Animals and induction of diabetes mellitus
A total of 38 adult male Fischer 344 rats (300 g, 16 week-old, Charles River, MA) were studied. Diabetes mellitus was induced with a single tail vein injection of STZ (Sigma-Aldrich Chemicals, St. Louis, MO, 35 mg/kg; Week 0, baseline). The control (CON) group received an equal volume of vehicle (0.1 mol/l citrate buffer, pH 4.5). Both groups received standard rat chow and water ad libitum. Animals were weighed at weekly intervals, and blood glucose levels were measured using the Freestyle glucose monitoring system (Therasense®, Alameda, CA). One week following STZ injection, all animals developed hyperglycemia with glucose values exceeding 300 mg/dL (STZ-DM group). Diabetic animals were monitored for polydipsia, polyuria, and weight loss. A onetime high dose of STZ in adult animals kills a proportion of pancreatic beta cells rendering the animal hypoinsulinemic and hyperglycemic but with sufficient residual insulin for short-term survival. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (National Institutes of Health, Publication No. 85–23, revised 1996) and approved by the University of Illinois at Chicago Institutional Animal Care and Use Committee.
Development of sensory neuropathy was measured by two methods in CON (n = 12) and STZ-DM animals (n = 13). Animals were tested for behavioral responses to noxious thermal stimuli using the Hargreaves method  and to non-noxious sensory stimuli using flexible Von Frey filaments . Both tests were performed before injections and weekly thereafter through Week 8. Animals were placed in individual cages, and behavioral accommodation was allowed until there was cessation of cage exploration and major grooming activities. Stimuli were presented to mid-plantar sections of both hind paws. Briefly, for Von Frey methods a floorless fiberglass box (18 × 8 ×8 cm) was placed on top of an elevated mesh floor for paw access. Von Frey threshold was calculated using the up-and-down method of Dixon . For the Hargreaves method, rats were placed under an inverted clear fiberglass box (18 × 8 × 8 cm) on top of an elevated glass floor and allowed to acclimate. The radiant heat source (Ugo Basile Biological Research Apparatus Model 737150, Comerio, Italy) was positioned under the glass floor directly beneath the hind paw, and the thermal latency was measured in seconds.
All echocardiograms were performed as previously described by our laboratory and according to the American Society of Echocardiography guidelines . Echocardiograms were performed in CON (n = 6) and STZ-DM animals (n = 7) at 4, 8, and 12 weeks by the same experienced sonographer using the Sequoia C256 Echocardiography System (Acuson Corporation, Mountain View, CA) and a 15.0 MHz transducer. Before the procedure, animals were anesthetized with an initial dose of methoxyflurane, and sedation was maintained thereafter via intubation with 1% isoflurane, using a Harvard small-animal ventilator (respiratory rate 80 breaths per minute, respiratory volume 2.5 ml). Body temperature was maintained at 37°C using a warming plate perfused with a water circulating bath. The transducer was placed on the left thorax, and M-mode and 2-dimensional echocardiography images were obtained in the papillary muscle level.
The measurements listed were obtained after a well-defined, continuous interface of the anterior and posterior walls was visualized. All parameters were measured with electronic calipers, and mean calculations were obtained from three or more consecutive cardiac cycles. After good visualization, left ventricular end-diastolic dimension (LVDD), left ventricular end-systolic dimension (LVSD) and interventricular septal thickness in diastole (IVSD) and in systole (IVSS) were measured by the leading edge method . The former two parameters reflect intraventricular volume and therefore dilation of the left ventricle and IVSD and IVSS, reflect the wall thickness of the septal wall during diastole and systole. In all animals, three to four beats were recorded using the same transducer position. Fractional shortening (FS) and relative wall thickness (RWT) were calculated as previously described . LV mass was calculated using the following equation: LV mass = 1.04 × [(LVDD + posterior wall thickness + IVSD)3 - LVDD3]. Using pulse-wave Doppler echocardiography, we also measured signals from the left ventricular inflow and outflow track and obtained indices of diastolic function. These included isovolumic relaxation time (IVRT), which reflects the time between the closure of the aortic valve and opening of the mitral value, deceleration time (DecT), and E/A ratio, in which the E wave represents early rapid filling of the ventricle and the A wave represents late filling of the ventricle. Mean values were used for statistical analysis.
Urine and myocardial norepinephrine levels
At 8 weeks, in a subgroup of CON (n = 5) and STZ-DM (n = 6), we measured urinary and myocardial NE levels. Twenty-four hour urine samples were collected for the measurement of NE and creatinine . The total 24 h volume was measured, and 1-ml aliquots (containing 6 mol/l hydrochloride) were stored at -20°C until assay. Norepinephrine was measured by enzyme-linked immunosorbent assay (Rocky Mountain Diagnostics, Colorado Springs, CO). All samples were run in duplicate and normalized to urinary creatinine to correct for urine volume . Norepinephrine levels were expressed as mmol/l 24 h NE/creatinine.
Following the 24 hour urine collection, animals were anesthetized with CO2 and then euthanized by cervical dislocation. Hearts were excised, rinsed in phosphate buffered solution, weighed and snap frozen in liquid nitrogen and stored at -70°C. Catecholamine levels in biological samples can change over time due to auto-oxidation, exposure to light, air oxidation and/or exposure to high pH and temperature . Therefore as noted below certain procedures/techniques were used when handling, storing and preparing samples to avoid degradation of NE. Frozen myocardial tissue samples were pulverized in liquid nitrogen and homogenized in glass homogenizers to minimize oxidation from metal homogenizers on ice in buffer (2 ml/g tissue) containing 0.1 N perchloric acid and 0.02 mmol/l EDTA. To control for oxidation of NE two homogenization buffers were tested, with and without sodium metabisulfite (see Additional file 1). Sodium metabisulfite, a commonly used antioxidant, quenched the NE signal and therefore was not used for the homogenization of samples. Homogenates were centrifuged (15,000 g, 20 min, 4°C), and the resulting supernatants were used for NE extraction and aliquots stored at -80°C. As previously described, samples were analyzed using a high-performance liquid chromatograph (Agilent 1200 series high performance liquid chromatography system, Santa Clara, CA) and Agilent Zorbax Rx-C8 (150 × 2.1 mm I.D., 5 μm) column, along with a pre-column filter (used in the isocratic, reverse-phase, ion-paring mode) coupled with tandem mass spectrometry detection . The mobile phase consisted of water–methanol-heptafluorobutyric acid (85/15/.13, V/V/V), with a flow rate of 0.2 ml min-1. The entire chromatographic effluent (10 μl) was passed through the mass spectrometer interface for subsequent detection. Under these conditions, NE retention time was about 4.4 min resulting in a total time (injection-to-injection) of 6 min. Deuterium-NE (d-NE) (Fisher #NC9750934, Pittsburgh, PA) was used as an internal standard. NE/d-NE area ratios were determined for the SRM chromatographic peaks using Agilent’s MassHunter software. Calibration curves were generated by plotting peak area ratios (NE/d-NE) obtained from working standard versus NE concentrations and fitting these data to a weighted (1/x) linear regression curve.
Evaluation of neuronal markers
The myocardial neuronal markers calcitonin gene-related peptide (CGRP) and PGP9.5 were also evaluated at 8 weeks in a separate subgroup of CON (n = 3) and STZ-DM (n = 4) animals. After euthanasia as described above, hearts were removed and pulverized with a ceramic grinder under liquid nitrogen and homogenized on ice in cold buffer (10 ml/g tissue) containing: 50 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.4), 5 mmol/l ethylenediaminetetraacetic acid, 150 mmol/l NaCl, 1% Triton X-100, 1X phosphatase inhibitor cocktail set II (Calbiochem #524625, Billerica, MA), 1X protease inhibitor cocktail (Sigma #P8340, St. Louis, MO), and 50 nmol/l okadaic acid. Homogenates were centrifuged at 14,000 rpm for 15 min at 4°C, and supernatants were used for blots. A small aliquot was reserved for protein quantification by bicinchoninic acid (Pierce #23225, Thermo Scientific, Hanover Park, IL). Supernatants with sample buffer were loaded onto a 4-12% Bis/Tris gradient gel (Invitrogen, Grand Island, NY) and separated under reducing conditions. Samples were incubated overnight at 4°C with primary antibodies against calcitonin gene-related peptide (CGRP, Millipore AB15360, Billerica, MA, 1:5,000), protein gene product 9.5 (PGP 9.5, Millipore AB1761, Billerica, MA, 1:2,000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abcam 341048, Cambridge, MA, 1:5,000). Corresponding secondary antibodies conjugated with horseradish peroxidase enzyme goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA, #111-035-045) and goat anti-mouse (Jackson ImmunoResearch, West Grove, PA, #115-035-044) were added to samples, and membranes were washed of unbound secondary antibodies, followed by incubation with peroxidase substrate for enhanced chemiluminescence and detection by film processor. Using Image J software (National Institutes of Health), optical densitometry was calculated and normalized to GAPDH.
All data are expressed as mean ± SEM. Normally distributed data (body weight, glucose measurements, Hargreaves, and echocardiograms) were compared using two-way repeated measures analysis of variance (RM-ANOVA), followed by the Holm-Sidak method for multiple comparison. Student's t test was used for two-group parametric comparisons (urinary and myocardial NE, Immunoblots). The Mann–Whitney test was used to compare non-parametric data at each interval (von Frey filaments). A p value less than 0.05 was considered significant. Statistical analyses were performed using Sigmaplot (Systat Software, San Jose, CA).