Morphometric Investigations Research Articles

Glomerular Expression of α-Smooth Muscle Actin (α-SMA) in Idiopathic Mesangiocapillary Glomerulonephritis Type I. A Quantitative Study

Eighteen renal biopsy specimens from patients with mesangiocapillary glomerulonephritis type I (MCGN-I) evaluated by both light and electron microscopy as well as immunofluorescence microscopy and whose full clinical data were available were examined quantitatively. As control 10 biopsy specimens of kidneys removed because of trauma were used. Morphometric investigations were performed by means of a computer image analysis system to evaluate the glomerular expression of α-SMA in MCGN-I, as well as to determine whether this parameter could correlate with quantitatively analysed glomerular cells and mesangial areas. Another purpose of this study was to verify if the expression of α-SMA correlated with the intensity of glomerular leukocyte infiltrates in this glomerulopathy. The morphometric study revealed that mean values of the expression of α-SMA, total glomerular cells per total glomerular area, mesangium (% of total glomerular area), CD 45RB+, CD 43+, CD 20+ and CD 68+ cells were in MCGN-I patients increased in comparison with normal controls. Moreover, in the MCGN-I group, but not in controls, significant positive correlation existed between the glomerular expression of α-SMA and total glomerular cells per total glomerular area, mesangium (% of total glomerular area), as well as CD 45RB+ cells and CD 68+ cells. Significant positive correlation between α-SMA staining and total glomerular cells and mesangial areas suggested that increased α-SMA expression might be in MCGN-I an indicator of mesangial cell activation and mesangial matrix production. The significant positive correlation between the glomerular expression of α-SMA and glomerular CD 68+ cells requires further investigations to elucidate whether monocytes/macrophages play a role in the process of inducing myofibroblast phenotype in mesangial cells.

Histological biomorphosis of human heart valve. II. Morphometric studies

Previous polarization-microscopical measurements combined with silver-staining methods at histological sections of the bicuspid valve and the tricuspid valve in the region of fibrosa showed (similar to that of the corresponding tendinous cords) for middle-aged persons a distinct difference in the collagen composition in comparison between high pressure vs. low pressure system. The aim of the current study was the morphometric investigation of the collagen composition, of the relative content of total collagen (measured as relative number of total collagen fibers per measuring area), and of the cell-collagen relation in dependence of age and possibly on sex in the high and low pressure system, respectively. Tissue samples of atrioventricular valves of 67 probands of both sexes were available. The probands were healthy with regard to heart and circulation and distributed in 3 age groups (1st to 2nd decade, 3rd to 5th decade, and 6th to 9th decade). After corresponding preliminary treatment, the histological sections of tissue samples were evaluated by a suitable combination of polarization-microscopical, immuno-histochemical, and morphometrical methodology. The thickness of the heart valves was measured by microscopic image analysis. A rise of the number of fibers per measuring area (ma) for both sexes and both valves was observed with increasing age. The slopes of the linear regression curves and the mean values of the numbers of collagenous fibers/ma (for each age group) were different between both sexes. They depend upon the valve considered. Analogous to these results, the differentiated statements were possible to the number of fibers/ma and to the percentages of the fiber species by the distinction between the collagen types I and III in the fibrosa of the heart valves. Whereas the relative numbers of fibers/ma rose with increasing age, the number of cell (fibroblasts, fibrocytes) nuclei for both valves and sexes slightly decreased. For the latter, there was no difference between high and low pressure system in contrast to the collagenous fibers. The opposite age-dependent behavior of the collagenous fibers and of their producing cells during the biomorphosis of the heart valves could be explained with the collagen turn-over.

Morphometric investigation on osteocytes in human auditory ossicles

An osteocyte lacunae differential count (1-lacunae with live osteocytes, 2-lacunae with degenerating osteocytes, 3-empty lacunae) was carried out on ear ossicles and clavicles from cadavers as well as on stapes removed by stapedotomy. The distance of the three types of lacunae from the vascular source was also determined by a computer-assisted light microscope. Results showed that the delayed fixation of bone from cadavers does not significantly interfere with osteocyte preservation, at least with the scope of this investigation. The results of osteocyte differential count show that the number of empty lacunae and lacunae with degenerating osteocytes: (a) is significantly higher in ear ossicles than in clavicles, (b) increases with age, (c) is higher in stapes than in incuses and mallei, (d) increases with the distance from the vascular sources in both ear ossicles and clavicles. Additionally it appeared that the process of osteocyte degeneration in ear ossicles is very rapid and widespread, over 40% of the cells being dead within the 2nd year of age. In the light of the recent literature and personal findings, which ascribe to osteocytes the function of mechanical detectors, and considering that bone remodeling occasionally occurs in ear ossicles, it is postulated that osteocyte death in these bones could be a programmed phenomenon (apoptosis?), due to which they lose the ability to react to strains and stresses and achieve the structural stability they need to perform their peculiar stereotyped function.

Development of basal membrane of villi epithelium. Immunohistochemical, ultrastructual and morphometric investigation

Development of basal membrane of villi epithelium. Immunohistochemical, ultrastructual and morphometric investigation

Glomerular basement membrane thickness in primary diffuse IgA nephropathy: ultrastructural morphometric analysis.

Sixteen renal biopsy specimens from adult patients with IgA nephropathy (IgAN) for whom both light and electron microscopy as well as immunofluorescence microscopy and full clinical data were available were examined quantitatively and compared with six cases of normal controls. The electron micrographs of the glomeruli were scanned in a Primax flatbed A4 scanner and then morphometric investigations were performed by means of a computer image analysis system to evaluate glomerular basement membrane (GBM) thickness and to study whether this parameter could correlate with the degree of the haematuria, which is thought to be a main renal symptom in this glomerulopathy. The study revealed that the mean value of the GBM thickness was in IgAN patients significantly lower in comparison with normal controls (268.6 nm versus 338.8 nm). The authors also noted the tendency to negative correlation between GBM thinning and haematuria, although this relationship was not significant. It is suggested that deposits located in the vicinity of the capillary loops may play a role in this process.

Characterizations of heterotopic neurons in the spinal cord of amyotrophic lateral sclerosis patients.

This report concerns a comparative immunocytochemical, ultrastructural and morphometric investigation on heterotopic neurons in the white matter of the spinal cords of 19 patients with amyotrophic lateral sclerosis (ALS) and 18 age-matched neurologically normal individuals. The study revealed that the heterotopic neurons were scattered in the white matter, often adjacent to gray matter, that they immunoreacted with the antibody to synaptophysin, and that there were synaptic apparatuses on the surface of their somata and their neuronal processes. Bunina bodies and ubiquitin-positive inclusions such as Lewy body-like inclusions and skein-like inclusions, characteristic of anterior horn neurons of ALS, were present in the cytoplasm of the patients' heterotopic neurons in the anterior or lateral column of the white matter. These findings suggest that heterotopic neurons in the anterior or lateral column have the characteristics of alpha motor neurons. The average number of heterotopic neurons observed in ALS patients was generally less than in normal subjects. This reduction was correlated with the severity of neuronal loss. The heterotopic neurons in ALS were less susceptible to the degenerative process as compared with spinal cord anterior horn cells. We assume that in this disease the heterotopic neurons may be degenerated and their number diminished after or concomitantly with the depletion of anterior horn neurons.

Coalinga Fibre: A Short, Amphibole-Free Chrysotile

We previously demonstrated, on the basis of detailed morphological and morphometric investigations of a lifetime inhalation study of F344 rats exposed to three sizes of chrysotile fibre, that a short, amphibole-free chrysotile from Coalinga, California failed to induce pathological effects whilst the other two, both long fibres from Canada, were both fibrogenic and tumourigenic. Here, we demonstrate that, as one would predict from their size differences, the Coalinga fibre was almost totally cleared whilst the Canadian fibres were largely retained 1 year post exposure. We therefore propose on the basis of these observations that the observed absence of biological effects noted with Coalinga is potentially attributable to its lack of biopersistence.

Coalinga Fibre: A Short, Amphibole-Free Chrysotile

We previously demonstrated, on the basis of detailed morphological and mor phometric investigations of a lifetime inhalation study of F344 rats exposed to three sizes of chrysotile fibre, that a short, amphibole-free chrysotile from Coalinga, California failed to induce pathological effects whilst the other two, both long fibres from Canada, were both fibrogenic and tumourigenic. Here, we demonstrate that, as one would predict from their size differences, the Coalinga fibre was almost totally cleared whilst the Canadian fibres were largely retained 1 year post exposure. We therefore propose on the basis of these observations that the observed absence of biological effects noted with Coalinga is potentially attributable to its lack of biopersistence.

Morphometric evaluation of stromal vascularization in the endometrium in adenomyosis.

A computerized morphometric investigation of stromal vascularization in the endometrium during adenomyosis was performed retrospectively. Using a polyclonal antibody and a peroxidase-antiperoxidase method, formalin-fixed paraffin sections of the tissue were stained for von Willebrand factor, a marker for endothelium. The subjects were divided into two groups: 42 patients with histologically proven adenomyosis and 29 fertile control subjects, 12 in the proliferative phase and 17 in the secretory phase. Objective quantitative colour image analysis was used to assess the staining intensity and hence the degree of vascularization. In the control group, stromal vascularization increased in the secretory phase. In adenomyosis, vascularization increased markedly, up to 11.6 times that of the controls, in terms of the in total surface area of capillaries per mm2 in the endometrium in the proliferative phase. These findings suggest clinical relevance to severe functional disturbances such as hypermenorrhoea or iron deficiency anaemia.

Morphological adaptations of human liver peroxisomes in cholestasis.

Part of the bile acid synthesis takes place in peroxisomes. An altered enterohepatic circulation of bile acids might influence peroxisomal beta-oxidation enzymes and peroxisomal morphology. We performed a morphological and morphometric investigation of peroxisomes in liver biopsy samples of eight patients with cholestasis of different origin: graft versus host reaction (n = 1), obstruction of the bile flow (n = 3), and drug-induced cholestatic hepatitis (n = 4). Peroxisomes were identified using catalase cytochemistry. They were regularly shaped and showed individual differences in electron density. A perinuclear distribution was observed in a variable number of hepatocytes in each sample. Morphometric analysis of peroxisomes revealed an increase in numerical density and surface density in all, and a decreased mean diameter in four liver samples. Based on previously obtained data in experimental animals, we hypothesize that the observed alterations in peroxisomal morphology indicate an enhanced metabolic activity of the enzymes in the peroxisomal matrix. Among them are enzymes involved in bile acid synthesis.

Anatomy of Microvascular Anastomosis in the Neck

Structural and morphometric investigations of the vessels of the neck region were carried out on 30 cadavers (15 male and 15 female in the age range of 45 to 93 years old) to gain more knowledge of the anatomy for microvascular surgery. Our results briefly are as follows: (1) The non-common-truck type of the external carotid artery (in which each branch arises separately from the external carotid artery) was found in 76.6 percent of cases, the truncus linguofacialis type was found in 21.7 percent, and the truncus thyrolingualis type was found in 1.7 percent. (2) The smallest internal diameter (the average was 1.2 mm) was found in the superficial cervical artery. (3) The largest internal diameter (4.4 mm) was measured in the external carotid artery. (4) The longest arterial section (127.8 mm) was measured between the clavicle and mandibular margin. This vascular section or stem consisted of three parts: the supraclavicular part of the common carotid artery, the proximal section of the external carotid artery, and the first 3 cm of the facial artery. (5) In 46.6 percent of cases, the facial, lingual, and superior thyroid veins joined together and formed a thyrolinguofacialis vein. (6) The type with one external jugular vein accounted for 83.0 percent of cases, and the type with one anterior jugular vein for 67.4 percent of cases. (7) The middle thyroid vein exhibited the smallest internal diameter (average of 2.0 mm). (8) The largest internal diameter (7.9 mm) was measured in the internal jugular vein. (9) The longest vessel to receive a vascular pedicle vein was the external jugular vein, the average length of which was 99.7 mm. (10) The frequency and location of valves in the facial vein were also determined.

Diffuse thalamic degeneration in fatal familial insomnia. A morphometric study

A morphometric investigation disclosed most thalamic nuclei severely degenerated in two patients with fatal familial insomnia. Associative and motor nuclei lost 90% neurons, and limbic–paralimbic, intralaminar and reticular nuclei lost 60%. These findings point to the disorganization of most thalamic circuits as a condition necessary for the sleep–wake rhythm being affected.

Neuronal changes in the basolateral complex during development of the amygdala of the rat

Neuronal changes in the amygdala basolateral complex were studied during development and maturation in fetal and postnatal rat brains using morphometrical methods. Forty brains of animals of various ages were fixed in formalin, frozen and cut into 25 μm thick sections and stained with cresyl violet or haematoxylin and eosin (H&E). In cresyl violet preparations, the complex appeared for the first time on embryonic day (E)17 and was composed of two homogeneous nuclei—lateral and basolateral. On about the seventh postnatal day, each of these nuclei was divided into two parts—the first one into the dorsolateral and ventromedial and the second one into the anterior and posterior. Morphometric investigations showed a different increase of the neuronal and nuclear size in various parts of the basolateral complex up to postnatal day (P)14; after that time these parameters did not change significantly. The neuronal density and the total number of neurons stabilized at P7 in all parts of this complex, except for the dorsolateral part of the lateral nucleus in which a 30% decrease of the total number of cells was observed. From P14, in all nuclei under study, the total number of neurons did not change significantly.

P069 Testing program of morphometric and stereometric investigations of malignant skin lymphomas

P069 Testing program of morphometric and stereometric investigations of malignant skin lymphomas

Cerebellar pathology in sporadic and familial Alzheimer's disease including APP 717 (Val→Ile) mutation cases: A morphometric investigation

Familial Alzheimer's disease (FAD) tends to present with more prominent neurological symptoms including cerebellar signs than sporadic Alzheimer's disease (SAD). In order to elucidate the pathological differences in the cerebellum, which may be associated with the cerebellar symptoms, we have investigated morphometrically β-amyloid deposits, atrocytosis, Purkinje cells and dentate neurons in the cerebellum of 10 FAD patients including two cases with the β-amyloid precursor protein (APP) gene mutation (APP717 Val→Ile), 10 SAD patients and 10 non-demented age-matched controls. The regions examined included the molecular, Purkinje cell and granular cell layers, the cerebellar white matter and the dentate nucleus. Purkinje cell density in FAD was significantly lower than in SAD. There were no significant differences in the density of dentate neurons among the three groups. The density of astrocytes in FAD was significantly greater than that in SAD in the granular cell and Purkinje cell layers and in the white matter. There were no significant differences in the amount and subtypes of β-amyloid deposits (extracellular, vascular and perivascular) between FAD and SAD in all the regions investigated. In two cases with the APP mutation, both Purkinje cell loss and β-amyloid deposition in the cerebellum were greater than the mean for FAD and SAD cases. Astrocytosis in the mutation cases was not greater than the mean for FAD cases except for the dentate nucleus in one case. Extracellular β-amyloid deposits were not seen in any of the control cases although amyloid angiopathy was observed in one case. This study demonstrates for the first time that Purkinje cell loss and reactive astrocytosis of the cerebellum in FAD are more severe than in SAD, but that β-amyloid deposition in the cerebellum in both FAD and SAD are similar. The more prominent neurological signs observed in FAD may be explained by more severe neurodegeneration than are found in sporadic cases.

O-19-276 - Inhibition of astrogliosis by methylthioadenosine

The demyelination (dmy) rat is a unique mutant exhibiting severe myelin breakdown in the central nervous system (CNS). In this study, we conducted immunohistochemical and morphometrical investigations in the dmy rat. From around 6 weeks of age, the affected rats developed ataxia especially in the hindlimbs. Afterwards, ataxia worsened rapidly, resulting in complete paralysis of the hindlimbs and recumbency. Histopathology at 7 to 10 weeks of age revealed myelin destruction throughout the white matter of the CNS in the dmy rats. The most severely affected lesions were distributed in the corpus callosum, capsula interna, striatum, subcortical white matter, cerebellar peduncle, and ventral and lateral parts of the spinal cord. Immunohistochemistry demonstrated prominent astrogliosis and many ED-1 positive macrophages in the myelin-destructed areas. Until the 4th week, no significant differences in myelin thickness and fiber diameter were found between dmy and control rats. However, from 5 weeks of age, myelin thickness of residual myelinated fibers in dmy rats became significantly less than that in controls. These data indicated that the dmy phenotype shows a prolonged period of myelin destruction, suggesting that dmy mutation affects the adequate maintenance of myelin.

Ultrastructural and Morphometric Investigation of Human Brain Capillaries in Normal and Peritumoral Tissues

Capillaries of peritumoral and normal brain tissues were ultrastructurally and morphometrically investigated to evaluate the changes in peritumoral capillaries connected with the tumor-associated vasogenic edema. The endothelial cells of peritumoral capillaries showed varying thickness, electron-lucent cytoplasm, and structurally normal tight junctions. The basal lamina was thickened, rarefied, and vacuolated. The pericytes were provided with pinocytotic vesicles and phagocytic bodies. The astrocytic glia appeared empty or swollen, with few glycogen granules and a disarranged cytoskeleton; well-preserved glia was occasionally observed. The brain tissue was slightly edematous. No statistically significant differences were observed between normal and peritumoral capillaries as regards diameter, wall thickness, endothelial thickness, and endothelial vesicle density. Instead, the peritumoral capillaries displayed three times as many endothelial surface-connected vesicles, a markedly thicker basal lamina, and significantly reduced extension of pericytic and glial investments. The kind and severity of the vascular modifications, compared with the slight edematous appearance of the nervous tissue, strengthen the hypothesis that peritumoral capillaries could be involved in the edema resolution process.

Proliferation of Myocardial Peroxisomes Caused by Several Agents and Conditions

In view of considerable gaps in our knowledge of myocardial peroxisomes, the aim of the present study is, on the basis of extensive electron-microscopic investigations, to provide reliable results on the inducibility of a proliferation (increase in the number) of these organelles in rodents' heart by several agents and conditions. As far as possible, we compared the response of heart and liver peroxisomes. Morphometric investigations were performed to assess the effectiveness of the hypolipidemic agent HL 41, erucic acid, ethanol, nifedipine, chlorpromazine, two cardiotonic drugs, isoprenaline, adriamycin, and physical exercise. The study also included spontaneously hypertensive rats (SHR). A further objective was to determine synergistic or additive effects that might occur when two or three peroxisome-proliferating stimuli act simultaneously. In every case we observed a clear peroxisome proliferation, which was found to increase by between 10 and 97% under the influence of an additional inducer. The observed increase in peroxisome number ranged from almost 200% to nearly 400%. Our results suggest that very different agents and conditions can induce myocardial peroxisome proliferation when they lead to metabolic alterations associated with an increased need for a peroxisomalβ-oxidation of fatty acids as an energy source and/or for preventing toxic effects. Regulatory mechanisms of these adaptive processes are apparently also present in the heart via peroxisome proliferator-activated receptors (PPARs) and their activation by fatty acids, which can also stimulate the PPARs gene expression. The assumption that stimulated catalase gene expression might be responsible for the induction of peroxisome proliferation as a cellular response to an extraperoxisomal oxidative stress situation (isoprenaline, adriamycin, or physical exercise) poses some critical questions. These questions pertain especially to: (a) quantitative aspects with regard to the possible effectiveness of an increase in catalase activity by two-, three-, or four-fold enhanced peroxisome numbers; (b) the role of cytoplasmic catalase; (c) the existence and importance of a myocardial mitochondrial catalase; and (d) the co-operation between the two H2O2-destroying enzymes catalase and glutathione peroxidase.

Preoperative creatine kinase blood levels and myopathological changes in malignant hyperthermia

In malignant hyperthermia (MH) the fundamental lesion is located in skeletal muscle fibres. The underlying cause of MH is suggested to be an increase in myoplasmic calcium, which is released from the sarcoplasmic reticulum. Although MH is believed to represent myopathy, patients susceptible to MH (MHS) rarely have clinical evidence of muscle diseases [1]. Creatine kinase (CK) as muscle cell enzyme is reported to predict MH disposition when blood levels at rest are raised [2]. The purpose of this study was to determine, whether any clinical prediction could be made of abnormal pre-operative CK blood levels or whether there is a correlation between raised CK values and histological abnormalities. After institutional approval and written informed consent, 196 patients aged 3–71 years with clinical suspicion for MH were enrolled in this prospective study. The patients were first classified as MHS or MHN (MH non susceptible) by the caffeine halothane contracture test (CHCT) according to the European MH protocol [3]. Patients tested equivocal (MHE) were excluded from this study. Additionally, small muscle specimens (approximately 3×3×10 mm) were excised from each patient for histological and morphometric investigation. Preoperative levels of CK in blood were measured and compared both with the CHCT and the histological results. Data are expressed as mean ± SEM or percentages, respectively. Statistical analysis was performed using the Mann-Whitney U-test. P<0.05 was considered significant. 80 from the 196 patients in this study (40.8%) were diagnosed MHS and 116 (59.2%) MHN. In the MHS patients 26 from 80 (32.5%) had elevated CK values compared to 13 from 116 (11.2%) in the MHN group. 52 (65%) of the MHS patients showed myopathological changes (9 specific, 43 unspecific), whereas only 49 (42.2%) of the MHN patients had any myopathological abnormalities (3 specific, 46 unspecific). Preoperative CK blood levels were increased in 39 patients (19.9%) and two thirds of these were classified MHS. While 88 of all patients were female (44.9%) the distribution was a little different in the two diagnostic groups with 26 (32.5%) female MHS and 62 (53.4%) female MHN patients. It was noticed that only 7 of the 39 patients with raised CK were female (17.9%) and all of them showed myopathological abnormalities both in the MHS group (4 female) and in the MHN group (3 female). In contrast only 50% of the male patients with preoperative elevated CK values showed myopathological changes with no differences between MHS and MHN. The results demonstrate that it is not possible to distinguish MHS from MHN individuals by determining the CK blood levels at rest or using histological techniques. At present, the CHCT remains the only reliable test method for the diagnosis of MH susceptibility. There were significantly more male than female patients with elevated CK values. A further significant difference was that 100% of the females but only 50% of the male patients with elevated CK values showed myopathological abnormalities. We recommend that all patients with increased CK levels at rest should have muscle biopsy both for MH diagnostic and histological investigation.

Assessment of the vascularization in prostatic carcinoma: A morphometric investigation

Prostatic carcinoma obtained from 41 patients (pT2N0, 5; pT2N+, 2; pT3N0, 16; pT3N+, 16; pT4N0,1; and pT4N+, 1) ranging from 45 to 79 years of age were investigated in the present study. A total of 410 tumor areas of different grades were analyzed (G1, n = 116; G2, n = 98; and G3, n = 196). Vascular structures were labeled immunohistochemically using factor-VIII-associated antigen. The vascular surface density (VSD), the microvessel number (NVES), and the maximum microvessel number (NVES-MAX) were assessed by means of stereology, and the results were related to tumor stage, nodal status, and grade of differentiation. NVES and NVES-MAX showed a significant increase with rising pT stage ranging from 25.5 ± 1.48 in controls to 135.0 ± 5.5 microvessels/mm 2 in pT4 tumors. Discrimination of different pT stages was more accurate with NVES-MAX. The VSD was significantly higher in pT2 tumors compared with controls, whereas there were no significant differences between pT3 tumors, pT4 tumors, and controls, although the values in pT3 and pT4 tumors were distinctly lower than in pT2 tumors ( P < .05). The VSD and the NVES were not able to discriminate between the pN0 and the pN+ group. Both parameters were slightly higher in patients with metastatic disease. Only NVES-MAX values differed between the two groups with an average of additional 21 microvessels/mm 2 in the pN+ group ( P < .05). Concerning the grade of tumor differentiation the VSD continuously decreased from G1 (14.58 ± 2.24 mm −1) to G3 tumor areas (5.41 ± 1.46 mm −1). Only Gl tumors showed significant differences compared with controls (6.65 ± 0.38 mm −1). The NVES increased with rising tumor grade with significant differences between all four groups ranging from 25.5 ± 1.5 in controls to 136.9 ± 37.2 microvessels/mm 2 in pT4 tumors.