Immunohistochemical Study on Perinatal Development of Rat Superoxide Dismutases in Lungs and Kidneys

0031-3998/91/2905-0487$03.00/0 PEDIATRIC RESEARCH Copyright © 1991 International Pediatric Research Foundation, Inc. Vol. 29, No.5, 1991 Printed in U.S.A. Immunohistochemical Study on Perinatal Development of Rat Superoxide Dismutases in Lungs and Kidneys KOHTARO ASAYAMA, HIDEMASA HAY ASHIBE, KAZUSHIGE DOBASHI, NORIHIKO UCHIDA, MAKIO KOBAYASHI, AKIRA KAWAOI, AND KIYOHIKO KATO Department (if Pediatrices [KA., H.H., KD., N. u.. KK] and Pathology [A.K.]. Yamanashi Medical College, Yamanashi 409-38, Japan and Department ofPathology, Tokyo Woman's Medical College, Tokyo 162, Japan [M.Kj ABSTRACT. It has been reported that levels of antioxidant enzymes are low in fetal rat lungs and kidneys, and that they increase rapidly during late gestation. Among the antioxidant enzymes, both copper-zinc and manganese superoxide dismutases (CuZnSOD and MnSOD) are assumed to play a key role in protection against oxidative tissue injury. To determine the nature ofthe rapid perinatal increase in CuZnSOD and MnSOD, immunoenzyme staining was performed in the lungs and kidneys of fetal (d 18 and 20 of gestation) and neonatal (d 22) rats. The CuZnSOD and MnSOD in the homogenates were assayed by RIA, and they were found to be higher in the neonatal organs than in the respective fetal organs. The neonatal bronchiolar epithelium was stained for both CuZnSOD and MnSOD more intensely than the fetal one. The CuZnSOD staining in the neonatal alveolar wall was more intense than that in the fetal one. There was a significant reactivity for MnSOD in the neonatal, but not in the fetal, alveolar walls. In the kidneys, the reactivities for CuZnSOD and MnSOD were confined to the undifferentiated tubules. Although the tubules were increased in numbers in the neonatal kidneys, the intensity of the staining for both CuZnSOD and MnSOD was unchanged. The histochemical study disclosed that CuZnSOD and MnSOD increased in the kidneys in a manner different from that in the lungs. The low concentration of both CuZnSOD and MnSOD in the fetal lung tissues may contribute to the vulnerability to oxygen toxicity. Such changes in the concentrations in specific tissues were not delineated in the kidneys. (Pediatr Res 29: 487-491,1991) system (1-4), and this is postulated to contribute to the development of bronchopulmonary dysplasia in preterm neonates. Superoxide anion radical, which is generated by one electron reduction of molecular oxygen, is considered to be an initiator of free radical chain reactions leading to oxidative cell injury in certain experimental models (5). Thus, SOD (EC 1.15.1.1), a scavenger of superoxide, may playa key role in the protection against free radical injury. Intracellular SOD is found in two forms: CuZnSOD and MnSOD. Using specific RIA (6) for both rat CuZnSOD and MnSOD, we have demonstrated that both SOD increase markedly during the late gestational period (i.e. from d 18 to 22 of gestation) in fetal rat lungs and kidneys (7). The immunoenzyme staining for rat CuZnSOD and MnSOD, which has been developed by us recently (8), revealed that there was a wide range of variability in the expression of both SOD from cell to cell in adult rat tissues. To our knowledge, immunohistochemicallocalization of CuZnSOD and MnSOD in fetal and neonatal rat tissues has not been reported previously. To further determine the nature of the rapid perinatal increase in SOD, lungs and kidneys of both fetus and neonate were stained for CuZnSOD and MnSOD, and the immunoreactive SOD in these organs were also measured. Our present study revealed that the increase in SOD occurred in certain specific tissues of these rapidly growing organs, Different characteristics of the increase between lungs and kidneys were also shown. MATERIALS AND METHODS Animal treatment. Breeding was accomplished by placing male and female Sprague-Dawley rats (Japan SLC Inc" Shizuoka, Japan) together overnight. The midpoint of cohabitation period was considered as the onset of pregnancy. Premature pups were Abbreviations delivered by hysterotomy with the dam under pentobarbital CuZnSOD, copper-zinc superoxide dismutase anesthesia (20 I I1mol/kg; 50 mg/kg) on d 18 and 20 of gestation, MnSOD, manganese superoxide dismutase and decapitated immediately after the delivery. Neonates were SOD, superoxide dismutase killed within 24 h after delivery under light ether anesthesia. The lungs and kidneys were excised, washed with PBS, and blotted with gauze. RIAfor SOD. The organs were weighed and pooled to obtain at least 80 mg of tissue for each sample, and then kept frozen at In recent years, cell injury mediated by reactive oxygen species -80°C. The tissues were homogenized with 20 times their volume has drawn increasing attention from neonatologists. Abrupt in- of 10 mM potassium phosphate buffer containing 81 11M digicrease of tissue oxygen concentration after delivery may induce tonin (pH 7.4), and then sonicated on ice for 30 s. The sonicate oxidative injury to vulnerable tissues. There is evidence suggest- was centrifuged at 13 000 x g for 5 min, and the supernatant ing that fetal lungs have a low-capacity antioxidant defense was obtained. This served as the sample for RIA. The methods of RIA for rat CuZnSOD and MnSOD have been described (6). Received May 29,1990; accepted December 17,1990. Correspondence and reprint requests: Kohtaro Asayama, M.D., Department of The data were expressed as means ± SEM. The statistical signifPediatrics, Yamanashi Medical College, 1110 Shimokato, Tamahocho. Nakakoicance was determined by the method of least significant differrnagun, Yarnanashi 409-38. Japan. ence calculated after one-way analysis of variance. Supported in part by Grant-in-Aid 63570431 from the Ministry of Education. Immunoenzyme staining for SOD. The specificity of each Science and Culture of Japan. 487 488 A SAY AMA E T AL. Table I. Immunoreactive SOD in hom ogenate of lung and kidney* Lung 18 d fetus Kidney 20 d fetus (n = 9) (n= 9) Neonate (n.= 8) 18 d fetus (n = 4) 20 d fetus (n = 9) CuZnSOD 1.41 ± 0.06 1.99 ± 0.05t 1.l 3 ± 0.07 1.80 ± 0.03t 2.90 ±0.12* 89 ± 12 154 ± IS MnSOD 77 ± 3 364 ± 9t 218 ± 16* * Data are means ± SEM. Values are nrnol/g wet tissue for CuZnSOD and pm ol/ g wet tissue for MnSOD. t p < O.OOI ( vs 18d ). t P < 0.001 (vs 20 d). Neonate (n = 8) 2.51 ± 0.08* 503 ± 14* TB a ,', ,, ." . ., ,< • I , .'b A "'-" . " '" . '. ...:?o' .j I ' -, - Fig. I. Photomicrographs of fetal and neonatal rat lungs stained for CuZnSOD. a, Fetal lung (d J 8); b, fetal lung (d 20); and c, neonatal lungs. The CuZnSOD reactivity is higher in the neonatal lungs than in the fetal lung. Original magnification x 200. Bar indicates 10 JLm. A , alveolus; TB, terminal bronchiole. antiserum was established when the RIA was developed (6). Th e immunoenzyme staining for both CuZnSOD and MnSOD was performed as described previously (8). The tissues were fixed with 10% buffered formali n and embedded in paraffin. The specimens were c (...truncated)