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ltered nucleolar morphology in substantia nigra dopamine neurons following-hydroxydopamine lesion in rats

ichelle Healy-Stoffel a, S. Omar Ahmadb, John A. Stanfordc, Beth Levanta,∗

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USADepartment of Occupational Therapy, Doisy College of Health Sciences, 221 N. Grand Blvd., St. Louis University, St. Louis, MO 63103, USADepartment of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA

i g h l i g h t s

Unilateral intrastriatal 6-OHDA lesions in rats were analyzed with stereology and TH-AgNOR stain.SNpc TH-AgNOR+ nucleolar volume was decreased by 16%.There was no change in the ratio of nucleolar volume to neuronal volume in lesioned SNpc neurons.SNpc TH+ planimetric volume, neuronal number and body volume were decreased after 6-OHDA lesions.

a r t i c l e i n f o

rticle history:eceived 22 January 2013eceived in revised form 30 March 2013ccepted 24 April 2013

eywords:ilver nucleolar stain-Hydroxydopaminetereologyubstantia nigra pars compactaeuronal morphology

a b s t r a c t

The nucleolus, the site of ribosomal ribonucleic acid (rRNA) transcription and assembly, is an importantplayer in the cellular response to stress. Altered nucleolar function and morphology, including decreasednucleolar volume, has been observed in Parkinson’s disease; thus the nucleolus represents a potentialindicator of neurodegeneration in the disease. This study determined the effects of a partial unilateralintrastriatal 6-hydroxydopamine (6-OHDA) lesion, which models the dopaminergic loss found in Parkin-son’s disease, on the nucleoli of dopaminergic cells in the substantia nigra pars compacta (SNpc). Adultmale Long–Evans rats underwent unilateral intrastriatal infusion of 6-OHDA (12.5 �g). Lesions were ver-ified by amphetamine-stimulated rotation 7 days later, and rats were euthanized 14 days after infusion.Coronal sections (50 �m) were stained for tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain usingMultiBrain Technology (NeuroScience Associates), which resulted in clearly defined nucleoli and neuronal

ucleolar volume outlines. Stereological methods were used to compare dopaminergic morphology between lesioned andintact hemispheres in each rat. In cells exhibiting a definable nucleolus, nucleolar volume was decreasedby 16% on the ipsilateral side. The ipsilateral SNpc also exhibited an 18% decrease in SNpc planimetric vol-ume, a 46% decrease in total TH-positive neuron number, and an 11% decrease in neuronal body volume(all P < 0.05 by paired t-test). These findings suggest that the 6-OHDA lesion alters nucleolar morphologyand that these changes are similar to those occurring in Parkinson’s disease.

. Introduction

Please cite this article in press as: M. Healy-Stoffel, et al., Altered nucleohydroxydopamine lesion in rats, Neurosci. Lett. (2013), http://dx.doi.org/1

The nucleolus is a likely participant in the neurodegenerativerocess of Parkinson’s disease. In addition to ribosomal ribonucleic

Abbreviations: rRNA, ribosomal ribonucleic acid; 6-OHDA, 6-hydroxydopamine;OR, nucleolar organizing region; AgNOR, silver nucleolar; SNpc, substantia nigraars compacta; TH, tyrosine hydroxylase; TH-AgNOR, tyrosine hydroxylase-silverucleolar; VTA, ventral tegmental area.∗ Corresponding author at: Department of Pharmacology, University of Kansasedical Center, Mail Stop 1018, 3901 Rainbow Blvd., Kansas City, KS 66160, USA.

el.: +1 913 588 7527; fax: +1 913 588 7501.E-mail addresses: mstoffel@kumc.edu (M. Healy-Stoffel), sahmad13@slu.edu

S.O. Ahmad), jstandford@kumc.edu (J.A. Stanford), blevant@kumc.edu (B. Levant).

304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.neulet.2013.04.033

© 2013 Elsevier Ireland Ltd. All rights reserved.

acid (rRNA) transcription and assembly, the nucleolus is involved indirecting the cellular response to stress [3], thus implicating it in theprocess of neurodegeneration. Notably, altered postmortem nucle-olar size and nucleolar damage have been observed in Parkinson’s,as well as in several other neurodegenerative diseases [13,20,24].Despite the importance of the nucleolus in neuronal function, theexact mechanisms of its dysfunction in degenerating neurons arestill poorly understood, justifying the need for better knowledgeof the roles of this influential organelle in neurodegenerative dis-ease.

lar morphology in substantia nigra dopamine neurons following 6-0.1016/j.neulet.2013.04.033

The partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) model of Parkinson’s disease destroys SNpc neurons byneurotoxic oxidative stress mechanisms [9,17]. In contrast tomore extensive dopaminergic ablation models, this method causes

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oderate SNpc neuronal loss resembling that found in early Parkin-on’s [2,6,9,15,17]. In addition to decreases in neuronal number,orphological changes to dopaminergic neurons and their nucleoli

fter 6-OHDA may also reflect the processes occurring in Parkin-on’s disease [10,13,19]. Nucleolar function has been associatedith nucleolar size; [3] hence a decrease in nucleolar volume

n the lesioned SNpc could reflect a loss of nucleolar functionfter 6-OHDA lesion, and thereby model the processes involved inarkinson’s disease. Design-based stereology is a well-establishedethod for obtaining robust data, and has been extensively used to

etermine neuronal number and morphology of the SNpc in Parkin-on’s disease and in animal models [1,4,7,8,15,25,26]. Accordingly,his study employed the tyrosine hydroxylase-silver nucleolar (TH-gNOR stain), which combines TH staining for catecholaminergiceurons with silver-binding nucleolar AgNOR staining [12], andtereological analysis techniques to determine the effects of a par-ial unilateral 6-OHDA model on the SNpc TH-positive neuronal anducleolar morphology in adult male Long–Evans rats.

. Materials and methods

All experiments were performed in compliance with the NIHuide for the Care and Use of Animals and were approved by theniversity of Kansas Medical Center Institutional Care and Useommittee.

.1. Animals, 6-OHDA lesion procedures and lesion validation

Animal husbandry protocol and experimental surgical andesion validation procedures are previously described in detail [12].riefly, male Long–Evans rats (90–100 days old) (n = 11) were giveningle-site unilateral intrastriatal microinjections with 6-OHDA12.5 �g in 5 �l). Amphetamine-stimulated rotation was assessed 7ays later. After an additional 7 days to allow clearance of drug [>10

Please cite this article in press as: M. Healy-Stoffel, et al., Altered nucleohydroxydopamine lesion in rats, Neurosci. Lett. (2013), http://dx.doi.org/1

1/2s, tissue t1/2 = 5–9 h for the elimination phase [18], rats wereuthanized by transcardial perfusion under pentobarbital anes-hesia. Brains were extracted and fixed as previously described12].

ig. 1. High-power and low-power micrographs of TH-AgNOR stained sections of contrf contralateral and ipsilateral SN qualitatively show depletion of the lesioned dopaminpsilateral neurons and the AgNOR-stained nucleoli (C and D). Representative sections fro

PRESSe Letters xxx (2013) xxx– xxx

2.2. Histological analysis

2.2.1. TH-AgNOR stainingBrains were sent to NeuroScience Associates (Knoxville, TN) for

embedding, sectioning and staining. Brains were prepared usingMultiBrain® Technology, in which multiple brains are embeddedwithin gelatin blocks, providing uniform exposure of the brainsto sectioning and staining conditions. Free-floating 50 �m sec-tions were stained with a modified TH-AgNOR stain as previouslydescribed [27], and mounted on gelatinized (subbed) glass slides.

2.2.2. StereologyEvery sixth section containing SNpc was selected for stereolog-

ical quantitation (Bregma −4.70 to −6.30 mm). TH-AgNOR-stainedcells were quantified using the Microbrightfield Stereoinvestigatorsoftware package combined with a Nikon Eclipse TE2000-U micro-scope coupled to a Heidenhein linear encoder unit and a QImagingRetiga-2000R color digital video camera. Using an atlas [22], theborders of the entire SNpc were carefully outlined in rostrocaudalsections at 4× magnification to exclude the pars reticulata, pars lat-eralis, pars medialis, and the ventral tegmental area (VTA). Seriescharacteristically contained between 5 and 7 slides per animal.The variations in brain positioning in the MultiBrain® embed-ding process provide a randomized sectioning start for each brainquantified. Cells were counted and volumes measured at 100×magnification using the simultaneous application of the opticalfractionator and a double nucleator method. The nucleus was usedas the unique marker for each neuron. The double nucleator wasthen employed by automatic software placement of four randomlyoriented crossed rays centered at the counting point, and four dis-criminately placed markers on the outside borders of the nucleolusand the outline of the cell body, respectively (Fig. 2). In some neu-rons (mean 20.1 ± 1.5% on the ipsilateral side and 17.4 ± 2.3% onthe contralateral side, with no significant difference between thetwo by paired t-test [P = 0.342]) the nucleolus was visible withinthe nucleus, but dark staining of the nucleus made it impossible to

lar morphology in substantia nigra dopamine neurons following 6-0.1016/j.neulet.2013.04.033

accurately distinguish the borders of the nucleolus for placement ofthe nucleator probe. These neurons were included in the total cellcount, but were not included in the volumetric analyses becauseit was not possible to employ the double nucleator technique. The

alateral and ipsilateral substantia nigra. 4× images of TH-AgNOR-stained sectionsergic regions (1A and B). 100× images show the outlines of the contralateral andm the same rat are shown.

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Fig. 3. Effects of unilateral intrastriatal 6-OHDA lesion on TH-AgNOR-stained plani-metric volume (A), neuron number (B) and neuron volume (C) in the SNpc. All ratsshowed decreased SNpc planimetric volume between the ipsilateral and contralat-eral hemispheres. The mean within-subject decrease was 18.3 ± 2.2% (P < 0.05 bypaired t-test, n = 11). All rats showed decreased neuronal number between the ipsi-lateral and contralateral SNpc. The mean within-subject decrease was 45.7% ± 5.1%

ig. 2. The double nucleator technique. Markers are placed on the nucleolus centernd where the nucleator rays intersect with the borders of the nucleolus and theeuronal body outline.

ean measured thickness was 31.2 ± 0.4 �m. A maximum coeffi-ient of error of 0.15 (m = 1) was accepted for all results [11].

.2.3. Data analysisData are presented as the mean ± S.E.M. The effects of 6-OHDA

etween the ipsilateral and contralateral hemispheres were testedor statistical significance using paired t-tests (SigmaPlot v.11.0).

ithin-subject changes in any parameter were expressed as aercentage relative to the contralateral side. Correlation between-amphetamine-stimulated rotational behavior and percent ofeuronal loss was determined by linear regression and significanceas tested by the Pearson product–moment correlation (SigmaPlot,

.11.0).

. Results

.1. Amphetamine-stimulated rotation

The mean number of amphetamine-stimulated rotations com-leted by lesioned rats during the 60-min observation period was54 ± 47. The relationship between rotation and percent of SNpceuronal was poorly correlated (r2 = 0.135).

.2. Stereological analysis

Low-and high-power photomicrographs show the morphologyf the ipsilateral and contralateral SNPC TH-positive neurons. Low4×) magnification demonstrates the loss of TH-positive neuronsypical of neurodegeneration of the lesioned SNpc (Fig. 1A and B).igh-power (100×) magnification (Fig. 1C and D) shows the out-

ines of nucleolar bodies, which are heavily pigmented comparedo the surrounding cytoplasm.

.3. TH-AgNOR-stained SNpc region volume

Regional planimetric volume of the ipsilateral SNpc was smallern all rats compared to the contralateral SNpc (Fig. 3A). The mean

ithin-subject decrease in planimetric volume between the ipsi-ateral and contralateral SNpc was 18.3 ± 2.2% (P < 0.05).

Please cite this article in press as: M. Healy-Stoffel, et al., Altered nucleohydroxydopamine lesion in rats, Neurosci. Lett. (2013), http://dx.doi.org/1

.4. TH-AgNOR-stained SNpc neuron number

The mean total number of TH-positive SNpc neurons was631 ± 355 on the ipsilateral and 9062 ± 781 on the contralateral

(P < 0.05 by paired t-test, n = 11). All rats showed decreased neuronal volumebetween the ipsilateral and contralateral SNpc. The mean within-subject decreasewas 11.2 ± 3.1% (P < 0.05 by paired t-test, n = 11).

sides (Fig. 3B), which is comparable with other stereological stud-ies [1,4,7,10,15]. All rats had decreased TH-positive cells betweenthe ipsilateral and contralateral SNpc, with a mean within-subjectdecrease of 45.7% ± 5.1% (P < 0.05).

3.5. TH-AgNOR-stained SNpc neuron volume

Average neuronal body volume was smaller in all rats in theipsilateral SNpc compared to the contralateral SNpc (Fig. 3C), witha mean within-subject decrease of 11.2 ± 3.1% (P < 0.05).

3.6. TH-AgNOR-stained SNpc nucleolar volume

lar morphology in substantia nigra dopamine neurons following 6-0.1016/j.neulet.2013.04.033

Average nucleolar volume was smaller in all rats in the ipsi-lateral SNpc compared to the contralateral SNpc, with a meanwithin-subject decrease of 16.4 ± 2.4% (P < 0.05) (Fig. 4A). However,the ratio of nucleolar volume to neuronal volume between the

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Fig. 4. Effects of unilateral intrastriatal 6-OHDA lesion on TH-AgNOR-stained nucle-olar volume in the SNpc. All rats showed decreased nucleolar volume betweenthe ipsilateral and contralateral SNpc (A). The mean within-subject decrease was1rc

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6.4 ± 2.4% (P < 0.05 by paired t-test, n = 11). The ratio of nucleolar volume to neu-onal volume in the ipsilateral SNpc was smaller in only 6 of the 11 rats theontralateral SNpc and there was no significant difference between sides (B).

psilateral and contralateral SNpc was smaller in only 6 of the 11ats and there was no significant difference between sides (Fig. 4B).

. Discussion

This study determined the effects of unilateral 6-OHDA neuro-oxic lesion on SNpc neuronal number, soma volume and nucleolar

orphology using stereology and TH-AgNOR staining. A functionalopaminergic deficit was confirmed in the ipsilateral striatum bymphetamine-stimulated rotations. The single-site injection of 6-HDA (12.5 �g) resulted in an 18% decrease in the planimetricolume of the TH-positive ipsilateral SN and a 46% depletion ofNpc TH-positive neurons (Fig. 3A and B). Appearance of clini-al signs in PD has been associated with SNpc neuronal losses ofround 50% [21] thus, cell loss of this magnitude reflects a clin-cally relevant model of moderate Parkinson’s. Neuronal volumeoss was 11% with a corresponding 16% decrease in nucleolar vol-me on the ipsilateral side. Overall, these findings suggest that theartial unilateral 6-OHDA lesion achieved a moderate depletion ofNpc neurons comparable to the threshold findings in parkinsonianuman brains.

Using the TH-AgNOR stain, the present data show a 16% decreasen nucleolar volume accompanied by an 11% decrease in neuronalody volume (Fig. 3C). These decreases in nucleolar and neu-onal size support morphological findings observed in postmortemarkinson’s brains [20,25]. This supports the validity of the 6-OHDAesioned rat as model of the early stages of parkinsonian neurode-

Please cite this article in press as: M. Healy-Stoffel, et al., Altered nucleohydroxydopamine lesion in rats, Neurosci. Lett. (2013), http://dx.doi.org/1

eneration, at least with respect to these parameters.Altered morphology is an informative tool in documenting the

hanges caused by the neurodegenerative process to neuronalnd nucleolar volume. Quantitation of morphological changes is

PRESSe Letters xxx (2013) xxx– xxx

an important first step in establishing differences between neu-rons, which can then be further explored with complementaryfunctional or biochemical assays to determine the mechanismsof change. The volumetric changes that occurred in this studycould be due to either neurodegeneration on the ipsilateral side,or to compensation-related hypertrophy on the contralateral side,since striatal sprouting after unilateral brain lesions has been pro-posed as a compensatory mechanism [5,8]. In future studies, itwould be interesting to determine the neuronal and nucleolarvolume in additional unlesioned rats for comparison; however,another study using a unilateral intrastriatal 6-OHDA (8 �g) andsolvent-injected control animals using stereological analysis foundno difference in contralateral TH-positive SNpc soma volumesbetween the 6-OHDA- and solvent-injected animals [10]. Nucle-olar size is associated with nucleolar activity [3], thus the 16%change in nucleolar volume in the ipsilateral hemisphere (Fig. 4A)may reflect a change in nucleolar function and provide a valuableindex of neuronal damage. Other changes to nucleolar morphol-ogy, such as nucleolar segregation and disruption, are associatedwith various cellular stressors, including DNA damage, hypoxia,viral infection, and nutrient stress [3]. Likewise, the stressors poten-tially involved in the etiology of Parkinson’s could also cause alterednucleolar morphology. Altered nucleolar morphology has beenreported in clinical Parkinson’s disease, as the volume of nucleoliwas decreased in post-mortem Parkinson’s brains by 16% [20]. Sinceneuronal and nucleolar function are closely interrelated, however,it may be more relevant to consider neuronal and nucleolar vol-ume together than separately. Indeed, in our study there was nodifference in the percentage of the nucleolus to total neuronalvolume in the ipsilateral and contralateral SNpc (Fig. 4B), suggest-ing a concomitant decrease of both neuronal body and nucleolusthat may indicate their interdependent morphology. Althoughthe morphological relationship between the nucleolar and neu-ronal body volumes in clinical Parkinson’s is not currently known,stereological assessment of neuronal and nucleolar volumes inpostmortem brains from Alzheimer’s patients showed both neu-ronal and nucleolar atrophy in the CA1 region of the hippocampus[14], implicating decreased nucleolar volume in neurodegenerativedisease.

How nucleolar morphology reflects the neurodegenerationin Parkinson’s disease is still uncertain, although there areseveral potential mechanisms for nucleolar damage and con-sequent morphology changes in Parkinson’s. Greater nucleolardamage to postmortem Parkinson’s neurons, assessed by lossof nucleolar integrity, has been found relative to controls [24].Oxidative damage to RNA has been a suspected factor in a num-ber of neurodegenerative diseases, including Parkinson’s [16],and may play a role in nucleolar damage. For example, inmice, 1,2,3,6-tetrahydro-1-methyl-4-phenylpyridine hydrochlo-ride (MPTP) treatment induced nucleolar damage marked bynucleophosmin staining localization to the cytoplasm and inhibitedmammalian target of rapamycin (mTOR) signaling, a regulator ofrRNA synthesis. [24]. The transcription initiation factor 1A (TIF-1A) regulates the nucleolus-specific RNA polymerase 1 (Pol1), andTIF-1A ablation in mouse embryonic fibroblasts results in nucleolardisruption and in upregulation of tumor-suppressing protein p53[28]. Adult mice with selective dopaminergic neuron ablation ofTIF-1A demonstrated progressive loss of SN neurons and locomotordeficits [24]. Nucleolar damage in Parkinson’s could also be pre-cipitated by DNA damage, as the DNA-topoisomerase-2 inhibitoretoposide inhibited Pol1 and induced nucleolar stress indicatedby staining for B23/nucleophosmin [23]. Future studies must

lar morphology in substantia nigra dopamine neurons following 6-0.1016/j.neulet.2013.04.033

determine specifically how nucleolar damage and correspondingnucleolar morphology changes contribute to the pathophysiologyof Parkinson’s disease and whether nucleolar morphology mayprove useful as an indicator of disease progression.

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In conclusion, TH-AgNOR staining combined with stereologi-al assessment indicated altered nucleolar morphology of SNpcopaminergic neurons in rats after a partial unilateral intrastriatal-OHDA. The observed decreased nucleolar volume suggests thathis organelle plays a critical role in neurodegenerative processesnd possibly also in the early clinical course of the Parkinson’sisease.

cknowledgements

The authors thank Drs. Robert Switzer and Stan Benkovic of Neu-oScience Associates for preparing the AgNOR stained sections andacilitating our use of this staining technique in this study. Sup-orted by NIH Grants NS067422 (BL), RR016475 (BL), HD02528BL, JAS), the Institute for Advancing Medical Innovation (MH-S),he Mabel A. Woodyard Fellowship in Neurodegenerative Disor-ers (MH-S), the University of Kansas Endowment (MH-S), andhe University of Kansas Medical Center Institute for Neurologicaliscoveries (MH-S).

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