Modern Histological Research

Introduction- IVTC as a Technique
It has taken decades to develop the in vivo cryotechnique (IVCT), which is currently being used for examination of the functioning of morphology in the living animals and other organisms. Using IVTC the animals and organisms are cryofixed while the blood is still circulating, this way all the soluble components in the body fluids and structural features of the organism in vivo are easily captured in the frozen tissue specimen. The morphological data from the experiment represents the living state of the organisms cells when it was cryofixed. It has emerged in the recent years that IVCT could be used for immunohistochemical studies and also in the analysis of translocated signal molecules. IVCT has not been used for analysis of liver in living organisms. But in the present study the ever changing liver of a mouse is examined using different hemodynamic conditions of IVCT. The results from this experiment were compared with the one from the conventional chemical-fixation and QF In Vivo Cryotechnique (IVCT) to View Mouse Liver.

Using In Vivo Cryotechnique (IVCT) to examine the mouse liver with ultrastructural, immunohistochemical and histochemical analyses sinusoids are widely opened with numerous erythrocytes flowing in samples prepared by IVCT, this was observed under normal circulation. It was also observed that they were collapsing in arrested mice. On the other hand the sinusoidal cavities were dilated by perfusion-fixation and were observed to have collapsed by immersion-fixation and quick-freezing (QF) methods. The immunoreactivity of the serum albumin and immunoglobulin G and intensity of periodic acid-Schiff-straining in hepatocytes were well preserved with the QF method and IVCT. Then the tissue resection was rapidly converted into hepatocytes as was demonstrated by immunoreactions on the QF tissue frozen 1or 5mm after resection. In the IVCT sample the translocation was not observed this is an indication that IVCT might be important in the examination of cell membrane permeability of hepatocytes in different pathological environments. Immunodistribution and dynamic morphology of the soluble parts in the living organisms (mouse liver) showing the pathological and the physiological states can be precisely examined by in vivo cryotechnique (IVCT) producing higher resolution/

Purpose of Experiment
Both immunohistochemical and Morphological in the analyses of the livers of organisms and animals under abnormal blood circulation and normal conditions would be necessary as they yield important results that reflect the physiology andor pathology of the human liver.

Technique and Procedure
Perfusion-fixation and the alcohol-dehydration (PF-DH) After four mice had been starved for one day, then they were anaesthetized with inhalation of diethyl ether. Then liver was resected and immersed in the same fixative for a night embedded in paraffin wax.

Immersion- fixation process followed by alcohol-dehydration (IM-DH) After the mice were removed from the embedment of the paraffin they were buffered with PFA for microscopy. The specimens were embedded in paraffin wax for magnification to be achieved and those that were to be viewed under electron microscope were embedded in epoxy resins.

Quick- freezing process resected tissues that were freshly obtained (FQF) After the above procedure was achieved, the left lobe of the four specimen were removed from the four specimen under anesthesia and were then chipped into small pieces which were immersed into isopentane for freezing to take place. This process was followed by imbedding the pieces in paraffin wax.

IVCT under varying conditions of hemodynamic In this step sixteen specimens were required and thus 16 mice were anaesthetized and their left lobes were exposed as previously described above. The first four mice livers were cryofixed with IVCT. Four other mice were used in another experiment before IVCT their portal veins and hepatic arteries were ligated at 80min (IVCT-IC), then sham control was performed on another four mice and lastly the four final group of mice were sacrificed by introduction of excessive presence of anesthesia before IVCT, heart arresting.

Preparation of the tissues by PF-DH, IM-DH, FQF and perfusion-diffusion followed by quick freezing (PF-QF) During this step four mice that had not been starved were anaesthetized, their four left lobes were resected.

Conclusions Based on the Experiment Findings
In conclusion, IVCT is used in examination of dead morphology but the dynamic and functional living morphology of mouse livers under various physiological or pathological conditions. It is thus necessary to preserve soluble components like proteins and glycogen in hepacytes which were lost through routine chemical-fixation methods and was also used in the analysis of changes of permeability in cell membranes in hepacytes (Ohao, Terada and Ohno 384).

Morphological Analysis of Sinusoids
In the experiment above the sinusoidal cavities were found to be opened and they contained flowing erythrocytes. For FQF 1min the erythrocytes were almost fully collapsed. It was more obvious here than it was in IVCT-IC and after the sham no results were reflected. With congested erythrocytes they were found to be wider when under heart arrest. PF-DH, there were no erythrocytes observed but the sinusoids were widely opened.

Immunohistochemistry Results
One important benefit for IVCT freezing for organs in living organism is that tissue resection can be achieved without stoppage of blood circulation. The functioning structures of the living tissues that change under varying hemodynamic conditions can be detected by IVCr.

Results of PAS Staining
In the first experiment PAS products from all lobular were preserved by FQF but were partially lost in the peripheral cytoplasm around the centrilobular sinusoids PF-QF. There was more glucose in the periportal areas (PF-DF) and the PAS staining in the cytoplasm was weak in the hepatocytes that are located in the mid centrilobular areas (Ohao, Terada and Ohno 386).

The specimens for PAS staining are prepared through varying methods. Examination of soluble glycogen in hepatecytes by PAS staining of specimens that were prepared by IVCT, FQF and PF-QF followed. To minimize the differences in glycogen content in the tissue blocks, the liver specimens were taken from the same lobe of the mouse and they were examined under the same conditions.

Conclusions Drawn from Results of Experiment
It can be concluded that, IVCT enables us to examine the functionality and dynamicity of the living morphology of the mouse livers under different physiological and pathological conditions and is also important in preservation of proteins and glycogen in hepatocytes.

Extracellular Space in Mouse Cerebellar Cortex under In Vivo Cryotechnique
The method for preparing tissue specimen to be used for morphological analysis of the central nervous the convectional method for preparing tissue specimen for the morphological analysis of the central nervous system. The current study is done by analysis of ultra structures of the cerebellar cortex of the mouse. Also, Purkinje cell layers and molecular structures of the mouse cerebellum are prepared among cellular profiles and synaptic clefts.

Purpose of Experiment
The purpose of features of IVCr for the morphological analysis in vivo is the essence to obtain the analysis from the experiment on the mouse cerebellar cortex with anoxia caused by few artifacts during tissue preparation steps by the use of IVCr.

Conclusions Based on Experiment Findings
There are not much differences in the mean perisynaptic ECS between opened and enclosed synapses. The difference that suggests that the glial processes ensheathing synapses might extend the entire open perisynaptic ECS with a few structural changes to the nearby structures area insignificant. The glial cells observed to affect synaptic neurotransmission, also related to the structural development and restructuring of synapses.

Morphological Comparison among IVCr, FQF, and PF-DH
The ECS that were obtained with IVCr were lucking ischemia and were observed to be bigger than those obtained from freezing method. But the results were smaller than those obtained from ischemia after 30 seconds and also smaller than those obtained from IVCr after synaptic clefts.

Purpose of Experiment
Some of the important Hemodynamic factors in a kidney are blood pressure and blood flow. They determine the native morphology of glomerular blood capillary loops in kidneys of a living mouse. The purpose of this experiment therefore is to examine the topographical changes in serum proteins, immunoglobulin and albumin passing through the capillary loops (glomerular) and how these proteins are taken back into the renal proximal tubules using immunohistochemistry and in vivo cryotechnique.

Technique and Procedure
The experiment method in use starts with the use of anesthesia on twelve to twenty gram adult mice using sodium pentobarbital. The second step involves opening of the abdominal cavity of four mice. The detection of left kidneys is done under normal circulation of blood. This group of mice is used as a control group. The next group of four mice is used to study glomerular leakage and the following reabsorption of the serum proteins in the renal tubules. For the purpose of use in this step, a mouse with acute renal hypertension is used. The mouse is prepared by the way of ligation of abdominal aorta. In vivo cryotechnique is performed using different blood flows.

Conclusions Based on Experiment Findings

HE staining with IVCT
The renal interstitium between the normotensive tubules had a wide open peritubular space in the normal state. Some Lumina spaces found in the proximal tubules in vivo were however not visible as they were too narrow. The bowmans space and the luminal spaces became wider in the renal cortices.

Immunolocalization of Albumin and IgG in Renal Proximal Tubules
The process revealed immunoreaction products of both IgG and albumin in the blood vessels and at interstitium in renal tubules. Albumin Immunolocalization was partially found in apical parts of renal tubules during the normotensive condition.

Immunolocalization of Immunoglobulin Light or Heavy Chain in Proximal Tubules
The Lambda and the Kappa light chains immunolabelling became easy to detect at hypertensive condition at the apical cell membrane of most proximal tubules. The antibody against IgG-1 heavy chain was however not immunoreacted in the proximal tubules but only in interstitium and peritubular capillaries.

Immunolocalization of albumin and IgG by conventional fixation
Both albumin and IgG were immunolocalized in the bowman spaces and also along apical membranes of the cell of the proximal tubules. The glomerular blood vessels were also partially washed and slightly immunostained due to fixation of perfusion.

Conclusions Drawn from Results of Experiment
The effects of Hemodynamic factors in a kidney in different conditions were observed. These factors have been identified among those that affect the abnormal leakage of the serum proteins, a situation which leads to proteinuria in pathological and experimental state in animal kidneys. In such a case, more proteins are reabsorbed in the renal proximal tubules. The above experiments have helped in establishing that the present in vivo cryotechnique can provide stopped images of functional renal tissues in a living mouse as well as distribution of filtered serum proteins and distribution in the renal proximal tubules.


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