Study of the myenteric and submucous plexuses after BAC treatment in the intestine of rats.

A morphological and quantitative study in the ileal and colonic myenteric and submucous plexuses of rats after BAC denervation was performed. Four groups were employed: SI--ileum control; CBI--denervated ileum; SC--colon control; and CBC--denervated colon. We used the Myosin-V immunohistochemistry technique to study the myenteric and submucous plexuses. In the submucous plexus of the ileum and colon there was not a significant decrease in the number of neurons/mm2 and of ganglia/mm2. The denervation of the myenteric plexus in the group CBI was 44.7% and in the group CBC, 68.3%. In the myenteric plexus there was also a significant decrease in the number of ganglia/mm2 (13.8% in group CBI and 52.14% in group CBC) and in the number of neurons/ganglion (33.9% in group CBI and 39.6% in group CBC). The morphological analyses showed that there was an alteration in the shape of the ganglia of the ileal and colonic myenteric plexus. The area of the cell bodies had a significant increase both in the myenteric and the submucous plexus in groups CBI and CBC. These data demonstrate that the BAC treatment causes morphologic and quantitative changes in the myenteric plexus and quantitative changes in the cell body area of the submucous plexus.     

Effects of aging on neurons and glial cells from the superficial layers of the superior colliculus in rats

The aim of this study was to investigate the effect of aging on glial cells and neurons from the superficial layers of the superior colliculus in rats. We used stereological methods to estimate the volume of the superficial layers, neuron size, and the number of neurons and glial cells in Wistar male rats aged 3, 24, 26, and 28 months. A 32.6% volume increase was found in the stratum griseum superficiale between the ages of 3 and 26 months, while in the 28-month-old animals a 19% decrease was observed. The stratum opticum did not show any changes in volume with age. Also, our analysis revealed a process of somatic and nuclear atrophy in the neurons of the superficial layers in animals aged 26 and 28 months. On the other hand, no statistically significant differences were found in the numbers of neurons. The number of glial cells in the stratum griseum superficiale showed an increase between the 3rd and 26th month, while the stratum opticum suffered no change.     

Creatine supplementation in trained rats causes changes in myenteric neurons and intestinal wall morphometry

Creatine is widely used by athletes as an ergogenic resource. The aim of this study was to evaluate the influence of creatine supplementation on the duodenum of rats submitted to physical training. The number and myenteric neuronal cell bodies as well mucosal and muscular tunic morphometry were evaluated. Control animals received a standard chow for 8 weeks, and the treated ones received the standard chow for 4 weeks and were later fed with the same chow but added with 2% creatine. Animals were divided in groups: sedentary, sedentary supplemented with creatine, trained and trained supplemented with creatine. The training consisted in treadmill running for 8 weeks. Duodenal samples were either processed for whole mount preparations or for paraffin embedding and hematoxylin-eosin staining for histological and morphometric studies of the mucosa, the muscular tunic and myenteric neurons. It was observed that neither creatine nor physical training alone promoted alterations in muscular tunic thickness, villus height or crypts depth, however, a reduction in these parameters was observed when both were associated. The number of myenteric neurons was unchanged, but the neuronal cell body area was reduced in trained animals but not when training and creatine was associated, suggesting a neuroprotector role of this substance .     

NADPH-diaphorase activity in the superficial layers of the superior colliculus of rats during aging

Neurons in the superficial layers of the superior colliculus are key elements in the visual system of rodents since they receive extensive afferent projections from retinal ganglion cells. The NADPH-diaphorase histochemical technique was used to detect differences in neuronal nitric oxide synthase (nNOS) in the superficial layers of the superior colliculus (sSC) of young adult (3 months) and aged (24 and 26 months) rats. The orientation of the dendritic processes of NADPH-diaphorase-positive neurons, cross-sectional area, and number of neurons per mm2 were analyzed. NADPH-d histochemistry revealed a high number of NADPH-d-positive cells in the stratum zonale and stratum griseum superficiale in adult and aged animals. NADPH-d-positive neurons were classified into the following morphological types: marginal, horizontal, pyriform, narrow-field vertical, wide-field vertical, and stellate. During aging, narrow field vertical and wide field vertical neurons present somatic atrophy and an increase in dendritic processes with dorsoventral orientation, whereas wide field vertical neurons show a decrease in those with lateromedial orientation. Marginal neurons undergo somatic hypertrophy at 26 months when compared with those at 3 months. The remaining types of neurons do not undergo size changes. Finally, the number of NADPH-d-positive neurons per mm2 in the various types of morphology does not significantly change with age. It is suggested to be likely that the aging process in the nitrergic neurons of the sSC does not lead to significant changes in the synthesis of NO from the constitutive NOS isoforms.     

Morphoquantitative evaluation of the duodenal myenteric neuronal population in rats fed with hypoproteic ration.

The purpose of this work was to analyze the morphoquantitative behavior of the neurons of the myenteric plexus, as well as the morphometry of the duodenal wall, in adult rats fed with normoproteic (22%) and hypoproteic (8%) rations, killed at the age of 345 days. For neuronal assessments duodenal whole-mounts stained with the Giemsa method were used, and for the evaluation of the duodenal wall routine histological processing and staining with Hematoxilin-Eosin were employed. The means of the number of neurons in 80 microscopic fields (12.72 mm2) and of the size of the neuronal cell bodies did not reveal statistically significant differences between the groups, but there was a greater incidence of large neurons in the protein restriction group (RP). The duodenum was markedly smaller in the RP group and, although there was no difference in the thickness of its wall, the mucosa was larger and the muscular layer was smaller in group RP. It was concluded that the neuronal and non-neuronal components of the duodenum adjust themselves to the nutritional condition, assuring the maintenance of their functions.     

Goblet cell number in the ileum of rats denervated during suckling and weaning.

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.     

Cell death in the choroid plexus following transient forebrain global ischemia in the rat

Following a complete disruption of blood flow to the brain, cerebral ischemia, a specific neuronal population, namely the CA1 pyramidal neurons in the hippocampus, will die a delayed type of cell death. This is often referred to as "delayed neuronal death" (DND). It is not known why it takes around 48 hours for these cells to die. It is very often speculated that events, intrinsic to the CA1 neurons, regulate their demise, whereas it is less often considered that extrinsic mechanisms also could play an important role for the development of DND. We discovered that in addition to the CA1 pyramidal neurons, cells in the choroid plexus were TUNEL (terminaldeoxynucleotidyl-mediated biotin-dUTP nick-end labeling)-positive following transient forebrain global ischemia. The time course and the number of TUNEL-positive cells were determined. A dramatic increase in the number of TUNEL-positive cells in the choroid plexus was seen at 18, 24, and at 36 hours of recovery, but not at 48 hours of recovery following 15 minutes of transient forebrain global ischemia. No TUNEL-positive cells were seen at 24 hours of recovery in the CA1 region. The cell death in the choroid plexus thus preceded the occurrence of cell death in the CA1 region. Massive cell death in the choroid plexus will inevitably lead to a leaky blood-CSF barrier, which in turn will allow substances to enter the ventricular system and from there reach the brain parenchyma. We, therefore, conclude that choroid plexus cell death may adversely affect the outcome of CA1 pyramidal neurons following transient forebrain global ischemia, through, e.g., a disruption of the blood-cerebro spinal fluid barrier. Alternatively, the choroid plexus may produce factors, which can affect the outcome of neurons.     

Morphometric and some immunohistochemical characteristics of human choroids plexus stroma and psammoma bodies

Psammoma bodies (PBs) are one of many choroids plexus aging changes. The aim of our research was to perform the quantification of PBs' presence in human choroids plexus stroma, as well as to evaluate the characteristics of choroids plexus stroma in cases in which PBs were present. Afterwards, the observations of the histochemical analysis would be confirmed by immunohistochemical analysis. Choroid plexuses of 30 cadavers were used for the histochemical and, choroids plexuses of 15 cadavers in which PBs' presence was confirmed during the histochemical analysis, were used as material for the immunohistochemical analysis. Light microscopy, histochemical, immunohistochemical, and morphometric method were applied during the study. Classification of the cases was performed by cluster analysis. We observed increase of choroids plexus PBs' presence during the aging process. But this increase is not linear. Their presence is the largest in the second cluster that is younger than the third and older than the first. Nuclear morphometric parameters of the stroma in these cases showed that the cellular composition in this cluster is different than in other two and, that contain larger number of lymphoid cells. Immunohistochemical analysis showed PBs' positive reaction on vimentin, CD45R0, and LCA markers, while in their vicinity, as well as inside them, numerous T-cells were observed. So, the presence of CD45R0 and LCA-positive T cells, PBs' positive reaction on the same markers, indirectly connect these cells with PBs' formation process.     

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats.

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate the neuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.     

Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat

To ascertain the possible implications of the nitric oxide (NO*) producing system in striatal senescence, and by using immunohistochemistry and image-processing approaches, we describe the presence of the enzyme nitric oxide synthase (NOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and nitrotyrosine-derived complexes (N-Tyr) in the striatum of adult and aged rats. The results showed neuronal NOS immunoreactive (nNOS-IR) aspiny medium-sized neurons and nervous fibres in both age groups, with no variation in the percentage of immunoreactive area but a significant decrease in the intensity and in the number of somata with age, which were not related to the observed increase with age of the striatal bundles of the white matter. In addition, NADPH-d activity was detected in neurons with morphology similar to that of the nNOS-IR cells; a decrease in the percentage of area per field and in the number of cells, but an increase in the intensity of staining for the NADPH-d histochemical marker, were detected with age. The number of neuronal NADPH-d somata was higher than for the nNOS-IR ones in both age groups. Moreover, N-Tyr-IR complexes were observed in cells (neurons and glia) and fibres, with a significant increase in the percentage of the area of immunoreaction, related to the increase of white matter, but a decrease in intensity for the aged group. On the other hand, we did not detect the inducible isoform (iNOS) either in adult or in aged rats. Taken together, these results support the contention that NADPH-d staining is not such an unambiguous marker for nNOS, and that increased protein nitration may participate in striatal aging.     

Morphometric analysis of human sciatic nerve perineurial collagen type IV content

Objectives: Aging is the process which unavoidably alters structure and function of the basal membranes in humans. Though, collagen type IV presents the most prominent component of the basal membranes, we estimated its presence in the perineurium of the human sciatic nerve samples during the aging process. Materials and methods: Material was 12 sciatic nerve samples, obtained from cadavers whose age ranged from 36 to 84 years. Cadavers were classified into three age groups: first which age ranged from 35 to 54 years, second which age ranged from 55 to 74 years and third which included cases older than 75 years. Tissue slices were further stained by labeled streptavidin–biotin method with collagen type IV monoclonal antibody and analyzed with light microscope under 100× lens magnification with oil immersion. Digital images of sciatic nerve perineurium were further processed and analyzed with ImageJ software. Results: Our results showed that there is statistically significant increase of perineurial area, perimeter, collagen type IV area, and collagen type IV area per perineurial perimeter unit in the third age group. These parameters also increased in the second age group, but this increase was not significant. Multiple regression analysis showed that beside fascicular size, age more significantly predict perineurial collagen type IV content. Conclusions: Results of morphometric and statistical analysis pointed to the conclusion that there is significant increase of sciatic nerve perineurial thickness during the aging process. This increase might represent the consequence of perineurial collagen type IV deposition with aging. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system

This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices. Organotypic spinal cord slices, normally cultured for 6 days, were divided into control group (Ctrl) and transplanted group (T). The choroidal plexus epithelial cells were dissociated and primary cultured (C group). The choroidal plexus epithelial cells cultured for 6–7 days were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanineperchlorate (CM-Dil), and were identified by transthyretin (TTR) in immunocytochemistry. They were adjusted to the density of 0.5–1 × 10⁷/ml, then 2 μl cells suspension were transplanted to the spinal cord slices in the T group. The same amount of basal medium was dripped on the spinal cord slices in the Ctrl group. After 14 days of transplantation, the differentiations into neurons and astrocytes, and the synapses were identified by immunofluorescence histochemistry. At the same time, the ratios of cell differentiations and synapses in new system, and the changes of MAPK signaling pathway were tested by western blotting. The choroid plexus epithelial cells were well labeled by CM-Dil and were immune-stained by TTR in immunocytochemistry. The choroid plexus epithelial cells bodies were small when transplanted on the spinal cord slices, but big when transplanted on the polyester membrane inserts. The transplanted cells could differentiate into astrocytes, and possibly differentiate into neurons, and there were a large number of synaptophysin positive vesicles between transplanted cells and organotypic spinal cord slices in immunofluorescence histochemistry. The levels of GFAP, TUB-III and synaptophysin in the T group were higher than which in the Ctrl and C groups in western blotting (P < 0.05). And the ratios of p-JNK/JNK and p-P38/P38 in the T group were significantly lower than which in the Ctrl and C groups (P < 0.05). But the ratio of p-ERK/ERK in the three groups was of no significant difference. The transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system.     

Effect of tissue non-specific alkaline phosphatase in maintenance of structure of murine colon and stomach

The gastrointestinal tract of mammals secretes a phospholipid-rich membrane that is enriched in alkaline phosphatase (AP) and surfactant proteins (surfactant-like particle, SLP). The production of this particle is stimulated in the small intestine by fat feeding and in cultured cells in vitro by transfection with intestinal alkaline phosphatase (IAP). To test whether tissue non-specific alkaline phosphatase (TNAP) was a factor in stimulating surfactant-like particle production in stomach and colon (tissues expressing TNAP), mice lacking this enzyme were studied. Mice were harvested at 8 days of life, when body weight of homozygous animals (TNAP -/-) was about half that of congenic controls (TNAP +/+) or heterozygotes (TNAP +/-), but before seizures had begun. No difference in content of the major SLP protein (65 kDa) by Western blotting or immunocytochemistry was seen in stomach or colon of TNAP -/- vs. TNAP +/+ animals, but the content was only about half in the IAP-expressing small bowel. Transmission electron microscopy of the TNAP -/- small bowel showed large dilated lysosomes and residual bodies. Colonocytes and gastric surface epithelial cells from the same animals showed mitochondria containing homogeneous dense inclusions, consistent with neutral lipid. In the underweight homozygous animals, there was a decrease in the neuronal content of submucosal ganglia in the jejunum and ileum and of myenteric ganglia in the jejunum of TNAP -/- animals. These findings suggest that (1) TNAP is not important in maintaining surfactant-like particle content of tissues that express TNAP, (2) normal fat absorption is important in maintaining SLP content in the small intestine, and (3) TNAP is important in the maintenance of some intestinal structures, and perhaps their function.     

Immunohistochemical and immunochemical characterization of the distribution of leptin-like proteins in the gastroenteric tract of two teleosts (Dicentrarchus labrax and Carassius auratus L.) with different feeding habits

Leptin is a modulator of food intake and energy homeostasis both in mammals and in some species of nonmammals vertebrates. In this study, we reported for the first time, using an immunohistochemical and immunochemical approach, the presence and distribution of immunoreactivity to leptin-like protein in the gastroenteric tract of Dicentrarchus labrax (bass) and Carassius auratus (goldfish), two teleostean species with different feeding and different adaptative morphological organization of the gastroenteric tract. Bass stomach showed intense immunoreactivity to leptin-like protein in all regions, with immunoreactive cells located at the base of the mucosal plicae and at the apical margin of the gastric crypts. Immunoreactive fibers and neuronal cells were observed close to vascular structures in the pyloric region. In bass and goldfish intestine, rare immunoreactive cells were observed along the mucosal epithelium mostly at the base or the apex of intestinal folds in the proximal and medium intestine; numerous immunoreactive nerve fibers in the circular and longitudinal layers of the tunica muscolaris as well as in the myenteric plexus were observed. Western blot analysis recognized a ～15 kDa signal with a similar expression pattern for goldfish and sea bass. Our results could contribute to confirm the evolutive conservation of leptin-like proteins and their probably precocious functional diversification in fish.     

Tumors of the choroid plexus

Choroid plexus tumors are rare intraventricular papillary neoplasms derived from choroid plexus epithelium, which account for only between 0.4-0.6% of all intracranial and 2-3% of pediatric neoplasms. Plexus papillomas outnumber choroid plexus carcinomas by a ratio of 5:1 and around 80% of choroid plexus carcinomas arise in children. Plexus tumors are most common in the lateral and fourth ventricles; while 80% of lateral ventricle tumors present in children, fourth ventricle tumors are evenly distributed in all age groups. Clinically, choroid plexus tumors tend to cause hydrocephalus and increased intracranial pressure. Histologically, choroid plexus papillomas correspond to WHO grade I, choroid plexus carcinomas to WHO grade III. Immunohistochemically, cytokeratins and vimentin are expressed by virtually all choroid plexus papillomas and most choroid plexus carcinomas while transthyretin and S-100 protein are present in 80-90% of cases, less frequently, though, in choroid plexus carcinomas. Glial fibrillary acidic protein can be found focally in about 25-55% of choroid plexus papillomas and 20% of choroid plexus carcinomas. The mean Ki67/MIB1 labeling index for choroid plexus papillomas is 1.9%, for choroid plexus carcinomas 13. 8%. Choroid plexus papillomas typically show hyperdiploidy with gains particularly on chromosomes 7, 9, 12, 15, 17, and 18 while one choroid plexus carcinoma showed rearrangements of chromosomes 7p11-12, 9q11-12, 15q22, and 19q13.4. Choroid plexus papillomas can usually be cured by surgery alone with a 5-year survival rate of up to 100% with occasional recurrences while choroid plexus carcinomas grow more rapidly and have a less favorable outcome with a 5-year survival rate of 26-40%.     

Structure and expression of the transthyretin gene in the choroid plexus: a model for the study of the mechanism of evolution

Thyroid hormones are key regulators of brain differentiation and function. They permeate strongly into lipid membranes. However, a substantial portion of thyroid hormone is retained in the intravascular/extracellular compartments by binding to plasma proteins. In the brain, transthyretin is the most important of these proteins. This transthyretin is synthesized in the epithelial cells of the choroid plexus and exclusively secreted towards the brain. A net movement of thyroid hormones from the blood to the brain ensues. During evolution, transthyretin synthesis in the choroid plexus and the beginnings of a neocortex first appeared at the stage of the stem reptiles. The affinity of transthyretin for thyroxine increased and that for triiodothyronine decreased during evolution. This could augment the importance of deiodination for regulation of metabolism and gene expression by thyroid hormones in the brain. Successive shifts of the splice site at the 5' end of exon 2 of transthyretin precursor mRNA in the 3' direction led to a shortening of the N-terminal sections and to an increase in hydrophilicity of the N-terminal regions of transthyretin. This shift can be explained by a sequence of single base mutations. It could be an example for a molecular mechanism of positive Darwinian evolution. The selection pressure, which led to the expression of the transthyretin gene in the choroid plexus during evolution, might have been the maintenance of thyroid hormone homeostasis in the extracellular compartment of the brain in the presence of the greatly increasing volume of the lipid phase.     

Proliferative characteristics of epithelial cells in the pregnant rat uterus

In the pregnant rat, killed at about mid gestation and 1 h after injection of tritiated thymidine, 40% of the cells in the epithelium lining the uterine lumen at the implantation site were labelled. Between implantation sites fewer than 20% of the surface epithelial cells were labelled. A series of rats was given tritiated thymidine on day 12 of pregnancy and killed at intervals in the next 30 h. A percentage labelled mitoses analysis of the epithelium between implantation sites (interconceptual) and within the implantation site (conceptual) showed that cells in either region spent 7 h in DNA synthesis and 1.5 h in the G₂ + ½ mitosis phases. The epithelial cells in the conceptual region spent 1.5 h in the G₁ + ½ mitosis phases whereas cells in interconceptual regions spent at least 11.5 h in these phases. The average cycle times of cells in conceptual regions was 10 h: in interconceptual regions minimum cycle time was 20 h and the average appeared to be considerably longer. The grain count of the epithelial cells in the conceptual region was rapidly reduced during the 30 h after injection of tritiated thymidine suggesting successive rounds of cell division. In contrast the grain count distribution of cells in interconceptual regions changed only slowly during this time. The percentage of labelled epithelial cells was determined in the animals killed up to 30 h after injection of tritiated thymidine. In both conceptual and interconceptual regions these percentages increased initially as labelled cells produced labelled progeny. In the conceptual region the increase was not maintained after 7 h as cells initially in G₁ divided to give unlabelled progeny. In the interconceptual region the increase in the percentage of labelled cells continued for 14 h; thereafter the percentage did not significantly alter. The interpretation of these results is discussed in relation to the differences in the kinetic characteristics of the epithelial cells in the two regions and in relation to the morphology of the epithelium lining the uterus during pregnancy.     

铌欠缺对掺镧PZN基陶瓷的有序微区和介电性能的影响

研究了铌欠缺对掺镧 PZN基陶瓷相结构、有序微区以及介电性能的影响。铌欠缺消除了掺镧所产生的焦绿石相 ,使陶瓷试样恢复为百分之百的钙钛矿相结构。 Ram an散射光谱分析表明 ,铌欠缺使掺镧 PZN基陶瓷的有序微区增大 ,同时铌欠缺也提高了掺镧 PZN基陶瓷的介电常数。铌欠缺使介电常数增大的原因归结于焦绿石相的消除和“有效有序微区”尺寸的扩大