Splenic Macrophage Aggregates Methods page This web page highlights methods used in portions of the St. Lucie Estuary Silver Porgy Health Study, focusing on splenic macrophage aggregates. Silver porgy are part of this study because they are common inhabitants throughout the St. Lucie Estuary, have an ecology that is closely tied to the St. Lucie environment (generally benthic; feeds upon algae, mulloscs, crabs), and this species appears susceptable enviromental stress based on observations of external pigmented foci and ulceration.
Silver porgy were collected by hook and line from two locations in the St. Lucie River Inlet: Site 10 (South of Sailfish Point docks; West of Sailfish Point heliport) and Site 30 (South jetty of St. Lucie Inlet State Park); see interactive Google Map, below:
Fish were humanely euthanized in the field, necropsied, and the spleens preserved in neutral-buffered formalin. Spleen tissues were processed for routine histology, and 5 micron sections were stained with hematoxilin and eosin. Preliminary studies were conducted to determine the number of microscopic fields and the appropriate magnification needed to support quantative evaluation of the average area of splenic tissue occupied by macrophage aggregates. Initial evaluations indicated that 3 fields of 0.9 x 0.9 mm (0.81 square mm) per spleen (per case) were sufficient to reasonably represent the entire tissue. This is consistent with previous studies by Fournie et al. (2001). Spleen tissues from each of 26 cases (as available: ten from site #10 and ten from site #30; see google map) were examined using light microscopy to discern macrophage area per square millimeter spleen tissue, number of macrophages per square millimeter, and size distribution of macrophage aggregates. Data for each case was presented as an average from each of the 3 microscopic fields evaluated for that case. The two images below help to describe the methods used to evaluate splenic macrophage aggregates.
Spleen sections (above) were examined using light microsopy to determine the area, number, and size distribution of macrophage aggregates. The left image shows a photomicrograph of spleen tissue from silver porgy that is photographed 100x magnification under the microscope. This entire left box represents a square field of view (under the microscope) that measures 0.90 mm x 0.90 mm (0.81 sq mm). The multiple, amorphous brown blotches, that are more concentrated on the left two-thirds of the image, are aggregates of macrophages. They are brown and sometimes speckled with black since they contain the pigment melanin. These macrophage aggregates are, hence, also known as melanomacrophage aggregates. In many fish species, accumulation of clumps of macrophage cells, known as macrophage aggregates, can serve as a biological indicator of exposure or stress. The present study is examing macrophage aggregates silver porgy, a common species in the St. Lucie known to be suceptable to a variety of stressors and stress outcomes. Image quantification: Three high resolution images were photographed using a 10x objective lenes from each spleen, using an Oympus D70 camera attached to an Olympus BX51 light micscrope. Areas of 0.81 sq mm were assessed for macrophage aggregates using Adobe Photoshop CS5 by manually coloring them using a hard-edge paintbrush tool (in a separate file layer). Painted areas (illustrated in the right image, above) were assessed for percent area, number and size distribution using the measurement tool. Measurement data was exported to Excel for management and graphical presentation. Differences between age, gender and site categories were evalauted. These data will be used to evaluate the potential utility of quantitative macrophage aggregate analysis in silver porgy in future studies. Semi-quantitative assessment: In addition to quantative measurements macrophage aggregate area and number was also assessed using a ranking scheme. Comparison between the quantitative and qualitative approaches will provide validation for the ranking approach and support use of the ranking system to assess other organ systems. Below are examples of the ranking scheme, using a severity index scale of 0-5, where 0=no observation (of macrophage aggregates), 1="minimal" observation, 2="mild" observation, 3="moderate" observation, 4="marked" observation, and 5="severe" observation. For each rank example below, two different images (side by side) are presented to illustrate the variability that may be seen within a given rank. These photomicrographs were taken with a 10x objective lens to provide imagry of what might be seen under the microscope. Actual rankings, however, are conducted at 4x - 10x to get "an impression" of the overall rank for that organ tissue sample.
|
|||||||||||||||||||||
|
Website developed through the University of Florida
|
