better. The following variables were investigated: (1) exchange of Na+ in the incubation medium for K+, and exchange of Cl- for the less permeable gluconate anion; (2) incubation at 4 degrees C rather than at 37 degrees C; and (3) addition of dextran to the incubation medium. Brief exposure (a few seconds) of liver slices to a buffer causes changes in the intracellular Na, Cl and K concentrations, depending on the ionic composition of the buffer. Incubation in a normal physiological (high NaCl) buffer at 37 degrees C results in a further increase of Na and Cl and a further decrease in K in liver cells. The changes reach a maximum at 30 min and the concentrations then remain stable throughout a 2-h incubation. Incubation in sodium gluconate medium or addition of dextran to the physiological buffer somewhat reduces the changes in the intracellular ion composition (compared to the standard physiological incubation medium). Incubation in potassium gluconate medium results in a decrease in cellular Na and an increase in K. Quantitative morphological studies show that tissue oedema is observed to the same extent in hepatocytes incubated in sodium gluconate, potassium gluconate and physiological buffer containing 10% dextran. However, these buffers cause significantly less cell oedema than the physiological (high NaCl) buffer. Incubation of liver, cerebral cortex or submandibular gland slices in physiological (high NaCl) solutions at 4 degrees C for 4 h caused a more extensive increase in Na+ and decrease in K+ than incubation at 37 degrees C for 2 h. This suggests inhibition of the Na+, K(+)-ATPase under these conditions. As compared to incubation at 37 degrees C for 2 h, tissues incubated in potassium gluconate buffer at 4 degrees C for 4 h have a cellular K concentration closer to the in situ value. Cholinergic stimulation of tissue slices from cerebral cortex and submandibular gland at room temperature for 1 min shows the best physiological response in tissue slices preincubated at 4 degrees for 4 h in high KCl, potassium gluconate and high NaCl, in this order. The response can, however, only be seen, when cholinergic stimulation is carried out in a standard physiological buffer with a high NaCl concentration. It is concluded that in vitro storage of tissue for X-ray microanalysis is best carried out at 4 degrees C in a solution with a high K+ concentration.

Quantitative morphology of mast cells in skeletal muscle of normal and genetically dystrophic mice.

Mast cells are indigenous connective tissue cells that function in the process of inflammation and edema. Their numbers were studied in a quantitative morphological study of the soleus muscles from 32-week-old and 56-week-old normal and genetically dystrophic dy2J and mdx mice to determine the incidence of mast cells in muscle to increasing age and to normal and myopathic conditions. Soleus muscles from normal C57B1/J and from dystrophic C57B1/SnJ (dy2J/dy2J) and C57BL/10ScSn mdx mice were processed for examination by light and electron microscopy. Quantitation of mast cells was performed on semi-thick sections and expressed as an average of cells per millimeter squared of muscle tissue. Mast cells were observed in the connective tissue interstitium that normally separates skeletal muscle into fascicles. Their cytoplasmic granules stained metachromatically with toluidine blue and often obscured the single, centrally placed nucleus. They occurred singly or in small groups and were most frequently seen adjacent to neurovascular elements within the muscle, and in many cases were closely associated with the outer capsular regions of muscle spindles. Between the 32- and 56-week-old groups in each strain, an age-related increase in mast cell numbers was observed. In the dystrophic conditions, the dy2J and mdx skeletal muscles exhibited a two- to four-fold increase in mast cells when compared to normals in both age groups. Extensive connective tissue proliferation and sites of necrotic and regenerating muscle were common features in both myopathies. Results of this study indicate that a significantly higher number of mast cells which exist in dy2J and mdx murine skeletal muscles may be related to the high amount of connective tissue infiltration and extensive muscle fiber remodelling in these conditions. Moreover, the close proximity of mast cells to muscle spindles and nerve fascicles suggests that these cells may play a role in modulating their activities.

Quantitative morphology of mast cells in skeletal muscle of normal and genetically dystrophic mice

Background Mast cells are indigenous connective tissue cells that function in the process of inflammation and edema. Their numbers were studied in a quantitative morphological study of the soleus muscles from 32-week-old and 56-week-old normal and genetically dystrophic dy2J and mdx mice to determine the incidence of mast cells in muscle to increasing age and to normal and myopathic conditions. Methods Soleus muscles from normal C57Bl/J and from dystrophic C57Bl/SnJ (dy2J/dy2J) and C57BL/10ScSn mdx mice were processed for examination by light and electron microscopy. Quantitation of mast cells was performed on semi-thick sections and expressed as an average of cells per millimeter squared of muscle tissue. Results Mast cells were observed in the connective tissue interstitium that normally separates skeletal muscle into fascicles. Their cytoplasmic granules stained metachromatically with toluidine blue and often obscured the single, centrally placed nucleus. They occurred singly or in small groups and were most frequently seen adjacent to neurovascular elements within the muscle, and in many cases were closely associated with the outer capsular regions of muscle spindles. Between the 32- and 56-week-old groups in each strain, an age-related increase in mast cell numbers was observed. In the dystrophic conditions, the dy2J and mdx skeletal muscles exhibited a two- to four-fold increase in mast cells when compared to normals in both age groups. Extensive connective tissue proliferation and sites of necrotic and regenerating muscle were common features in both myopathies. Conclusions Results of this study indicate that a significantly higher number of mast cells which exist in dy2J and mdx murine skeletal muscles may be related to the high amount of connective tissue infiltration and extensive muscle fiber remodelling in these conditions. Moreover, the close proximity of mast cells to muscle spindles and nerve fascicles suggests that these cells may play a role in modulating their activities. Anat. Rec. 247:341–349, 1997. © 1997 Wiley-Liss, Inc.

Image processing analysis in reconstructing fragmentation and transportation mechanisms of pyroclastic deposits. The case of Monte Pilato-Rocche Rosse eruptions, Lipari (Aeolian islands, Italy)

A quantitative study of particle morphology of samples from Monte Pilato-Rocche Rosse deposits, Lipari (Aeolian islands, Italy) shows a significant variability in particle structure and texture. The study consisted of image processing techniques and multivariate statistical methods in analysis of the quantitative particle morphology data, with the goal of identifying the genetic processes responsible for the formation of the deposits. From these investigations we numerically define the form of the clasts, thus avoiding subjectivity in the classification of clast morphology. Starting from this clast definition, we then construct diagrams in which clasts due to different genetic processes (i.e., magmatic vs. hydromagmatic fragmentation), are discriminated based on simple nondimensional parameters. The application of the methods used seems to be of general interest in discriminating the fragmentation and transportation dynamics responsible for the formation of pyroclastic deposits.

Signs of aging are apparent in the testis interstitium of Sprague Dawley rats at 6 months of age

The present study investigated the effects of aging in the testis interstitium in Sprague Dawley rats. Rats of 3, 6 and 24 months of age were used. Testes of rats ( n = 5) were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in eponaraldite. Using 1 μm sections stained with methylene blue, qualitative and quantitative morphological studies were performed. Purified Leydig cell preparations, obtained by collagenase digestion followed by elutriation and density gradient centrifugation, were used to determine luteinizing hormone (LH; 100 ng/ml) stimulated testosterone secretory capacity per Leydig cell in vitro. Testosterone levels in the incubation medium, and testosterone and luteinizing hormone levels in serum of these three groups of rats were determined via radioimmunoassay. Morphological studies revealed that Leydig cells were more abundant in the testis interstitium at 6 and 24 months when compared to 3 months. Moreover, collagen fiber bundles were more frequently observed in the testis interstitium at older ages. Blood vessels of the testis interstitium in 24-month-old rats frequently showed partial and complete occlusion of their lumen and thickening of vessel walls. This feature was also present at 6 months, but less frequently. The results of the sterological studies revealed that the volumes of seminiferous tubules, interstitium and Leydig cells per testis was significantly higher ( P < 0.05), at 6 and 24 months of age than those at 3 months. Moreover, volume of macrophages per testis was observed to be significantly higher ( P < 0.05) at 6 months when compared to 3 and 24 months, and volume of connective tissue cells per testis was observed to be significantly lower ( P < 0.05) at 6 and 24 months when compared to 3 months of age. No significant difference ( P > 0.05) was observed for the volume of lymphatic space per testis in the three age groups studied. Volume of interstitial blood vessels per testis was not significantly different at 3 and 6 months of age, but a significantly greater ( P < 0.05) volume was observed at 24 months. However, at 6 and 24 months, only 71% and 31% of the total blood vessel volumes respectively had completely open lumen in them; the rest of the blood vessels were either partially (12.5% at 6 months and 17% at 24 months) or completely (16.5% at 6 months and 52% at 24 months) occluded. The number of Leydig cells per testis was doubled at 6 and 24 months of age compared to 3 months. The average volume of a Leydig cell was not significantly different between 3 and 6 months of age, however, at 24 months a significantly lower ( P < 0.05) value was observed. LH stimulated testosterone secretory capacity per Leydig cell in vitro was reduced by 50% at 6 months of age compared to 3 months; a further significant ( P < 0.05) reduction was observed at 24 months. Serum testosterone and LH levels were not significantly different between 3 and 6 months of age but at 24 months a significantly lower ( P < 0.05) value was observed for both of these hormones. In summary, the present study demonstrated many changes in the components of the testis interstitium in the aged Sprague Dawley rat. Modifications in the blood vessels and the occurrence of abundant collagen fibers in the interstitial space could possibly contribute to the reduced testosterone secretory capacity per Leydig cell with advancing in age. The observed Leydig cell hyperplasia could be suggested as a compensatory effort to maintain the normal androgen status of the aged rat, which is rather successful at 6 months but unsuccessful at 24 months. This investigation further revealed that these characteristic changes in the aged testis interstitium at 24 months are also present to some extent at 6 months of age in Sprague Dawley rats, suggesting that aging of the testis in this strain of rats commences early in life.

An ultrastructural analysis of the epithelial-fiber interface (EFI) in primate lenses

The purpose of this study was to conduct a comprehensive ultrastructural analysis of the epithelial-fiber interface (EFI) in normal adult primate (Macaque nemestrina and fascicularis; 6-9 years old, n = 10) lenses. Scanning electron microscopy (SEM) was used to initially characterize the gross size, shape and three-dimensional organization of central zone (cz) epithelial cells and the anterior ends of elongating fibers beneath these cells. This fiducial information was essential to properly orient lens pieces in freeze fracture specimen carriers for the production of replicas with unambiguously identifiable EFI. Transmission electron microscopy (TEM) of replicas and thin-sectioned material were used to ultrastructurally analyse the cz EFI. TEM thin-sectioned material was also used to ultrastructurally analyse the pregerminative (pgz), germinative (gz) and transitional zone (tz) EFI. Correlative SEM and TEM of cz EFI components revealed that the apical membrane of both epithelial and elongating fiber cells were irregularly polygonal in shape, and aligned in parallel as smooth, concave-convex surfaces. However, whereas epithelial cell apical surfaces had minimal size variation, elongating fibers were larger and considerably variable in size. Quantitative analysis of > 10000 micron2 cz elongating fiber apical surfaces failed to detect any gap junctions defined in freeze fracture replicas as complementary aggregates of transmembrane proteins (connexons) conjoined across a narrowed extracellular space. However, a comparable frequency of vesicular events was noted in this region as quantified previously in adult and embryonic chick lens. Correlative TEM analysis > 1500 linear micrometers of thin-sectioned EFI from this region confirmed the presence of epithelial-epithelial gap junctions, elongating fiber-elongating fiber gap junctions, and an extreme paucity of epithelial-elongating fiber gap junctions. In contrast, TEM analysis of > 1000 linear micrometers of thin-sectioned pgz, gz and tz EFI, confirmed the presence of epithelial-epithelial gap junctions, elongating fiber-elongating fiber gap junctions, numerous epithelial-elongating fiber adherens junctions and a few epithelial-elongating fiber gap junctions. Thus, the results of this and previous quantitative morphological and physiological studies (electronic and dye coupling) demonstrate that there is limited coupling between cz epithelial cells and underlying elongating fibers. Furthermore, the absence of gap junctional plaques in cz EFI freeze-fracture replicas and either pentalaminar or septalaminar profiles in correlative thin-sections, suggests that this limited coupling could be mediated via isolated gap junction channels. However, the results of this and previous quantitative studies further show that a greater degree of coupling exists across the pgz, gz and tz regions of the EFI and that this coupling is likely to be mediated by gap junction plaques. Finally, this and other studies continue to demonstrate that transcytotic processes play a role in lens physiology at the EFI.

A Quantitative Study of Long Saphenous Vein Morphology in Patients Undergoing Arterial Surgery

Objective: To quantify the incidence and extent of structural changes present in the long saphenous vein of patients with arterial disease. Design: Observational study of saphenous vein morphology. Setting: Departments of Surgery and Pathology, Leicester Royal Infirmary. Patients: Sixty vein biopsies from patients undergoing arterial surgery. Main outcome measures: Intimal and medial thickness and morphology. Results: Smooth muscle cell hyperplasia, elastosis and fibrosis contributed to intimal thickening (&gt; 10 μm) in 87% of veins. This was frequently associated with medial longitudinal muscle hypertrophy. Intimal thickness had a skewed distribution with a median (range) of 33 (8–381) μm, The upper limit of the normal range was 200 μm. The median (range) medial thickness was 293 (131–468) μm. Conclusions: Intimal thickening is common in the long saphenous vein of patients undergoing arterial surgery but is extensive in only a small proportion. The upper limit of the normal range was 200 μm.

Vasculature in Nontumorous Hypophyses, Pituitary Adenomas, and Carcinomas: A Quantitative Morphologic Study.

Vascular supply is essential for tumor proliferation and metastasis formation. Correlation was noted between vascular density and tumor size as well as metastases in several tumor types. The aim of the present study was to assess vascular density in nontumorous hypophyses, pituitary adenomas, primary pituitary carcinomas, and carcinomas metastatic to the pituitary. Twenty nontumorous hypophyses, 87 endocrinologically active or inactive pituitary adenomas, 8 primary pituitary carcinomas, 8 metastatic carcinomas, and 10 randomly selected noninvasive and 6 invasive adenomas were included in the study. Tissues were fixed in formalin, embedded in paraffin, cut, stained with hematoxylin and eosin, PAS, and immunostained for adenohypophysial hormones as well as Factor VIII-related antigen using the streptavidin-biotin-peroxidase complex method. Four counts were performed: percentage of capillary area, number of vessels per field, percentage of endothelial cells, and number of endothelial cells per field. The results show that pituitary adenomas have significantly lower vascular densities as compared to nontumorous adenohypophyses. Prolactin-producing adenomas removed from untreated patients have the highest counts and growth hormone-producing adenomas the lowest counts. However, the observed differences among adenoma types are not of statistical significance. No differences are noted between noninvasive and invasive tumors. Primary pituitary carcinomas show no significant increase in vascular densities. Some metastatic tumors exhibit high vascularity. It can be concluded that pituitary adenomas have a limited capacity to induce angiogenesis. Lack of significant angiogenesis may play a role in the slow pace of pituitary tumor growth and rarity of metastases.

Effects of continuous hyperfractionated accelerated and conventionally fractionated radiotherapy on the parotid and submandibular salivary glands of rhesus monkeys

Radiotherapy is a major treatment modality for head and neck cancer. It is often not possible to exclude the salivary glands from the treatment fields. The unique susceptibility of the serous cells of the salivary glands to irradiation often results in xerostomia with ensuing secondary complications and discomfort to the patients. Recent reports have suggested that continuous hyperfractionated accelerated radiotherapy (CHART) can lead to considerably less reduction in salivary flow of the parotid salivary gland than conventional radiotherapy. This study was undertaken to assess histologic changes of salivary glands induced by CHART and conventional radiation fractionation schedules. The parotid and submandibular salivary glands of adult rhesus monkeys were irradiated with cobalt-60 γ radiation at 50 Gy/20 fractions/4 weeks, 55 Gy/25 fractions/5 weeks, or 54 Gy/36 fractions/12 days (CHART). Salivary tissues were harvested at 16 weeks following irradiation and evaluated histopathologically. Microscopically, the glands receiving 50 Gy, 55 Gy, or CHART were virtually indistinguishable. There was severe atrophy and fibrosis of all glands. Quantitative analysis revealed that 50 Gy, 55 Gy, and CHART induced a reduction of serous acini in parotid glands by 86.4%, 84.8%, and 88.8%, respectively. In submandibular glands, serous acini were reduced by 99.4%, 99.0%, and 100%, respectively. The corresponding reduction in mucous acini were 98.4%, 98.4%, and 99.2%, respectively. These histopathologic and quantitative morphologic studies show that the magnitude of serous gland atrophy in the parotid and submandibular salivary glands of rhesus monkeys was similar at 16 weeks after receiving 50 Gy in 20 fractions, 55 Gy in 25 fractions, or CHART.

Microflora modulates endocrine cells in the gastrointestinal mucosa of the rat

Background/Aims: Gastrointestinal peptides and biogenic monoamines participate in the regulation of gastrointestinal functions. The aim of this study was to examine the influence of the microflora on the distribution of endocrine cells and on the release of gastrointestinal peptides. Methods: A quantitative morphological study using stereological methods was performed in gastrointestinal sections of conventional and germfree rats. Tissue and plasma concentrations of peptides were measured. Results: The total volumes of gastrin- and serotonin-immunoreactive cells were significantly increased in the gastric mucosa of germfree rats (P < 0.05), as well as the total volumes of serotonin-and motilin-immunoreactive cells in the ileum (P < 0.05) and serotonin-immunoreactive cells in the colonic mucosa (P < 0.05). The tissue concentration of somatostatin was significantly higher in the jejunum (P < 0.05) and lower in the ileum of germfree rats than in controls (P < 0.05). Plasma glucagon was significantly increased in germfree rats (P < 0.05). The total volume of the fundic mucosa was enlarged in germfree rats (P < 0.05), whereas the total volume, the mucosal thickness, and the number of crypt cells of the colonic mucosa were significantly reduced in these rats compared with controls (P < 0.05). Conclusions: Our findings suggest that the intraluminal microflora influences the release of biologically active peptides and that it participates in the regulation of gastrointestinal endocrine cells and the epithelial structure.

The endocrine pancreas in nonimmune hydrops fetalis

Objective: Our purpose was to compare the endocrine pancreas of hydropic infants of immune and nonimmune origin.Study Design: A quantitative morphologic study was performed on the pancreas in 10 infants with immune and nonimmune hydrops. The volume density of the endocrine pancreas in the gland was calculated, as was the numerical density of the endocrine cells in the islets. The percentage of the different cell types was measured on immunohistochemical stained slides. Ten normal infants were used as controls.Results: An increased amount of endocrine tissue and an increased total number of islet cells of immune and nonimmune origin is present in hydropic infants. However, an increased proportion of the insulin-producing B cells is not present.Conclusions: The endocrine pancreas in hydrops fetalis of immune and nonimmune origin is equal, excluding hemolysis as the stimulating factor for equilibrated islet overgrowth. Metabolic stimuli, as seen in infants of diabetic mothers, are probably not involved, because they preferentially stimulate the insulin-producing B cells.

Quantitative morphologic study of intimal thickening at the human carotid bifurcation: II. The compensatory enlargement response and the role of the intima in tensile support

Arteries enlarge where intimal plaques form, tending to preserve lumen cross sectional area but causing an increase in mural tangential tension due to the increase in radius. To characterize the compensatory enlargement process at the carotid bifurcation and to evaluate the possible contribution of intima thickening to mural tensile support during the enlarging process, we assessed the relationships among intimal thickening, artery size and estimated tensile stress at 9 sequential axial levels in 42 human carotid bifurcations obtained during post-mortem examinations of 36 adults with no clinical or anatomical evidence of cerebrovascular disease. Right and left bifurcations were available for 6 patients. The arteries were fixed under conditions of controlled pressure distention and histologic sections were prepared at 0.5 cm axial intervals. We determined vessel radius ( r), intima thickness (IT), media thickness (MT), intima area (IA), lumen area (LuA) and the area encompassed by the internal elastic lamina (IELA), i.e. the lumen area if there were no intimal thickening. Although IT, IA and r were greatest in the proximal sinus region, there was a highly significant linear relationship between IA and IELA at each axial level; correlation coefficients ranged from 0.64 to 0.97 with P < 0.001 at each level. Stenosis (IA/IELA × 100) ranged from 10.8 ± 8.0% at the common carotid level immediately proximal to the bifurcation angle to 22.3 ± 17.9% at the level immediately distal to the angle, but LuA remained nearly constant at each level regardless of IA. When wall tensile stress (TS) was computed using only media thickness (MT), values ranged from 8.2 ± 2.5 to 17 ± 7.0 × 10 5 dyn/cm 2 (1 dyn = 10 −5 N) with the maximum occurring at the proximal sinus. When, however, total wall thickness (IT + MT) was used, TS was similar at all levels with a mean of 6.5 ± 2.5 × 10 5 dyn/cm 2, (range, 5.5 ± 1.8 to 7.3 ± 2.7 × 10 5 dyn/cm 2) with no significant differences among the 9 levels, including those with little or no intimal thickening. We conclude that during stages of minimal or moderate intimal thickening and plaque formation at the carotid bifurcation, artery size (IELA) at each level increases with IA, thereby preserving LuA. The sum of intima and media thickness corresponds to the increase in radius such that estimated tangential mural tensile stress remains normal at each level.

Quantitative morphologic study of intimal thickening at the human carotid bifurcation: I. Axial and circumferential distribution of maximum intimal thickening in asymptomatic, uncomplicated plaques

The spatial distribution of intimal thickening was determined for each of 42 carotid bifurcations removed at autopsy from patients with no clinical or anatomic evidence of cerebrovascular disease. Both right and left specimens were available for six of the individuals. Each bifurcation was removed intact and included a 1.5–2.9-cm length of the common carotid artery and a 1.5–2.5-cm length of the internal carotid artery. The specimens were restored to in situ length, fixed under conditions of controlled-pressure perfusion at 100 mmHg, filled with a radio-opaque mixture, radiographed and sectioned at 0.5-cm intervals. Computer assisted contour tracing of projected images of histologic sections was used to determine intimal thickness, intimal cross sectional area and lumen area within each of eight equal 45° polar sectors with 0° indexed at the flow divider, 90° at the outside wall, 180° opposite the flow divider and 270° at the inner side wall. Intima occupied 0.9–42% of the area encompassed by the internal elastic lamina, i.e. the potential lumen area if no intimal thickening were present, but there was no lumen narrowing on lateral X-ray projections. Intimal thickening was eccentric at each level of section but the circumferential location of maximum intimal thickness (MIT) shifted in a continuous helix from level to level. At the common carotid artery level 1.0 cm proximal to the bifurcation, MIT tended to be at the flow-divider side at 15 ± 59°. Immediately proximal to the flow divider, MIT was at the lateral side wall. In the mid-sinus region of the internal carotid artery MIT was opposite the flow divider at 179 ± 64°. At the distal internal carotid just beyond the sinus, MIT was at the inner side wall. The distal internal carotid was minimally involved or free of intimal thickening. Comparison of right and left bifurcations revealed that the helical spatial distribution of MIT was in mirror-image symmetry for the two sides. The findings correspond closely with previous demonstrations of a helical flow pattern in the region of the bifurcation. Although locations of MIT just proximal and just distal to the bifurcation are similar and tend to be at the ‘far wall’, individual differences in the shifts of MIT with axial location should be taken into account when sites of interrogation by non-invasive clinical methods are selected for detection of intimal thickening.

Quantitative morphology and shape classification of neurons by computerized image analysis

We describe a new image processing method for semiautomatic quantitative analysis of neuronal morphology. It has been developed in a specific image analysis environment (IBAS 2.0), but the algorithms and the methods can be employed elsewhere. The program is versatile and allows the analysis of histological preparations of different quality on the basis of different levels of evaluation and image extraction. Some significant algorithms have been implemented (i.e. one for multiple focus image acquisition and one for automatic cell body shape recognition and classification). A wide set of specific morphological parameters has been defined to allow a better mathematical characterization of neuronal morphology as regards both dendrite trees and cell bodies. Cell bodies' shapes can be classified automatically, defining different neuronal populations. This is done by evaluating the number of main dendrites and perykaria shapes through a multi-valued-decision-tree based method, tested on somatostatin-positive cells in mouse brain. The methods presented have been applied to analysis of neurons, but they can well be used for any quantitative morphological study of other cell populations.