1. Materials and Methods (continued)
Expression of S100B, GFAP, and Nestin in rats after soman-induced brain injury Grace Capacio Mentored by Dr. Robert Kan and Jessica Leuschner
Introduction
Materials and Methods (continued)
Results (continued)
Soman is an organophosphorus acetylcholinesterase (AChE) inhibitor used as a chemical nerve agent which causes seizures and, ultimately, brain injury in an exposed victim. This leads to severe neuroinflammation, evident through the expression of Glial-Fibrillary Acidic Protein (GFAP) by reactive astrocytes. Although a majority of published studies use GFAP to examine astrocytic populations, GFAP alone does not accurately describe all reactive astrocytes after soman challenge. The present study was developed to determine the expression of GFAP in comparison to S100B and Nestin. S100B is part of a family of calcium-binding proteins commonly found in the brain and has been dubbed a classical astrocyte marker (Ludwin, Kosek, & Eng 1976). GFAP marks “both protoplasmic and fibrous astrocytes” but “is more prominent in the latter” (Ludwin et al., 1976). It is used commonly in neuropathology to survey protein expression in the brain. Nestin, “an embryonic intermediate filament protein” (Kaya, Mahmood, Li, Yavuz, &Chopp, 1999), is a traditional pathological marker which is used to identify dividing cells and is useful in identifying astrocytic proliferation.
The following morning the incubation chamber was removed from the 4 °C and the slides placed in a PBS rinse. Then the slides were removed from the rinse and placed back into the incubation chamber, and each tissue sample was then covered in a mixture of AlexaFluor® 488 anti-mouse and AlexaFluor 594 anti-rabbit. The chamber was then closed and the slides left at room temperature. After incubation, the slides were removed from the chamber and placed in a PBS rinse. After rinsing, each slide was cover-slipped using a 2-(4-Amidinophenyl)-1H-indole-6-carboxamidine (DAPI) mounting media. The slides were scanned into a computer using a Hamamatsu© NanoZoomer and the images imported into Visiopharm, a pixel quantifying program, for analysis (see Figure 1).
In the GFAP stained animals, there was a significant increase in the amount of staining at the 3, 5, 6, 7, and 8 week time points. Significance in the 3 week animals was observed in the Medial Dorsal Thalamus (MDT) and LDT, in the 5 week animals in the MDT and LDT, in the 6 week animals in the Hippocampus, DC and LC, and the MDT and LDT, in the 7 week animals in the Hippocampus and the MDT and LDT, and in the 8 week animals in the Hippocampus, DC and LC, and the MDT and LDT. In the Nestin stained animals, there was a significant increase in amount of staining in the 6 and 8 week time points. Significance in the 6 weeks animals was observed in the MDT and LDT, and in the 8 week animals in the MDT.
Results
A Grubbs outlier test was performed to eliminate outliers and significance testing was performed using a two-way ANOVA with a Bonferroni post-test at a 95% confidence level. Each time point was compared to 2 weeks to determine significance (see Graph 1). In the S100B stained animals, there was a significant increase in the amount of staining at the 3, 4, 5, 6, and 7 week time points. Significance in the 3 week animals was observed in the Hippocampus and Lateral Dorsal Thalamus (LDT) regions, in the 4 week animals the Dorsal Cortex (DC), in the 5 week animals the Piriform Cortex, in the 6 week animals in the DC, the Piriform Cortex, and the LDT, and in the 7 week animals the Lateral Cortex (LC).
Conclusions
Protein expression across the time course proved to change significantly. The significant increase in the S100B accompanied by the significant increase in GFAP confirms that the S100B is acting as an astrocyte marker. Some astrocytes expressed both GFAP and S100B, but there seems to be more cells expressing S100B than the GFAP. It appears that only fragments of astrocytes expressed Nestin, unlike the S100B and GFAP, which seemed to label the entire astrocytic cell. The GFAP and S100B remained elevated at the end of the time course and continued studies with longer time points will tell if the GFAP and S100B may eventually return to normal levels. Further studies will compare S100B to a neuronal marker and a microglia marker to determine if other cell types also express this protein. Quantification of the stains proved difficult and inconsistent without repeated trials and may have played a role into the final values obtained. In the future, it would be important to have more than an n of three at each time point.
Materials and Methods
Rats were given the oxime HI-6 (125 mg/kg, ip) thirty minutes prior to soman challenge and treated with atropine methyl nitrate (AMN) (2.0 mg/kg, im) one minute after soman administration. At 2, 3, 4, 5, 6, 7, or 8 weeks after seizure onset, brains were collected, paraffin processed, and sectioned. The sectioned tissues were then placed on positive-charged slides, deparaffinized, and hydrated using a series of xylene baths followed by ethanol-distilled water dilutions in decreasing concentrations. The slides were then placed in Phosphate-Buffered Saline (PBS; pH 7.4) followed by a 5% Hydrogen Peroxide bath to suppress endogenous peroxidase activity and rinsed. To perform antigen retrieval, a coplin jar with pH 6.0 citric acid was heated in a coplin jar. The slides were then placed inside and heated again. After heating, the slides cooled at room temperature, rinsed, and placed in an incubation chamber and covered in 2% Horse/Goat serum to block nonspecific staining from the secondary antibody. The slides were rinsed, placed in the incubation chamber, and covered with either a dilution of S100B and a dilution of GFAP or a dilution of S100 and a dilution of Nestin. All antibodies were diluted in 2% Horse/Goat serum. The chamber was covered and left at room temperature before being placed in the 4 °C refrigerator overnight.
Graph 1: Pictured above is a graph of GFAP expression across the time course by region. The overall trend depicts an increase in GFAP expression from 2 to 8 weeks. There was significance at the 3, 5, 6, 7, and 8 week time points. mm
Figure 1: Pictured to the left is an image taken on the Hamamatsu© NanoZoomer. The image shows a lesion in the Piriform Cortex (white arrow) at 2 weeks, typical in animals exposed to nerve agent. The red staining indicates a presence of S100B and the green indicates a presence of GFAP.
References
Kaya, S. S., Mahmood, A., Li, Y., Yavuz, E., & Chopp, M. (1999) Expression of nestin after traumatic brain injury in rat brain. Elsevier Science, 840,
Ludwin, S. K., Kosek, J. C., & Eng, L. F. (1976). The topographical distribution of S-100 and GFA proteins in the adult rat brain: and immunohistochemical study using horseradish peroxidase-labelled antibodies. Journal of Comparative Neurology, 165,