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106J. Luo et al. / Journal of Neuroscience Methods 120 (2002) 105/112We have found that a fixation step introduced afterthe dye loading and before the dissociating proceduresignificantly increased the post-injury ROS signalsdetected by flow cytometry compared to that withoutthe fixation step. The data obtained from this newmethod was in overall better agreement with two otherstandard ROS detection methods, image analysis-basedfluorescence microscopy and LPO assay. This methodmay facilitate the more frequent use of flow cytometryin free radical research in nervous tissue.2. Methods and materials2.1. In vitro spinal cord injury modelAll animals used in this study were handled in strictaccordance with the NIH guide for the Care and Use ofLaboratory Animals and the experimental protocol wasapproved by the Purdue Animal Care and UsageCommittee. In these experiments, every effort wasmade to reduce the number and suffering of the animalsused. Adult Hartley guinea pigs, weighing 300/500 gwere anesthetized deeply with a mixture of ketamine (60mg/kg) and xylazine (10 mg/kg). They were thenperfused through the heart with 500 ml of oxygenated,cold Krebs’ solution to remove blood and lower coretemperature. The vertebral column was excised rapidlyand a complete laminectomy was performed. The spinalcord was removed from the vertebrae and immersed incold Krebs’ solution. The cord was separated into twohalves by midline sagittal division. The composition ofthe modified Krebs’ solution was as follows: NaCl, 124mM; KCl, 2 mM; KH 2 PO 4 , 1.2 mM; MgSO 4 , 1.3 mM;CaCl 2 , 2 mM; dextrose, 20 mM; NaHCO 3 , 26 mM,equilibrated by bubbling with 95% O 2 /5% CO 2 toproduce a pH of 7.2/7.4. The spinal cords weremaintained in oxygenated Krebs’ solution at 37 8C for1 h before the onset of the experiment to ensure recoveryfrom dissection before experimentation. All the sampleswere bubbled with 95% O 2 /5% CO 2 throughout theduration of the experiment.The spinal cord strips were randomly divided intothree groups: control-uninjured (control), compressionuntreated(injury), and compression-treated (treated).The control-uninjured group did not receive any compression.The compression-untreated group received thecompression injury which was induced by a constantdisplacement of 5 s compression of the spinal cord usinga modified forceps possessing a spacer. The compression-treatedgroup was placed in the modified Krebs’solution containing 100 mM manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) immediately aftercompression. MnTBAP is a cell-permeable superoxidescavenger. All experiments were performed at 37 8C.One hour after injury, the spinal cord strips weretaken out of the Krebs’ bath and the center of the injurysite (about 6 mm long) was obtained and the ROSmeasurements were performed.2.2. Detecting ROS using flow cytometryHydroethidine (HE) was used as a superoxide indicator.HE is oxidized selectively by superoxide toethidium, whose fluorescence intensity within the cell isproportional to the total production of superoxide(Rothe and Valet, 1990; Bindokas et al., 1996). Theoxidation of HE is not accomplished by hydroxylradical, singlet O 2 ,H 2 O 2 or nitrogen radicals (Bindokaset al., 1996).2.2.1. Isolating cells for flow cytometry2.2.1.1. Method A. This method was optimized forinjured spinal cord from the cell dissociation methodsused in most literature (Grogan and Collins, 1990;Watson, 1991; Visscher and Crissman, 1994; Diamondet al., 2000). The isolation procedure was performed onfresh tissues using a mixture of enzymes after mechanicalmincing. Briefly, spinal cord strips, from the centerof the injury site, were minced with a surgical blade. Theminced tissue was disaggregated first by an automaticmechanical procedure using BD Medimachine (BDBiosciences, San Jose, CA) for 15 s. The resultant tissuewas then incubated in 1 ml of previously oxygenated(95% O 2 /5% CO 2 ) dissociation solution for 20 min andkept oxygenated during the dissociation process. Thedissociation solution contained 0.5 mg/ml collagenaseand 0.1 mg/ml trypsin in Krebs’ solution. The concentrationsof the enzymes and duration of incubation wereoptimized to enable effective dissociation of cells and atthe same time cause minimal cell damage based on theobservation of production of subcellular debris. Duringdissociation, the tissue was gently triturated with a 2 mlpipette (10 strokes). After 20 min dissociation, theremaining tissue was removed and the suspensionswere spun at 300/g for 10 min, the dissociationsolution was aspirated and the cells were washed twicewith PBS before dye loading. Pelleted cells were washedwith 2 ml PBS and centrifuged as above. Undisaggregatedtissue debris and doublets were removed byfiltration through a mesh (60 mm). The resultant cells(10 6 ) were then incubated in 1 ml of 1 mM HE solutionat 37 8C for 5 min in the dark. Cells were then washedtwice in PBS by two additional spinning/washing cyclesand resuspended in 1 ml of PBS for flow cytometricanalysis.2.2.1.2. Method B. The major differences betweenmethods A and B were that the fluorescence dye wasloaded before the dissociation procedure and the tissue
J. Luo et al. / Journal of Neuroscience Methods 120 (2002) 105/112 107was immediately fixed after dye loading; therefore, thedissociation was performed on the fixed tissues. Thespinal cord strips were incubated with 1 ml of PBS withHE at final concentrations of 1 mM for 5 min at 37 8Cin the dark. The strips were then fixed with 3.7%formaldehyde for 2 h. After fixation, the spinal cordstrips were subjected to rehydration by incubation inPBS overnight. The aim of the rehydration procedurewas to remove the formaldehyde to prevent the inactivationof enzymes used to digest the tissue (Gibellini etal., 1995). After rehydration, the spinal cord strips wereminced manually. The minced tissue was disaggregatedusing BD Medimachine (BD Biosciences, San Jose, CA)for 25 s and incubated in enzymatic dissociationsolution for 20 min. The dissociation mixture contained5 mg/ml collagenase and 1 mg/ml trypsin in Krebs’solution. During dissociation, the tissue was gentlytriturated with a 2 ml pipette (maximum of 20 strokes).After the remaining tissue was removed, the suspensionswere spun at 300/g for 10 min and the dissociationsolution was aspirated. Pelleted cells were washed with 2ml PBS and centrifuged as above. Undisaggregatedtissue debris and doublets were removed by filtrationthrough a 60 mm metal mesh. The final cells (10 6 ) wereresuspended in 1 ml PBS and were analyzed by flowcytometry immediately.2.2.2. Flow cytometry analysisFlow cytometric data acquisition and analysis wereperformed on an EPICS XL cytometer (Beckman-Coulter, Miami, FL). The flow cytometer was equippedwith an argon ion laser with excitation at 488 nm and a15 mW output power. The red fluorescence of ethidiumwas monitored using a 610 nm long pass filter. Electronicgating was used to exclude doublets and subcellulardebris. Specifically, a forward scatter (FSC) thresholdwas set to eliminate most of the subcellular debris. Foreach parameter investigated, at least 2/10 4 events(cells) were analyzed per sample. The following datawere collected: forward scatter, 908 scatter, and themean fluorescence intensity of ethidium signals (aslogarithmically amplified data).Since we introduced a fixing step in method B, wewanted to know the effect of the fixation procedure onthe fluorescence intensity of HE. This was done bycomparing the fluorescence intensity in the same cellpopulation with and without fixation.As compared with unfixed samples, the fixed samplesshowed an average 6/10% increase of fluorescenceintensity in both control and injured groups (data notshown). The fixation did not significantly affect themean coefficient variation (data not shown), and theincrease was cancelled out when expressed as a percentageof the control.2.3. Morphological studies using fluorescence microscopyIn order to observe the morphology change afterisolation, the isolated single cells were observed under afluorescence microscope. This was done immediatelywhen the cells were ready for flow cytometry (beforeresuspension) by dropping the pellets directly onto theglass slides and observing immediately under an OlympusVanox fluorescence microscope (excitation filter: BP545 nm; barrier filter: 0/590 nm).2.4. Detecting ROS using fluorescence microscopyIn order to compare the accuracy of the flowcytometry data obtained from the cells dissociated bymethods A and B, we used fluorescence microscopy tomeasure the fluorescence intensity. For the fluorescencemicroscopy experiment, the dye loading and fixationwas the same as described above in method B. Afterfixation, the segments were cut into sections of 40 mm,using an EMS-4000 Tissue Slicer (Electron MicroscopySciences, Fort Washington, PA). The sections weredehydrated and mounted on glass slides with DPX.The sections were then observed with an OlympusVanox fluorescent microscope (excitation filter: BP 545nm; barrier filter: 0/590 nm). HE fluorescence imageswere captured through a 2/ objective, using a highresolutionCCD camera (DEI-750, Optronic Eng.,Goleta, CA). At this magnification, we were able tocapture the whole section with just one image. Theimages were saved for later analysis and quantification.Camera exposure and light settings were kept constantduring each experiment.Quantification was performed from coded slides by anindividual who did not know the treatment history ofthe specimen. The fluorescence intensity measurementswere carried out by an image analysis program, SigmascanPro (SPSS Science, Chicago, IL). For each tissuesample, which was about 6 mm long, five sections werechosen randomly and the fluorescent intensity wasmeasured separately. An average of the values fromthese five sections was calculated and used as the finalfluorescent value of the cord sample. For each sectionimage, the area occupied by the spinal cord tissue wascarefully delineated manually along the edge and thefluorescent intensity of the tissue was taken. Foursmaller areas (5% of the total field) were randomlyselected at each corner (outside of the spinal cord tissue)and the average fluorescence intensities of these fourareas were used as background intensity. The fluorescenceintensity of the spinal cord samples was obtainedby subtracting the background fluorescence from that ofthe spinal cord image and expressed in arbitrary units.
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