AN 12

The electrical resistance of a cellular monolayer, measured in ohms, is a quantitative measure of the barrier integrity.^{26 (link)} The classical setup for measurement of TEER, as shown in Figure 1, consists of a cellular monolayer cultured on a semipermeable filter insert which defines a partition for apical (or upper) and basolateral (or lower) compartments. For electrical measurements, two electrodes are used, with one electrode placed in the upper compartment and the other in the lower compartment and the electrodes are separated by the cellular monolayer. In theory, the ohmic resistance can be determined by applying a direct current (DC) voltage to the electrodes and measuring the resulting current. The ohmic resistance is calculated based on Ohm's law as the ratio of the voltage and current. However, DC currents can damage both the cells and the electrodes. To overcome this issue, an alternating current (AC) voltage signal with a square waveform is applied. In the widely used and commercially available TEER measurement system known as an Epithelial Voltohmmeter (EVOM),²⁷ an AC square wave at a frequency of 12.5 Hz is used to avoid any charging effects on the electrodes and the cell layer. The EVOM system has a measurement range of 1-9,999 Ω with a 1Ω resolution and uses a pair of electrodes popularly known as a STX2/“chopstick” electrode pair. Each stick of the electrode pair (4 mm wide and 1 mm thick) contains a silver/silver chloride pellet for measuring voltage and a silver electrode for passing current. The measurement procedure includes measuring the blank resistance (R_BLANK) of the semipermeable membrane only (without cells) and measuring the resistance across the cell layer on the semipermeable membrane (R_TOTAL) The cell specific resistance (R_TISSUE) in units of Ω, can be obtained as:
Where resistance is inversely proportional^{28 (link)} to the effective area of the semipermeable membrane (M_AREA) which is reported in units of cm².
TEER values are typically^{28 (link)-29} reported (TEER_REPORTED) in units of Ω.cm² and calculated as:
The TEER readings with EVOM2 are highly dependent on the electrode positions and a careful handling of the electrodes is required while introducing them into the well under test to avoid any disturbance to the cells. The uniformity of the current density generated by the electrodes across the cell layer has a significant effect on the TEER measurements. The STX2/chopstick electrode cannot deliver a uniform current density over a relatively large membrane,²⁷ such as the ones used in 24 mm diameter tissue culture inserts, and leads to an overestimation of the TEER value. As an alternative to STX2/chopstick electrodes, an EndOhm chamber³⁰ which allows the cups from culture wells to be inserted can be used. In an EndOhm chamber, both the chamber and the cap contain a pair of concentric electrodes: a voltage-sensing silver/silver chloride pellet in the center plus an annular current electrode. The symmetrical arrangement of circular disc electrodes on both sides of the membrane in an EndOhm chamber generates a more uniform current density across the membrane when compared to STX2/chopstick electrodes. Also, with Endohm's fixed electrode geometry, variation of measurements on a given sample is reduced³⁰ to 1-2 Ω when compared to 10-30 Ω with STX2 electrodes. Apart from the EVOM/STX2/EndOhm systems, a few of the other commercial systems available for TEER measurements include Electric Cell-Substrate Impedance Sensing (ECIS)^{31 (link)} (Applied BioPhysics Inc., Troy, NY), REMS AutoSampler (World Precision Instruments, Sarasota, FL), Millicell-ERS system^{10 (link), 32 (link)} (Millipore Corp., Bedford, USA) and Ussing Chamber Systems^{33 (link)} (Warner Instruments, Hamden, CT).

Whole-genome fragment libraries were prepared using a modification of Illumina’s genomic DNA sample preparation kit. Briefly, 3 μg of human genomic DNA (Coriell) was sheared for 4 min. on a Covaris E210 instrument set to duty cycle 5, intensity 5 and 200 cycles per burst. The mode of the resulting fragment-size distribution was ~250 bp. End repair, non-templated addition of a 3′-A, adapter ligation and reaction clean-up followed the kit protocol except that we used a generic adapter for libraries destined for shotgun sequencing after hybrid selection. This adapter consisted of oligonucleotides C (5′-TGTAACATCACAGCATCACCGCCATCAGTCxT-3′ with “x” denoting a phosphorothioate bond resistant to excision by 3′-5′ exonucleases) and D (5′-[PHOS]GACTGATGGCGCACTACGACACTACAATGT-3′). The ligation products were cleaned up (Qiagen) and size-selected on a 4% NuSieve 3:1 agarose gel followed by QIAquick gel extraction. A standard prep starting with 3 μg of genomic DNA yielded ~500 ng of size selected material with genomic inserts ranging from ~200 to ~350 bp, i.e., enough for one hybrid selection. To increase the yield we typically amplified an aliquot by 12 cycles of PCR in Phusion High-Fidelity PCR master mix with HF buffer (NEB) using Illumina PCR primers 1.1 and 2.1, or, for libraries with generic adapters, oligonucleotides C and E (5′-ACATTGTAGTGTCGTAGTGCGCCATCAGTCxT-3′) as primers. After QIAquick clean-up, if necessary, fragment libraries were concentrated in a vacuum microfuge to 250 ng per μl before hybrid selection.

The Clinformatics™ Data Mart captures administrative health claims across the United States for members of a large national managed care company affiliated with OptumInsight (Eden Prairie, MN). We examined claims from January 1, 2012 to June 30, 2015 among adults ages 18 to 64 to capture surgical procedures performed between 2013 and 2014 to account for the 12-month preoperative and 6-month postoperative study period. We included only individuals with continuous medical and prescription drug coverage to evaluate the complete health care experience. We excluded patients ages 18 and younger, as well as patients older than 64 years due to incomplete capture of Medicare Part D prescriptions claims data. The study was deemed exempt from review by the University of Michigan Institutional Review Board.
We selected 13 common elective surgical procedures, and categorized these into minor and major groups based on prior literature. Minor surgical procedures included varicose vein removal, laparoscopic cholecystectomy, laparoscopic appendectomy, hemorrhoidectomy, thyroidectomy, transurethral prostate surgeries, and parathyroidectomy. Major surgical procedures included ventral incisional hernia repair, colectomy, reflux surgery, bariatric surgery, and hysterectomy. We identified patients undergoing surgery using Current Procedural Terminology (CPT) or International Statistical Classification of Diseases and Related Health Problems (ICD9_ procedure codes (Supplemental Table 1).
We sought to determine new persistent opioid use after surgery, and included only patients who filled an opioid prescription fill either in the month prior to surgery or within two weeks after discharge. Comparable to previous studies of opioid naïve surgical populations,^{7 (link),8} patients who had filled one or more prescriptions for opioids 12 months to 31 days prior to their surgical procedure were excluded from the analysis (Figure 1). To account for prescriptions provided preoperatively for postoperative pain control, patients filling opioids in the 30 days prior to surgery were included, and prescriptions filled in this time was included as a covariate in the analyses. Lastly, we excluded patients who underwent additional surgical procedures during the study period using subsequent procedural codes for anesthesia in the 6-month postoperative period.
As a comparison cohort of patients who did not undergo surgery, we identified a random 10% sample patients ages 18 to 64 years of age who did not undergo surgery in the study period We included only patients in the nonoperative group who did not fill an opioid prescription during a 12 month period and did not have any codes for surgical procedures or anesthesia during this period. These patients were then given a random date of surgery. No patients had an opioid fill in the year prior to their fictitious surgery date nor did they have any anesthesia codes in the 6 months following their fictitious surgery date.