Examination of Morphologic Changes in Liver

Associated with Exposure to Cadmium Selenide Quantum Dots

Opt307/407 - Spring 2008

Gayle Schneider

 

Introduction

This study had three objectives - first and foremost was to determine if quantum dots that were intravenously administered to rats could be seen within the liver tissue using the electron microscope. Second - If an accumulation of particles could be seen, could they be positively identified as cadmium with Energy Dispersive X-ray Spectrometry (EDS)? The third objective was to determine if the cadmium quantum dots caused morphologic changes to the cells due to the toxicity of cadmium.

The presence of quantum dots was confirmed with fluorescence microscopy before the tissue was processed for electron microscopy. However, the encouraging display of fluorescent points within suspected Kupffer cells did not translate to masses of dark particles when viewed under the electron microscope. Very few lysosomes within Kupffer cells contained dark contents and those that were discovered were unable to be examined with EDS because the equipment broke down and was sent away for repair.

Morphologic changes were observed with both SEM and TEM. An interesting observation was made when viewing the tissue under SEM - Unusual blebs within the sinusoids and central veins of the liver were present. These blebs occured in the control tissue as well as the two experimental conditions, so they could not be attributed to cadmium toxicity. Varying degrees of cellular damage were observed under TEM. The most extensive damage was seen in the liver that had been dosed with carboxyl-coated quantum dots.

 

Background

Cellular Organization of the Liver

The liver is composed of cells called hepatocytes that are arranged in lobules. Within each lobule, the hepatocytes are arranged in thin layers in a radial pattern around a central vein. Between each layer of hepatocytes are spaces called sinusoids that are connected to a hepatic venule also referred to as a central vein. Blood carrying absorbed compounds from the digestive system enters the sinusoids from branches of the hepatic portal vein and hepatic artery. From there solutes in the blood pass between endothelial cells separating the sinusoids from the hepatocytes. The hepatocytes then process the solutes accordingly and the blood goes on to the hepatic venule and out of the liver. Within the sinusoids are specialized cells called Kupffer cells. Kupffer cells are macrophages that clean the blood of bacteria and other foreign substances by engulfing the invaders and digesting them with lysosomes.

 

The two images above show the arrangement of sinusoids (S), Kupffer cells and hepatocytes (H)

 

Quantum dots

Quantum dots are nanocrystals that absorb photons of light and then re-emit those photons, making it possible to view them with a fluorscence microscope. Invitrogen Corporation Qdots are manufactured with a cadmium selenide core surrounded by a shell of zinc sulfide, then coated with an amphiphilic polymer coating. The Qdots can then be further modified by attaching a biomolecular coating such as amine or carboxyl to facilitate uptake by the cells.

 

Materials and Methods

Qdot ITK quantum dots (~15 nm dia.) in both carboxyl-coated (#Q21331) and amino (PEG)-coated (#Q21531) were obtained from Invitrogen Corp. Three rats were used in the study - one rat was injected with amine-coated Qdots, one with carboxyl-coated and one rat served as a control and was not injected with Qdots. The liver was harvested 24 hours after injection and frozen at -60 degrees C for 6 months prior to examination.

A section of frozen tissue was cut with a cryostat and examined under a fluorescence microscope. Once it was established that the Qdots were taken up by the cells, the rest of the frozen liver tissue was processed for viewing under SEM and TEM. Processing included fixation in 2.5% glutaraldehyde and post-fixation in 1% osmim tetroxide followed by a graded ethanol dehydration. The samples for SEM were dried with CPD, viewed uncoated with Variable Pressure SEM then the control sample was sputter-coated with Au/Pd and viewed with conventional SEM. The liver tissue for TEM was taken from 100% ethanol, transitioned to epoxy resin with propylene oxide, embedded in Epon/Araldite resin and polymerized for 2 days at 60 degrees C. Sections (1 um thick) were cut and examined under a light microscope to establish orientation then the sample was thin sectioned (80 nm) with a microtome and placed on grids for viewing with TEM and STEM on SEM.

 

Results

The fluorescence microscope images below show the deposition of Qdots inside cells within the sinusoids (dark spaces) of the liver. These deposits were thought to be contained within lysosomes of the Kupffer cells.

Left: Amine-coated quantum dots; Right: Carboxyl-coated quantum dots

 

Once the Kupffer cells were examined under TEM it was apparent that these brightly fluorescing areas did not correspond with identifiable lysosomes in the quantity shown under fluorescence. A few areas of dark deposits were found in both experimental conditions. These deposits contained smaller diffuse particles in the size range of the coated quantum dots (~15 nm) but because the EDS unit was not operating, they could not be positively identified as cadmium selenide quantum dots. Another type of deposit that was present in both of the experimental conditions and the control is shown below on the right.

Left: Kupffer cell and deposit of dark particles; Right: Kupffer cell with concentric myelin-like layers

 

The concentric figures were found throughout the hepatocytes and Kupffer cells and could be an artifact of freezing the tissue before processing. There is also mention in Bloom and Fawcett's "A Textbook of Histology" that lysosomes may take on a form with "concentric systems of lamellae interpreted as myelin forms of phospholipid". So, it is possible that these forms may be lysosomes and that they may contain Qdots.

When the liver tissue was viewed under SEM it was discovered that blebs were protruding into the sinusoidal spaces. These blebs could be caused by low oxygen conditions within the liver tissue as noted by Lemasters and Stemkowski. They concluded that the blebs could be hepatocyte microvilli that swell under conditions of hypoxia.

 

Both SEM images above are of the control liver - without Qdots

Variable Pressure SEM of sinusoid

 

Control liver with blebs and Kupffer cell in sinusoid

 

Viewing a Kupffer cell on a TEM grid with a STEM unit on SEM was attempted twice, but because the Qdots were not packaged neatly in easy-to-locate lysosomes, the lengthy search for dark particles caused a hydrocarbdon deposit to be laid down each time.

 

Conclusion

Cellular damage and artifacts were noted in all samples. The unusual concentric myelin forms as well as the formation of blebs and ruptured nuclear membranes could be caused by freezing or tissue processing . It would be interesting to try this again with samples that had been fixed immediately without prior freezing to see how much damage can be attributed to freezing.

Qdot location noted in the fluorescence images did prove to be elusive with TEM due to the scarcity of defined lysosomes within the Kupffer cells. The few particle deposits discovered as well as the ubiquitous concentric forms should be examined with EDS when the unit is repaired.

 

References

Cell Surface Changes and Enzyme Release During Hypoxia and Reoxygenation in the Isolated, Perfused Rat Liver. LeMasters, Stemkowski, Ji, Thurman. The Journal of Cell Biology, Vol. 97, Sept. 1983, Rockefeller University Press

A Textbook of Histology, Bloom and Fawcett, Chapman & Hall, 1994

 

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