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Tissue preparation for assays                       Total protein
          Tissue preparation for hydroxyproline estimation    The total protein  content of the  wound tissue
          The granulation tissue specimens were dried at 60°C for   homogenate  was determined  according to the method
                                                                           [19]
          24 h and then were weighed and kept in glass‑stoppered   of Lowry  et  al.   Absorbance  was  measured  at  540  nm
          test  tubes.  6N HCl was added to each tube  for a total   and expressed  in  mg/g  of tissue.  Standards were  treated
          of 40  mg  of dried granulation tissue  per ml  of acid.   similarly using BSA at concentrations of 0, 20, 40, 60, 80,
          The  tubes  were  kept  in  boiling  water  bath  for a  total   and 100 µg/mL in 0.1 M phosphate buffer at pH 7.4.
          of 24  h (12  h  and each for 2  days) for hydrolysis.  The   Matrix metalloproteinase‑2 and ‑9
          hydrolysate was then  cooled, and the  excess acid was   A total of 100  mg of tissue was homogenized in 1 mL
          neutralized by 10N sodium hydroxide  (NaOH) using   of  ice‑cold  lysis  buffer.  Subsequently,  homogenates  were
          phenolphthalein/methyl red as an indicator. The volume   centrifuged at 3,000 g for 5 min at 4°C, and supernatants
          of neutral hydrolysate was diluted with distilled water to   were stored at  ‑80°C until use. MMP‑2 and MMP‑9 were
          a concentration of 20  mg/mL  of dried granulation tissue   measured using prefabricated ELISA kits, according to
          in the final hydrolysate. The hydrolysate was used to   manufacturer protocol (R and D Systems, USCN Life Science
          estimate the level of hydroxyproline. [18]          Inc., USA). Plates were read at 450 nm and 540 nm, and

          Tissue preparation for estimation of antioxidants and   concentrations were calculated using a 4‑point standard
                                                              curve and expressed as ng/mg of protein.
                                                                                                 [20]
          oxidative biomarkers
          The wet weight of the granulation tissue  samples was   Nitric oxide estimation
          noted and homogenized by a Rotex homogenizer  in    The level of NO was estimated as NO , an NO metabolite
                                                                                              −
                                                                                              2
          ice‑cold 0.2 M phosphate buffer  (pH  7.4). Homogenates   in tissue samples. NO is a highly reactive free radical gas
          were centrifuged at 15,000  rpm for 30  min in a    which is a ready oxidizer and remains stored in tissues as
                                                                                  −
          cooling centrifuge, and the supernatant was then used   nitrates  (NO )  or  NO .  Thus,  NO  concentration can be
                                                                         −
                                                                                 2
                                                                         3
                                                                                                              −
                                                                                                     −
          to determine  total protein,  GSH,  and an oxidative   estimated by measuring concentrations of NO  and NO
                                                                                                     3
                                                                                                             2
          biomarker (MDA).                                    in  combination.  The  simplest  technique  is  to  monitor
                                                              the  reduction of NO  to NO  by  nitrate  reductase or
                                                                                 −
                                                                                         −
          For the MMP‑2 assay, the granulation tissue was rinsed   a metallic catalyst, followed  by the colorimetric Griess
                                                                                        2
                                                                                3
          in ice‑old phosphate buffer saline  (PBS)  (0.1 mol/L,   reaction to measure NO  levels (nitrite levels).
                                                                                   −
          pH  7.0‑7.2) to remove all traces of excess blood and                   2
                                                                        −
          weighed prior to homogenization. The tissues were   Sample NO  levels were estimated by a colorimetric assay
                                                                       2
          minced into small pieces and homogenized on ice in   which is  based on the Griess  reaction. Using a two‑step
                                                                                           −
          5  mL of PBS. The resulting suspension was sonicated   diazotization reaction, acidified NO  produces a nitrosating
                                                                                           2
          with an ultrasonic cell disrupter or subjected to two   agent  which  reacts  with  sulfa  nitric  acid  to  produce  the
          freeze‑thaw cycles to further break the cell membranes.   diazonium ion. This ion is then coupled to N‑(1‑naphthyl)
          The homogenates were then centrifuged for 5  min at   ethylenediamine dihydrochloride to form a red azo derivative
          50  g, removing the supernatant and aliquot store at   which  is  measured  at  550  nm  using  a  spectrophotometer.
                                                                                    −
          no more than ‑80°C.                                 The concentrations of NO  are calculated from a standard
                                                                                    2
                                                              curve established with serial dilutions of sodium NO .
                                                                                                         − [21]
          For the NO assay, the tissue was homogenized  in an                                            2
          isotonic  solution  of PBS  containing  10 mM  of NEM   Glutathione estimation
          and 2.5 mmol ethylenediaminetetraacetic  acid  (EDTA).   The  reaction  mixture  was  prepared  by  adding 80  µL  of
          The  addition  of NEM/EDTA  blocks the  SH‑groups   tissue supernatant to 0.9 mL of 0.2 M sodium phosphate
          while inhibiting the transition  of metal‑catalyzed   buffer of pH  7.00  and 20  µL of 10 mM 5.5’‑dithiobis
          trans‑nitrosation reactions, preventing artificial nitrosation   (2‑nitrobenzoic acid) (DTNB) solution.  The yellow‑colored
          and thiolate‑ and ascorbate‑mediated degradation of   substance formed by the reaction of GSH and DTNB
          endogenous Nitrosothiols and nitrite  (NO ). The protein   was measured at 412  nm. The results were expressed as
                                              −
                                             2
          concentration was determined according to Lowry et al.    GSH µmol/mg protein. [22]
                                                         [19]
          using purified BSA as the standard.                 Malondialdehyde
          Estimation of biochemical parameters                The MDA levels of wound tissue  homogenate  were
                                                                                                           [23]
          Hydroxyproline                                      measured according to the method of Ohkawa et al.  To
          The  dried  tissue  was  added  to  10  mL  of  6N  HCl   0.1 mL of the test sample, 1 mL of 10% TCA and 1 mL of
          and stored at 110°C for 24  h. The neutralized acid   0.67% TBA were added and then heated in a boiling water
          hydrolysate of the dry tissue was used to determine   bath at 100°C for 30 min. The mixture was cooled under
                                                              tap water and centrifuged at 12,000 rpm for 10 min. Clear
          the level of hydroxyproline. The reaction mixture   supernatant was determined by the absorbance at 535 nm
          contains  0.05  M  copper  sulfate,  2.5N  NaOH,  6%  H O ,   and expressed as nmol/mg protein.
                                                        2
                                                          2
          3N sulfuric acid, and 5% p‑dimethylaminobenzaldehyde
          using L‑hydroxyproline as the standard. Absorbance was   Statistical analysis
          measured  at  540  nm  and  expressed  as  µg/mg  of  dry   Statistical analysis for biochemical parameters was
          tissue weight. [18]                                 performed by Student’s t‑test, and data were expressed as
           268                                                           Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015
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