Abstract: 

Reliance on hydrocarbon fuel has led to health risks that are largely unrecognized. These include damage to the brain’s central auditory centers. The purpose of this study was to determine the optimum NBT/BCIP staining time for future immunohistochemistry research to localize c-AMP activity in the brains of 20 Fischer344 rat brains that have been exposed to chronic noise, JP-8 jet fuel, and jet fuel plus noise. Previous research on the 20 rats has shown that auditory brain function was damaged in the auditory pathways (Guthrie et al., 2015). Future work will attempt to locate the extent of damage to central auditory brain structures. 

 

Introduction:

c-AMP is an element binding protein that initiates the CREB pathway. The CREB pathway is a regulator of gene expression that is stimulated through phosphorylation. CREB alters the transcription of specific genes whose DNA contains sequences termed c-AMP responses. In addition, CREB plays a role in adjusting levels of neuronal excitability. Neurons present in the central auditory nervous system respond to changes in auditory stimuli by adjusting their potassium channels. Previous studies have shown that within 30 minutes of physiological auditory stimulation, changes in potassium channel proteins can be detected, which are most likely controlled by alterations in protein synthesis (Strumbos et al., 2010). Furthermore, by examining c-AMP activity in the Fischer 344 brains damaged inflicted by aromatic hydrocarbon fuel may show damage in the central auditory nervous system. A study done on CBA mice, which are bred to study neurochemistry and brain development (River, n.d.) has suggested that c-AMP activity was present when hearing levels were normal; however, when hearing levels were impaired the c-AMP activity was abolished (Hehn et al., 2004). Ultimately, by studying c-AMP activity in Fischer 344 rat brains aromatic hydrocarbon fuel may show damage in the central auditory nervous system, which may induce hearing loss.

 

Methods:

The purpose of this study was to locate c-AMP activity in Fischer 344 rat brains to observe the effects of aromatic hydrocarbon fuel on the brain through immunohistochemistry. Immunohistochemistry (IHC) is a research process that images selected proteins in the cells by staining tissues. This method is intended to detect specific proteins in cells through an antigenantibody reaction that then can be observed through a microscope. The success of the IHC process depends on antigens in the cells of the brain tissue to bind specifically to c-AMP activity. The IHC technique requires several steps prior to staining with the NBT/BCIP stain. By altering the concentration of the antibodies as well as increasing the citrate acid antigen retrieval time, the results showed the NBT/BCIP stain binding specifically to c-AMP. By increasing the concentration of both the primary and secondary antibody, the cells could bind to the antigens to bring out c-AMP activity. This preliminary study focused on the immunohistochemistry procedure to further examine the effect of aromatic hydrocarbon exposure on the brains of Fischer 344 rats

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Results:

Opitmum exposure time for NBT/BCIP was 4 minutes. This exposure time produced the best visualization for c-AMP protein activity. 

 

 

Conclusion:

Overall, the NBT/BCIP staining procedure works best through adjusting the antibodies, increasing the citrate acid heating, and staining with NBT/BCIP for 4-minutes.The positive staining result could show that the IHC procedure is effective in amplifying the c-AMP activity in the Fischer 344 rat brains. Previous attempts showed high background activity and over stained the tissues, which made it hard to identify true c-AMP activity from other protein activity in the brain. Further research will be done using this procedure to examine neuron damage that potentially was induced by noise exposure, hydrocarbon jet fuel, or both noise and hydrocarbon jet fuel together. Additionally, further research studies will be pursued such as what the physiological effects of hydrocarbons in jet fuel on glial cells specifically, astrocytes in auditory brain stem neurons might suggest. 

 

References:

Guthrie, O. W., Brian, A. Wong, McInturf, S. M., Reboulet, J. E.,…Mattie, D. R.(2015). Inhalation of hydrocarbon jet fuel suppress central auditory nervous system function. Journal of Toxicology and Environmental Health, Part A, 78, 1154-1168 DOI: 10.1080/15287394.2015.1070389

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 VonHen, C.A., Bhattacharjee, A., Kaczmarek, L.K. (2004). Loss of Kv3.1 tonotopicity and alterations in cAMP response element binding protein signaling in central auditory neurons of hearing impaired mice. Journal of Neuroscience, 19, 36-40. DOI:10.1523/JNEUROSCI.4554-03.2004 

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Song, P., Yang, Y., Barnes, D. M., Bhattacharjee, A, Hamann, M., Forsythe, I. D., …Kaczmarek, L. K. (2005). Acoustic environment determines phosphorylation state of the Kv3.1 potassium channel in auditory neurons. Nature Neuroscience, 13, 35-42 DOI 10.1038/nn1533 

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Lu, Y., Monsivais, P., Tempel, B. L., Rubel, E. W. (2004). Activity dependent regulation of the potassium channel subunits Kv1.1 and Kv3.1. Journal of Comparative Neurology, 470, 93-106 DOI: 10.1002/cne.11037 River, C. (n.d.). CBA mouse CBA/CaCrl. Retrieved from Charles River