# (-)-Fenchone Ameliorates TNBS-Induced Colitis
## 4 Materials and Methods
### TNBS-Induced Intestinal Acute Inflammation in Rats
#### 4.4.1 Histological Analysis and Morphometric Analysis  
Fragments of colon tissue from the TNBS-induced ulcerative colitis acute and recurrence-phase protocol from the cysteamine-induced ulcer protocol were preserved in a 10% buffered formaldehyde solution until histological processing. The tissues were embedded in histological paraffin and sectioned with a thickness of 4 \(\upmu\)m. From the sectioned tissues, 2 stains were performed: hematoxylin and eosin (HE) and Masson's trichrome (MT). The histological sections stained by Masson's trichrome were visualized through the 40x objective of the Olympus microscope (Tokyo, Japan) and 20 random images were digitalized through the same microscope and Q-Color3 microscope, in each image, all pixels with shades of blue (Masson's trichrome) were selected to create a binary image, digitally processed and calculate the area in \(\upmu\)m\({}^{2}\). 4.2 Determination of Reduced Glutathione (GSH), Malondialdehydedehyde (MDA), Myeloperoxidase (MPO), and Superoxide Dismutase (SOD)  
GSH Determination  
The protocol used by [PERSON] (1995) [PERSON] (1995) [PERSON] (1995) was followed to determine the GSH levels. The tissue samples were suspended in 0.02 M 1:10 (\(v/v\)) EDTA. From this homogenate, 400 \(\upmu\)L was removed, and 320 \(\upmu\)L of distilled water and 80 \(\upmu\)L of 50% trichloroacetic acid were added, being centrifuged at 3000 rpm at 4 \({}^{\circ}\)C for 15 min. Then, 100 \(\upmu\)L of the resulting supernatant was pipetted into a 96-well microplate, and 200 \(\upmu\)L of Tris and 25 \(\upmu\)L of DTNB were added. This microplate was incubated at room temperature and after 15 min, a reading on a spectrophotometer (Polaris) was performed at a wavelength of 412 nm. The calibration curve was made with reduced L-glutathione. The GSH values of the samples were calculated by interpolating the values with the standard curve and expressed in nmol GSH/mg of protein. MDA Determination  
The samples were suspended in Trizma(r) buffer (Tris HCl) 1:5 (\(w/v\)), homogenized, and centrifuged at 11,000 rpm at 4 \({}^{\circ}\)C for 10 min. Soon after, 300 \(\upmu\)L of the supernatants was transferred to Eppendorf tubes; 750 \(\upmu\)L of the chromogenic compound (10.3 mM of 1-methyl-2-phenylindol) and 225 \(\upmu\)L of hydrochloric acid (37%) were added, and incubated at 45 \({}^{\circ}\)C in a water bath for 40 min and then were centrifuged again at 11,000 rpm at 4 \({}^{\circ}\)C for 5 min. Then, 300 \(\upmu\)L of the supernatant was transferred to a 96-well microplate, and absorbance was determined by colorimetry (586 nm), using a plate reader (Polaris'spectrophotometer). The data were interpolated with the standard curve, and the results were expressed as nmol MDA/g tissue [58]. MPO Determination  
The tissue sample fragments were homogenized in the hexadecyltrimethylammonium bromide (HTAB) buffer, which has a detergent function, lysing the granules of the neutrophils that contain the myeloperoxidase. After homogenization, the material was subjected to sonication for 5 min. Then, the sample was subjected to a double freezing and thawing process to facilitate the disruption of cellular structures and, consequently, the release of the enzyme. The homogenate was centrifuged at 5,000 rpm at 4 \({}^{\circ}\)C for 20 min and concentrated for 24 h. On the following day, 7 uL of the supernatant was collected, to which 200 uL of the reading solution (o-dianisidine hydrochloride, potassium phosphate buffer, and 1% H\({}_{2}\)O\({}_{2}\)) was added. The reading was performed using a spectrophotometer at 450 nm wave, at times 0 and 1 min. The results were expressed as units of myeloperoxidase per gram of tissue [59]. SOD Determination  
The tissue samples were homogenized in phosphate buffer (0.4 M, pH 7.0) and centrifuged for 15 min at 10,000 rpm at 4 \({}^{\circ}\)C. The supernatant was removed and used in the assay.