The images were analyzed by the image analysis program Optimas 6.5 (Optimas, Bothell, WA).
The diameters of nerve fascicles were measured with the Optimas 6 Image Analysis Suite (Optimas Corp).
OGF (10 mg/kg), the opioid antagonist naltrexone (NTX; 30 mg/kg), or saline solution (0.2 mL) was injected intraperitoneally daily for 28 days into the rats, and restenosis of the carotid artery was examined with morphometric analysis using Optimas software.
Images were subsequently obtained at 10-min intervals and analyzed using a motion analysis package (Optimas 6) (Optimas Corporation, Bothell, Washington, USA).
The gel was then stained overnight (0.5 μg ml−1 ethidium bromide), destained overnight in ddH2O and CCD camera (Sony XC-75CE) equipped with an image analysis system (Optimas, Silverspring, MD, USA) was used to quantify the FDR.
In these images, each vessel was marked and the mean density of perfused vessels, the mean distance between neighbouring vessels and the area of vascular domains were calculated using image analysis software (Optimas Ver. 6.2, Media Cybernetics, Silver Spring, MD, USA).
Autoradiographs were quantified using an image analysis optical software (Optimas 5.2, Optimas Corporation, Seattle, WA).
Inductively coupled plasma atomic emission spectrometer [ICP-AES] analysis utilizing PerkinElmer OPTIMA 4300 DV (PerkinElmer, Waltham, MA, USA) was performed to confirm the compositions of the obtained materials.
Alternate optima analysis was conducted as described in [ 23], using a mixed integer linear programming algorithm that is a modification of that previously used in [ 22], which prevents revisiting of already identified optimal solutions.
Fluxes of interest used in our alternate optima analysis were restricted to those observed to be involved in redox balancing or for which we had experimental measurements for comparison (RXN1205, RXN0222, RXN0109, RXN1427, RXN1308, RXN1425, RXN1441, RXN1121, and RXN0681 - see Additional File 2 - Table S1 contains reaction details).
