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Examining the induction of limb regeneration through modulation
of Stump currents by the sodium ionphore monensin in non-
Regenerating post-metamorphicRana pipiensfrogs
An honors thesis
Submitted byDavid Patterson Hessler Jr.
In partial fulfillment of the requirements
For the degree of
Bachelor of Science
In
Biomedical Engineering
May !""
Advisor# Professor David $aplan% Ph.D.
&ufts 'niversity
Acknowledgments
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&han( you to the &ufts 'niversity )iomedi*al +ngineering Department for providing me
,ith this in*redible opportunity to intera*t ,ith an early stage regenerative investigation. I ,ould
also li(e to than( the -evin lab at &ufts enter for /egenerative and Developmental )iology for
providing assistan*e throughout.
I ,ant to than( Prof. $aplan for allo,ing me to 0oin on this pro0e*t ,ith 1i(. I ,as very
e2*ited at the possibility to ,or( on a regeneration study% and Prof. $aplan helped me to find a
pro0e*t that my efforts ,ould be *riti*al to its su**ess.
I also ,anted to than( 1i( $o0i* for the opportunity to ,or( alongside of his resear*h. It
,as a pleasure to spend so mu*h time in the frog room ,ith 1i(% and I ,ish him the best on his
*urrent and future endeavors. In addition% Punita $oustubhan deserves re*ognition for all her
help and ,or( ,ith this entire pro0e*t.
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Table of ontents
&able of
Figures33333333333333333333333333333333333333333... p.iv
".
Abstra*t333333333333333333333333333333333333.. p."
. )a*(ground 4
Introdu*tion333333333333333333333333....p.a. /egeneration
pro*ess33333333333333333333333.. p.
b. Stumpurrents333333333333333333333333333. p.5
*. lassi*al
Studies333333333333333333333333333p.6
d. urrent/esear*h33333333333333333333333333.p.""
e.
Monensin333333333333333333333333333333.p."7
f. 8ur
Approa*h333333333333333333333333333... p."97.
Hypothesis3333333333333333333333333333333333..p.":
9. Spe*ifi*Aims333333333333333333333333333333333.. p.";
5.
Methods33333333333333333333333333333333333
3 p."roups33333333333. p.6
ii.
Surgery33333333333333333333333333.. p.:iii. &reatment
)aths333333333333333333333. p.:
iv. ?isual
inspe*tion333333333333333333333. p.;v.
Histology3333333333333333333333333.. p. BSodium >lu*onateC in Poland Spring @ater. &his is an e2tremely high
*on*entration of sodium *ompared to the normal salt levels in Poland Spring. &herefore the
treatment solution provides the pharma*ologi*al ingredient% monensin% as ,ell as the
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ne*essary ion sour*e of sodium to in*rease the
intra*ellular *on*entration and thus drive stump
*urrents. Frogs are allo,ed to soa( for
minutes. After the initial soa(ing% the solution is
*ontinually diluted ,ith additional Poland
spring. &he solution is then poured out% and ne,
Poland Spring ,ater is added to the *ontainers.
Frogs are (ept in these *ontainers until they
begin to move around normally% at ,hi*h point
they are returned to their *age. &he t,o *ontrol
treatments are performed in the same e2a*t
manner.
I?. ?isual inspe*tion
Frogs ,ere inspe*ted at least on*e a ,ee( for any *hange in appearan*e to the amputation
site. Pi*tures ,ere ta(en of ea*h frog in three different poses so that the amputation site
*ould be vie,ed from several angles. &he frogs ,ere *ompletely a,a(e and uninhibited
during the pi*ture ta(ing% be*ause anesthetiGing them for su*h a short period ,as
unne*essary.
?. Histology
Proto*ols are *urrently being submitted so that ,e *an run histology samples on the
sa*rifi*ed frog. &he right forearm ,ill be fi2ed and histology ,ill be performed loo(ing
for gro,th of tissue% bone and nerves. Many regeneration events do *ause native tissue%
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bone% and nerves to gro,% but rather result in a hypomorphi* spi(e *hara*teriGed by some
tissue surrounding a *artilaginous outgro,th.
/$ Results"""""""""""""""""""""""""""""""""""""""""""""""
"
Although it is still very early in the regenerative pro*ess% as the first group of e2perimentally
treated frogs is only t,o months post treatment% a fe, initial results have sho,n the potential for
regeneration indu*tion. As e2pe*ted amputated frogs *ompleted a normal ,ound healing
response that *aused the amputation site to slo,ly *lose up ,ith regular epithelium. @ithout any
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treatment% the frogs sho,ed no regenerative *apability nor did they demonstrate any signs of
developing a bud. In *ontrol group frogs treated only ,ith Poland spring% the thi*( epithelium
*ontinued to *lose over the ,ound site% and 7 days after the treatment BQ months post
amputationC the ,ound site ,as *ompletely *losed over BFig "6C. &his is the typi*al ,ound
healing response of adultRana pipiens% and *onfirms that these frogs do not retain their
regenerative *apabilities. In addition% frogs treated ,ith Monensin ,ithout sodium ultimately
healed similarly to the untreated frogs. As seen in Figure ":% some redness appears after
treatment ,ith monensin% but it never develops into an outgro,th and by Day 56 the ,ound is
*ompletely healed over. &his agrees ,ith the results a*hieved by A. &seng et al. ,hi*h indi*ated
that treatment ,ith monensin or high levels of sodium alone is in*apable of indu*ing
regeneration.
In *omparison% frogs sub0e*ted to the monensin and sodium treatment have demonstrated a
distin*t *hange in appearan*e of the ,ound site. A ,ee( after treatment the healed ,ound site
be*ame reddish in *olor and a distin*t bulge develops. &his small outgro,th % although only a
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fe, mm in e2tension from the original plane of amputation% is thought to be a blastema and is
representative of a limb development phenomenon% an event not e2perien*ed by the *ontrol
frogs. In Figure ";% this regeneration bud *an be *learly seen by Day "5. &his outgro,th is
*learly absent ,hen *ompared to the nonregenerative frogs in Figure "6O":. &he head=on vie,
used in Figure "< *learly demonstrates the *hange in appearan*e of the amputation site as the
regenerative outgro,th matures. After the initial appearan*e of the regenerative outgro,th% the
bud *ontinued to *hange in stru*ture and appearan*e as further time passed post amputation.
&hese frogs ,ill *ontinue to be monitored until all gro,th has halted. At this point% the spe*imen
,ill be sa*rifi*ed and the regenerated arm ,ill undergo histologi*al e2amination.
)e*ause this study is still at a very early timepoint% ,e do not *urrently have any
histologi*al or quantitive results in ,hi*h to present. Solutions to these issues are presented in
the dis*ussion and future ,or( se*tions.
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0$ 1iscussion""""""""""""""""""""""""""""""""""""""""""""
"
Although no definitive *on*lusions *an be dra,n at this point due to the la*( of
histologi*al data as ,ell as the need for more samples for reprodu*ibility% the monensin and
sodium treated frogs are *learly demonstrating some type of regenerative outgro,th. &his is even
more *on*lusive ,hen *ompared to the untreated frog BFig. "6C and the frog that ,as only
sub0e*ted to monensin BFig. ":C. Interestingly% the appearan*e of the monensin treated frog limb
,as slightly different from the untreated group. &his is most li(ely due to minute amounts of
sodium ions that ,ere present in the solution. Ho,ever% at su*h small quantities% the ,ound site
only slightly *hanged in appearan*e% but did not present any sort of gro,th. In *omparison% the
images seen in Figures "; 4 "< demonstrate a *lear outgro,th from the amputation site. &his
blastema formation is indi*ative of the early stages of a regenerative response. Although it is too
early in this study to *on*lude that these outgro,ths are indu*ed by monensins ability to rapidly
transport sodium ions into the *ell% the initial results are promising. &his regeneration indu*tion
is most li(ely the result of a *as*ade of signaling that is enhan*ed by the in*reased ioni* flu2
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B-evin !!
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be*ame very vo*al. &hese sounds seemed to alert the other frogs in the room that food ,as
*oming. 8ne (ey aspe*t of our approa*h for frog *are ,as to populate the tan(s ,ith "!="5 frogs.
Most previous studies have housed ea*h frog in their o,n individual *ontainer. In our
e2perien*e% frogs sequestered to individual *ontainers did not demonstrate the same vora*ity and
liveliness of the grouped frogs% and often times ,ould go ,ithout eating. Ho,ever% on*e they
,ere returned to the group they resumed normal a*tivity. Although a more detailed study of this
response ,ould be appropriate% it ,as of potential interest due to the differen*e in animal *are of
this study as *ompared to previous ones.
I ,as very fortunate to be*ome a part of this investigation at a very early stage in the
resear*h pro*ess. I 0oined ,ith 1i( $o0i* in the Spring of !"!% and it ,asnt until the late fall of
that same year that our first frog spe*imens arrived. It has been a very long and time *onsuming
pro*ess from starting off ,ith a fe,Xenopus laevisfrogs to transitioning to in*lude over !!
Rana pipiens. Ho,ever% the *ontinued effort has led to a fantasti* learning e2perien*e%
spe*ifi*ally for e2perimentation using live animals% as ,ell as a great opportunity to learn from
1i( and Punita. Although my ,or( on this pro0e*t is shortly *oming to a *lose% there remains a
great amount of ,or( and resear*h that ,ill be *ontinue to be *ondu*ted on this on=going study.
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treatment are to be impli*ated in the regeneration of the limbs% similar treatments should result in
similar outgro,ths from the study organisms. -arge variations in the regenerative response
,ould indi*ate that additional fa*tors are at play. &his *an be strengthened by further
demonstration that sodium treatment or monensin treatment alone is in*apable of produ*ing a
regenerative response. In addition% histologi*al results ,ill provide information regarding the
quality and amount of regeneration that is o**urring. Although visual inspe*tion allo,s for basi*
information regarding the outgro,th% it is very minimal and is in*apable of identifying the
me*hanisms responsible for this response. &herefore% the monitoring of the *hange in
*on*entration of sodium and other important ions ,ill provide *ru*ial data at the mole*ular level
BNhang B!!
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limb formation. In addition% they ,ere able to produ*e developmental defe*ts in these limbs by
alteration of the out,ard stump *urrent. Although the pro*esses may not be *ompletely
understood% this indi*ates that stump *urrents are *riti*al to the development of vertebrate spe*ies
and thus may represent a unique possibility. If the *ells in a non=regenerative vertebrate *an be
triggered in su*h a manner to mimi* developmental *onditions% then regeneration might o**ur.
@e hope that our resear*h ,ill provide further information regarding the regenerative path,ays
and ,ill further our (no,ledge regarding regeneration.
*. @hat I have learned
@or(ing on this pro0e*t has been a unique opportunity and an amaGing learning e2perien*e.
Despite spending *lose to ten hours a ,ee( ta(ing *are of the frogs% I feel privileged to have had
this opportunity. Although it is disappointing to not have more results after ,or(ing for over a
year a on this resear*h% it has provided several important lessons that I am *onfident ,ill be
benefi*ial for my future endeavors.
&he first of ,hi*h is fle2ibility. @hen I first started to ,or( ,ith 1i(% the fo*us of our
efforts ,as on using Sil( fibroin e=gels on the amputated limbs ofXenopusfrogs ho,ever% that
fo*us ,as shifted to our *urrent effort on limb regeneration using monensin. In addition%
shipments of frogs or *ri*(ets *ould arrive at anytime during the day therefore it ,as important
to be able to ad0ust the ,or( s*hedule to ,hen the shipments arrived.
Patien*e ,as another important lesson that ,as stressed by ,or(ing on this pro0e*t. @e
had to ,ait for several months before our proto*ols ,ere a**epted. &herefore% although ,e ,ere
ready to start our e2perimentation last spring% the long ,aiting pro*ess for approval pushed the
start until the fall. @hen our frog room ,as finally approved in early fall% ,e ,ere e2*ited to
finally begin the study. Ho,ever% as previously mentioned%Rana pipiensare not available
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throughout the year% usually they be*ome available by the start of 8*tober. Ho,ever due to
de*lining population and alteration in the environmental *onditions% ,e re*eived our first
shipment of frogs by early 1ovember. Although our start time ,as greatly delayed% it is *riti*al
not to *ompound mista(es by attempting to rush through to a*hieve results. Patien*e is espe*ially
important in live animal studies% ,here *ontrolling all fa*tors is *hallenging. In *omparison to in
vitrostudies% be*ause the frogs are living organisms they are not al,ays going to respond as
desired. It is *riti*al to remain gentle and *alm ,hen handling the frogs% as rough handling *an
*ause damage and dis*omfort to the organism.
&his resear*h opportunity has been an amaGing e2perien*e for s*ientifi* learning.
Although animal studies are very different from some of my past resear*h e2perien*es% I
thoroughly en0oyed the responsibility asso*iated ,ith *aring for over !! animals. 1ot only did
it require lots of hard ,or( but also a *ommitment to a high standard of quality and *are% a
ne*essity ,hen handling live organism. I hope that all my efforts% and the *ontinued ,or( of the
resear*h team ,ill ,or( to demonstrate the regenerative abilities indu*ed by monensin and
sodium treatment.
3$ !uture
4ork"""""""""""""""""""""""""""""""""""""""""
&he future of this study appears to be very promising ho,ever% there is still a great deal of ,or(
that needs to be *ondu*ted. 1ot until the end of the regro,th pro*ess for the intial treatment
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group ,ill ,e have a better understanding of the regenerative *apa*ity indu*ed by treatment ,ith
monensin and sodium. In addition in the span of several months our operation siGe has in*reased
from 7! frogs in early ,inter to over !! frogs at the *urrent time. Although this mar(ed in*rease
in siGe is largely due to the fa*t thatRanapipiensare prote*ted during their spa,ning time in the
summer and are unattainable% it is also out of ne*essity for reprodu*ible results. For e2ample% Dr.
Smiths results should be *onsidered truly remar(able% espe*ially due to the fa*t that one of his
study organisms demonstrated a *omplete regeneration of a lost limb. Ho,ever% many details
regarding the methods used by Dr. Smith are missing% and he never repeated his e2perimental
pro*ess. &herfore% it is *riti*al that ,e are *apable of demonstrating *onsistent gro,th ,ith our
regenerates. At the *urrent time% additional *ontrol treatments are being *ondu*ted to ensure that
no environmental fa*tors B*o*onut substrate% ro*(s% or Poland Spring ,aterC ,ere a*tive in
indu*ing the regeneration event. &hese *ontrols ,ill help to strengthen the results of the
monensin and sodium treated spe*imens. In addition% repeat e2periments sub0e*ting the frogs to
treatments of monensin only and sodium only should be *ondu*ted to provide additional support.
Finally% depending upon the up*oming results% an attempt to repeat the Smith and )orgens
e2periments should be made to guage ,hat s*ale of regro,th ,e *an a*hieve. &his ele*tri*al
stimulation *ould be used in *on0u*tion ,ith monensin and sodium treatment to *reate very
strong *urrents% ho,ever it is un*ertain ,ether e2*essive *urrents ,ould be benefi*ial or
detrimental.
Monitoring of these *urrents ,ill be important to demonstrate the flu2 of sodium ions.
)orgens used vibrating probes that allo,s them to measure e2tra*ellular voltage differen*es.
&his allo,s for inferen*e about the e2tra*ellular *urrent densities% but provides no means for
visualiGation. 1. 8G(u*ur et al. have re*ently demonstrated that this visualiGation is possible
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through the use of ion spe*ifi* vital dyes. &hese dyes allo, for the in vivomonitoring of several
spe*ifi* ions during the regeneration of a2olotol limb. &his real=time monitoring ,ould enable us
to visualiGe the movement of ions during the regeneration of the Rana.
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"!. References""""""""""""""""""""""""""""""""""""""""
AltiGer A% et al.B!!"C. +ndogenous ele*tri* *urrent is asso*iated ,ith normal development of
the vertebrate limb. Developmental Dynami*s "# 7
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)ro*(es J. B"ardiner D% +ndo &% 4 )ryant S. B!!C. &he mole*ular basis of amphibian limb regeneration#
integrating the old ,ith the ne,. Seminars in ell 4 Developmental )iology "7# 795=75.
He*havarria D% De,ilde A% )raunhut S% -evin M% 4 $aplan D . )ioDome regenerative sleeve
for bio*hemi*al and biophysi*al stimulation of tissue regeneration.
Halliday &. 4 Adler $. B"
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$rag M et al.B!!
Developmental dynami*s 7
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Smith S.B"
