ARTICLE |
Correspondence to: Bruno De Luca, Dept. of Experimental Medicine, Seconda Università degli Studi di Napoli Via Costantinopoli, 16, 80138 Naples, Italy. E-mail: bruno.deluca@unina2.it
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copperzinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis. (J Histochem Cytochem 51:865871, 2003)
Key Words: free radicals, cerium, DAB, quantitative histochemistry, image analysis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SUPEROXIDE ANIONS (O2-) are highly reactive radicals (ROS) generated in cells and tissues by enzymes such as xanthine oxidase and the mitochondrial electron chain (
Different mechanisms protect cells from O2·- and related radicals such as H2O2 and OH-. The most important one is the dismutation of O2·- to H2O2 + O2 and the subsequent dismutation of H2O2 to O2 and H2O. The first reaction is catalyzed by superoxide dismutase (SOD), the second one by catalase.
In the nervous system, SOD activity is unbalanced during neuronal apoptosis (
The localization and quantification of SOD activity in tissues is of great interest and can give information about the tissue responses to oxidative stresses. At present it is possible to localize and quantify the amount of SOD enzyme immunohistochemically by use of specific antibodies, but not its activity (
Previous experience led to histochemical techniques able to localize SOD activity in situ, but these techniques could not be used to quantify the signal (
This method is based on riboflavin-UV as a source of O2·- (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Twelve adult male SpragueDawley rats from our own colony (200250 g body weight) were housed in groups of two on standard conditions with a 12-hr lightdark cycle (lights from 0700 to 1900) and kept on a laboratory diet and water ad lib. The animals were handled daily to reduce stress due to the manipulation. All other parameters fulfilled the requirements of the "Guide for the Care and Use of Laboratory Animals" by the National Research Council, implemented by EU and local rules.
Brain Removal, Freezing, and Sectioning
The animals were sacrificed by decapitation during the light phase of the cycle to minimize circadian variation. The brain was quickly removed and either directly frozen by immersion in 2-methylbutane cooled in dry ice or first homogenized on ice for 23 min (tissue standards) and then transferred into small tubes and frozen in dry ice. Frozen brains and brain pastes were then stored at -80C until sectioning with the cryostat. Cryostat sections (50 µm thick) were cut at -20C and mounted on precleaned glass slides (single frosted; Sigma, Milan, Italy).
Histochemical Staining Procedure
After washing in PBS, sections were dehydrated in ethanol 85%, 95%, and 100% for 5 min each, cleared in xylene, and coverslipped with Permount.
Immunohistochemistry
Alternate sections were stained with the histochemical procedure as above or with immunohistochemistry for the Zn/CuSOD isoform as detailed here.
Sections were fixed for 5 min in formaldehyde 4% in PBS. After washing in PBS, sections were treated for 30 min with H2O2 0.3% in methanol, washed again in PBS, and incubated in mouse anti-SOD (Sigma) 1:400 in PBS + 10% normal bovine serum (NBS) overnight at 4C. After three washes in PBS, sections were incubated in biotinilated anti-mouse antibody (Vector; Burlingame, CA) 1:100 + 10% NBS in PBS for 1 hr at room temperature (RT). Sections were then washed three times in PBS and then incubated with ABC (Vector) for 1 hr according to the supplier's instructions. After three washes in PBS, the signal was visualized with 0.1% DAB + 0.02% H2O2 in PBS for 10 min in the dark at RT. The reaction was then stopped with cold PBS, the sections dehydrated in alcohol, cleared in xylene, and coverslipped with Permount.
Control Experiments
The following variables were changed during the histochemical staining of sections of tissue standards to test the specificity and the optimal conditions for SOD histochemistry.
Fixation. Sections were stained without fixation in glutaraldehyde.
UV Irradiation. To reduce the formation of superoxide, sections were not exposed to UV irradiation during the incubation with riboflavin.
DAB. The intensification step with DAB was omitted to evaluate the intensity of the signal deriving from cerium ions.
Diethyldithiocarbamate.
Superoxide dismutase activity was inhibited adding 10 mM diethyldithiocarbamate (Carlo Erba; Milan, Italy) in the first incubation medium (
Methodological Validation of SOD Histochemistry
To validate the histochemical assay as a quantitative tool, the relationship between time of incubation, section thickness, and the densitometrically measured reaction product was studied to fulfill general quantitative methodological requirements detailed by
The relationship between time of incubation and OD was analyzed using 50-µm-thick tissue sections incubated at increasing times during the first step of UV exposure (0, 0.5, 1, 2, 5, 10, 15, and 30 min).
Image Analysis
Brain sections and tissue standards were analyzed at low magnification (obj. x0.5) with a Zeiss Axioskop 20 equipped with high-resolution analogic monochrome camera Hamamatsu C5405. Images were digitized (box size 1024 x 1024; pixel size 1.5 µm) using an MCID-2 board and analyzed with AIS imaging (Imaging Research; St. Catherines, Ontario, Canada). The regions of interest were outlined and the relative optical density [ROD=log (256/gray level)] calculated. The background ROD (unstained tissue) was also calculated. All data were then reported after subtraction of background OD to get an absolute scale (0 OD = unstained tissue).
The light intensity (Köler illumination) was adjusted to avoid saturation in blank fields and in the regions of interest. A blue filter ( = 440 ± 10 nm) was used to filter out green and red visible light. Light stability was checked over time using the illuminator device of an MCID system. Neutral glasses of defined transmission were used to calibrate the system and check the densitometric linearity.
The camera gain was set to 0 to minimize noise. Slices were always in focus during measurement and each image was averaged over 16 digitized images. Four different measurements were taken on each standard section and four different standard sections were used for each time/section thickness point: therefore, 16 measurements were obtained for each standard.
A blank field was digitized before every session for shading correction. All other parameters were adjusted according to the guidelines in
Statistics
Each measurement was repeated four to ten times on different sections. Measurements were statistically analyzed calculating the regression coefficient for thicknessOD and time of incubationOD relationships, or testing different conditions in control experiments by ANOVA and planned comparisons with Student's t-test for non-paired data. All data in the graphs have been reported as mean ± SE.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histochemical Staining Procedure
The three-step histochemical procedure described here is easy to perform and gives reproducible results. Moreover, the use of standards allows comparison of experiments executed at different times.
This technique requires a first step in glutaraldehyde to preserve morphological details and to fix SOD activity in situ. After the fixation, repeated washes of the tissue sections in PBS before the incubation step effectively remove red blood cells that could interfere during the intensification step with DAB.
Sections are then incubated in a solution containing an O2·- -producing system (riboflavinUV) and a capture system for H2O2 (cerium). Oxidized riboflavin is reduced under UV irradiation and then undergoes autoxidation to release superoxide radicals (
The morphological details of the neurons, due to the prevalent localization of the enzyme in the soma, are very good (Fig 1). SOD activity is localized mainly in neuronal soma, whereas the neuropil is less reactive (Fig 1). The staining is more evident in the neuronal cytoplasm than in the nucleus (Fig 1). Myelin and white matter appear unstained (Fig 2).
|
|
Positive neurons are widespread in the nervous system, across different regions and different neuronal types in the same region. However, some neurons are more intensely stained than others. For example, pyramidal cells of the hippocampus, granule cells in the cerebellum, deep cerebellar nuclei, and some neuronal subsets in the reticular nuclei of the brainstem neurons showed the highest intensity (Fig 2 and Fig 3). Endothelial and ependymal cells, and cells of choroid plexus, are also intensely stained. White matter was generally unstained.
|
Immunohistochemistry
Alternate sections treated with the histochemical reaction (SOD activity) or immunostained for SOD protein displayed the same staining pattern (Fig 3). The immunohistochemical localization of the cytoplasmic SOD isoenzyme shows a higher definition of cell contours, whereas the histochemical SOD reaction shows some signal also in the neuropil, possibly due to the identification of the extracellular SOD isoenzymes (see also
Specificity and Optimal Conditions of the Histochemical Reaction
A balance is needed between optimal enzyme activity and good tissue preservation, because a totally unfixed brain shows several artifacts and is poorly stained (control = 0.0969 ± 0.008 OD; no fixation = 0.0169 ± 0.008 OD; t-test p<0.05; Fig 4) due to the loss of SOD enzyme in the reaction buffer. The best conditions were no perfusion-fixation and post-fixation on slides for 5 min with 0.5% glutaraldehyde.
|
The reaction product was significantly reduced without UV irradiation in the incubation step, although residual staining persisted in both paste standards and brain sections (control = 0.0969 ± 0.008 OD; no UV = 0.0437 ± 0.004 OD; t-test p<0.05; Fig 4). The absence of the intensification step resulted in a significant decrease of the reaction product (control = 0.0969 ± 0.008 OD; no DAB = 0.0290 ± 0.003 OD; p<0.05, Fig 4).
Superoxide dismutase inhibitors such as diethyldithiocarbamate (DTC) significantly inhibited the signal (control = 0.0969 ± 0.008 OD; DTC = 0.02 ± 0.006 OD; p<0.05).
Methodological Validation
As shown in Fig 4, there is a high correlation between section thickness and OD (r=0.98; p<<0.05) and between time of incubation and OD (r=0.935; p<<0.05).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A great deal of work in the literature is based on the immunolocalization of SOD enzyme, a few papers focusing on the localization of SOD activity in tissue sections.
Previous work by
Another histochemical technique was based on blotting the tissue sections on a filter and then staining the filter using the principle of inhibition of autoxidation of nitroblue tetrazolium in the presence of SOD (
The present study shows that SOD activity can be determined and quantified by autoxidation of riboflavin in the presence of UV light as the source of substrate (superoxide radicals) for SOD activity (
The advantage of the technique reported here is the possibility of visualizing SOD activity with high resolution and of quantifying the reaction product. Our technique showed high SOD activity in hippocampal pyramidal and granular cells and in the granular layer of cerebellum, in agreement with previous data (
It is interesting to note that without UV irradiation (therefore without an external source of superoxide anions) there is weak residual staining in both paste standards and brain sections, possibly due to ROS production in the section (
The optical density (OD) of the sections was highly correlated with the duration of incubation time and the section thickness, fulfilling general quantitative methodological requirements detailed by
However, because cerium ions have a low degree of penetration, detergents (Triton X-100 in our case;
Some differences between SOD histochemistry and immunohistochemistry, particularly from a quantitative point of view, may depend on the activity of different forms of SOD enzymes (Cu/ZnSOD, MnSOD, and extracellular SOD) with the histochemical method, whereas immunolocalization allows the determination of one type of SOD isoform due to the specificity of the antibody (see also
Moreover, the histochemical method could be sensitive to the enzymatic activity/inactivity of SOD in the section, modulated by oxidative substances (
![]() |
Acknowledgments |
---|
We are grateful to anonymous referees for useful comments.
Received for publication August 1, 2002; accepted March 12, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angermuller S, Fahimi HD (1988) Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases. J Histochem Cytochem 36:23-28[Abstract]
Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276-287[Medline]
Böcking A, Giroud F, Reith A (1997) Consensus report of the ESCAP task force on standardization of diagnostic DNA image cytometry. European Society for Analytical Cellular Pathology (ESCAP). http://www.biochem.mpg.de/valet/esacimag.html
Briggs RT, Drath DB, Karnovsky ML, Karnovsky MJ (1975) Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol 67:566-586[Abstract]
Cheeseman KH, Slater TF (1993) An introduction to free radical biochemistry. Br Med Bull 49:481-493[Abstract]
Deng HX, Hentati A, Tainer JA, Iqbal Z, Cayabyab A, Hung WY, Getzoff ED et al. (1993) Amyotrophic lateral sclerosis and structural defects in Cu/Zn superoxide dismutase. Science 261:1047-1051[Medline]
Frederiks WM, Bosch KS (1997) Localization of superoxide dismutase activity in rat tissues. Free Radic Biol Med 22:241-248[Medline]
Freeman BA, Crapo JD (1982) Biology of disease: free radicals and tissue injury. Lab Invest 47:412-426[Medline]
Fujimura M, MoritaFujimura Y, Kawase M, Copin JC, Calagui B, Epstein CJ, Chan PH (1999) Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. J Neurosci 19:3414-3422
Halliwell B, Chirico S (1993) Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 715S724S
Halliwell B, Gutteridge JM, Cross CE (1992) Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 119:598-620[Medline]
Hammond B, Kontos HA, Hess ML (1985) Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage. Can J Physiol Pharmacol 63:173-187[Medline]
Hardie DC, Gregory R, Hebert PDN (2002) From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry. J Histochem Cytochem 50:735-749
Jewett SL, Cushing S, Gillespie F, Smith D, Sparks S (1989) Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H 2O2(and HO2- leading to loss of copper. Eur J Biochem 180):569-575
Jonker A, Geerts WJC, Chieco P, Moorman AFM, Lamers WH, Van Noorden JF (1997) Basic strategies for valid cytometry using image analysis. Histochem J 29:347-364[Medline]
Kehrer JP (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 23:21-48[Medline]
Kontos CD, Wei EP, Williams JI, Kontos HA, Povlishock JT (1992) Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo. Am J Physiol 263:H1234-1242[Medline]
Luetjens CM, Bui NT, Sengpiel B, Munstermann G, Poppe M, Krohn AJ, Bauerbach E et al. (2000) Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production. J Neurosci 20:5715-5723
Marklund SL (1982) Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci USA 79:7634-7638[Abstract]
Moreno S, Nardacci R, Ceru MP (1997) Regional and ultrastructural immunolocalization of copper-zinc superoxide dismutase in rat central nervous system. J Histochem Cytochem 45:1611-1622
Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, Chan PH (1998) Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci 18:205-213
Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA (1992) Oxygen radicals in cerebral ischemia. Am J Physiol 263:H1356-1362[Medline]
Okabe M, Saito S, Saito T, Ito K, Kimura S, Niioka T, Kurasaki M (1998) Histochemical localization of superoxide dismutase activity in rat brain. Free Radic Biol Med 24:1470-1476[Medline]
Robinson JM (1985) Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization. J Histochem Cytochem 33:749-754[Abstract]
Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D et al. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59-62[Medline]
SeguraAguilar J (1993) A new direct method for determining superoxide dismutase activity by measuring hydrogen peroxide formation. Chem Biol Interact 86:69-78[Medline]
Stoward PJ (1980) Criteria for the validation of quantitative histochemical enzyme techniques. Trends in Enzyme Histochemistry and Cytochemistry. Amsterdam, CIBA Foundation, Excerpta Medica, pp. 1131
Traber R, Kramer HE, Hemmerich P (1982) Mechanism of light-induced reduction of biological redox centers by amino acids. A flash photolysis study of flavin photoreduction by ethylenediaminetetraacetate and nitrilotriacetate. Biochemistry 21:1687-1693[Medline]
Troy CM, Shelanski ML (1994) Down-regulation of copper/zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells. Proc Natl Acad Sci USA 91:6384-6387[Abstract]
Ukeda H, Maeda S, Ishii T, Sawamura M (1997) Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'1(phenylamino)-carbonyl3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase. Anal Biochem 251:206-209[Medline]
Van Noorden CJF, Frederiks WM (1993) Cerium methods for light and electron microscopical histochemistry. J Microsc 171:3-16[Medline]
Weisiger RA, Fridovich I (1973) Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J Biol Chem 248:4793-4796
Wentworth P, Jr, Jones LH, Wentworth AD, Zhu X, Larsen NA, Wilson IA, Xu X et al. (2001) Antibody catalysis of the oxidation of water. Science 293:1806-1811