Toluidine Blue-stained Semithin Sections Research Articles

Quercetin ameliorates acute lung injury in a rat model of hepatopulmonary syndrome

BackgroundCommon bile duct ligation (BDL) is a rat experimental model to induce biliary cirrhosis. Lung fibrosis and pulmonary vascular angiogenesis and congestion are the most common complications of biliary cirrhosis that is known as hepatopulmonary syndrome. The aim of the present work is to investigate the acute lung injury in a BDL model and to investigate the possible protective effect of quercetin on this injury.MethodsTwenty-four adult male albino rats of the Wister strain (weighing 150–250 g). Animals were divided into 3 groups, with 8 rats each: Group I: Sham-operated group (control). Group II: Bile duct ligation group (BDL) sacrificed after 28 days from the surgery. Group III: Quercetin-treated bile duct ligation group (Q-BDL) was given orally by gastric gavage in a dose of 50 mg/kg/day, starting from the 4th day of the operation until the 28th day. At the end of the experiment, at day 28, all rats were sacrificed. Lung specimens were processed to measure Endothelin B receptor gene expression by PCR, lung surfactant by ELISA, “eNO” s by immunohistochemistry. Histological assessment was done using; H&E, Masson’s trichrome, PAS, toluidine blue-stained semi-thin sections, transmission electron microscope. Histomorphometric and statistical studies were done.ResultsBDL group showed significant increase in lung index together with mononuclear cellular infiltration denoting lung inflammatory state. Also, the significant increase in pulmonary endothelial nitric oxide synthase ("eNO" s) area percent and endothelin B receptor (ETB) gene expression indicates enhanced angiogenesis. Pulmonary surfactant concentration was significantly decreased together with thickening of interalveolar septa denoting lung injury and fibrosis. Quercetin led to significant decrease in lung index, pulmonary "eNO" s area percent, ETB gene expression and significant increase in pulmonary surfactant concentration. Quercetin treatment improved histological changes and morphometric measurements, limited mononuclear cellular infiltration and decreased perivascular and perialveolar collagen deposition.ConclusionQuercetin ameliorates the hepatopulmonary syndrome-induced lung injury through its anti-inflammatory, antioxidative and antifibrotic effects.

Ultrastructure of UVR‐B‐induced cataract and repair visualized with electron microscopy

The aim of the study is to investigate and visualize the ultrastructure of cataract morphology and repair, after in vivo exposure to double threshold dose UVR-B in the C57BL/6 mouse lens. Twenty-six-week-old C57BL/6 mice received in vivo double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-source had λMAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was visualized with electron microscopy techniques. Induced, cataract was quantified as forward lens light scattering. Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine blue-stained semi-thin sections, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dark field illumination photography. UVR-B-exposed lenses developed anterior subcapsular and/or cortical and nuclear cataract after 1 day. Lens light scattering peaked 2 days after exposure. Lens epithelial cell damage was seen in TEM as apoptotic cells, apoptotic bodies, nuclear chromatin condensation, and swollen and disrupted anterior cortex fibres throughout the sections of the whole anterior lens surface. These morphologic changes were also visualized with SEM. Within 8 days, anterior subcapsular cataract was repaired towards the anterior sutures. UVR-B exposure of double cataract threshold dose induces a subtotal loss of epithelial cells across the whole anterior surface of the lens. This damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation.

Ultrastructural changes in the equine colonic mucosa after ischaemia and reperfusion

Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformaldehyde and osmium tetroxide, and embedded in epon. Morphological and ultrastructural changes were evaluated in toluidine blue-stained semithin sections by light microscopy and in thin sections stained with uranyl acetate/lead citrate by transmission electron microscopy. Ischaemia caused swelling of epithelial cells and their organelles, opening of tight junctions, detachment from the basement membrane, early apoptosis and single cell necrosis. Autophagy was a prominent feature in epithelial cells after ischaemia. Reperfusion was characterised by apoptosis, epithelial regeneration and restoration of apical cell junctions. Phagocytic-like vacuoles containing cellular debris and bacteria were evident in epithelial cells after reperfusion. Paracellular and subepithelial clefts formed, accompanied by infiltration of neutrophils, lymphocytes and eosinophils into the epithelium. Subepithelial macrophages and luminal neutrophils had increased phagocytic activity. Ischaemia caused ultrastructural damage to the colonic epithelium, but epithelial cells recovered during reperfusion. Transmission electron microscopy can demonstrate subtle ultrastructural damage to epithelial cells and evidence of recovery after I/R in equine colon.

A fractal analysis of human fetal pituitary blood vessels

Fractal analysis allows a non‐Euclidian evaluation of the tissue‐blood vessels ratio and distribution. The fractal dimension varies from 1 to 2. Di Ieva et al have shown that fractal dimension is a good quantitator for microvasculature of normal (2007) and adenomatous (2008) pituitaries.We present here a fractal analysis study of pituitary vasculature in human fetuses of 9‐24 weeks obtained from terminations with ethical approval, performed on digital images taken from toluidine blue‐stained semithin sections and fluorescence microscopy after lectin binding or propidium iodide.Both light microscopy after toluidine blue and fluorescence microscopy are suitable for fractal analysis; fluorescence in red gave the best results. The fractal indices varied from 1.41 to 1.86, progressively decreasing from 9 to 24 weeks; the distribution of individual values varied following a logarithmic curve, thus demonstrating that in each case, there is a limit, over which the vessels fractal dimension does not increase; the ratio cells/blood vessels varies from 1.15 (9‐10 weeks) to 0.91 (18 to 24 weeks).These data show that fractal analysis is suitable for the study of dynamic changes in blood vessels in the human fetal pituitary, during ontogenesis, giving a quantitative image of its progressive development and time‐related evolution, complementary to the data derived from morphometric vascular density measurements.

Maturational changes in diffusion anisotropy in the rat corpus callosum: Comparison with quantitative histological evaluation

To determine the main histological components that affect fractional anisotropy (FA) in postnatal development of the rat corpus callosum and compare FA values with histological changes evaluated quantitatively. Diffusion tensor image (DTI) data of the rat (postnatal 1-10 weeks) corpus callosum were obtained with a 7.0 T MR scanner. Histological parameters were quantitatively assessed in toluidine blue-stained semithin sections. Simple and multiple linear regression analyses were performed to investigate relationships between FA values and histological variables. The mean FA value (mFA) increased significantly in the early growth stages, whereas the change became smaller after postnatal week 4. Simple regression analysis showed a high correlation between the area of myelin sheath and mFA (r = 0.856; P < 0.01). The area of extracellular space correlated negatively with mFA (r = -0.813; P < 0.01). In a forward stepwise analysis, the area of myelin sheath had the strongest influence on mFA (P < 0.001), followed by the number of unmyelinated axons (P = 0.113). Multiple linear regression analysis revealed that both parameters predicted mFA with a highly significant adjusted correlation coefficient (r(2) adj. = 0.738, P < 0.001). During the early development stage in the rat corpus callosum, the strongest contribution to FA value is the area of myelin sheath.

SERCA1 and calsequestrin storage myopathy: a new surplus protein myopathy

We describe four patients, from four different families, affected by a mild myopathy or asymptomatic elevated serum creatine kinase levels, in whom toluidine blue-stained semithin sections of muscle specimens revealed inclusions of different size and shape. The inclusions did not stain by routine histochemical studies. The sarcoplasmic or endoplasmic reticulum calcium 1 (SERCA1) ATPase and/or calsequestrin reactivity of inclusions, by immunohistochemistry, and the SERCA1- and calsequestrin-increased expression, by immunoblot, suggested that inclusions were constituted by an excess of proteins normally present in the terminal cisternae of sarcoplasmic reticulum. Our cases, both sporadic and familial, represent a new type of surplus protein myopathy.

Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord

The phagocytic cell response within the injured spinal cord is inefficient, allowing myelin debris to remain for prolonged periods of time within white matter tracts distal to the injury. Several proteins associated with this degenerating myelin are inhibitory to axon growth and therefore prevent severed axons from regenerating. Inflammatory agents such as lipopolysaccharide (LPS) can stimulate both the migration and phagocytic activity of macrophages. Using in situ hybridization, we found that the expression of the LPS membrane receptor, CD14, was enhanced in the mouse dorsal column following a dorsal hemisection. Double labeling studies showed that microglia and macrophages are the two major cell types expressing CD14 mRNA following spinal cord injury (SCI). We therefore tested whether systemic injections of LPS would increase the number and phagocytic activity of macrophages/microglia in the ascending sensory tract (AST) of the mouse dorsal column following a dorsal hemisection. Mice were treated daily via intraperitoneal injections of either LPS or phosphate-buffered saline (PBS). At 7 days post-SCI, greater numbers of activated mononuclear phagocytes were present in the AST undergoing Wallerian degeneration (WD) in LPS-treated animals compared with controls. Animals treated with LPS also exhibited greater Oil Red O staining, which is specific for degenerating myelin and macrophages phagocytosing myelin debris. Myelin clearance was confirmed at 7 days using Luxol Fast Blue staining and on toluidine blue-stained semi-thin sections. These results indicate that it is possible to manipulate the innate immune response to accelerate myelin clearance during WD in the injured mouse spinal cord.

Effects of chronic oestrogen treatment are not selective for uterine noradrenaline-containing sympathetic nerves: a transplantation study.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

Development and maturation of noradrenaline-containing nerves of the rat uterine artery. Effects of acute and chronic oestrogen treatment.

The development and maturation of noradrenaline-containing nerves of the rat uterine artery was investigated, histochemically and biochemically, at seven different postnatal age-stages and following acute and chronic treatment with oestradiol. Morphological changes in the vessel were quantitatively evaluated on toluidine blue-stained semithin sections and low magnification electronmicrographs. In summary, the uterine artery is innervated at birth; the adult pattern of innervation is established at two weeks of age; the innervation density increases progressively between the infantile and prepubertal periods, accompanying proliferation and growth of smooth muscle cells in the tunica media; changes in the innervation are followed by an increase in the tissue concentration of noradrenaline and neither the endocrine changes characterizing puberty nor acute or chronic treatment with oestradiol have an effect on the pattern of development of the uterine artery and associated noradrenaline-containing nerves. Results are interpreted considering the differential susceptibility of urinogenital organs to sex hormones.

Insulin-like growth factor-I treatment reduces immune cell responses in acute non-demyelinative experimental autoimmune encephalomyelitis.

To test the effects of insulin-like growth factor-I (IGF-I) on clinical deficits, lesion severity, and immune cell response in acute, non-demyelinative experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats by passive transfer of an MBP-reactive T lymphocyte line. Four days after receiving 5 x 10(5) MBPL-1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 micrograms IGF-I i.v. twice daily for 6 days, and the other 10 received the same volume of 0.89% NaCl. Pairs of rats were sacrificed after 4 days and 6 days of IGF-I and placebo treatment and spinal cord sections were processed for immunostaining, in situ hybridization, and morphological examination. IGF-I treatment decreased clinical deficits, lesion numbers, and lesion areas significantly. Numbers of CD4-positive T cells, alpha/beta TCR-positive cells, and ED-1-positive macrophages were also significantly reduced by IGF-I treatment. Similar reductions were found in our second trial, when 11 days of placebo and IGF-I injections began the day after transfer. No demyelination was observed in either toluidine blue-stained semithin sections or sections immunostained with an antibody raised against myelin basic protein (MBP). We conclude that IGF-I-induced reductions in immune cell responses can occur in the absence of demyelination and are of major importance in decreasing clinical deficits and lesion severity in EAE. If IGF-I has similar effects in multiple sclerosis, we think that it will be useful therapeutically.

Neuronal and glial characteristics of central neurocytoma: electron microscopical analysis of two cases.

We have identified two central neurocytomas which contained cells co-expressing glial fibrillary acidic protein and synaptophysin defined by double-label immunostaining. Dual-positive cells were mostly polygonal in shape and with a morphological appearance similar to that of reactive astrocytes. This distinct morphology could be used to distinguish cells expressing glial fibrillary acidic protein from cells with round and clear cytoplasm which did not express glial fibrillary acidic protein and which composed the majority of the tumor. Samples containing polygonal cells were selected for electron microscopy from toluidine blue-stained semithin sections. Ultrastructural findings were similar in both neurocytomas, with both being composed predominantly of round cells with clear cytoplasm corresponding to the clear cells identified by light microscopy. Dense-core vesicles and clear vesicles were frequently observed in the cell processes. Apart from these clear cells, polygonal cells with electron-dense cytoplasm were noted. Paralleling the result of double immunostaining, these polygonal cells contained both dense-core vesicles and intermediate, presumably glial filaments. Microtubules and lipofuscin granules were also observed. These results suggest that cells expressing glial fibrillary acidic protein in central neurocytoma include tumor cells with both neuronal and glial characteristics.

An experimental painful peripheral neuropathy due to nerve constriction: I. Axonal pathology in the sciatic nerve

A constriction injury to the sciatic nerve of the rat produces a painful peripheral neuropathy that is similar to the conditions seen in man. The pathology of the sciatic nerve in these animals was examined at 10 days postinjury, when the abnormal pain sensations are near maximal severity. The nerves were examined with (1) complete series of silver-stained longitudinal sections of pieces of the nerve (3 cm or more) that contained the constriction injury in the center, (2) toluidine blue-stained semithin sections taken at least 1 cm proximal and 1 cm distal to the constriction, and (3) EM sections taken adjacent to those stained with toluidine blue. One centimeter or more proximal to the constriction, both myelinated and unmyelinated axons were all normal. Nearer to the constriction, extensive degeneration of myelinated axons became increasingly common, as did signs of endoneurial edema. Distal to the constriction, the nerve was uniformly edematous and full of myelinic degeneration. There was a profound loss of large myelinated axons and a distinctly less severe loss of small myelinated and unmyelinated axons. These observations show that at 10 days postinjury the constriction produces a partial and differential deafferentation of the sciatic nerve's territory. The absence of degeneration in the nerve 1 cm proximal to the constriction indicates the survival of the primary afferent neurons whose axons are interrupted.

Glutamate decarboxylase-immunoreactive neurons in the aging rat hippocampus are more resistant to ischemia than CA1 pyramidal cells

Glutamate decarboxylase (GAD)-immunoreactive, supposedly GABAergic inhibitory, neurons in various fields of the rat hippocampus and pyramidal cells in area CA1 were quantified 1 week after transient cerebral ischemia by 4-vessel occlusion. Whereas the number of CA1 pyramidal cells in Toluidine blue-stained semithin sections were found reduced by 50% when compared with controls there was no loss of GAD-immunoreactive cells in vibratome sections of hippocampus proper and fascia dentata. These data suggest that GABAergic hippocampal neurons are more resistant to ischemia than CA1 pyramidal cells.

Mixed Sertoli-Leydig cell tumor and rete testis adenocarcinoma in rats treated with CdCl2.

Sertoli cell tumors are, except in dogs,' rare testicular neoplasms; two have been reported in rats.' Tumors of the rete testis are even rarer. Not reported in rats, they have been described to occur spontaneously in old JCL : ICR mice,'O in Mustornys,y or in mice treated prenatally with diethylstilbestrol.' This report describes the morphology of a mixed SertoliLeydig cell tumor and a rete testis adenocarcinoma in Wistar rats [Crl : (WI)BR] from two long-term experiments on the carcinogenic potential of cadmium in males. A single subcutaneous (s.c.) injection of cadmium can induce acute vascular lesions in the testis and ischemic necrosis of the seminiferous tubules. Following resolution of the necrotic debris, periand intratubular fibrosis and mineralizations occur that may also involve the rete testis. Leydig cells, however, survive and give rise to interstitial cell t ~ m o r s . ~ The first study on cadmium carcinogenesis, started to determine the dose-response relationships for cadmium chloride (CdCl,) injected subcutaneously at approximately 8 weeks of age, involved 315 rats and lasted for 104 weeks. The second study, designed to investigate effects of zinc pretreatment on cadmium carcinogenesis, involved 364 rats for 104 weeks. Most organs and all altered tissues were fixed in neutral buffered formalin, trimmed, and processed for light microscopic investigation of hematoxylin and eosin (HE)-stained paraffin sections. Periodic acid-Schiffs (PAS) reaction with or without diastase pretreatment and the alcian blue-periodic acid-Schiff's reaction were also used. Toluidine blue-stained semithin sections were obtained from the Sertoli-Leydig cell tumor that was reembedded in epoxy resin. The mixed Sertoli-Leydig cell tumor was found in a 112week-old rat that had been treated with a single S.C. injection of 30 pmol CdClJkg and three S.C. injections of 0.1 mmol Zn-acetate 6 hours before, at the same time, and 18 hours after the CdCI, injection. At necropsy, the testes appeared atrophied. A red-brown nodule, 1-cm in diameter, was in one testis. By light microscopy, both testes had bilateral diffuse, severe peritubular fibrosis and intratubular mineralizations. One testis had a single Leydig cell tumor and the other had two separate neoplasms: a small Leydig cell tumor and a larger mixed Sertoli-Leydig cell tumor (Fig. 1). The tumor consisted mainly of variably sized and shaped tubuli expanding beyond the tunica vaginalis via the rete testis (Fig. 1). Intermixed were areas of round cells with eosinophilic cytoplasm that appeared to be of Leydig cell origin. Larger, more mature tubules were lined by spindle-shaped Sertolilike cells (Figs. 1-4) with basally oriented nuclei and pale vacuolated cytoplasm extending towards a lumen. Vacuola-

Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride: Morphologic and physiologic characteristics

Frog fundic mucosas mounted in Ussing chambers between HCO3−-buffered nutrient and unbuffered secretory solutions were exposed to 1 M NaCl on the mucosal surface for 10 min. After washing and return to control solutions, transmucosal potential difference, short-circuit current, and tissue electrical resistance decreased markedly, but within 6 h these measurements had gradually returned to almost control values. A net luminal alkalinization occurred during the first 4 hours, changing into a net acid secretion of ~1.1 μmol · cm−2 · h−1 at 6 h. Histamine increased H+ secretion in all tissues at 8 h. In seven metiamide-treated tissues, an average alkaline flux of ~0.75 μmol · cm−2 · h−1 was obtained during the first 4 h after damage, decreasing to ~0.40 μmol · cm−2 · h−1 during the ensuing 4 h. With HCO3−-free nutrient solution (n = 7) luminal alkalinization was decreased by about 80% 2–4 h after injury. After 1 M NaCl, the surface epithelium and gastric pit cells were destroyed and partially lifted from the gastric glands. The lamina propria between the remaining intact glands was open to the lumen or contiguous with the damaged mass of cells and mucus. During the 6 h after damage, there was a gradual process of restitution of epithelial integrity, beginning with squamous-shaped cells that appeared to be migrating from the glands and ultimately concluding with complete epithelialization by cuboidal and columnar cells. Typical junctional complexes were present between adjacent epithelial cells. The uniformity of the restoration process was such that it was possible to predict blindly in toluidine blue-stained semithin sections whether the recovery stage was short (30 min or less), intermediate (1–2 h), or advanced (4–6 h). These observations indicate that there is a very intimate correlation between the restoration of epithelial continuity and the reestablishment of secretory and electrical activity of frog gastric mucosa damaged by hypertonic NaCl in Ussing chambers.

Do oculomotor neuroblasts migrate across the midline in the retal rat brain?

Toluidine blue-stained semithin sections and Cajal-Castro preparations are used to study in rat fetuses whether oculomotor neuroblasts migrate across the midline at a certain period of development. In confirmation of previous studies, a group of oculomotor neuroblasts was detected which first grow cytoplasmic processes into the mesencephalic midline, and afterwards translocate their somata towards the midline, between the 12th and the 15th days of gestation. At this moment a midline mass of neuroblasts characterizes the meeting at this landmark of both left and right migrating neuroblastic groups. No crossing oculomotor axons yet are demonstrable with reduced silver techniques. In further stages of development the neuroblasts continue their migration until they arrive at the contralateral nucleus at the 16th and 17th day of gestation. At the midline the mass of neuroblasts disappears gradually and crossed oculomotor axons become visible. The electron microscope was then used to study ultrastructurally the migrating motoneurons. It was discovered that no preexisting structure guides their movement by contact. Their leading processes show no filopodial activity, and contain abundant microtubules and thick bundles of neurofilaments in eccentric position. The neuroblasts carry their axon across the midline as a trailing process.