Ismail Eddafali05/03/2014

The Liver

The liver has many different structures that are quite uniform in their

arrangement. The comparison can be made to a suburb where houses are aligned

uniformly one after another, with gas stations and convenience stores found in equal

distances from each other. The liver is divided into classic lobules, which are somewhat

hexagonal in shape. The lobule can be thought of as a neighborhood. The liver lobule is

structurally made up of the parenchymal cells called the hepatocytes. The hepatocytes are

aligned in tube like arrangement much like the houses of a suburb. The area containing

the hepatocytes also contains structures called sinusoids, which can be compared to roads

or yards of the suburban houses. The classic lobule contains a central vein at the center

and a portal triad consisting of a portal vein, hepatic artery, and bile duct at the six

vertices. These structures are comparable to the gas stations and convenience stores as

they supply certain materials for the function of the liver. Each classic lobule is divided

by connective tissue septa comparable to the main roads that divide neighborhoods of a

city.

Our experiment consisted of labeling the liver using two different staining

methods. The first method was an antibody immunohistochemistry stain. We decided to

stain connexins, laminin, and cell nuclei, as these features are prominent and distinct in

the liver. The connexin stain allows us to view the gap junctions between the hepatocytes

of the liver as shown in figure 1. We used indirect immunohistochemistry, which requires

the use of a primary antibody to bind our proteins and a secondary antibody that will be

fluorescent and bind our primary antibody. This method allows for a higher fluorescence

to be present in our tissue. To bind connexins, we used an anti-connexin antibody from a

rabbit as the primary antibody. To bind and fluorescently stain our primary antibody, we

used the FITC conjugate-goat anti-rabbit IgG antibody that stained fluorescent green. The

nuclei were stained with a DAPI stain that strongly binds adenine-thymine regions of

DNA. Laminin is a major protein found in the basal lamina which is a prominent

structure found in many cell types as a layer of the basement membrane of a cell. To bind

laminin, our primary antibody was anti-laminin from a mouse. To bind our primary

antibody, we used a secondary antibody, which was anti-mouse IgG that stained

fluorescent red. The structures we predicted to be stained include the hepatocytes, the

portal triad, the central vein, and all cell nuclei. After staining was complete we saw that

The connexin stain labeled gap junctions between hepatocytes as shown in figure 1. The

laminin stain labeled portal triads, central veins and connective tissue septa, a feature that

we had not predicted would be stained as shown in figure 1. Finally, DAPI stained all cell

nuclei present.

The second method was a toluidine blue stain that stained nucleic acids blue and

polysaccharides purple as it is a basic dye staining highly acidic tissue structures. This

method allowed for similar structure staining as the immunohistochemical method. The

stain was able to distinguish many features of the liver including the connective tissue

septa, the hepatocytes, portal triads, central veins, sinusoids, and miscellaneous

vasculature. Figure 2 illustrates the cords of hepatocytes arranged around the central vein

and the sinusoids that align in a similar fashion.

We faced many challenges in our journey that came about from every stage in the

process. Some of our tissue was damaged in the process of fixation and some of it was

because of the age of the tissue. Tissue damage was present as a result of sectioning, as

knife marks were prominent in some mounted slides. In addition, certain areas exhibited

folding over, thickness variation and freeze fracturing. In the washing process due to

friction forces, before staining was conducted, there was some tissue loss this was

especially present in our immunohistochemistry stained slides. In the immunofluorescent

method, the antibody stain for connexin showed a prominent background, which

interfered in distinguishing the gap junctions between hepatocytes. During the toluidine

staining process, certain tissue areas stained longer and thus darker than others, which

created a gradation along the tissue. The mounting process may have moved tissue

around and caused folding over. Moreover, the mounting allowed air bubbles to

accumulate and this caused distortion of the tissue under the microscope.

Primary biliary cirrhosis is a disease that affects the bile ducts of the liver and is

believed to be a chronic autoimmune disease. The cause of the disease is unknown but

research has found a link to an immunological response against components of the

mitochondria. The symptoms associated with the disease include jaundice, itchy skin,

enlarged spleen, edema around the abdomen, and hepatic encephalopathy. Histologically,

the area of the portal triad will become inflamed which is caused by the gradual

destruction of the bile ducts. The area becomes highly concentrated with lymphocytes

and macrophages and granulomata appear. In addition, fibrosis around the triad and a

proliferation of small bile ducts begins to develop. The septa of the liver will become

more pronounced. Eventually the damage caused will become irreversible and the disease

will lead to liver failure.

Figure 1. Immunochemistry stained image at 20x magnification showing liver hepatocytes (H) with distinctive connexon (C) stained gap junctions. Connective tissue septa (S) is also prominent outlining the hexagonal classic liver lobule with a possible portal triad (PT) visible.

Figure 2. Toluidine stained image at 20x magnification showing twin central veins (CV). The sinusoids (S) and hepatocytes (H) align around the central vein in a semi-linear fashion.