Cyclosporine and hyperoxia-induced lung damage in neonatal rats

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Respiratory Physiology & Neurobiology 187 (2013) 41– 46

Contents lists available at SciVerse ScienceDirect

Respiratory Physiology & Neurobiology

j ourna l ho me pa ge: www.elsev ier .com/ loca te / resphys io l

eview

yclosporine and hyperoxia-induced lung damage in neonatal rats�

ndrea Porzionatoa,∗, Patrizia Zaramellab, Veronica Macchia, Gloria Sarasina, Camillo Di Giulioc,ntonella Rigonb, Davide Grisafib, Arben Dedjab, Lino Chiandettib, Raffaele De Caroa

Section of Anatomy, Department of Molecular Medicine, University of Padova, ItalyDepartment of Pediatrics, University of Padova, ItalyDepartment of Neurosciences and Imaging, University G. D‘Annunzio, Chieti, Italy

r t i c l e i n f o

rticle history:ccepted 20 February 2013

eywords:ronchopulmonary dysplasiaevelopmentrematurity

a b s t r a c t

Cyclosporine effects on hyperoxia-induced histopathological and functional changes in the rat adultlung are controversial and the newborn lung has not been studied. Thus, we evaluated the effects ofcyclosporine in young rats after 60% hyperoxia exposure postnatally. Experimental categories included:(1) room air for the first 5 postnatal weeks with daily subcutaneous injections of saline from postnatalday (PN)15 to PN35; (2) room air with daily injections of cyclosporine from PN15 to PN35; (3) 60% oxygenfrom PN0 to PN14 and then daily saline injections during the following three weeks; (4) 60% oxygen from

D31PN0 to PN14 followed by cyclosporine treatment from PN15 to PN35. Hyperoxia significantly reducedthe number of secondary crests and microvessel density, and it increased the mean alveolar size andsepta thickness. Cyclosporine treatment did not significantly modify the hyperoxia-induced changes.Conversely, in normoxia, cyclosporine reduced microvessel density and the number of secondary crests.In conclusion, cyclosporine did not modify alveolar and microvascular parameters in hyperoxia exposure,

chang

although it caused some

. Introduction

Cyclosporine is an immunosuppressive drug used in the treat-ent for organ transplantation and autoimmune diseases. It acts

y blocking activation and proliferation of T lymphocyte and bynhibiting their cytokine production. Cyclosporine may also indi-ectly affect B-cell immune response through inhibition of T helperymphocytes (Osadchy and Koren, 2011). Cyclosporine is excretednto human milk and women in treatment with cyclosporine aresually advised not to breastfeed (American Academy of Pediatricsommittee on Drugs, 2001). The literature describes cases ofreast-feeding without adverse effects and with undetectableyclosporine concentrations (Nyberg et al., 1998; Munoz-Flores-hiagarajan et al., 2001), but therapeutic concentrations have also

een observed in infant blood (Moretti et al., 2003). Cyclosporinehows controversial effects on the adult lung, but the effects in therst postnatal period are largely unknown. Moreover, cyclosporineas been reported to show some therapeutic effects on lung

� This paper is part of a special issue entitled “Immunopathology of the Respiratoryystem”, guest-edited by Professor Mietek Pokorski.∗ Corresponding author at: Section of Anatomy, Department of Molecularedicine, University of Padova, Via A. Gabelli 65, 35121 Padova, Italy.

el.: +39 049 8272314; fax: +39 049 8272328.E-mail address: [email protected] (A. Porzionato).

569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.resp.2013.02.018

es in normoxia.© 2013 Elsevier B.V. All rights reserved.

hyperoxic damage in adult experimental animals (Matthew et al.,1999, 2003; Pagano et al., 2004), but there are no reports addressingits possible role in experimental models of bronchopulmonary dys-plasia (BPD). BPD is the most common chronic lung disease ofprematurity. The classic severe form of BPD is strongly relatedwith oxygen toxicity and mechanical injury, whereas the milderforms which are seen nowadays, mainly in small premature infantssurviving after prolonged mechanical ventilation, relate more toimmaturity, perinatal infection and inflammation, persistent duc-tus arteriosus, and disrupted alveolar and capillary development(Bancalari et al., 2003). BPD mainly results in impaired alveo-lar growth and dysmorphic vascular architecture (Thebaud andAbman, 2007). An experimental model of BPD involves exposureto hyperoxia of rats and mice in the early postnatal period. Hyper-oxia disrupts postnatal alveolar development, leading to smallernumbers of enlarged and simplified alveoli, thicker septa, increasednumbers of alveolar macrophages, and changes in microvasculardevelopment (Dauger et al., 2003; Balasubramaniam et al., 2007).Hyperoxia may also cause injury to alveolar type II cells and impairsurfactant phospholipids synthesis and decrease surfactant pro-teins and mRNA (Minoo et al., 1992). The mechanisms of action ofcyclosporine could, theoretically, modify some of these pulmonarychanges. Thus, the aim of the present work was to assess the effects

of postnatal cyclosporine exposure in young rats and, specifically,in a rat model of BPD, to ascertain whether cyclosporine exposureworsens hyperoxia-induced changes or whether it may show sometherapeutic effects.

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. Methods

.1. Materials

Female wild-type Sprague-Dawley rats (Harlan, Udine, Italy)nd their offspring were housed and handled in accordance withhe guidelines of the Helsinki Declaration and the recommenda-ions of the Italian public health authorities. The study had approvalrom the Ethics Committee of the University of Padua for experi-

ents on animals. The study was conducted on male or femaleat pups kept together with their nursing mother in clear polishedcrylic chambers, where oxygen and CO2 were continuously mon-tored (BioSpherix, OxyCycler model A84XOV, Redfield, NY). Thenimals were maintained under standardized conditions of lightalternate 12-hour cycles of light and dark, starting with light ont 08.00 h) at room temperature of 22 ◦C and humidity of 50%.fter term gestation, the pups were randomly distributed between

he following four experimental groups: (1) neonatal rats (n = 7)aised in room air for the first 5 postnatal weeks with subcutaneousaily injection of saline (NaCl 0.9%) from postnatal day (PN)15 toN35 (21%+ Saline); (2) neonatal rats (n = 6) raised in room air withaily subcutaneous injection of 15 mg/kg cyclosporine from PN15o PN35 (21%+ Cyclosporine); (3) neonatal rats (n = 6) raised in 60%xygen from PN0 to PN14 and then given daily injection of salineuring the following three weeks (60%+ Saline); (4) neonatal ratsn = 5) raised in 60% oxygen from PN0 to PN14 followed by dailynjection of cyclosporine from PN15 to PN35 (60%+ Cyclosporine).he nursing dams were rotated every one or two days to preventny negative effects of hyperoxia on nursing. The pups’ body weightas measured daily. On postnatal day 35, the animals were euth-

nized with an overdose of tiletamine-zolazepam (Zoletil®) andylazine (Rompun®). The lungs were then removed in toto and fixedn buffered formalin, serially dehydrated in rising concentrations ofthanol, and embedded in paraffin.

.2. Morphometric analysis

For each rat, two 4-�m sections of the lungs were stainedith hematoxylin and eosin, two sections were stained with

zan-Mallory and two with Weigert–van Gieson staining. Allssessments were conducted with a Leica DM 4000B microscopeLeica, Solms, Germany) integrated with a camera (Leica DFC 280).

orphometric analyses were performed according to Grisafi et al.2012). Briefly, photomicrographs were obtained on a field of68 �m × 422 �m with a LeicaDM4000B microscope (Leica, Solms,ermany) integrated with a camera (Leica DFC 280). Lung morpho-etric analyses were performed by two independent researchers

linded to the treatment strategy, using ImageJ, a public domainava image processing program created by Wayne Rasband athe Research Services Branch, National Institute of Mental Health,ethesda, MD (http://rsb.info.nih.gov/ij). In particular in each sec-ion, from a specific plug-in which leads to the evaluation of thekeletonized air spaces into each high powered field (hpf), the meanlveolar size was evaluated by considering the alveolar minimumnd maximum diameter and excluding the areas of large airwaysr vessels from analysis. A cell counter was applied for assessinghe secondary crests number/hpf.

.3. Immunohistochemistry

Microvessel density was calculated according to Porzionatot al. (2004) and Grisafi et al. (2012) in two 4-�m-thick sec-

ions immunostained with anti-CD31 antibody. Lung sectionsere hydrated gradually and were incubated in 0.03% hydrogeneroxide in deionized H2O, to eliminate endogenous peroxidasectivity and to enhance antibody penetration in the tissue. Antigen

& Neurobiology 187 (2013) 41– 46

unmasking was performed with 10 mM sodium citrate buffer, pH6.0, at 96 ◦C for 30 min. Sections were incubated for 30 min in block-ing serum (0.04% bovine serum albumin (A2153, Sigma–Aldrich,Milan, Italy) and 0.5% normal goat serum (X0907, Dako Corpora-tion, Carpinteria, CA, USA) to eliminate unspecific binding. Sectionswere then incubated for 1 h at room temperature with a mousemonoclonal antibody against CD31 (Dako, Milan, Italy) diluted1:50 in PBS. Primary antibody binding was revealed by incubationwith anti-rabbit/mouse serum diluted 1:100 in blocking serum for30 min at room temperature (DAKO® EnVision + TM Peroxidase,Rabbit/Mouse, Dako Corporation, Glostrup, Denmark) and devel-oped in 3,3′-diaminobenzidine for 3 min at room temperature.Lastly, sections were counterstained with hematoxylin. Sectionsincubated without primary antibodies showed no immunoreactiv-ity, confirming the specificity of the immunostaining. For each case,we examined ten fields per section at a Leica DM 4000B micro-scope (Leica, Solms, Germany) integrated with a camera (LeicaDFC 280). The number of CD31-positive vessels (<100 �m in size)was counted per hpf. The mean values of microvessel density werecalculated for each case and for the entire experimental groups.

2.4. Statistical analysis

Results are expressed as mean values ± SD. Statistical analysiswas performed with one-way anova and Tukey’s multiple com-parison test. A P < 0.05 was considered statistically significant.Statistical calculations were conducted with Prism 3.0.3 (GraphPadSoftware Inc., San Diego, CA).

3. Results

No statistically significant differences in body weight werefound between the different experimental groups. Histopatho-logical examination of the lung sections showed an impairedalveolar development in the two hyperoxic groups in compar-ison with the control animals grown in room air. The distalairspaces were fewer in number and enlarged, with reduced sep-tation. Patchy areas of marked interstitial thickening were alsoappreciable, with increased content of collagen and elastin at azan-Mallory and Weigert–van Gieson stainings. Cyclosporine treatmentin hyperoxia-exposed animals did not significantly modify theabove-mentioned hyperoxia-induced changes in alveolarization.Conversely, in normoxic groups, cyclosporine treatment induceda slight reduction in septation and an increase in the thickness ofthe alveolar septa. Azan-Mallory and Weigert–van Gieson stainingsalso showed a slightly increased content of collagen and elastinin the alveolar septa of normoxic rats treated with cyclosporine(Figs. 1 and 2).

The results of the lung morphometric analysis are shownin Figs. 3 and 4. The mean number of secondary crests perhigh-powered field was higher in the control rats (21%+ Saline:12.7 ± 0.8) than in all the other experimental groups (21%+Cyclosporine: 9.5 ± 0.9, P < 0.05; 60%+ Saline: 8.3 ± 0.8, P < 0.01; and60%+ Cyclosporine: 6.3 ± 0.7, P < 0.001). The mean alveolar sur-face in the rats exposed to hyperoxia and saline (60%+ Saline:1837.0 ± 96.6 �m2) was higher than in both normoxic groups(21%+ Saline: 958.2 ± 64.7 �m2, P < 0.01; 21%+ Cyclosporine:1107.0 ± 224.3 �m2, P < 0.05). Conversely, cyclosporine treatmentwas found not to significantly modify the mean alveolar surfaceafter hyperoxia exposure (60%+ Cyclosporine: 1399.0 ± 310.5 �m2,

P > 0.05). The pulmonary microvessel density was also higher in thecontrol rats (21%+ Saline: 22.9 ± 1.3) than in all the other experi-mental groups (21%+ Cyclosporine: 18.5 ± 1.0, P < 0.05; 60%+ Saline:16.8 ± 0.9, P < 0.01; 60%+ Cyclosporine: 17.6 ± 1.2, P < 0.05).

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Fig. 1. Representative lung sections of the four study groups stained with hematoxylin and eosin, showing impaired alveolar development (enlarged airspaces and areaso treatmt : 60%

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f parenchymal thickening) in hyperoxia-exposed rats irrespective of cyclosporinehickness of alveolar septa. (A: 21%+ Saline; B: 21%+ Cyclosporine; C: 60%+ Saline; D

. Discussion

Hyperoxia exposure has mainly been used in the literatureo develop experimental animal models of bronchopulmonaryysplasia in prematurely born infants; hyperoxia disrupting post-

atal alveolar and microvascular development (Dauger et al.,003; Balasubramaniam et al., 2007). The present study con-rmed the impairment of alveolarization caused by exposure to

ig. 2. Representative lung sections of the four study groups stained with Weigert–vanlastin fibers (arrows) in hyperoxia- and/or cyclosporine-exposed rats. (A: 21%+ Saline; B

ent. In normoxia, cyclosporine also induced reduction of septation and increased+ Cyclosporine). Scale bars: 150 �m.

hyperoxia, resulting in fewer, larger-diameter alveoli with reducedseptation and areas of parenchymal thickening. Morphometricanalyses also showed a reduced number of secondary crests andan increased mean alveolar surface after hyperoxia. Moreover, asignificant reduction in pulmonary microvessel density was also

found, being consistent with hyperoxic damage to angiogenesisand integrity of the vascular network. It is important to stressthat the above hyperoxia-induced changes in alveolarization and

Gieson, showing increased thickness of alveolar septa and an increase content in: 21%+ Cyclosporine; C: 60%+ Saline; D: 60%+ Cyclosporine). Scale bars: 37.5 �m.

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In conclusion, cyclosporine has a potential to adversely affect

ig. 3. Mean values (±SEM) of secondary crests (A) and alveolar surface (B) in theour study groups. *p < 0.05; **p < 0.01; ***p < 0.005.

icrovascularization were still present after the three weeks oformoxic recovery, supporting the chronicity of the histopatho-

ogical modifications.One of the aims of the present study was the evaluation of

yclosporine effects on lung hyperoxic damage of the neonate rat,s in adult mice exposed to hyperoxia, cyclosporine treatmenthows some therapeutic effects and its use has been suggested foratients undergoing prolonged high-oxygen therapy. Pretreatmentnd simultaneous treatment with cyclosporine has been reportedo attenuate the hyperoxia-induced reduction in lung compliance.his was mainly ascribed to increased amount of surfactant pro-ein B in alveolar type II cells and stimulation of surfactant protein

secretion from type II cells. Neutrophil infiltration, capillary con-estion, edema, and hyaline membrane formation have also beeneported to be reduced in mice treated with cyclosporine (Matthewt al., 1999). Immunohistochemical analysis of cyclin B1 expres-ion also showed a decrease in the number of positive type II cellsfter 72 h of >95% hyperoxia and a return to a normal value withyclosporine treatment during hyperoxia (Matthew et al., 2003).yclosporine treatment was also found to prevent cytochrome celease and mitochondrial damage in type II cells of adult ratsxposed to 100% O2 for 72 h (Pagano et al., 2004). In the above study,yclosporine treatment also reduced the hyperoxia-induced lung

amage, consisting of alveolar septa thickening, edema, and hyalineembrane formation. In the present study, however, cyclosporine

reatment did not significantly modify hyperoxia-induced changes

& Neurobiology 187 (2013) 41– 46

in alveolar parameters and microvessel density, revealing neithera therapeutic nor exacerbating effect on hyperoxic changes. Thesedifferent results with respect to the reports on adult animals may beascribed to the different pathogenetic mechanisms of the hyperoxicdamage in adult vs. newborn/infant. Further exploration is requiredto address the effects of pretreatment or simultaneous treatmentwith cyclosporine.

Apart from the above actions on hyperoxia-related changes,some studies also considered the effects of cyclosporine on normallung structures, suggesting some detrimental effects. For instance,cyclosporine is known to induce renal tubular interstitial fibro-sis also through epithelial-mesenchymal transition associated withupregulation of TGFbeta1 (Slattery et al., 2005) and the capabil-ity of cyclosporine to induce bronchial epithelial-mesenchymaltransition has also been investigated. The bronchial epithelial cellline RL-65, after 3–5 day long cyclosporine treatment, has beenreported to increase the expression of transforming growth fac-tor, fibronectin, collagen, and vimentin and to lose E-cadherin atcell membranes. Moreover, the above cells also showed morpho-logical changes consistent with epithelial-mesenchymal transition(Felton et al., 2011). Adult rats daily treated with cyclosporine for8 weeks have been reported to show inflammatory cellular infil-tration of the lung, with bronchiolar associated lymphoid tissuehyperplasia and perivasculitis. These pulmonary histopathologiclesions were ascribed to the activation of the local immunore-sponse mechanisms by opportunistic pathogens (Böhmer et al.,2011). In adult rats, cyclosporine treatment has also been observedto reduce the mucus production from goblet cells and the mucocil-iary transport velocity (Pazetti et al., 2007). In rats, the effects ofcyclosporine treatment during the first three weeks of postnatallife have also been considered. Cyclosporine was found to increasethe DNA, collagen, and elastin content, although the elastin/DNAand collagen/DNA ratios were reduced (Roos et al., 1991). In thepresent study, cyclosporine exposure in normoxic rats from PN15to PN35 produced some changes in the alveolar and microvascularstructures which could be at least partially explained with refer-ence to the above mechanisms. Cyclosporine reduced the numberof secondary crests, although statistically significant changes in thealveolar dimensions were not found. It must be considered that,although in the rat secondary alveolar septa are usually consideredto be mainly formed during the first three postnatal weeks (Randellet al., 1989), a late alveolarization has also been reported, withformation of new secondary crests up to young adulthood (PN60)(Schittny et al., 2008). Moreover, alveolar septa appeared thicker,with increased content of collagen and elastin. These histopatho-logic changes are consistent with the above literature data aboutthe role of cyclosporine in inducing epithelial-mesenchimal transi-tion and in increasing the absolute content of collagen and elastin.In our study, cyclosporine was also found to reduce the microvesseldensity. This finding could indirectly derive from the above increasein the connective component or it could be a consequence of antian-giogenic action of the drug (Sharpe et al., 1989). It is also intriguingthat, in normoxia, cyclosporine induced lung changes which havesome characteristics in common with those induced by hyperoxiaalone. Although some papers report reduced oxidative stress insome tissues after cyclosporine treatment (e.g., Gill et al., 2012),others note enhancement in reactive oxygen species production invarious cell types, such as renal mesangial cells (Han et al., 2006),hepatocytes (Kwak et al., 2002), glial cells (Mun et al., 2008) andbronchial epithelial cells (Jeon et al., 2012). Thus, the cyclosporine-induced lung changes in the present work may have been partlyproduced by generation of oxygen free radicals.

the neonatal lungs. Cyclosporine does not seem to be useful in ther-apy of bronchopulmonary dysplasia in premature infants. Changesin the lungs of neonatal rats exposed to cyclosporine, described

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ig. 4. Representative lung sections of the four study groups immunostained withan in the other experimental groups. (B: 21%+ Cyclosporine; C: 60%+ Saline; D: 60xperimental groups. *p < 0.05; **p < 0.01; ***p < 0.005. Scale bars: 37.5 �m.

n the present study, do not permit to exclude the induction oftructural modifications in breast-fed infants by mothers treatedith cyclosporine. Thus, breast-feeding should be avoided dur-

ng cyclosporine therapy. It must be considered, however, that thetudy has some limitations as, for instance, the administration ofyclosporine directly to the pups and not to the mother. In this sensehis study must be considered a preliminary report which will needurther confirmations. The kind and degree of the specific immuno-ogic modifications in the lung tissue, due to the controversy of theiterature data on the matter, will also have to be addressed.

onflicts of interest

The authors declare no conflicts of interest in relation to thisrticle.

-CD31 antibody, showing higher microvessel density in controls (A: 21%+ Saline)closporine). (E) Mean values (±SEM) of pulmonary microvessel density in the four

Acknowledgements

The authors are grateful to Anna Rambaldo for skillful technicalassistance.

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