Arachidonic Acid

We compared the effects of the three aforementioned euthanasia methods on metabolic biomarkers in serum (n = 8 per protocol). All rats were individually handled during the week prior to testing and killed by one experienced person. The rats in the first group were killed by decapitation, using a guillotine. The rats in the second and third groups were killed by CO₂ inhalation – 2 min. 30 sec., fixed time and gradually increased concentration – and an overdose of pentobarbital – 120 mg/kg intraperitoneal injection, in a volume of 1 ml/100 g of body-weight – respectively. Blood samples from all groups of animals were collected using a standardized protocol. After decapitation, 1 ml trunk blood was collected at the decapitation site and allowed to coagulate before centrifugation at 1000 × g for 10 min. and the serum was stored at −80°C until analysis.
The analysis of corticosterone and insulin was conducted using the Coat-A-Count Rat Corticosterone ¹²⁵I RIA kit (Siemens Medical Solutions, Los Angeles, CA, USA) and the Mercodia Rat Insulin ELISA (Mercodia, Uppsala, Sweden) following the instructions of the manufacturer. Triglycerides, cholesterol and glucose were analysed with enzymatic colorimetric methods using an automated chemistry analyser Architect c4000 (Abbott Diagnostics, Lake Forest, IL, USA). FFAs were extracted from serum by protein precipitation and quantified by mass spectrometry. In short, 10 μl of serum was added to an equal volume of an internal standard mix (²H₂-16:0, ¹³C₁₆-16:1n-7, ²H₂-18:0, ²H₂-18:1n-9, ²H₄-18:2n-6, ²H₆-20:3n-6, ²H₈-20:4n-6, prepared in methanol), and 80 μl of methanol. Samples were vortexed, and precipitated proteins were removed by centrifugation. Supernatants were diluted in 10 volumes of methanol in glass autosampler vials, and immediately quantified by liquid chromatography–tandem mass spectrometry as previously described [9 (link)]. Twelve FFA – myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid – were analysed. The analytes were separated on a Kinetex 2.6-μm core shell pentafluorophenyl column (100 × 2.1 mm, 100 Å; Phenomenex, Macclesfield, UK) using a Prominence UFLC_XR system (Shimadzu, Milton Keynes, UK), and detected by ‘pseudo-molecular’ scheduled multiple reaction monitoring transition on a QTRAP 5500 hybrid triple quadrupole mass spectrometer (AB Sciex, Warrington, UK). Analyst software version 1.5.1 (AB Sciex) was used for data acquisition and analysis.

Two sample groups: LPV-afflicted cases and age / race-matched, pain-free controls were recruited from the Division of General Ob/Gyn clinical practice of the University of Rochester between December 2012 and February 2014. All subjects provided informed consent and the research project was approved by the University of Rochester Institutional Review Board (RSRB # 42136). Cases needed to fulfill “Friedrich’s Criteria” for the diagnosis of LPV which included clinical evidence of tenderness localized within the vulvar vestibule confirmed by cotton swab test (CST) using the modified diagnostic criteria of Bergeron et al.[4 (link)] Anatomically, the vestibule is situated between the external vulva (consisting of the labia majora, labia minora, perineum, and mons pubis) and the vagina. Anatomic landmarks include Hart’s line, between the external vulva and vestibule and the hymeneal ring between the vestibule and vagina (see further illustration in Figure 1D–E below). This project involved a small number of cases and controls, based upon the need to develop and maintain fibroblast strains (two strains per subject) and the overall complexity of the experimental design. The limitation in subject number necessitated a careful selection and characterization of cases and controls with matching on particular pivotal characteristics such as age and race. A decision to match by age (within 3 years) was based on reports of increased IL-6 production by fibroblasts from older donors[65 (link)]. A decision to racially match was based on reported racially-related pain threshold differences[17 (link)] and the potential difference in pro-inflammatory mucocutaneous reactivity based on melanin concentration[57 (link)]. Other research has found histopathologic differences in vulvar nerve density, hormonal receptors, and inflammatory infiltrate based upon the classification of primary and secondary vulvodynia[30;38]. Primary vulvodynia is characterized by a clinical history of pain experienced at first introital touch and may be recognized during attempted tampon insertion or insertional dyspareunia. Secondary vulvodynia is characterized by insertional pain development following a pain-free time interval[64 (link)]. LPV Cases entering the study were categorized as primary and secondary and this study permitted recruitment of either category. All subjects were selected from patients previously scheduled for gynecologic surgery.
Subjects underwent a structured health history and three published methods of vulvar mechanical pain measurement and assessment (Figure 1A–C): the “cotton swab test” (CST), Morrision algesiometer, and Wagner^™ algometry. The CST (Figure 1A), a direct scaling measure, conformed to the technique of Bergeron et al.[4 (link)]. The CST applied a consistent, but uncalibrated, force on 12 defined points of the labia majora, minora, and lower vagina. Precise measurement of the defined anatomic sampling sites is described below. Following a consistently “light force”, manually applied by cotton swab, study subjects rated evoked pain on a numeric rating scale (NRS) ranging from 0 = “no pain” to 10 = “worst possible pain”. The Morrison algesiometer method (Figure 1B), a threshold measure using Method of Constant Stimuli, conformed to previously described method[20;25]. Probe force ranged from 176 mN to 1868 mN in 8 increments. A random staircase method was performed on four anatomic sites of the vestibule and external vulva: 5:00 external vulva and vestibule and 7:00 external vulva and vestibule. Based upon the particular paired site (5:00 or 7:00 in position) that displayed the widest threshold differential by Morrison algesiometry, the external vulvar-vestibular pair subsequently underwent threshold assessment by Wagner^™ algometry. The Wagner^™ algometer method (Wagner Instruments, Greenwich, CT) (Figure 1C), a threshold method using Method of Limits, followed the technique described by Zolnoun et al.[70 (link)] and tested the previously defined external vulvar and vestibular sites corresponding those tested by the Morrison algesiometer. Using the Wagner^™ algometer, an increasing 0.5 N per second force (range 0 to 5 N) was applied perpendicular to the mucocutaneous surface by a moistened, dacron tipped swab affixed to the Wagner^™ algometer. Force was terminated at point of pain development (signaled by hand-held clicker) or when the test reached 5 N force. Consistently increasing 0.5 N per second force change was assured by pre-test practice using MESUR^™ gauge software, (Mark-10 Corp. Copiague, NY). The threshold testing alternated between external vulvar and vestibular sites until the median value of 3 tests within each test site varied less than 10%. The interstimulus interval at each paired site approximated 2 minutes. The Wagner^™ algometer was considered the primary threshold measure for the study with the CST and the Morrison algesiometer available for comparative construct validity assessment. The identical anatomic locations of the paired sites undergoing pain threshold assessment were re-confirmed by measurement and subjected to biopsy on the day of surgery, usually 3 to 5 days following pre-operative exam. Location of sampling was confirmed by digital photography. The three selected techniques reflected different dimensions of vulvar pain assessment and thereby provided a chance to assess both the relationship of the particular pain assessment method to fibroblast behavior and the relationship to the other pain assessment methods.
As illustrated in Figures 1D–F, pain threshold testing and subsequent biopsies were performed from two sites: a) lower 1/3 of the vulvar vestibule (at 5:00 or 7:00) in close approximation to the hymen and b) the adjacent external vulva. As seen in Figure 1D, a line was created from the midpoint of the glans clitoris to center of the anal orifice with an intersection of the base of hymen to be designated as (0,0). In Figure 1E, sampling sites were measured in mm using the defined (X, Y) axes with X axis rotation tangential to the hymenal remnants. The two biopsy sites were separated by the region of color and reflectance change known as Hart’s line (dotted outer line, Figure 1D and 1E) which is considered the embryologic interface of endodermal derived (vestibule) and ectodermal derived (external vulva) tissue. Figure 1F, following measurement and marking, photographs were taken to document location of threshold testing/sampling. The distance between the center of each of the testing/sampling sites (“painful” vestibule and “pain-free” external vulva) ranged 18 to 30 mm with a mean of 27 mm. At surgery, biopsy sites corresponding to pain threshold sites, were confirmed by repeat measurement. From each of the two sites, two--6 mm biopsies were processed one sample for fibroblast strain development and one sample for in situ IHC study.
Fibroblast strains were established according to our published methods using RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), followed by subsequent passaging in Minimum Essential Medium (MEM) + 10% FBS[36 (link)]. Early passage vulvar and vestibular fibroblast strains were seeded at 5 × 10⁴ cells/well. After achievement of confluence, fibroblasts were serum-reduced for 48 h in fresh media containing 0.5% FBS. Fibroblast cellular identity was confirmed by microscopic inspection. Prior confirmation of cells as fibroblasts was performed with cell type-specific markers (vimentin positive, collagen expressing) and epithelial cell marker (cytokeratin negative), smooth muscle and myofibroblast marker (α-smooth muscle actin negative), endothelial cell marker (CD34 negative), and bone marrow derived cell marker (CD45 negative)[3 (link)]. The fibroblasts were used for analysis following early passage (4 to 7 passages). Cultures of fibroblast strains were seeded to 24-well tissue culture plates at roughly half confluence and were allowed to grow until completely confluent (~3–4 days) at 37°C and 5% CO₂ in Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS), GlutaMAX, gentamycin, and antibiotic/antimycotic solution (Gibco Life Technologies, Grand Island, NY). Once confluent, cells were transitioned to serum-reduced media (supplemented with 0.05% FBS) and incubated for 48 h.
The evening prior to infection, yeast cells were inoculated into 10 ml cultures of yeast peptone dextrose broth (YPD; Fisher Scientific) from YPD plate cultures less than two weeks old. Yeast cultures were incubated overnight at 37°C and 220 rpm for Candida albicans, Candida glabrata, and Candida tropicalis, while Saccharomyces cerevisiae was incubated at 30°C and 220 rpm. After ~18 h growth, cultures were diluted to OD₆₀₀ = 1.0 in fresh YPD broth. Inoculums were prepared by diluting these yeast cultures to ~1 × 10⁴ CFU/ml in antibiotic/antimycotic-free MEM supplemented with 0.05% FBS and GlutaMAX. Confluent fibroblast wells were then infected with 1 ml (1 × 10⁴ blastoconidia) of yeast inoculum each and incubated for 24 h at 37°C and 5% CO₂. At the same time, zymosan challenged wells were treated with 100 μg/ml zymosan (Sigma-Aldrich, St. Louis, MO), which was diluted in MEM from a 250X stock dissolved in 100% EtOH. Zymosan is a commercially available preparation of the cell wall of Saccharomyces cerevisiae, which is a mixture of beta;-glucan and mannoprotiens, both of which are highly stimulatory[28 (link)]. A corresponding vehicle control was also prepared. At the end of the experiment, supernatants were collected to determine fibroblast viability (Cytotox One kit, Promega Corp., Madison, WI) and cytokine release. Standard sandwich ELISAs were performed for IL-6 (BD Biosciences, Franklin Lakes, NJ) and competitive EIA assays were performed for PGE₂ quantification (Cayman Chemical Company, Ann Arbor, MI).
The yeast strains, C. albicans SC5314, C. glabrata BG2, C. tropicalis 20336, and S. cerevisiae, were all originally from the American Type Culture Collection (ATCC), provided from the microbiology lab of author C.H. All Candida strains selected are clinical isolates that have been sequenced and are available from the ATCC and these strains have been proven virulent in mouse models (http:/www.candidagenome.org). Although S. cerevisae is regarded as largely non-pathogenic, its cell wall shares greater that 90% similarity with known pathogens C. albicans and C. glabrata[35 (link)]. These particular strains have been selected because their genotypic and phenotypic information is available and their behaviors are comparatively predictable versus new uncharacterized clinical isolates. We also cultured the tissues in tandem, with the preparation of fibroblast strains and histological examination, tissues were processed for microbiological testing specifically attempting to isolate relevant yeast strains.
Vestibular and external vulvar tissue samples for in situ IHC microscopic study were received in additional separate containers. Tissue samples were oriented, bisected, and formalin-fixed. Paraffin-embedded tissue was cut into 5 micron sections for IHC staining. Monoclonal antibodies to IL-6 (1:1000 dilution), (Leica Biosystems, Buffalo Grove, IL), and Cox-2 (1:200 dilution), (Cell Marque, Rocklin, CA) were used during staining procedures conforming to previously published techniques[18 (link)]. During a pro-inflammatory process, like the mucocutaneous assault by pathogenic yeast, arachidonic acid product increases with enzymatic clevage of tissue lipids by cytosolic lipooxygenase A2. Arachidonic acid is, in turn, converted into intermediary Prostaglandin H₂ by Cox-2, and finally converted into PGE₂ by microsomal Prostaglandin E Synthetase (mPGES-1)[59 ]. Although PGE₂ antibody is available for IHC, Cox-2 IHC is felt to be a better and more widely published marker of inflammation. Density of fusiform-shaped fibroblasts expressing these proteins in tissue sections from both vestibule and external vulva were evaluated by counting total number of cells in 10 high power fields (HPF) at 400x magnification using an Olympus BX-41 microscope,(Olympus Corp. Tokyo, Japan). Evaluation of cell morphology and enumeration was restricted to mesenchymal areas in close proximity to the epithelium, within 50 μm of basal epithelial cell layer, to include primarily the sub-epidermal neural plexus region[63 (link)] in the evaluation. Slide identifiers were masked to the evaluator (K.S.) with respect to case/control and vestibule/external vulva.