Application for Graduate Research and Creative Activities (GRACA) Funding Student: Jacquelyn Davis Faculty Mentor: Suzanne Sollars, PhD

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1 Application for Graduate Research and Creative Activities (GRACA) Funding Student: Jacquelyn Davis Faculty Mentor: Suzanne Sollars, PhD Title: Characterization of Innate Immune Response in the Neonatal Rat Tongue Following Peripheral Nerve Injury I am currently a graduate student in the Psychology department (Neuroscience and Behavior area) and am working under Dr. Suzanne Sollars, who shall also serve as the advisor for the research outlined in this application. I completed my Master s degree in 2012 and am currently continuing my graduate career as a PhD student. As such, I intend to enroll in classes for Fall 2014 and beyond, until the completion of my PhD. If approved, it is my intention to use this funding to purchase supplies essential for my Doctoral dissertation, as well as to provide a summer stipend enabling me to continue my research beyond the standard academic year. The techniques proposed here, while previously used in the field of taste research, are new to me and represent a significant contribution to my existing research skills. Furthermore, this project represents a significant contribution to the field; there is currently a nearly complete lack of information regarding innate immune system functioning in the immature taste system. Project Description The taste system is one of the least studied sensory systems and as such, it is possible to yet make substantial gains in understanding regarding some of the more basic aspects of function and recovery in the system. Previous studies have found developmental differences in morphology, functionality and nerve recovery (e.g. Hard af Segerstad, Hellenkant & Farbman, 1989; Sollars, 2005; Sollars & Bernstein, 1996) but the mechanisms behind these differences are as yet unknown and immune factors remain an excellent candidate for investigation. On the tongue, the taste buds (which contain the taste receptor cells responsive to the four known tastes; sweet, salty, bitter and sour) are contained within structures called papillae. There are several types of papillae on the tongue and palate; of particular interest to this study are the fungiform papillae, located on the dorsal, anterior two-thirds of the tongue. The taste buds within these papillae are maintained by the chorda tympani (CT) nerve, which is responsible for transmitting taste information from the tongue to the brain. When the CT nerve is cut (CTX) taste receptor cells and associated taste buds and papillae disappear (Sollars, 2005). When animals undergo CTX as adults, these changes in taste buds are transient; after approximately 45 days the nerve regenerates and the system returns to normal (Hendricks, Brunjes & Hill, 2004). Compared to adults, CTX in young animals (performed between 5-10 days of age; P5-P10) results in nearly complete taste bud loss and permanent damage to the system, as a result of the CT s failure to regenerate (Sollars, 2005). Such developmentally-dependent outcomes have also been supported across species by behavioral investigations in humans. Patients who experienced a high number of childhood inner-ear infections (which may damage the CT as it runs along the malleus of the inner ear) had preferences towards foods with higher fat and sugar content than did control patients without a history of inner-ear problems, suggesting decreased functionality in the CT (Bartoshuck, Canatalanotto, Hoffman, Logan & Snyder, 2012). Differential outcomes across development also appear in studies of the lingual nerve which innervates the fungiform papillae themselves. Similar to results seen with CTX, severing the lingual nerve (LX) in young animals results in taste bud loss (Gomez & Sollars, 2006). While these effects are more severe following surgery at a younger age (as with CTX) the changes are transient in nature and even the youngest animals eventually experience regeneration of the lingual nerve and subsequent return of taste structures. Thus, nerve regeneration is not always prohibited following neonatal injury but is a distinct characteristic of CT damage. The mechanisms underlying this difference in nerve regeneration capacity (both between varying ages and between differing nerves) are not yet understood. One proposed possibility is that differences in

2 innate immune function across development influence nerve regeneration. The immune response following adult nerve transection consists of a stereotyped two-stage process. Neutrophils, a type of white blood cell, are the primary response of the innate immune system and become activated within hours of system insult (McCluskey, 2004). Neutrophil presence has been shown to have a deleterious effect on surrounding, healthy tissue and leads to decreased functioning in the nearby nerves in the tongue following CTX (Stein, Shi, He, & McCluskey, 2010). Approximately 48 hours after injury, infiltration by macrophages leads to the down-regulation of neutrophil response and the recovery of nearby nerve function (Wall & McCluskey, 2008). Dietary sodium restriction has been shown to reduce macrophage response and leads to reduced nerve functioning and recovery (McCluskey, 2004). Injection of the endotoxin lipopolysaccharide (LPS), which causes an up-regulation of immune response, has been shown to counteract the depressive effects of sodium-deprivation on both immune and nerve functioning (Cavallin & McCluskey, 2005). In conjunction, these findings led Steen and colleagues (2010) to conclude that innate immune response was directly responsible for the nerve regeneration seen in the adult taste system. Additional findings suggest that developmental differences in immune response to injury exist in the taste system and that these factors play a role in nerve regeneration. An investigation of immune response following CTX in older-aged animals found that aged animals experienced both an elevated neutrophil response and drastically slower nerve regeneration than adult animals (He, Yadgarov, Sharif, & McCluskey, 2012). It remains to be seen if such differences exist in the taste system pre-maturation and if so, the extent to which they may be implicated in the impeded nerve regeneration. In order to directly assess the innate immune response to peripheral nerve injury a comparison will be made between the neutrophil presence in animals which undergo CTX pre- and post-taste system maturation (which occurs at P40; Sollars, 2005). It is hypothesized that increased neutrophil response (such as is seen in aged animals; He et al, 2012) contributes to the failure of the CT to regenerate in young animals, possibly by suppression or degradation of surrounding healthy neural tissue. Results differing from these expectations would still allow for a characterization of peripheral immune response in neonatal animals, a critical consideration given the present dearth of information in this area. Methodology The presence of immune factors will be assessed using standardized immunohistochemical staining protocols previously outlined in Stein et al., Briefly, female, Sprague-Dawley rats (bred at UNO) will undergo unilateral CTX at P5 or P40. The CT is bilaterally represented, meaning separate, parallel nerves run along each side of tongue to the brain. Thus, unilateral CTX (cutting only the nerve on one side) allows for a comparison of the cut and uncut sides of the tongue within a single animal. Animals will be sacrificed with i.p. injection of ketamine (40-78mg/kg) mixed with xylazine (5-13 mg/kg) and tongue will be excised at 12, 24, or 48 hours post-surgery. These time points coincide with the observed neutrophil response in adult CTX animals, which peaked and then normalized between sectioned and intact sides by 2 days post-surgery (Steen et al, 2010). There will be three animals per condition (e.g., three animals which undergo CTX on P5 and are sacrificed at 12 hours post) for a total of 18 animals. Following flash freezing and sectioning at 8 m, neutrophil presence will be evaluated across the CT innervated field, which shall include sections from the anterior, mid and posterior tongue. Sections of spleen tissue will also be collected as and stained in conjunction with tongue tissue, to serve as a positive control (ensuring immunohistochemical processing was successful). Tissue will be fixed in 0.2% glutaraldehyde in phosphate-buffered saline (PBS) followed by incubation in rabbit anti-rat myeloperoxidase antibody (1:100; Abcam), and then incubation in biotinylated goat anti-rabbit IgG (1:100; Sigma-Aldrich), avidin-biotin complex (Sigma-Aldrich) and diaminobenzidine (Sigma-Aldrich). Following staining, tissue will be examined with a Leica microscope and photographed at 50x magnification with MagnaFire software (Olympus). Color images will be analyzed for immunopositive

3 neutrophil presence across a standardized area and cell counts will be performed (staining selectively binds to activated neutrophils and allows them to be visualized as brown cells against a pale background). Cell counts will be compared between surgical and control sides of the tongue as well as compared across surgical age and post-surgical time points, resulting in a 2 (surgical versus intact side) x 2 (neonatal versus adult CTX) x 3 (time post-surgery; 12, 24, or 48 hours) ANOVA. Differences in neutrophil response are expected as a function time post-surgery, with peak neutrophil invasion around 24 hours on the cut side of the tongue. As some neutrophil presence is expected throughout the tissue, it is the relative variation in overall neutrophil presence which is of interest, particularly as a function of surgical age. All experimentation involving live animal subjects will be carried out in accordance with the Institutional Animal Care and Use Committee (IACUC), following the approval of the protocol outlined here. My research aims to characterize the innate immune response in the tongue of young animals after nerve transection surgery. Elevated or prolonged neutrophil response is predicted to occur in neonatal CTX animals, particularly on the injured (cut) side of the tongue. Such a response would suggest that immune response is implicated in the lack of neural regeneration seen in young animals. Results differing from these expectations would suggest that either later immune response (i.e., macrophage response) may be different in the immature taste system or that immune responding is not directly related to CT regeneration. This study represents the first step in understanding the immune system in the immature taste system. Future research will focus on manipulating immune response through LPS injections (shown to up-regulate macrophage response in adult animals) and comparisons with other types of taste system injury (such as lingual nerve cuts, from which even the youngest animals demonstrate full nerve recovery). Research results will be submitted for presentation at national conferences such as the Association for Chemoreception Sciences (AChemS) and the Society for Neuroscience (SfN). Additionally, results will be submitted for publication to appropriate peer reviewed journals to allow dissemination to the pertinent scientific communities. Results will also be presented at the 2015 Student Research and Creative Activity Fair, in accordance with the requirements of the GRACA funding. Spring/Summer 2014 February March May June- August September October Project Timeline Submit IACUC protocol Purchase needed supplies, train on necessary procedures Surgery, tissue removal, cryosectioning Immunohistochemical processing, cell counts Finish data collection, conduct mathematical analyses Prepare manuscript for publication Role of Student and Mentor All animal breeding, maintenance, sacrifice, tissue removal, staining, and analysis will be completed by me. The innate immune response in the neonatal tongue is currently uncharacterized, likely because of the limited number of investigators able to perform the relevant surgical manipulations on young rats. Fortunately, Dr. Sollars has a well-established record of successfully performing such procedures, thus enabling the proposed investigation. In addition to conducting surgeries, Dr. Sollars s role will be that of advisor; providing protocol supervision and training on pertinent techniques, particularly the immunohistochemcial procedures.

4 References Bartoshuck, L.M., Canatalanotto, F., Hoffman, H., Logan, H. & Snyder, D.J. (2012). Taste damage (otitis media, tonsillectomy and head and neck cancer), oral sensations and BMI. Physiology & Behavior, 107(4), Cavallin, M.A. & McCluskey, L.P. (2005). Lipopolysaccharide-Induced up-regulation of activated macrophages in the degenerating taste system. Journal of Neuroscience Research, 80, Gomez, A. M., & Sollars, S. I. (2006). Developmental effects of lingual nerve transection on taste bud volumes in rat [Abstract]. Association for Chemoreception Sciences Annual Meeting, 445. Härd af Segerstad, C., Hellekant, G. & Farbman, A. I. (1989). Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chorda-lingual nerve. Chemical Senses, 14(3), He, L., Yadgarov, A., Sharif, S. & McCluskey, L.P. (2012). Aging profoundly delays functional recovery from gustatory nerve injury. Neuroscience, 209: Hendricks, S. J., Brunjes, P. C., & Hill, D. L. (2004). Taste bud cell dynamics during normal and sodiumrestricted development. Journal of Comparative Neurology, 472, McCluskey, L.P. (2004). Up-regulation of activated macrophages in response to degeneration in the taste system: effects of dietary sodium restriction. Journal of Comparative Neuroscience, 479, Sollars, S.I. (2005). Chorda tympani nerve transection at different developmental ages produces differential effects on taste bud volume and papillae morphology in the rat. Journal of Neurobiology, 64(3), Sollars, S.I. & Bernstein, I.L. (1996). Neonatal chorda tympani transection alters adult preference for ammonium chloride in the rat. Behavioral Neuroscience, 110(3): Steen, P.W., Shi, L., He, L. & McCluskey, L.P. (2010). Neutrophil response to injury or inflammation impair peripheral gustatory function. Neuroscience, 167(3) Wall, P.L., McCluskey, L.P. (2008) Rapid changes in gustatory function induced by contralateral nerve injury and sodium depletion. Chemical Senses, 33,

5 Budget Justifications Despite the high significance of the proposed experiments, the supplies needed for implementation are potentially prohibitively costly. GRACA funding would be necessary and sufficient for the purchase of all immunohistochemical components required for project completion. The proposed experiments represent a considerable time commitment (animal care and management pre- and post-surgery, sacrifice and tissue removal, tissue preparation, sectioning and staining, tissue examination, plus additional time to learn and practice relevant procedures). As such, the ability to work over the summer months will be critical to the timely completion of this project. Funding provided by the GRACA would allow me to devote significant time in the lab over the summer without the financial restraints of needing outside employment. The amount of compensation requested is comparable to the amount paid for a teaching or research assistantship during the academic year. An average of hours per week over the summer is likely necessary to complete the project in accordance with the proposed timeline. Funding Request Expense Cost Vendor Quantity Total Funding Requested Rabbit anti-rat $389 + S&H, tax Abcam 100 g $448 myeloperoxidase antibody Biotinylated goat $ S&H, tax Sigma Aldrich 0.5mL $49 anti-rabbit IgG Diaminobenzidine $ S&H, tax Sigma Aldrich 5g $68 Glutaraldehyde $ S&H, tax Sigma Aldrich 10mL $127 Summer Stipend $1231/month 3.5 months (May-mid August) $4308 $5000