Protective effect of growth hormone on neuronal apoptosis after hypoxia–ischemia in the neonatal rat brain

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  Protective effect of growth hormone on neuronal apoptosis after hypoxia–ischemia in the neonatal rat brain
  Protective effect of growth hormone on neuronal apoptosis after hypoxia–ischemia in the neonatal rat brain Dong Hoon Shin a , Eunju Lee a , Jong-Wan Kim b , Bum-Sun Kwon c , Mi Kyung Jung d , YounHee Jee d , Jaehyup Kim a , Su-ryeon Bae a , Young Pyo Chang d, * a  Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea b  Department of Biology, Dankook University, Chonan, South Korea c  Department of Rehabilitation Medicine Dankook University, Chonan, South Korea d  Department of Pediatrics, Dankook University, Anseo-Dong San 29, Chonan 330-714, South Korea Received 25 July 2003; received in revised form 24 September 2003; accepted 25 September 2003 Abstract Recent studieshave shownthat growth hormone (GH) canreduce neuronal loss after hypoxic-ischemic injury (HI)in neonatal and juvenilerat brains. Here, we investigated whether GH exerts its neuroprotective role through an anti-apoptotic effect in neonatal rat brains damagedby severe HI. Gross and histological observations showed that the extent of brain damage was found to be reduced in GH-treated brain at E7after injury. In a terminal transferase-mediated dUTP nick-end-labeling (TUNEL) study, TUNEL-positive apoptotic cells were localizedonly at the damaged region inanimals treated withsaline, which was confirmed by anelectron microscopy. Inan immunohistochemical studywith anti-bcl-2, -bax, -bad, -neuronal nitric oxide synthase (nNOS), -inducible NOS (iNOS) and -endothelial NOS (eNOS) antibodies, weobserved that bax, bad, iNOS and eNOS were elevated in the saline-treated group. This study thus suggests that the protective role of GHagainst HI injury is mediated thorough an anti-apoptotic effect, which offers the possibility of a GH application for the treatment of neonatalHI encephalopathy. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords:  Growth hormone; Hypoxia–ischemia; Nitric oxide synthase; Bcl-2; Terminal transferase mediated dUTP nick end labeling The major role of growth hormone (GH), which issynthesized by somatotrophic cells in the anterior pituitarygland, is to stimulate the postnatal growth of the skeletonand of soft tissue. Moreover, there is growing evidence thatGH might influence the functions of the central nervoussystem (CNS), like other neurotrophins [12,13]. Such a relation could explain the presence of growth hormonereceptor (GHR) in various types of CNS cells, such asneurons, astrocytes, oligodendrocytes, and in microglia inthe neonatal rat brain [5,9,12].Other studies have shown that GH administration mayinhibit neuronal death during perinatal hypoxia–ischemia(HI) damage, and that GH may have a neuroprotective effectin the cerebral cortex, hippocampus and thalamus [8,17]. Inview of the fact that apoptosis occurs frequently in theimmature brainsubjected to mild to moderate HI injury [3,7,20],wereasonedthattheinvolvementofGHintheinhibitionof apoptosismay be ofimportance [8,11,14,16,17],althoughthe mechanisms of the role of GH against HI injury have notbeen fully explained. Thus, we investigated whether theneuroprotectiveeffectofGHduringHIinjuryintheneonatalrat brain is due to an anti-apoptotic effect of GH.The experiments were performed according to the GuidefortheCareandUseofLaboratoryAnimals(NIHpublicationNo. 86-23, 1985 edition). Unilateral common carotid arteryocclusion combined with hypoxia was used to administer HIdamage in the neonatal rat brain. Briefly, after the rightcommon carotid artery of a postnatal 7-day-old Sprague–Dawley rat had been cut between sutures ligated with 4/0surgical silk, animals were exposed to a gas mixture of 6%oxygen and 94% nitrogen in an airtight, humidified chamberat 37  8 C for 2 h. Pups were killed 3, 5, and 7 days after theadministration of HI injury. Half of the pups ( n ¼ 15, theGH-treated group) received three subcutaneous injections of recombinant human growth hormone (50 mg/kg, Grotropin,Dong-Ah Pharmacy Co., Seoul, South Korea) just before HIand at 12 and 36 h after HI. The other pups ( n ¼ 15, thesaline-treated group) were used as controls, and receivedsubcutaneousinjectionsofthesameamountofsaline.Tissuepreparation for immunohistochemistry was performed as Neuroscience Letters 354 (2004) 64–$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.neulet.2003.09.070* Corresponding author. Tel.: þ 82-2-740-8203; fax: þ 82-2-6230-9160. E-mail address: (Y.P. Chang).  previouslyreported[18].The general morphology ofcontrol and GH-treated tissues was evaluated with the hematoxylinand eosin (H&E) staining method [1,19]. Terminal transfer- ase-mediated dUTP nick-end-labeling (TUNEL) stainingwas performed using a TUNEL Apoptosis Detection Kit(Upstate Co., USA), according to the manufacturer’sinstructions, to detect the presence of apoptotic cells inbrain tissue. To observe apoptotic cells under an electronmicroscope (JEOL 1200 EX-II), the tissues were post-fixed,dehydratedandembeddedinEpon812(EMS,FortWashing-ton, PA). Ultrathin sections were cut and mounted on nickelgrids coated with Formvar film.For immunohistochemistry, the following primary anti-sera were used; rabbit anti-bcl-2 (1:300, Santa Cruz, CA,USA), rabbit anti-bax (1:300, Santa Cruz), rabbit anti-bad(1:300, Santa Cruz), mouse anti-neuronal nitric oxidesynthase (nNOS, 1:1000, Chemicon, CA, USA), mouseanti-inducible NOS (iNOS, 1:1000, Chemicon) and rabbit Fig. 1. The general morphologies of saline-treated and growth hormone(GH)-treated rat brains. (A,B) GH- and saline-treated brains at 3 days afterhypoxia–ischemia (HI) injury, respectively. (C,D) GH- and saline-treatedbrains at 7 days after HI injury, respectively. A and B showed fewmorphologic differences 3 days after HI injury. However, while the GH-treated brains (C) seemed apparently intact 7 days after HI injury, thesaline-treated brains (D) showed more severe cortical atrophy (rectangle) inthe hemisphere damaged by HI injury. (E,F) Sections of GH- and saline-treated brains (7 days after HI injury) stained by hematoxylin and eosin(H&E). While the cerebral cortex (Cx) in E showed no change after HIinjury, the same region in F showed severe atrophy. H, hippocampus. (G,H)H&E-stained sections of GH and saline-treated brains. GH-treated sections(G and right inset in G) exhibited a normal cellular appearances, as shownby a section of undamaged brain tissue (left inset in G). On the other hand,most of the cells in the saline-treated brains (H) showed pyknosis of nuclearchromatins (blank arrows in H). In some cases, minute specks of pyknoticchromatin (arrows in H) were observed within the cells (inset of H). (I)Thickness of Cx of GH (GH)- and saline-treated (saline) brains. (J) Thenumber of pyknotic cells within the Cx of GH-treated, normal and saline-treated brain. Scale bars: G and H, 50  m m; insets of G and H, 15  m m.Fig. 2. Terminal transferase-mediated dUTP nick-end-labeling (TUNEL)staining (A–F). (A) Diagram showing the orientation of micrographs. Thehemisphere indicated by the arrows was affected by unilateral commoncarotid artery occlusion. All TUNEL-positive signals were detected in theaffected hemisphere. (B) In the cerebral cortex of the saline-treated group,many TUNEL-positive cells were detected. (C) Magnified image of TUNEL-positive cells in B. (D) Hippocampus and (E) Thalamus of saline-treated animals also showed weaker TUNEL-positive signals than those of the cerebral cortex.(F) Thecerebral cortexin the affectedhemisphereof theGH-treated brain showed no TUNEL-positive signals. In addition to thisregion, the hippocampus and thalamus showed no TUNEL-positive signals.(G) Mean densities of apoptotic signals in the cerebral cortex (Cx),hippocampus (Hi) and thalamus (Th). Filled and hatched bars indicate theGH- and saline-treated groups, respectively. (H–J) Electron microscopicobservations. (H) Most of the cells in the GH-treated rat brain showed noremarkable ultramicroscopic structural change. Nu, nucleus. (I,J) In thesaline-treated rat brain, cells showed the typical apoptotic patterns of compaction and segregation of chromatin lying against the nuclearenvelope (asterisks in J), and of discrete nuclear fragments containingcompacted chromatin (arrows in I). Scale bars: A–F, 50  m m; H–J, 2  m m.  D.H. Shin et al. / Neuroscience Letters 354 (2004) 64–68  65  anti-endothelialNOSantibodies(eNOS,1:1000,Chemicon).Tissue sections were incubated sequentially for 1 h in eachof: (1) primary antiserum overnight at 4  8 C; (2) biotinylatedanti-rabbit or anti-mouse IgG (1:500, Vector, CA, USA) atroomtemperature;(3)Cy-3conjugatedstreptavidin(1:1000,Jackson Immunoresearch, PA, USA) at room temperature.The sections were observed under a Zeiss Axiophotphotomicroscope with a fluorescence attachment.Densitometry was performed using an NIH imageprogram (Scion Image, MD, USA). All densitometric dataare expressed as mean densities, defined as the sum of thegray values of all pixels in a selection divided by the numberof pixels.At 3 days after HI, the general appearances of the GH andsaline-treated brains were similar (Fig. 1A,B). However, at7 days after HI injuries, even though saline-treated groupsshowed remarkable damage to the hemispheres injured bythe unilateral occlusion of common carotid artery, the GH-treated groups did not show any changes in their appearance(cf. Fig. 1C vs. D). Differences between the morphologies of the GH- and saline-treated groups were also remarkable inthe cross sections. GH-treated brain (Fig. 1E) did not show Fig. 3. The sections of (A) GH-treated and (B) saline-treated cerebral cortex 5 days after HI injury. This immunohistochemical study was performed using bcl-2, bax, bad, nNOS, iNOS and eNOS antibodies. In the GH-treated cerebral cortex, lower levels of bax, bad, iNOS and eNOS immunoreactivities wereobserved. (C) The mean densities of bcl-2 family protein IRs in the GH-treated (filled bars) and saline-treated (hatched bars) cerebral cortices. (D) Mean IRdensities of NOS family proteins in GH-treated (filled bars) and saline-treated (hatched bars) cerebral cortices. Scale bars: 50  m m.  D.H. Shin et al. / Neuroscience Letters 354 (2004) 64–68 66  any changes while severe cortical atrophy was observed inthe damage affected sides of the saline-treated brains (c.f.Fig. 1E vs. F). In the light microscopic study of H&E-treated sections at 7 days after HI, the cells in both groupsshowed marked morphologic differences after HI injury.Cells in GH-treated sections showed few abnormal findings(Fig. 1G), whereas cells in the cerebral cortex of saline-treated animals showed pyknosis of nuclear chromatins,which is a characteristic of cell death (Fig. 1H). Thethicknesses of cerebral cortices, which reflect the degree of cortical atrophy, are summarized in Fig. 1I, and differencesin the numbers of pyknotic cells are summarized in Fig. 1J.In terms of the TUNEL staining used to detect thepresence of apoptotic cells (Fig. 2A–F), intensely stainedcells were observed in the cerebral cortex (Fig. 2A–C), andless staining was observed in the hippocampus (Fig. 2D) andin the thalamus (Fig. 2E) of saline-treated animals.However, it was not easy to detect TUNEL-positive cellsin GH-treated rat brain (Fig. 2F). The quantitative data forTUNEL study are summarized in Fig. 2G. The inhibitingeffect of GH on neuronal apoptosis was further supported byan electron microscope study because neurons in GH-treated sections did not exhibit any ultramicroscopicabnormalities (Fig. 2H). On the other hand, comparablesaline-treated sections showed apoptotic characteristics, the compaction of chromatin lying against the nuclearenvelope (Fig. 2I), the segregation of chromatin and theconvolution of the nuclear and cellular envelopes (Fig. 2J).Immunohistochemistry was performed in sections of GHand saline-treated brain tissues 5 days after HI injury, todetermine if the inhibition of apoptosis observed during GHtreatment was due to changes in the expressions of bcl-2family proteins. Though similar expression patterns of bcl-2IRs were observed both in GH (Fig. 3A) and saline-treated(Fig. 3B) brains, intense bax and bad immunoreactivities(IRs) were detected only within the cerebral cortices of thesaline-treated animals (cf. Fig. 3A vs. B). The meandensities of the IRs of bcl-2 family proteins are summarizedin Fig. 3C. Immunohistochemistry for NOS proteins in GH- treated (Fig. 3A) and saline-treated (Fig. 3B) sections at 5 days after HI injury showed that higher iNOS and eNOS IRswere observed in saline-treated tissues. On the other hand,nNOS IR was similar in the two groups of GH and saline-treated brains (cf. Fig. 3A vs. B). Quantitative results aresummarized in Fig. 3D.Recently, the protective role of GH after HI brain injuryhas become an important research issue [8,16,17]. Althoughprevious studies suggest that exogenous GH treatmentmight induce a neuroprotective pathway, these suggestionswere based only on observations of reductions in the extentof brain edema or of the extent of neuronal loss after GH hadbeen treated to HI-injured brains [8,17]. Therefore, we undertook the present study to determine whether theneuroprotective mechanism of GH is mediated by thesuppression of apoptosis related protein expressions after HIinjury.In TUNEL and electron microscopic studies, which arerecognized as the most effective tools for the detection of apoptosis in various tissues, no apoptotic neurons werefound in the GH-treated group, whereas the saline-treatedgroup showed profuse numbers of apoptotic cells in the HIinjured region. These findings suggest the possibility thatGH treatment could protect neurons from HI injury byinhibiting apoptosis. Immunohistochemistry for bcl-2, baxand bad showed that bcl-2 IRs were equally elevated in bothgroups, whereas bax and bad were up-regulated only insections of saline-treated animals, which is of some asrelevance as bax and bad accelerate apoptosis and bcl-2protects against apoptosis [2,6,10,15]. Here, the neuropro-tection afforded by GH seemed to be mediated by the down-regulation of the IRs of bax and bad, not by changes in bcl-2expression. In addition, we observed that iNOS and eNOSlevels were elevated in the saline-treated control sections,but that nNOS IR was similar in the two groups.In the brain HI model, some believe that nNOS and iNOScorrelate with neurotoxic effect by release of nitric oxide(NO) under uncontrolled circumstances whereas eNOSaffects on neuroprotective effect [4]. Accordingly, we think that the presumed neuroprotective effect of GH might is theresult of iNOS inhibition because GH did not affect nNOSand eNOS expressions. However, the issue requires furtherconsideration because the neurotoxic or neuroprotectiveroles of the NOS family proteins remain unresolved untilnow.Taken together, we provide the first evidence that GHtreatment following HI injury has a neuroprotective effect,and that this is probably achieved by modulating theexpressions of apoptosis related proteins. References [1] G. Anderson, K.C. Gordon, Tissue processing, microtomy andparaffin sections, in: J.D. Bancroft, A. Stevens (Eds.), Theory andPractice of Histological Techniques, Churchill Livingstone, NewYork, 1996, pp. 47–68.[2] A. Basu, S. Haldar, The relationship between BcI2, Bax and p53:consequences for cell cycle progression and cell death [review], Mol.Hum. Reprod. 4 (1998) 1099–1109.[3] E.J. Beilharz, C.E. Williams, M. Dragunow, E.S. Sirimanne, P.D.Gluckman, Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis duringselective neuronal loss, Brain Res. Mol. 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