In Vivo Immuno-Spin Trapping: Imaging the Footprints of Oxidative Stress

Abstract

A plethora of disease processes are associated with elevated reactive species formation and allied reactions with biomolecules that alter cell signaling, induce overt damage, and promote dysfunction of tissues. Unfortunately, effective detection of reactive species in tissues is wrought with issues that significantly limit capacity for validating species identity, establishing accurate concentrations, and identifying anatomic sites of production. These shortcomings reveal the pressing need for new approaches to more precisely assess reactive species generation in vivo. Herein, we describe an in vivo immuno-spin trapping method for indirectly assessing oxidant levels by detecting free radicals resulting from reaction of oxidants with biomolecules to form stable, immunologically detectable nitrone-biomolecular adducts. This process couples the reactivity and sensitivity of an electron paramagnetic resonance spin trap with the resolution of confocal imaging to visualize the extent of cell and tissue oxidation and anatomic sites of production by detecting resultant free radical formation.

INTRODUCTION

Numerous pathologies are allied to enhanced rates of reactive species formation. Reactive species are defined as highly biorelevant free radical and nonfree radical molecules that mediate redox reactions with surrounding biomolecules. These molecules include but are not limited to reactive oxygen species (ROS), which include hydrogen peroxide (H₂O₂), superoxide (O₂^•−), hydroxyl radical (^•OH), and singlet oxygen (¹O₂), as well as reactive nitrogen species (RNS), e.g., nitric oxide (^•NO), nitrogen dioxide (NO₂^•), and peroxynitrite (O=NOO⁻). Under disease states, these reactive species often initiate free radical formation on cellular proteins and lipids that, in turn, propagate further free radical events with neighboring molecules, ultimately resulting in abnormal signal transduction and cellular, tissue, and organ dysfunction. As such, a more incisive understanding regarding the timing of onset, extents, and anatomic localization of tissue free radical generation would facilitate the design of mechanistically targeted treatment strategies.

Assessment of free radical formation in tissues is routinely accomplished by measuring the accumulation of more stable secondary byproducts of redox reactions and/or the reduction in concentration of small molecule antioxidants such as glutathione (GSH). However, there are substantive shortcomings associated with these methods. First, the necessity of homogenizing or freezing and then sectioning samples before adding probes precludes the ability to identify anatomic sites of production. Second, the identity of the instigating species is often ambiguous because of promiscuous reactions of indicator molecules. Third, artificial amplification of signals are induced by redox cycling of indicator molecules (Tarpey and Fridovich, 2001; Tarpey et al., 2004; Kalyanaraman et al., 2012). Together, these shortcomings affirm the need for novel techniques to more precisely assess free radical formation in living tissues.

Coupling the affinity of the electron paramagnetic resonance (EPR) spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for protein and lipid free radicals with a polyclonal antibody against DMPO-octanoic acid, immuno-spin trapping (IST) allows for the visualization of DMPO-adducted molecules (Mason, 2004). The presence of DMPO-adducts in biological samples is not only indicative of the extent of reactive species generation (oxidative stress) but affords the capacity to visualize anatomic sites of production. The Basic Protocol describes the assessment of oxidative stress by detection of stable DMPO-nitrone adducts in vivo. The Alternate Protocol provides an additional method to quantitatively determine the amount of protein-DMPO-nitrone adducts by western blot (immunoblot) analysis.

BASIC PROTOCOL

IN VIVO IMMUNO-SPIN TRAPPING (IST)

In vivo IST is used to assess the overall extent of oxidative stress by detecting free radicals on proteins and lipids initially generated by reaction with reactive species. This process involves high-affinity reaction of free radicals with the nitrone EPR spin trap DMPO. For example, the rate constant for DMPO with carbon-centered radicals (C•) and many other biomolecular free radicals is ~10⁷ M⁻¹ s⁻¹, an affinity that allows DMPO to compete for reaction and covalent bond formation before a peroxide (COOH) is established by reaction with O₂ (K_oxygen/C• = ~10⁹ M⁻¹ s⁻¹; Mason, 2004; Chatterjee et al., 2009; see Fig. 12.42.1). The competition for reaction between DMPO and O₂ is further skewed in favor of DMPO by (1) diminished O₂ levels in inflamed, hypoxic tissues and (2) amplification of DMPO concentration. However, the key process for successful imaging is the formation of a covalent bond between DMPO and the biomolecule that serves to capture DMPO in the tissue, thus making it available for anti-DMPO antibody adduction (Fig. 12.42.1). It is also important to note that reaction of DMPO with reactive species, such as O₂^•− or ^•OH will form DMPO-OOH or DMPO-OH, respectively, which (1) does not result in subsequent adduction to biomolecules, (2) will be removed by perfusion and washes, and (3) therefore, will not be detected by the primary antibody.

Figure 12.42.1.

Immuno-spin trapping of biomolecular free radicals in pulmonary tissue of obese mice. (Top) The reactions involved in the immuno-spin trapping (IST) process. (Bottom) A representative image of an airway and surrounding tissue that was generated by exposing a diet-induced obese mouse (C57Blk/6 J, subjected to a diet consisting of 60% calories derived from fat for 20 weeks) to 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The presence of abundant DMPOadducted biomolecules (red) is seen in the pseudostratified ciliated columnar epithelial cells of the airway as well as the basolateral space, alveoli, and interstitium. This is indicative of elevated reactive species generation in these anatomic sites. This experiment was conducted with the approval of the University of Pittsburgh IACUC (protocol # 1009934 A). Red, DMPO; Blue, DAPI; Green, actin; 2°, secondary antibody.

In vivo IST is accomplished by exposure to and delivery of DMPO to the tissues to significantly elevate its local concentration. This is routinely achieved by injecting high doses of DMPO over a 24-hr time frame. Following the DMPO injection procedure, mice are sacrificed and perfused, and organs are harvested, fixed, sectioned, and probed with an anti-DMPO antibody. DMPO-biomolecular adducts are visualized by confocal microscopy, as described in detail below. However, it is very important to note that this protocol has been optimized for detecting tissue free radical formation associated with chronic, low-grade inflammation induced by obesity and, as such, care should be taken when adapting this method for alternative models where oxidative stress is more acute. See Commentary for additional considerations.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Materials

• Laboratory mice with diet-induced obesity (Khoo et al., 2012)

• 5,5-dimethyl-1-pyrroline N-oxide (DMPO) solution (see recipe)

• Phosphate-buffered saline (PBS; APPENDIX 2A)

• Liquid nitrogen

• 2% (v/v) paraformaldehyde (PFA)/PBS: dilute 4% (v/v) PFA (Santa Cruz Biotechnology, cat. no. SC-281692) with an equal volume of PBS

• 30% (w/v) sucrose (Sigma, cat. no. S7903)

• 2-methylbutane (Fisher Cat No. O3551-4)

• PBB: 20% (v/v) serum (from the species in which secondary antibodies are made)/0.5% BSA (v/v)/PBS

• Anti-DMPO polyclonal antibody (ALX-210-530-R050, Enzo Life Sciences)

• Secondary antibody: Alexa Fluor 488 anti-rabbit IgG (Life Technologies) Hoechst stain: 1 mg Hoechst stain (Sigma, cat. no. B-2883)/100 ml water Gelvatol (see recipe)

• Test tubes large enough for fixing and embedding samples, e.g., 15-ml conical, polystyrene or glass test tubes (Fisher)

• 250-ml plastic beaker

• Ice bucket, Styrofoam box, or equivalent

• Filter paper

• Forceps, small with blunt tips

• Cryovials, cryopreservation bags, or pasteboard sliding boxes (EMS; http://www.emsdiasum.com) of appropriate size

• Glass microscope slides (e.g., Fisher brand Superfrost Plus, Fisher Cat. No.12-550-15) and cover glasses

• Slide box

• Additional reagents and equipment for injecting and euthanizing mice (Donovan and Brown, 2006) and performing cryosectioning (Watkins, 2009a; UNIT 12.15), immunohistochemistry (Watkins, 2009b; UNIT 12.16), and scanning confocal laser microscopy (Smith, 2011)

Inject DMPO into mice and prepare samples

• 1
  Inject mice intraperitoneally with a total of 1.5 g/kg DMPO in three 0.5 g/kg doses at 18, 12, and 6 hr before sacrifice.

  • This approach has been optimized to afford maximum accumulation of DMPO-adducted biomolecules in tissue under a condition of chronic low-grade inflammation associated with obesity (Khoo et al., 2012). This injection protocol and dosing can and should be adapted to each individual model, especially when the oxidative insult is acute and not chronic.
• 2At sacrifice, perfuse organs and tissues with PBS (pH 7.4), in test tubes large enough to submerge them, to remove blood.
• 3aTo prepare organs and tissues for western blot (immunoblot) analysis: Remove them from the PBS and freeze in liquid nitrogen. Store indefinitely at −80°C. Proceed with western blot analysis, as in the Alternate Protocol.
• 3bTo fix organs and tissues for immunohistochemical analysis: Go to step 4

Fix and imbed samples

For additional details on fixation, cryosectioning, and general immunostaining guidelines see Watkins, 2009a,b (UNITS 12.15 and 12.16).

• 4
  Completely submerge the organs or tissues in 2% PFA for 1 to 2 hr.

  • NOTE: Larger tissues (e.g., a lobe of liver) require 2 hr in 2% PFA. For a small tissue (e.g., a mouse lymph node) or hollow organs (e.g., rodent intestines), 1 hr is sufficient.
• 5Completely submerge the fixed organs or tissues in 30% sucrose for 24 hr, changing the sucrose solution two to three times within the 24-hr time period.
• 6
  Place a plastic beaker containing 2-methylbutane in an ice bucket, Styrofoam box, or equivalent container containing a few inches of liquid nitrogen. Cool the beaker for several minutes.

  • NOTE: The beaker with 2-methylbutane is to remain in the ice bucket submerged in the liquid nitrogen during the entire freezing protocol.
• 7
  Place two small pieces of filter paper in between forceps to pick up fixed samples.

  • The paper is dry and the tissue is damp, so it adheres. The tissue often freezes to the forceps if picked up directly.
  • Alternatively, fixed tissue samples can be embedded in optimum cutting temperature (OCT) compound in a small cassette (see Watkins, 2009a).
• 8Pick up the sample, and completely immerse it in the cooled 2-methylbutane for 30 sec.
• 9Remove the sample from the beaker, and immerse it directly in the liquid nitrogen for an additional 10 sec.
• 10Immerse a properly labeled cryovial or cryopreservation bag in the liquid nitrogen for a few seconds to cool the container to the same temperature as the tissue.
• 11
  Place the sample into the cryovial or bag, and immerse the container with the sample in liquid nitrogen for 10 sec, and place it into −80°C storage.

  • NOTE: If there is any wait time before sectioning samples (e.g., transfer of samples between labs), the samples should be kept on dry ice to avoid thawing.

Section samples

• 12Obtain cryostat sections (~6 to 7 microns), and place them onto glass slides. Keep the slides at 20°C until ready for use.
• 13
  Rehydrate the tissue sections with two 5-min (minimum) washes of PBS, decanting or aspirating and replacing the solution.

  • Depending on the protein or cell type of interest (e.g., dense tissue like brain), detergent permeabilization may be necessary (10 min in 0.1% Triton X-100/PBS (see Watkins, 2009a,b).

Block samples and treat with antibodies

• 14
  Block the sections in PBB with the serum component from the species in which the secondary antibodies are made.

  • For example, if the secondary antibody is made in goat, use 20% goat serum. Likewise, if the secondary is made in donkey, use 20% donkey serum.
• 15Wash the sections five times with PBB for a minimum of 5 min each time.
• 16
  Dilute the primary antibody to the desired concentration in PBB. Vortex gently, and centrifuge 5 min at 2300 × g, 4°C.

  • It is very important to vortex and centrifuge the solution to remove any aggregates.
• 17
  Gently drop 70 to 100 μl of anti-DMPO antibody solution over the section, so the section is completely covered. Incubate 60 min at room temperature.

  • Different primary antibodies can be added together, but the host in which they are generated must be from different species, unless the primary antibodies are directly conjugated with the fluorophore.
• 18Wash the sections five times with PBB for a minimum of 5 min each time, after the addition of each antibody.
• 19Dilute the secondary antibody to the desired concentration in PBB. Vortex gently, and centrifuge 5 min at 2300 × g, 4°C, to get rid of aggregates.
• 20
  Gently drop 70 to 100 μl of antibody solution over the section, so the section is completely covered. Incubate 60 min at room temperature.

  • The secondary antibody step should be kept to within an hour.
  • You may add fluor-conjugated phalloidin (for F-actin counterstaining) to the secondary solution.
  • Secondary antibodies can be added together if using more than one, but they must be from different species.
• 21Wash the sections five times with PBS for a minimum of 5 min each time to remove the BSA that would bind Hoechst indiscriminately.

Stain and image samples

• 22Add Hoechst stain for and incubate 30 sec to stain the nucleus.
• 23Wash the sections three times with PBS for a minimum of 5 min each time.
• 24
  Adhere a cover glass over the sample with gelvatol, place the slides horizontally in slide box, and allow the cover glass to adhere to slide overnight at 4°C in the dark.

  • Gelvatol is water soluble; if necessary, the coverslip may be gently removed by submerging the sample in water and allowing the coverslip to lift without resistance from the slide.
• 25Obtain optical sections at Nyquist sampling density (see Smith, 2011), using point scanning confocal microscopy.

ALTERNATE PROTOCOL

ANTI-DMPO WESTERN BLOT

Western blot analysis can be used as an alternative approach for homogenized tissues to quantitatively determine the amount of protein-DMPO-nitrone adducts. Whereas not all DMPO-adducted proteins and lipids will be oriented in optimal configuration for ready access to the primary antibody, a tissue homogenate that is electrophoretically separated on a gel may provide a more comprehensive analysis of the total amount of protein adducts present. However, the drawback to this approach is that anatomical location cannot be determined.

When preparing samples for electrophoresis, use the western blotting protocol and gel percentage of your choice (e.g., see Gallagher et al., 2008). However, do not use 2-mercaptoethanol (2-ME) as a reducing agent. The presence of 2-ME can result in removal of the DMPO from the sample. Instead, the use of phosphine-based reducing agents is recommended. In addition, when loading supernatant, the exact amount (μg/lane) may vary, depending on the tissue of interest. It will require some trial and error to identify the optimal amount of total protein to use. Of course, this is also dependent on the gel percentage and physical size of the lane.

Materials

• Frozen organs or tissues from DMPO-treated mice (Basic Protocol, step 3a)

• Chelex-treated PBS, pH 7.4: prepare using PBS (APPENDIX 2A) and Chelex 100 (Bio-Rad) according to the manufacturer’s directions

• 100 μM diethylene triamine pentaacetic acid (DTPA; Sigma-Aldrich)

• BCA assay kit (Pierce)

• 5% (v/v) BSA (Sigma)/TBST (see recipe)

• TBST (see recipe)

• 1:1000 rabbit anti-DMPO polyclonal antibody (Enzo Life Sciences, cat. no. ALX-210–530-R050)/5% (v/v) BSA

• 1:5000 goat anti-rabbit IgG (Abnova, cat. no. PAB13665)/5% (v/v) BSA

• ECL-plus chemiluminescence kit (GE/Amersham)

• Phosphine-based reducing buffer (e.g., Bio-Rad XT, cat. no. 161-0792)

• Homogenizers

• Refrigerated centrifuge

• Software for measuring gel staining density (e.g., UN-SCAN-IT; Silk Scientific)

• Additional reagents and equipment for performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (Gallagher et al., 2008)

Prepare tissues for western blotting

• 1Homogenize tissues in Chelex-treated PBS, pH 7.4, and 100 μM DTPA.
• 2Centrifuge the homogenized tissue 20 min at 2300 × g, 4°C, to remove insoluble material.
• 3Determine protein concentrations using the BCA assay kit, according to the manufacturer’s directions.

Electrophorese samples and transfer to membrane

• 4
  Separate the samples by SDS-PAGE (e.g., Gallagher et al., 2008). Typically, use 10 μg protein per lane.

  • IMPORTANT NOTE: When preparing samples for electrophoresis, do not use 2-mercaptoethanol (2-ME) as a reducing agent.
  • We used the Bio-Rad Criterion System, Bio-Rad Ready Gels (10%), Bio-Rad XT sample buffer (cat. no. 161-0791), and Bio-Rad XT MOPS running buffer (cat. no. 161-0788).
• 5After SDS-PAGE, transfer proteins to PVDF membranes for western blotting (e.g., see Gallagher et al., 2008).

Stain samples

• 6Block membranes 2 hr in 5% BSA/TBST.
• 7
  Replace blocking solution with primary antibody solution (1:000 rabbit anti-DMPO), and incubate 1 hr at room temperature.

  • Primary antibody is diluted in 5% BSA.
• 8Wash the membranes three times with TBST, 5 min each time.
• 9
  Add secondary antibody solution (1:5000 goat anti-rabbit IgG), and incubate 1 hr at room temperature.

  • Secondary antibody is constituted in 5% BSA.
• 10Wash the membranes eight times with TBST, 5 min each time.
• 11Develop the membranes using an ECL-plus chemiluminescence kit.
• 12Assess total lane signal intensity (all bands) using software of your choice.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A.

DMPO stock solution

• Using a pipet, remove DMPO (Dojindo, 1 ml, neat; cat. no. D048-10) from the brown glass Dojindo vial in low light, and place it into a 50-ml polystyrene conical culture tube containing 2 ml of sterile, room temperature, pyrogen-free saline (e.g., Baxter), and wrap in aluminum foil.

  • DMPO is light sensitive.
• Deposit the DMPO into the saline to prevent DMPO associating with the plastic tube walls. Rinse the vial several times with 1.5 ml of sterile, pyrogen-free saline, and gently vortex. After each rinse, place the DMPO-containing saline into the 50-ml culture tube. Repeat this process several times.

  • Neat DMPO from Dojindo (1 ml = 1 g) has the consistency of honey. Therefore, several rinses are necessary to ensure residual DMPO in the manufacture’s vial is all removed.
• Adjust the final volume to 20 ml with sterile saline.

  • Note that the final dilution of the original DMPO is 1:20. At this point you have a 0.05 g/ml stock solution of DMPO.

• Place the 20 ml of stock solution on ice.

• Calculate the volume of the 0.05 g/ml stock DMPO solution, necessary to attain delivery of 0.5 g/kg body weight.

  • For example, a mouse weighing 20 g (or 0.02 kg) would require 0.01 grams of DMPO to achieve 0.5 g/kg. Injection of 200 μl (or 0.2 cc) would result in delivery of 0.5 g/kg.

• Calculate the total volume necessary for all the mice at each of the three injection time points, and store the three aliquots up to 6 months at −80°C until immediately before use. Do not thaw and refreeze.

Gelvatol mounting medium

• Add 21 g poly (vinyl alcohol) (PVA; Sigma, cat. no. P-8136) to 42 ml glycerol (Sigma, cat. no. G-9012).

• Add 52 ml water and a few crystals of sodium azide (Fisher, cat. no. S227-100).

• Then add 106 ml of 0.2 M Tris, pH 8.5 (see recipe).

  • You may need to add more PVA at this point until the solution is clear and slightly less viscous than molasses.

• Stir with low heat until solution reaches 65 °C (~1 hr).

• Refrigerate the beaker of gelvatol overnight at 4°C and observe in the morning to be sure that the viscosity is now that of molasses. If it is, continue with clarification (below). If it is too viscous, add a little more glycerol to bring the viscosity down. If it is not viscous enough, add more PVA with heat, and refrigerate for a few more hours to check the viscosity before proceeding.

• Clarify the mixture by centrifuging 15 min at 5000 × g, 4°C, to remove particulates.

• Dispense into 3-ml aliquots and store at 4°C.

TBST

• 10 ml of 10% (v/v) Tween 20

• 100 ml of 10× TBS (see recipe, but add 10× Tris and NaCl)

• 890 ml water

• Store up to 4 weeks at room temperature

Tris buffer (pH 8.5), 0.2 M

• Add 12.1 g of Trizma base to 500 ml water. Stir until dissolved.

• Adjust the pH to 8.5 with concentrated HCl

• Store up to 4 weeks at room temperature

Tris-buffered saline (TBS), 1×, pH 7.6

• 6.05 g Tris base

• 8.76 g NaCl

• 800 ml water

• Adjust the pH to 7.6 with 1 M HCl

• Adjust the volume to 1 liter with water

• Store up to 4 weeks at room temperature

COMMENTARY

Background Information

As discussed previously, in vivo IST combines the reactivity of the EPR spin trap DMPO with confocal microscopy to enable investigators to visualize the extent and anatomic sites of reactive species formation. For example, we demonstrated that IST affords the capacity to assess oxidative stress associated with low-grade inflammation induced by metabolic syndrome (Khoo et al., 2012). However, IST has also been used in vitro to identify specific sites of free radical formation on purified preparations of proteins and DNA (Siraki et al., 2008; Stadler et al., 2008; Chatterjee et al., 2009; Kojima et al., 2009; Narwaley et al., 2011). As such, IST is emerging as a powerful tool in the redox biology tool box for assessment of biomolecular free radicals generated from reactions of biomolecules and reactive species. Critical aspects of IST have been recently reviewed in detail (Gomez-Mejiba et al., 2014).

It is a common misconception that IST directly detects ROS such as O₂^•−. The reason for this assumption is most likely rooted in the history of DMPO as the seminal spin-trapping agent used for EPR-based O₂^•− detection and quantification. However, as noted previously, in vivo reaction of DMPO will not result in further reaction with lipids or proteins and thus, will not be retained by the tissue as antigen for the primary anti-DMPO antibody. IST is a measurement of down-stream reactions of reactive species that result in the generation of an unpaired electron on a biomolecule. It is this unpaired electron that is the target for the high-affinity DMPO reaction (k = 10⁵–10⁷ M⁻¹ s⁻¹) and the resultant DMPO-biomolecule adduct is the target for the primary antibody (Mason, 2004).

Critical Parameters

In vivo IST requires amplification of tissue DMPO concentrations via administration that is temporally compatible and concurrent with anticipated elevation of free radical generation to afford optimal potential for DMPO-free radical reactions and consequent accumulation of DMPO-adducts. This is a critical consideration when adapting this protocol, designed for chronic low-grade reactive species formation and resultant tissue free radical generation, to in vivo models of more acute and temporally restricted oxidative stress such as ischemia and reperfusion. One must take care to deliver a high dose of DMPO concurrent with the generation of biomolecular free radicals, as these species have an extremely short half-life because of intermolecular rearrangements, reaction with neighboring free radicals, and/or reaction with other molecules including O₂ and antioxidants such as GSH. Thus, it is optimal to attain double-digit high concentrations (preferably in the millimolar range) of DMPO in local microenvironments to facilitate the ability of DMPO to compete for reactions with the unpaired electrons on the biomolecule. It is unfortunate that the literature is devoid of reports describing the pharmacodynamics and pharmacokinetics of DMPO, and thus, considerable effort and trial and error must be pursued. Suffice it to say that the goal is to expose the target tissue to as much DMPO as possible with minimal effect on the system being studied. This concept, as well as other considerations, was recently reviewed (Gomez-Mejiba et al., 2014).

Troubleshooting

In addition to concentration considerations outlined above, there are two additional issues that may affect IST experimental outcomes. First, the presence of molecular oxygen (O₂) is inhibitory. For example, in models where there is a high concentration of O₂ such as a lung injury induced by the mechanical stress of a ventilator or dilatory responses of a vessel in a myograph system, the level of O₂ used in the ventilation or perfusate is typically 95%. This level of O₂ produces a concentration in solution approaching 1 mM and thus provides significant competition with DMPO for the reaction with free radicals on the biomolecule (see Fig. 12.42.1). To address this issue, one may consider intravenous delivery of DMPO and/or reducing the level of O₂ used in the model. Second, the anti-DMPO antibody was raised to a small epitope (DMPO-octanoic acid conjugate) and thus, may present issues regarding cross-reactivity (Gomez-Mejiba et al., 2014). To avoid misinterpretation of results, we recommend that controls always be performed using animals that have not been exposed to DMPO, as well as conditions where the primary antibody is not used for analysis.

Anticipated Results

As shown in Figure 12.42.1 and in previously published work (Khoo et al., 2012), IST is sensitive enough to detect the accumulation of tissue-associated free radicals over 24 hr induced by low-grade chronic inflammation associated with obesity. Therefore, it is reasonable to anticipate that model systems that result in an equivalent amount of reactive species generation would produce similar results. However, as discussed above, attention must be given to the time frame of oxidant generation, as well as both the local concentration of DMPO and competitive molecules such as O₂ and antioxidants.

Time Considerations

As described in the protocol, the overall time required for exposure to DMPO is 24 hr for this in vivo model of oxidative stress. During this time frame, the initial generation of diluted and injectable DMPO stock coupled to the actual intraperitoneal injections consumes time that is directly dependent upon the total number of animals in the study. Therefore, a study of 12 animals may take a total of 1.5 hr over the 24-hour time frame, whereas a study of 24 animals may take 3 hr. From the time of sacrifice to tissue freezing, the required time for tissue processing is similarly dependent upon the total number of animals in the study, but a minimum of 3 days can be expected for sectioning, immunological labeling, and confocal imaging.