Apoptosis in various organs of preterm infants: histopathologic study of lung, kidney, liver, and brain of ventilated infants

ABSTRACT Apoptosis, the well-characterized form of active programmed cell death, is a physiologic phenomenon in embryonal and fetal life in developing organs. Severe hypoxia, which occurs in

most preterm infants, also leads to cell death, which may be necrotic or apoptotic. The aim of our study was to examine the incidence of apoptosis in various organs (such as lung, kidney,

and brain) of preterm infants who suffered from clinically proven respiratory distress causing infantile respiratory distress syndrome (IRDS), cardiac failure, and periventricular

leukomalacia (PVL). Twenty-four autopsy cases were studied histologically to detect the apoptotic ratio, which was performed on the basis of hematoxylin and eosin staining and validated by

terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) reaction. Elevated apoptotic ratio was found in stages II, III, and IV of bronchopulmonary dysplasia (BPD) among

alveolar and bronchiolar cells. The apoptotic activity was very low in stage I of BPD. High apoptotic ratio was detected in hypoxic injuries of the central nervous system (CNS) of preterm

infants. Features of apoptosis were present in proximal and excreting tubules of the kidney. Significant elevation of apoptotic activity may play a role in the development of BPD, ischemic

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INDUCE LUNG AND BRAIN INJURY IN NEONATAL RATS Article Open access 22 April 2021 MAIN Apoptosis is the well-characterized form of active programmed cell death and plays an important role in

organ development and cell differentiation. Various pathogenic effects occurring in preterm infants after assisted ventilation, such as severe hypoxia, oxygen toxicity,

volutrauma/barotrauma, sepsis, and lowered levels of antiproteases and antioxidant enzymes lead to cell death, which may also be apoptotic. The aim of our study was to examine the incidence

of apoptosis primarily in the lung and in selected cases in the liver, kidney, and brain of preterm infants who suffered clinically proven respiratory distress causing IRDS. We hypothesized

that this form of programmed cell death plays an important role in the development of BPD and other pathologic sequels of assisted ventilation and hypoxia, such as renal and hepatic failure,

IVH, and PVL. METHODS Tissue samples were available from 24 preterm infants (24–36 wk gestation) who died of respiratory failure or IVH. All infants showed clinical and radiologic signs of

IRDS, and hyaline membrane disease or BPD was proven by routine postmortem histologic examination. The lungs of two neonates, 27 and 36 wk gestation, served as controls. These two infants

died during labor with severe forms of diaphragmatic hernia. Histologic and TUNEL reactions showed that apoptosis of alveolar and bronchiolar cells was rare and the number of apoptotic

figures in the brain, liver, and kidney was less than five per 10 microscopic fields (×20 magnification). The tissue samples were obtained between 1993 and 1999 from autopsies at the First

Institute of Pathology and Experimental Cancer Research of Semmelweis University of Medicine. The design of this study and methods, and the reported data have been approved by the

Institutional Review Board. Tissue blocks were fixed in 10% phosphate-buffered formalin, dehydrated through graded ethanol, and embedded into paraffin. Five-μm-thick sections were serially

cut and mounted on Superfrost Plus (Menzel-Glaser, Freiburg, Germany) glass slides. The processed, sectioned, and deparaffinated tissues were stained with hematoxylin and eosin (Fig. 1).

Sections from the same blocks were prepared for evaluation of apoptosis by applying the TUNEL method (ApopDETEK Cell Death Assay System; Enzo, New York, U.S.A.). The incorporated

deoxynucleotides were detected with a horseradish peroxidase-diaminobenzidine _in situ_ detection system (Simply Sensitive Horseradish Peroxidase-DAB _In Situ_ Detection System; Enzo, New

York, U.S.A.). The ratio of positively labeled pulmonary alveolar and bronchiolar epithelial cells, neural cells, renal tubular cells, and hepatocytes was determined microscopically. In the

lungs and kidneys, apoptotic activity was considered marked (++) if the ratio of TUNEL-positive cells exceeded 30% per terminal airway or per renal tubule. If this ratio was below 30%,

apoptosis was considered mild (+); if no apoptotic cells were found it was negative (-). In liver and brain tissue, the number of TUNEL-positive cells was counted in 10 microscopic fields

using a ×20 objective. Apoptotic activity was considered marked (++) if more than 10 cells were positive; 5–10 TUNEL-positive cells was considered mild activity. RESULTS APOPTOSIS IN

PULMONARY TISSUE OF PRETERM INFANTS. Lung tissue of 24 preterm infants was examined retrospectively. The neonates were born in gestational wk 24–37, their birth weight was 600–2000 g, and

their age at death was 10 h–27 wk. All had clinically proven respiratory distress; ventilation, infection, and medication data are summarized in Table 1. Marked apoptotic activity was found

in nine cases, and apoptosis was present in another eight cases. In seven cases and in the control case this type of cell death could not be detected. The reaction was strong in pyknotic

nuclei of dying cells, lining-expanded terminal sacculi, and in the injured bronchiolar epithelial cells (Fig. 2_A–C_). Cases with the most prominent activity were observed in stages II,

III, and IV. The phenomenon was not seen in the acute-exudative stage of BPD. Apoptotic cells of mesenchymal origin, such as fibroblasts and macrophages in the interstitial, peribronchial,

and perivascular connective tissue were present but not evaluated in this study. APOPTOSIS IN PREMATURE BRAIN. In 16 of the 24 cases clinically diagnosed IVH occurred, and seven patients

suffered from PVL. Routine histologic examination was performed in four cases, three of which showed both IVH and PVL upon postmortem examination. Histologically, disorganization of the

white matter and increased karyorrhexis were detected. A marked apoptotic activity (average 14.2; 15; 10.9 TUNEL-positive cells in 10 microscopic fields using a ×20 objective) could be

observed in damaged areas of the white matter and germinal layer in three cases, respectively (Fig. 3). In one case mild apoptotic activity was detected. APOPTOSIS IN THE KIDNEY AND LIVER OF

PRETERM NEWBORNS. Kidney tissue was examined in seven cases and liver tissue in four cases. In case 8 hematuria was observed and CMV nephritis was diagnosed microscopically. In one case

marked apoptotic activity, and in four cases mild activity was detected with TUNEL reaction in the proximal and distal tubular epithelium. In two cases no apoptotic activity was observed in

the kidneys. In case 2 centrolobular fatty change was present in the liver. In the same areas prominent apoptotic activity (average 13.3 TUNEL-positive cells in 10 microscopic fields using

×20 objective) was observed (Fig. 4). In three cases no apoptotic activity occurred in hepatocytes. DISCUSSION Severe complications of immaturity in preterm infants, such as pulmonary

insufficiency or cerebral IVH, are the most common causes of death in this age group. Great advances in neonatal intensive care have improved survival and led to prolongation of life, but

with an increased incidence of chronic respiratory difficulties, BPD, and disabilities of the CNS. These complications have both clinical and public health importance and are highly

researched territories in pediatric science (1). In spite of this, much remains to study regarding the role of apoptosis in lung and CNS injuries of preterm newborns. Data on the ontogeny,

and the qualitative and quantitative changes in relation to the spatial and temporal occurrence of apoptosis in fetal and postnatal rat lung have previously been reported (2). Apoptosis in

rat and human fetal lungs has been demonstrated as a normal feature of development (3). Antiapoptosis genes, such as survivin and bcl-2, are also present in human fetal tissue as the

regulators of tissue differentiation and contribute to tissue homeostasis during fetal life (4). Apoptosis is a major pathway in the destruction of type 2 pneumocytes in acute lung injury of

adults with clinical symptoms of ARDS. In chronic interstitial pneumonia, however, type 2 pneumocytes show minimal apoptotic activity (5). Endothelial as well as epithelial cells show

apoptosis, which plays a prominent role in lung remodeling after acute injury (6). Although apoptosis is prominent in the lungs of animals injured by inhaling 100% O2, hyperoxia was proven

to kill _in vitro_-cultured epithelial cells via necrosis, not apoptosis (7). Increased expression of stress proteins protects lung cells against endotoxin-mediated apoptosis and oxidant

injury (8). Our findings are supported by data from the literature, which emphasize the role of apoptosis in alveolar and bronchiolar epithelial cells in association with lung injury (5).

The apoptotic activity of the epithelial cells is increased during the early and late subacute phases of lung remodeling and still exists in the chronic lung disease (stage IV of BPD). The

possible causes of this process are multifactorial. The role of hypoxia, oxygen toxicity, steroid administration, lower level of superoxide dismutase, and the toxic effect of viral proteins

in the development of programmed cell death are well known (9, 10). In addition, the role of other factors in association with the prophylaxis and treatment of BPD, such as volutrauma,

surfactant administration, antenatal thyrotropin-releasing hormone administration, and permissive hypercapnia should also be considered in regard to apoptosis. The complications of

prematurity and life-sustaining mechanical ventilation have not only pulmonary but neurologic consequences. Both hypocapnia and hypercapnia may alter neurodevelopmental outcomes, represented

in the brain by severe periventricular echodensities, grade 3 or 4 IVH, or PVL (11). The presence of apoptotic phenomenon in the CNS has already been established. The role of hyperoxia and

inhibition of glutathione synthesis was examined in an animal model, and increased nerve cell death via apoptosis was detected (12). Experimentally induced intrauterine infection in rabbits

was described as the cause of fetal white matter lesions in which apoptosis was demonstrated (13). According to our preliminary results, apoptosis can be detected in white matter lesions of

preterm neonates. In our case, with marked apoptotic activity of neural cells, clinically intrauterine infection with connatal pneumonia, well-developed BPD, and bacterial sepsis were

diagnosed, which led to cerebral IVH and PVL. All of these, plus assisted ventilation, may be held responsible for the high apoptotic ratio in the brain lesions. We also detected programmed

cell death in the liver. The nuclei of several hepatocytes showed typical microscopic evidence of apoptosis. We could not find data in the literature about this phenomenon in the fetal

liver. Renal tubular damage may occur in preterm infants, causing electrolyte imbalance and poor growth (14, 15). Early stages of apoptosis, such as nuclear condensation, margination, and

clumping of chromatin were observed in the nuclei of proximal and excreting tubules; these changes could be the structural background of renal tubular dysfunction. CONCLUSIONS The occurrence

of apoptosis was examined in several organs of preterm neonates with acute and chronic lung disease (BPD). To the best of our knowledge this is the first report on the role of apoptosis in

complications associated with prematurity. We have shown this type of programmed cell death in type 2 pneumocytes in BPD and its importance in the disappearance of these cells during the

late subacute stage of BPD. Apoptosis of mesenchymal cells was also detected in the chronic stage of BPD. Our observations indicate that apoptosis in lesions of the CNS is a major mode of

cell death. Apoptotic features seem to contribute to the evolution of neurodevelopmental deficits and cerebral palsy of preterm infants. In the kidneys apoptosis was observed in tubular

epithelial cells, but further data are needed to clarify the association with tubular dysfunction and intrinsic renal failure. Our results suggest that apoptosis may play a prominent role in

the pathogenesis of several diseases of premature infants, and further investigations will concentrate on the potential clinical applications of our findings. ABBREVIATIONS * IRDS:

infantile respiratory distress syndrome * TUNEL: terminal deoxynucleotidyl transferase-mediated nick end-labeling * IVH: intraventricular hemorrhage * PVL: periventricular leukomalacia *

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_Curr Opin Pediatr_ 9: 148–153 Article  Google Scholar  Download references AUTHOR INFORMATION Author notes * B Hargitai: Beata Hargitai. M.D., Semmelweis University Budapest, First

Institute of Pathology and Experimental Cancer Research, Budapest, Üllõi út 26. H-1085 Hungary; e-mail: [email protected] AUTHORS AND AFFILIATIONS * First Institute of Pathology and

Experimental Cancer Research, Semmelweis University Budapest, Budapest, Üllõi út 26, H-1085, Hungary B Hargitai, V Szabó, P Farid, Z Papp & B Szende * First Department of Obstetrics and

Gynecology, Semmelweis University Budapest, Budapest, Baross u. 27, H-1085, Hungary J Hajdú & Á Harmath * Second Department of Pediatrics, Semmelweis University Budapest, Budapest,

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Hargitai, B., Szabó, V., Hajdú, J. _et al._ Apoptosis in Various Organs of Preterm Infants: Histopathologic Study of Lung, Kidney, Liver, and Brain of Ventilated Infants. _Pediatr Res_ 50,

110–114 (2001). https://doi.org/10.1203/00006450-200107000-00020 Download citation * Received: 29 February 2000 * Accepted: 18 August 2000 * Issue Date: 01 July 2001 * DOI:

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