| Objective: To observe the morphological changes of ependymal cells in lateral recess fourth ventricle(LR4V) of the type 1 diabetes mellitus rats at different times, as well as this place the NF-кB expression in ependymal cells, and the distribution of nNOS positive cells, and to evaluate the change and significance of this place in diabetes mellitus.Methods: Healthy male Wistar rats, weighing 190g~220g, were randomly divided into four groups:the control group,model 2 weeks, model 4 weeks, model 8 weeks; 20 animals were selected each group to carry on the light microscope, the electron microscope, NF-кB immunohistochemistry and the NADPH-d histochemistry observation.The experimental results were statistically analyzed to observe the relationships between each group.1 Preparation for the animal model of the type 1 diabetes mellitus rats: The animals were fasting and were unable to restrain the water 12 hours before the induction. The model group animals were injected intraperitoneally with 60 mg/kg streptozotocin once to induce hyperglycemia. The control group animals were injected with aseptic citric acid/sodium citrate buffer solution.2 Preparation for paraffin sections specimen: 10% chloral hydrate anesthesia, conventional cardiac perfusion and fixation were done, then the rat brain was taken out, through the conventional dehydration, the clearing, soaked in the wax, the embedding,the slicing. The sections were stained with HE staining, and with NF-кB immunohistochemical staining,respectively. After being sealed, both staining results were observed by microscope and recorded with photography.3 Preparation for electron microscope specimen: The procedure of anesthesia and perfusion were the same as above, but the perfusate solution contained 2% paraformaldehyde and 2.5% glutaraldehyde in PBS. The sample of LR4V was picked out and rinsed with PBS, gradient alcohol dehydration, alcohol replacement, vacuum drying, and sprayed gold. Finally the results were observed and recorded with Hitachi S-3500N SEM.Preparation for TEM sample was as the similar to that of SEM. Then dipped into 4% glutaraldehyde fixation, 1% osmium tetroxide post-fixed, embedded within resin, and ultrathin sections were got with ultrathin microtome. The results were observed and recorded with T-S7500 TEM.4 Preparation for frozen sections specimen: The procedure of anesthesia, perfusion and specimens were the same as the preparation of paraffin sections specimen. The specimen in turn soaks in 15% and 30% sucrose phosphoric acid buffer solution until they were impregnated completely. The coronal sections were cut with the freezing microtome 40 um in thickness. The sections were collected in PB and dipped in 1% Triton solution, clipped in incubation solution, washed in PB and mounted on slides.After air drying in room temperature, the neutral red staining was proceeded, then dehydrated, cleared and coverslipped with neutralized resin. The results were observed by microscope and recorded with photography.5 Statistical treatments: The measurements of average optical density of NF-кB-positive cells were carried out with the digital medical image analysis system (Motic Med 6.0). The obtained data were expressed by±s. SNK-q tests were carried out by comparing between two parts. P<0.05 stood for significant difference.Results:1 Before establishing the model, the blood glucose level of the rats were less than 8.9 mmol / L. And the blood glucose level were above 16.7 mmol/L after they were injected intraperitoneally with streptozotocin once to induce hyperglycemia. It suggested that the diabetes mellitus animal model was successfully prepared.2 The results of light microscope and NF-кB observation2.1 Control Group:The ependymal cells of LR4V were mostly single layer arranged, and the cell body looked low columnar or cubic, arranged even neat.The cell nucleus were oval or round; NF-кB-positive cells were not stained in cell nucleus, but cytoplasm in light brown-yellow color and the value of average optical density was 169.89±3.00.2.2 Model Group: In model 2 weeks group, ependymal cells arranged in varying density, spindle or oval-shaped nucleus; NF-кB-positive cells were also stained in cytoplasm in light brown-yellow color and were not stained in cell nucleus too. The regional value of average optical density was 177.46±2.05. In model 4 weeks group, the ependymal cells arrangement were crowded, and rugged like waves or displayed double layers; the local arrangement of surface cilia were dense, cell nuclears were diversity and arranged irregularly; NF-кB-positive cells were stained in cell nuclears in deep brown-gray color.Cytoplasm was weakly positive. The regional value of average optical density was 181.43±2.43. In model 8 weeks group, ependymal cells were monolayer and sparse. The cell body's shape was flat, cell nucleus were fusiform and cilias were sparse.Nuclear expression was disappeared,but stained in cytoplasm in light brown-yellow color. The regional value of average optical density was 177.21±2.30. The results showed that the value of average optical density stained were significantly different(P <0.05) between the model group and control group.And model 4 weeks group compared with 2 weeks group and model 8 weeks group were significantly different (P <0.05). Model 2 weeks group compared with 8 weeks group were no significant difference(P >0.05).3 The results of SEM observation3.1 Control Group:Under the low magnification observed that the direction of LR4V was a curved tubular structure which was along the foot of the brachium pontis, extended to the outer and the ventralis forms across the inferior cerebellar peduncle.Under the high magnification observation, ependymal cells were regular.The ependymal cells have a clear outline, and spindle or polygonal. Free surface were rich in cilia which were densy and microvilli could be seen between them. Surface of ependymal cells scattered a few spherical secretory granules, smoothy and varied in size.The fibers above the surface of ependymal cells were rare.3.2 Model Group: In model 2 weeks group, the overall outline of the ependymal cells is not clear, rugged and uneven. The cilia were densy and disordered. The large or small irregularly shaped secretory granules were densy and mostly flake or agglomerate distribution with smooth surface. The surface was some smoothy, some roughy or wrinkled. The single fiber with fusiform varicosities were rare and branched closing to the surface of ependymal cells or cilia roots. In model 4 weeks group, the cilia arranged disorder and the partial of them were sparse. The adhesion made them to be wispy. Microvilli were sparse and some regions without microvilli. The rared secretory granules were mostly flake or agglomerate distribution and scattered secretory granules. The fibers above the surface of ependymal cells were more common, distributed widely. They could be divided into two types: A coarse fibers with branches were wide and flat ranging of different lengths. The shape of them were flaky, bundle or network on the surface of cilia. Another cylindrical fibers were thinner,uniform thickness, smoothy and a small amount of them had fusiform varicosities. They closed to the surface of ependymal cells or among cilia roots.The branches of the fibers were rare, but they interweaved into network crowedly. In model 8 weeks group,the overall outline of the ependymal cells were not clear.The cilia and the microvilli were sparse. Surface of ependymal cells scattered a few spherical secretory granules. The fibers above the surface of ependymal cells were rare.4 The results of TEM observation4.1 Control Group: The ependymal cells were arranged in neat rows, with rich in cilia and microvilli, and the gap junctions were clearly visible. Mitochondria are more common in the cytoplasm and they were distributed below the free surfaces and both sides of the nucleus.Lysosomes were rare.4.2 Model Group:In model 4 weeks group,the volume of ependymal cells was varied in size.On the free surface of the ependymal cells, cilia and microvilli were rich. The cell nucleus were oval or round, occasional shrinkage. The chromatin stained to the edge. Mitochondria were more common in the cytoplasm. Number of mitochondria within the cytoplasm were more dense, and some vacuoles were seen in the phenomenon of them. Lysosomals were common,hading varied shapes and different shady particles. A large number of free ribosomes in the cytoplasm. The ribosome on the rough endoplasmic reticulum were irregular, and the stain was shady.5 The results of frozen sections observation5.1 Control Group: Ependymal cells are arranged in regularity, but the nNOS-positive cells, nNOS-positive fibers were rare.5.2 Model Group: At the entrance of LR4V, nNOS-positive cells were in the ventricular membrane, and cell body was spherical,lightly stained nucleus was clearly visible, the cytoplasm was stained purple-blue, more than a 1-2 streak protrusions deep into the lower of ependyma.Subependymal nNOS-positive cells, often had 3-4 aggregated display. Cell body was round, spindle or cone, which often had 1-3 neurites, and they parallel to the ependyma. Subependymal there were more common point-like particles of dark blue, with intensive. It could seen nNOS-positive fibers among the deep blue point-like particles and paralleled with the ependyma.Conclusions:1 Successfully prepared for the model of the diabetes mellitus.2 Under the scanning electron microscope, it was observed that massive secretions and the fibers were on the surface of the ependymal cells in the model 4 weeks group. And it had significant differences with other groups.3 Under the transmission electron microscope, there were a large number of lysosomes and free ribosomes,and some vacuoles were seen in the phenomenon of mitochondrion in the model 4 weeks group.4 NF-κB expression in the nucleus was strongly positive, weakly positive cytoplasm in the model 4 weeks group; in model 2 weeks and 8 weeks the expression of NF-κB was weak in the cytoplasm, the nucleus did not expressed.
|
PDF Full Text Request