Effectof Nuclei from Donors of Different Ages into ...downloads.hindawi.com/journals/np/1993/732456.pdfBethesda,MD20892-0008,USA INTRODUCTION The technique of transplantation was successfully

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Effect of Transplanting Suprachiasmatic Nuclei from Donors ofDifferent Ages into Completely SCN Lesioned Hamsters

Claire M. Kaufman and Michael Menaker

NSF Centerfor Biological Timing, Department ofBiologyUniversity of Virginia, Charlottesville, VA 22903, USA

ABSTRACT

The suprachiasmatic nucleus (SCN) is theprimary circadian pacemaker in mammals.Ralph and colleagues/14/provided recent newevidence for this by transplanting SCNs betweengolden hamsters with different geneticallydetermined periods and producing circadianrhythms of running wheel activity with periodscharacteristic of the donor. We have extendedthese studies in order to evaluate the age rangeof donor tissue that can be used for transplanta-tion. SCN of hamsters from embryonic day 11through postnatal day 12 can serve as functionalgrafts to restore rhythmicity to arrhythmic SCNlesioned animals. The time between SCN trans-plantation and onset of rhythmicity does notdepend on the age of the donor. The presence ofpatches containing vasoactive intestinal peptide(VIP) immunoreactive cells is a good indicatorof graft success, while its absence is correlatedwith a lack of transplant effect. The 18 day spanduring which SCN tissue can be harvested fortransplantation should expand the uses to whichthis technique can be put. Our results alsosuggest that it would be advantageous toexamine the age range of neural tissue that ca’nbe used in other transplantation models.

KEY WORDS

circadian rhythm, Syrian hamster, VIP

Reprint address:Claire M. KaufmanNIH/NIDDK-LNBuilding 8, Room 111Bethesda, MD 20892-0008, USA

INTRODUCTION

The technique of transplantation wassuccessfully used by Ralph and colleagues in 1990/14/ to show that the characteristics of thesuprachiasmatic nucleus (SCN) itself determinedcircadian period. The protocol employed involvedplacing the SCN of a mutant fetal hamster with acircadian period of 20 h into a 24 h wild-type adulthamster which had been rendered arrhythmic bylesion of its SCN. In other experiments donor andhost genotypes were reversed. SCN transplantswhich restored circadian rhythms of wheel runningactivity always did so with a period characteristic ofthe donor.

All previous attempts at SCN transplantation inrodents have utilized tissue harvested during lategestation or within a day of birth. It has beengenerally accepted that neuronal tissue should beused for transplantation when it is still undergoingneurogenesis or shortly thereafter. In the goldenhamster gestation lasts 16 days and thesuprachiasmatic nuclei themselves undergoneurogenesis from embryonic day (E) 9.5-13/2,3/.In the rat gestation takes 22 days and SCNneurogenesis occurs between E 14-17 /1/. Thereported age range of hamster SCN tissue used fortransplantation was from E 13 to 15/5,8/, and ratSCN tissue from E 17/6/ to postnatal day (P) 1/16/, where day 0 is the day of birth.

The research described here focuses on definingthe age range of donor SCN tissue that yieldssuccessful transplants. We tested early fetal tissue togain insight into the importance of graft maturation,and examined older postnatal tissue becauseworking with larger brain structures allows moreprecise excision and greater ease of manipulation ofdonor SCN prior to implantation. In addition, weexamined the characteristics of the restored rhythm,as well as the expression in the graft of two antigens

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258 C.M. KAUFMAN AND M. MENAKER

normally abundant in the intact SCN but not insurrounding hypothalamic areas: glial fibrillaryacidic protein (GFAP) /12/ which is found inastrocytes and vasoactive intestinal peptide (VIP)/9,17/.

METHODS

All methods were as described previously/14,19/, except where specifically noted. Briefly,adult male Lakeview Golden-Outbred hamsters(Mesocricetus auratus’, Charles Rivers, Wilmington,MA) were anesthetized, positioned stereotaxically(+0.6 mm AP, -8.3 mm V from bregma; nosebar-2.0 mm), and a platinum-iridium electrode waslowered through a hole drilled in the skull. Thelesion was made with a 4 mA current passed for 15sec. The electrode was removed, Gelfoam wasplaced over the hole, and the skin was closed withsurgical staples.

Implantation did not immediately follow thelesion, but was performed 17 to 37 weeks later.Implants were prepared as previously described/14/. However, in this case, all donors werehomozygous period mutants that had been bred inour colony and which would have displayedcircadian periods of about 20 h if allowed tomature. All hosts were wild-type hamsters. In orderto collect fetal SCN tissue, pregnant dams receiveda lethal dose of sodium pentobarbital. Fetuses wereexposed by caesarian section and rapidlydecapitated. Postnatal animals were decapitated.Heads were placed in medium [60% Basal Eagle’sMedium, with Earl’s Salts and without L-glutamine;40% Hanks’ Balanced Salt Solution (BME-HBSS)]at 36C and further dissected to isolate a block ofSCN-containing tissue (1-2 tl).

For implantation of SCN tissue, host animalswere once more anesthetized and placed in astereotaxic apparatus (David Kopf, Tujunga, CA).The tissue was implanted through the preexistinghole by means of a glass cannula and plunger intothe base of the third ventricle, the site of the SCNlesion. The wound was closed as after the lesion.

Locomotor activity was recorded from hostanimals individually housed in cages with runningwheels, given unlimited access to food and water,and kept in constant darkness in light-tight boxes.

Activity recordings were accomplished aspreviously described/19/. Data were analyzed usingthe chi-squared periodogram/18/. The criteria usedby Vogelbaum and Menaker /19/ were followedexcept that test periods ranges from 19 to 21 h (theperiod range of homozygous mutant hamsters/13/)in 6 min intervals, and rhythms were consideredsignificant only if periodogram peaks (each peakhaving a confidence level of p<0.05) continued formore than 5 consecutive days. Latency to the onsetof rhythmicity (i.e., the time from implantation toappearance of the donor rhythm) was determined bythree independent observers who analyzed activityrecords of animals assigned coded identifications toeliminate bias. These values were then statisticallyanalyzed using a one-way ANOVA followed by apost hoc Scheffe test. Only surviving animalshaving complete SCN lesions and grafts asdetermined histologically were included in thesestudies. For calculation of percentage survival aftertransplantation, only completely SCN lesionedanimals (as determined histologically) wereincluded; lesion success for those animals dyingprior to histological analysis was determined on thebasis of behavior (9/75; 8 of those 9 weretransplanted with tissue from E 10, E 11, or P 28donors).

Transplant histology was examined in allsurviving animals. After locomotor activity datawere collected, animals were anesthetized with alethal dose of halothane by inhalation. They wereperfused intracardially with physiological saline(0.9%) containing 150 IU heparin/10 ml, followedby Zamboni’s fixative (pH 7.4) containing 4%formaldehyde in 0.1 M phosphate buffered saline(PBS; 0.1 M phosphate, 0.9% saline) with picricacid (15%). After perfusion, brains were removedand postfixed at 4C. One day before sectioning,brains were cryoprotected by immersion in 25%sucrose in 0.01 M PBS. Brains were frozen and cutat a section width of 50

Free floating sections were washed in PBS-T(0.01 M phoshpate, 0.9% saline, 0.2% Triton X-100; pH 7.4) and incubated for 30 min in a 0.01 MPBS solution containing 0.003% hydrogenperoxide followed by several washes in PBS.Sections were incubated for 1 h at roomtemperature in normal goat serum (1:30 PBS) andplaced directly into anti-VIP (RIN 7161; Peninsula

JOURNAL OF NEURAL TRANSPLANTATION & PLASTICITY

TRANSPLANTING SCN FROM DIFFERENT AGE DONORS 259

Laboratories, Belmont, CA) diluted 1:1000 or anti-GFAP (ICN Bio-medicals, Costa Mesa, CA)diluted 1:500 in PBS-T for 72 h (24 h 22C: 48 h4C). Sections were washed in PBS and sites ofantibody:antigen binding were tagged by an avidin-biotin peroxidase procedure (Elite ABC Kit, VectorLabs, Burlington, CA). Following this, sectionswere washed in 0.05 M Tris buffer (pH 7.3) beforeincubation with 0.0025% diaminobenzidine (DAB)containing 0.003% hydrogen peroxide for about 4min. Sections mounted onto gelatin-coated slideswere dehydrated through graded alcohols intoxylene where they remained for a minimum of 4 h.Sections were then rehydrated in distilled waterand immersed in 0.2% osmium tetroxide 2 min tointensify staining. Histological analysis andphotography were performed with a ZeissAxiophot photomicrograph system.

RESULTS

As can be seen in Figure 1, donor SCN fromE 11 to P 12 produced functional grafts incompletely SCN lesioned hosts (Table 1). The ageof the donor SCN producing the largest percentagesof host animals with transplants that displayed

TABLE 1

Age (days) Mortalitya Donor Rhythmb

E 10 4/5 0/1E 11 2/7 2/5E 12 1/5 1/3E 14 0/11 9/10P 2 0/6 5/6P 3 1/8 4/5P 4 0/8 5/8P 6 0/5 4/5P 8 0/5 2/5P 10 0/5 3/5P 12 0/6 1/4P 28 0/4 0/1aNumber of deaths per total number of transplanted hostswith complete SCN lesionsbNumber of hosts displaying a donor rhythm per totalnumber of hosts having a discernable transplant andcomplete SCN lesionE embryonic, P postnatal

donor periods were E 14 and P 2. Even transplantedSCN from P 10 animals yielded clear circadianrhythms of wheel running activity with theapproximately 20 h period of the donor (Fig. 2).

Fig. 1:

8 10 12 14 0 2 4 6 8 10 12 14 26 28 30Embryonic Postnatal

DONOR AGE (days)

Effect of 20 h mutant SCN transplanted into wild-type host hamsters with complete SCN lesions.E 11 to P 12 hamsters SCN produced functional transplants. Percentages of completely SCN lesioned hosts withhistologically detected grafts having a period characteristic of the period mutant hamster varied with age of donor([]; regression line was calculted with a 5th order polynomial equation). Mortality of all transplanted hosts was higherwith the use of younger donors (O).

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260 C.M. KAUFMANAND M. MENAKER

T

HOURS20 40

.,__-_,.

-30

40

50

60

-70

Fig. 2: Activity rhythm of a host before and after SCNlesion and transplantation with a P 10 SCN. Theactivity is doubled-plotted such that twoconsecutive 20 h days (the approximate length ofthe donor’s period) are placed next to each other onone line and the latter day is repeated beneath theformer. L day of lesion. T day of transplant.

Mortality occurring after the day oftransplantation was highest when donors were fromthe youngest groups (Fig. 1 and Table 1). Tissuefrom E 10 fetuses was extremely difficult to isolatesurgically both because the brain was in a primitivestate and because the head was quite soft and small.As a result, the graft often contained cartilage fromthe skull as well as a larger area of the brain. In allcases (N--5) this led to illness or death. Histologicalanalysis revealed volume increase of the graft.

Several grafts from animals older than P 10appeared to have undergone degeneration withinthe host. As neonatal animals mature andparticularly around the time the optic chiasmbecomes myelinated (P 10 in the rat /10/), theexcised SCN (which inevitably contains part of theoptic chiasm) increases in firmness. This may havecontributed to the poor graft success seen whenolder donors were used. The characteristics of thesemore mature implants may result in physicalisolation from nutrients, hormones, or necessaryneural connections.We compared the presence of patches of cells

and fibers immunopositive for VIP with thepresence of a circa-20 h rhythm characteristic of thedonor SCN as determined either by periodogramanalysis or by visual observation by threeindependent observers. Though there was somedisagreement between the computational and visualmethods of period analysis in terms of whichtransplants yielded donor activity rhythms, thepercentage correlation of donor rhythm presenceand of VIP patches within the graft was similar forthe two period analysis methods. Patches of VIPimmunopositive cells could be seen in all but threesuccessful transplants as defined by periodogramanalysis (N=36; Fig. 3 immunocytochemistry; Fig.4 behavior). In one of the exceptions thetransplant clearly conferred a 20 h rhythm, thoughno VIP stained c.ells could be detected. Theremaining two grafts without apparent VIP alsoconferred an activity rhythm having a periodconsistent with the donor phenotype according tochi-squared periodogram analysis; however, thethree observers did not find such a rhythm in therecords. Of the 22 grafts in animals with completeSCN lesions that showed no statistically significantdonor rhythm by periodogram analysis (e.g., Figs. 5and 6), only three had patches of VIP immuno-reactive cells and fibers. One of these appeared(visually) to be totally arrhythmic. Although chi-squared periodogram analysis also failed to detect asignificant circa-20 h rhythm for the other twotransplanted animals, visual inspection of thelocomotor activity revealed apparently clear donorrhythms. If one were to use only visual observationsof activity records and compare the results with theassumption that grafts having patches of VIPstained cells were indicative of successful



.............,.::.........,......,.:.’.., ......:.:,.’..v.:’.:.’..

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Fig. 3: Photomicrographs showing GFAP and VIP stainedalternate sections from successful SCN grafts asdetermined by periodogram analysis. Two differentgrafts of P 4 SCN (A-C and D-F) are represented.Corresponding behavioral data is shown in Fig. 4Aand B, respectively. A and D: GFAP staining neargraft borders which are indicated by arrows. "OC"indicates the optic chiasm. B and E: Patches ofVIP immunoreactive cells and fibers typical offunctional transplants. * marks the location of VIPimmunoreactive patches in B and E. These patchesare seen at an 8x higher magnification in C and F,respectively. Scale bar 200 tm for A,B,D,E; 25

tm for C and F.

transplants, there would be disagreement in onlytwo out of 58 cases one animal with no VIP patchbut having a visually evident rhythm and one with aVIP patch but no visually evident rhythm. Therewas a 97% correlation between the presence of VIPpatches and of rhythmicity characteristic of thedonor as determined by visual inspection, and thiscorrelation is a little higher than that between thepresence of VIP patches and of a donor rhythm asdetermined by chi-squared periodogram analysis(90%). Though the three possible criteria fortransplant success (i.e., the presence of a patch ofVIP positive cells, visual analysis of running wheelactivity, and chi-squared periodogram analysis) didnot agree in every case, the percentage of functionalgrafts identified by each criterion was similar. Whileit is difficult to select one method of determiningtransplantation success as being superior, it may bethat combining immunocytochemical analysis of thegraft with a method of period analysis wouldprovide the best means of assaying transplantSuccess.

We noted several transplanted animals thatappeared to have complete SCN lesions as judgedhistologically, but in which 24 h host activityrhythms were still detectable. Four such animals hadactivity rhythms characteristic of only the host withno evidence of donor rhythm, while one displayedrhythms characteristic of both the host and donor.Using a protocol similar to the one described in thispaper, Vogelbaum and Menaker (manuscript inpreparation) have found that if a host rhythm isdetectable after transplantation, it is less likely that adonor rhythm will appear.

GFAP was detectable in all grafts, bothsuccessful (Fig. 3A and C) and unsuccessful (Fig.5A). Levels of GFAP seemed to have nocorrespondence to restoration of rhythmicity, butwere difficult to quantify. The GFAP staininginduced after trauma resulting from implantation,however, made the graft borders more easilydefinable.

Latency to the onset of rhythmicity wasmeasured from the time of implantation and scoredby three independent observers and by the chi-squared periodogram. Mean latency for all agesdetermined visually was 20.2 days and determinedby periodogram was 17.1 days. Latency showed nocorrelation to donor age (p=0.1, Fig. 7).

VOLUME 4, NO. 4, 1993

262 C.M. KAUTMAN AND M. MENAKER

A HOURS20 40

40

-50

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Fig. 4: Activity rhythms from the successful transplantsdepicted in Fig. 3. A and B: Locomotor behavior intwo animals that received P 4 SCN grafts (Fig. 3A-C and 3D-F, respectively) double-plotted aspreviously described with a day-length of 20 h. Lday of lesion. T day of transplant.

HOURS0 20 40

-40

-60

DISCUSSION

We were able to transplant successfully the SCNof period mutant (20 h) hamsters, aged fromembryonic day 11 through postnatal day 12, into thethird ventricle of wild-type SCN lesioned hosts.This 18 day span begins during SCN neurogenesisand lasts approximately until the number ofsynapses per unit area reaches adult levels (P 10 forthe rat /10/). Since it was possible to transplanttissue that was in the midst of active neurogenesis,maturation of the transplanted SCN may havecontinued within the host. Based on the previousfindings of Ralph and colleagues/14/, we have noreason to believe the results would be different ifthe host and donor genotypes were reversed.

The age of donor tissue had no relationship tothe latency to rhythm onset after transplantation.Romero and Silver/15/have suggested that latencyto rhythm onset may be due to the time required torenew peptidergic expression after a developmentaldelay resulting from transplantation. Our findingssuggest this is not the case; if it were, older SCNtissue in which levels and patterns of neuropeptidesresemble those of the adult/4,7,11/should producerhythmicity with a shorter latency than youngertissue. On the contrary, younger fetal SCN tissuedoes not appear to require more time than olderpostnatal grafts to develop or to make appropriateconnections with the host. The possibility exists,however, that a small difference in latency related todonor age is obscured because of disruption of



Fig. 5: Photomicrograph of an unsuccessful graft asdetermined by chi-squared periodogram analysis.A: GFAP staining of an unsuccessful P 10 graftand its borders. Arrows point to position of graftborders. "OC" indicates the optic chiasm. B: Thealternate section showing a lack of VIP staining inthe graft. Scale bar 200 l.tm. Locomotor activityfrom this animal is seen in Fig. 6.

running wheel activity caused by trauma induced bythe transplantation process.

The levels of VIP staining did not appear tocorrespond to either age of the donor SCN ortransplant success. Nevertheless, there was a highcorrelation between transplant success and presenceof at least one patch of VIP immunoreactive cellsand fibers which resemble intact SCN. Thesefindings suggest that VIP may be necessary for

0HOURS20

40

-20

.40

-50

-60

Absence of donor phenotype as determined by chi-squared periodogram analysis. Activity of ananimal bearing the P 10 transplant pictured in Fig.5, double-plotted with a day length of 20 h as inprevious figures. L day of lesion. T day oftransplant.

transplant function. This type of VIP stainingappears to be as sensitive an assay of transplantsuccess as chi-squared periodogram analysis ofbehavior. GFAP staining aided in the localization ofgraft borders.

Our findings extend the age range over whichdonor SCN tissue from hamsters can be expected toconfer rhythmicity successfully. The extended rangeadds flexibility to breeding and surgery schedules.Because of their larger size, SCN from olderpostnatal animals can be excised more preciselythan can SCN from fetuses or young neonates.Better results should be attainable by labeling

VOLUME 4, NO. 4, 1993

264 C.M. KAUFMAN AND M. MENAKER

Fig. 7:

, 35-

u) 28-zo-r- 21-

0 14-

z 7-

010 12 14 0 2 4 6 8 10 12Embryonic Postnatal

DONOR AGE (days)

Latency to onset of rhythm conferred by SCN transplants. There was no significant correlation(p-:0.1) between donor age and the number of days from transplantation to donor activity rhythm appearance. Symbolsrepresent mean __. SEM for values estimated by 3 independent observers.

postnatal rather than embryonic transplant tissue inorder to trace transplant efferent fibers. Sincemothers need not be killed if neonates are used asdonors, results from transplantations usingsuccessive litters from the same dam can becompared. Additionally, the light:dark cycle couldbe used over a matter of weeks to impart anidentifiable characteristic to the rhythm other thanperiod (e.g., phase of activity) which might then betransplantable.

The 18 day span during which donor SCN maybe harvested from hamsters for successfultransplantation suggests that the SCN may becapable of undergoing changes and adaptationsduring a lengthy maturation. This may be importantfor normal calibration of the developing clock bymaternal cycles (daffy rhythms of maternalhormones such as melatonin) and environmentalcycles (e.g., light:dark).

Note added in proof." Romero et al. (Dev Brain Res1993; 71" 45-52) have shown that SCN from P 3hamsters can restore rhythmicity to SCN lesionedhosts.

ACKNOWLEDGEMENTS

The research reported was supported by AirForce Office of Scientific Research grant 90-0098to M.M.C.M.K. Was also supported by the NSFCenter for Biological Timing.

REFERENCES

1. Altman J, Bayer SA. The development of the rathypothalamus. Adv Anat Embryol Cell Biol 1978;100: 1-173.

2. Crossland WJ, Uchwat CJ. Neurogenesis in the centralvisual pathways of the golden hamster. Dev Brain Res1982; 5: 99-103.

3. Davis FC, Boada R, LeDeaux J. Neurogenesis of thehamster suprachiasmatic nucleus. Brain Res 1990;519: 192-199.

4. De Vries GJ, Buijs RM, Swaab DF. Ontogeny of thevasopressinergic neurons of the suprachiasmaticnucleus and their extrahypothalamic projections in therat brain presence of a sex difference in the lateralseptum. Brain Res 1981; 218: 67-78.

5. DeCoursey PJ, Buggy J. Restoration of circadianlocomotor activity in arrhythmic hamsters by fetalSCN transplants. Comp Endocrinol 1988; 7: 49-54.



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6. Drucker-Colfn R, Aguilar-Roblero R, Garcfa-Hern,Sndez F, Fern,:Sndez-Canino F, Rattoni FB. Fetalsuprachiasmatic nucleus transplants’ diurnal rhythmrecovery of lesioned rats. Brain Res 1984; 311: 353-357.

7. Ishikawa K, Frohman LA. Ontogenesis of peptide-histidine-isolucine (PHI)-containing neurons in thesuprachiasmatic nucleus (SCN) of the rat. Brain Res1987; 407: 144-148.

8. Lehman MN, Silver R, Gladstone WR, Kahn RM,Gibson M, Bittman EL. Circadian rhythmicity restoredby neuronal transplant. Immunocytochemicalcharacterization of the graft and its integration withthe host brain. J Neurosci 1987; 7: 1626-1638.

9. Loren I, Emson PC, Fahrenkrug J, Bj6rklund A,Altumets J, Hakanson R, Sundler F. Distribution ofvasoactive intestinal polypeptide in the rat and mousebrain. Neuroscience 1979; 4: 1953-1976.Moore RY, Bernstein ME. Synaptogenesis in the ratsuprachiasmatic nucleus demonstrated by electronmicroscopy and synapsin immunoreactivity. JNeurosci 1989; 9: 2151-2162.Moore RY, Shibata S, Bernstein M. Developmentalanatomy of the circadian system. In: Reppert SM, ed,Development of Circadian Rhythmicity andPhotoperiodism in Mammals. Ithaca: PerinatologyPress, 1989; 1-24.Morin LP, Johnson RF, Moore RY. Two brain nuclei

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controlling circadian rhythms are identified by GFAPimmunoreactivity in hamsters and rats. Neruosci Lett1989; 99: 55-60.Ralph MR, Menaker M. A mutation of the circadiansystem in golden hamsters. Science 1988; 241: 4225-4227.Ralph MR, Foster RG, Davis FC, Menaker M.Transplanted suprachiasmatic nucleus determinescircadian period. Science 1990; 247: 975-978.Romero M, Silver R. Time course of peptidergicexpression in fetal suprachiasmatic nucleustransplanted into adult hamsters. Dev Brain Res 1990;57: 1-6.Sawaki Y, Nihonmatsu I, Kawamura H.Transplantation of the neonatal suprachiasmaticnucleus into rats with complete bilateralsuprachaismatic lesions. Neurosci Res 1984; 1’ 67-72.Sims KB, Hoffman DL, Said SI, Zimmerman EA.Vasoactive intestinal polypeptide (VIP) in mouse andrat brain: an immunocytochemical study. Brain Res1980; 186: 165-183.Sokolove PG, Bushell WN. The chi-squareperiodogram: its utility for analysis of circadianrhythms. J Theor Biol 1978; 72: 131-160.Vogelbaum MA, Menaker M. Temporal chimerasproduced by hypothalamic transplants. J Neurosci1992; 12: 3619-3627.

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Effectof Nuclei from Donors of Different Ages into ...downloads.hindawi.com/journals/np/1993/732456.pdfBethesda,MD20892-0008,USA INTRODUCTION The technique of transplantation was successfully