UA005.019.019.00001.archival

Examining the induction of limb regeneration through modulation
of Stump currents by the sodium ionphore monensin in non-
Regenerating post-metamorphic Rana pipiens frogs
An honors thesis
Submitted by
David 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
&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.
Table of ontents
&able of
Figures333333333333333333333333333333333333
33333... p.iv
".
Abstra*t33333333333333333333333333333333333
3.. p."
. )a*(ground 4
Introdu*tion333333333333333333333333....p.
a. /egeneration
pro*ess33333333333333333333333.. p.
b. Stump
.urrents333333333333333333333333333. p.5
*. .lassi*al
Studies333333333333333333333333333p.6
d. .urrent
/esear*h33333333333333333333333333. p.""
e.
Monensin333333333333333333333333333333.
p."7
f. 8ur
Approa*h333333333333333333333333333... p."9
7.
Hypothesis3333333333333333333333333333333333..
p.":
9. Spe*ifi*
Aims333333333333333333333333333333333.. p.";
5.
Methods33333333333333333333333333333333333
3 p."<
a. +2perimental
Pro*ess33333333333333333333333.. p."<
b. Animal
.are33333333333333333333333333333.
p."<
i. Study
8rganism3333333333333333333333. p."<
ii. .age
Setup333333333333333333333333.. p.!
iii.
Feeding33333333333333333333333333.. p."
iv. Frog &an(
.are3333333333333333333333.. p.
v. .ri*(et
.are333333333333333333333333. p.9
vi. Post=8perative
.are3333333333333333333. p.6
*. +2perimental
Setup333333333333333333333333. p.6
i. .ontrol and +2perimental
>roups33333333333. p.6
ii.
Surgery33333333333333333333333333.. p.:
iii. &reatment
)aths333333333333333333333. p.:
iv. ?isual
inspe*tion333333333333333333333. p.;
v.
Histology3333333333333333333333333.. p.<
6.
/esults333333333333333333333333333333333333
.. p.7!
:.
Dis*ussion3333333333333333333333333333333333..
p.77
;.
.on*lusion3333333333333333333333333333333333.
p.76
a. &his
pro0e*t33333333333333333333333333333 p.76
b. Potential of
/egeneration33333333333333333333.. p.76
*. @hat I
learned333333333333333333333333333. p.7:
<. Future
@or(333333333333333333333333333333333. p.7<
"!.
/eferen*es3333333333333333333333333333333333
p.9"
Table of
!igures"""""""""""""""""""""""""""""""""""""""""""""
Figure " &ypes of
/egeneration33333333333333333333333333333333.
333..p.
Figure Phases of
/egeneration333333333333333333333333333333333
3.3p.7
Figure 7 -imb /egeneration
.artoon333333333333333333333333333333.33..p.9
Figure 9 Stump .urrents from 1e,ts and
Frogs333333333333333333333333..33p.6
Figure 5 Dr. Smith implanted
ele*trode3333333333333333333333333333333.p.:
Figure 6 )orgens implanted
ele*trode33333333333333333333333333333333p.<
Figure : Stump *urrent
batteries33333333333333333333333333333333333
p.""
Figure ; /esults of regeneration indu*tion using monensin in
tadpoles333333333333..p."
Figure < Stru*ture and me*hanism of
monensin33333333333333333333333333p."9
Figure "! +2perimental
Pro*ess33333333333333333333333333333333333
3.p."<
Figure "" 1orthern -eopard
Frog33333333333333333333333333333333333.p.!
Figure " Frog
Habitat333333333333333333333333333333333333
33333..p."
Figure "7 Frog
Habitat333333333333333333333333333333333333
33333..p.7
Figure "9 .ri*(et
$eeper333333333333333333333333333333333333
3333.p.5
Figure "5 Amputation
Site3333333333333333333333333333333333333
33.p.;
Figure "6 Poland Spring &reated
Frog333333333333333333333333333333333p.7!
Figure ": Monensin &reated
Frog33333333333333333333333333333333333p.7"
Figure "; Monensin A Sodium &reated
Frog33333333333333333333333333333.p.7
Figure "< Monensin A Sodium &reated Frog B&op Do,n vie,C
333333333333333333..p.7
#$ Abstract""""""""""""""""""""""""""""""""""""""""""""""
"
-imb regeneration in mammals has long been a notion better belonging to the s*ien*e
fi*tion realm than in reality. Humans are able to demonstrate some regenerative *apabilities in
the liver and in the regenerating fingertips of young *hildren BIllings,orth "<:9C. Here ,e
demonstrate a study aimed to indu*ing regeneration of an amputated limb by an adult non=
regenerative frog. /egenerative medi*ine is an in*redibly interesting field of resear*h and its
potential for therapeuti* treatment is unmat*hed.
@hile previous studies have fo*used on ele*tri*al stimulation BSmith "<:9 4 )orgens
"<::C% ,e e2amined a pharma*ologi*al means of in*reasing the stump *urrent. Rana are a non=
regenerative spe*ies of frog that serve as a model organism to study regenerative gro,th.
Previous studies by the -evin -ab have demonstrated that regeneration in vertebrates *an be
indu*ed by transient sodium *urrent% spe*ifi*ally by the sele*tive sodium ionphore monensin.
Frogs ,ere amputated at the right forelimb and allo,ed to fully heal before treatment ,as
applied. &he ob0e*tive of this pro0e*t ,as to e2amine the regenerative abilities of the Rana
pipiens frogs in response to monensin treatments in sodium baths. &herefore% this study sought to
determine the ability of monensin to indu*e a regenerative event to post=morphogeni* non=
regenerative frogs
Although the study is still at an early time point and full analyti*al data is not yet
re*orded% early indi*ation sho,s that treatment ,ith monensin and sodium is *apable of indu*ing
a regeneration event in adult Rana pipiens. @hen *ompared to ,ater treated and monensin only
treatment groups% the monensin A sodium treatment produ*ed results that appear indi*ative of
blastema formation and propagation. After all gro,th has halted and animals are sa*rifi*ed%
histologi*al data ,ill reveal ,hat stru*tures% if any% ,ere regenerated.
%$ Background"""""""""""""""""""""""""""""""""""""""""""
"
a. /egenerative pro*ess
/egeneration is a ubiquitous pro*ess that fun*tions throughout the life *y*le in all
spe*ies. &hese regenerative pro*esses *an be bro(en do,n into three distin*t events as sho,n in
Figure ". 8ur resear*h fo*uses dire*tly on Dedifferentiation BFig ".C% ,hi*h allo,s for the lo*al
*ells around the ,ound site to be transformed into a stem=*ell=li(e state% a *riti*al initial step in
the regeneration pro*ess and formation of the blastemaBFig C.
After amputation% a layer of epithelial *ells (no,n as the ,ound epidermis *overs the
,ound area BSto*um !!6C. &his ,ound epidermis does not *ontain a basement membrane and
differs from the epidermis that is seen in normal ,ound response. &his initial pro*ess is very
*riti*al to regenerative su**ess% as for*ed ,ound *losure by se,ing a s(in flap over the
amputated area halts regeneration BIlling,orth "<:9 4 )orgens !!C. In addition% this
resistan*e of the ,ound epithelium *an dire*tly alter the stump *urrentsD stump *urrent and
,ound epithelium thi*(ness are in an inverse relationship. @hen the ,ound epithelium ,as
gently removed from a limb ,ith a lo, stump *urrent% the *urrent ,as immediately transformed
to a higher level BM*>innis "<;6C. After the amputated limb is *overed in a thin epidermal layer%
the blastema begins to form under the surfa*e.
&he blastema% a mass of dedifferentiated mesen*hymal *ells% is formed from underlying
tissues% in*luding mus*le% *artilage% dermis% and S*h,ann *ells B&ana(a !!7C. &he tissue *ells
dedifferentiate into a Estem=*ellF li(e state% allo,ing them to differentiate into bone% tissue% or
nerve. /e*ent resear*h by $rag et al. in !!< demonstrated that these *ells do not need to rea*h a
fully pluripotent state in order to su**essfully regenerate. &hey e2hibited that positional identity
,as retained by *artilage=derived blastema *ells% but not from S*h,ann *ell=derived blastema
*ells% indi*ating that most *ell types are restri*ted to their o,n tissue identity. &hese results *all
indi*ate that the blastema is a not 0ust a EmassF of dedifferentiated *ells% but rather retains some
original identity. Many fa*tors are believed to be responsible for signaling these *ells to
dedifferentiate. It has been demonstrated that the rate of ,ound healing and the e2tent of
proliferation are dire*tly regulated by ele*tri*al *ues BSong B!!CC.
&he final step in this pro*ess is the re=development of the amputated limb. &his stage is
*hara*teriGed by the gro,th and differentiation of the blastema. At this stage% studies have
indi*ated that the blastema is fun*tionally equivalent to a developing limb bud% seen in larval
development B>ardiner et al. !!C. +arly stage limb buds *an regenerate perfe*tlyD ho,ever% by
the end of limb development% this regenerative ability is *ompletely lost for mammals. Indu*ed
regeneration for organisms su*h as Henopus% results in the gro,th of a stru*ture (no,n as a
hypomorphi* spi(e instead of a fully fun*tional limb B)ro*(es "<<:C. Histologi*al analysis has
sho,n that mus*le% bone% tendons% ligaments and dermis are not *ontained ,ithin the spi(e% only
s(in% nerves and blood vessels
reform B)e*( et a., !!<C. In
order for proper re*onstru*tion%
these blastema *ells must
remember information about their
original positioning and features
of the lost limb. &his positional
identity is demonstrated in the
yearly regeneration of antlers in
deer B-evin !!<C. Figure 7
depi*ts the events of regeneration
from initial limb loss through to
full regeneration.
b. Stump .urrents
&he alteration of *ellular transmembrane potentials% due to ion flo,s% is thought to be a
triggering fa*tor for the *ells to dedifferentiate and enter a highly mitoti* state BHe*havarria et
al.C In fa*t a great deal of resear*h has indi*ated that stump *urrents play a *ru*ial role in the
regenerative and developmental pro*ess B)orgens% M*.aigC. &his *urrent must be pulled out of
the stump% as in,ard *urrent is *apable of indu*ing degeneration B)orgens B"<::% "<<6CC.&hese
stump *urrents originate in the subdermal area of the organisms% indi*ating that nervous system
signals are not responsible for driving ioni* flo, B)orgens B"<:<CC and that the battery is s(in
driven. &herefore% alteration of the s(ins permeability *an in*rease the flo, of ions. -arge steady
*urrents leave the end of the amputated stump in ne,t limbs for up to t,o ,ee(s after an
amputation. &hese *urrents li(ely in*rease the signaling path,ay pro*ess and e2pose the
amputation site to a variety of fa*tors that signal *ells to dediferrentiate B-evin B!!<CC. &hese
ne,ts are *apable of *omplete regeneration throughout their lifetimeD ho,ever% most frog spe*ies
do not e2hibit the same type of regenerative *apability. &he *urrent density leaving the stump of
Rana pipiens is greatly depressed Bless than a quarterC in *omparison to ne,ts. It has been
suggested that these depressed *urrents are due to shunts *aused by the subdermal sinuses of the
frogs as seen in Figure 9 B)orgens B"<:<CC. Rana pipiens tadpoles retain the ability to regenerate
until the adult phase% mat*hing the development of the subdermal sinuses B)orgens B"<:<CC.
&hese greatly depressed *urrents are in*apable of indu*ing regeneration. &herefore% the
development of a large and uniform *urrent out of the stump is *riti*al for the regeneration
pro*ess. Sodium ions play a *riti*al role in this pro*ess as they are largely responsible for
*ontrolling membrane potentials. In fa*t% the magnitude of the stump *urrent is often dependent
upon the *on*entration of sodium in the e2ternal medium B)orgens B"<;9CC. In addition% adult
frogs are more responsive to *hanges in Sodium *on*entration than larval stage organisms B/ose
B"<99CC. As su*h% the fo*us of this and many other studies are to determine effe*tive methods to
in*rease this stump *urrent.
*. .lassi*al Studies
High degrees of frog limb
regeneration have been produ*ed
through resear*h studies. For
instan*e% implantation of an
additional adrenal glands in
hypophyse*tomiGed frogs resulted
in a very high degree of
regenerationBS*hotte 4 @ilber
"<5;C. 8ur resear*h is fo*used on
in*reasing the regenerative
abilities of Rana pipiens by
stimulating the stump to produ*e a
larger stump *urrent. Previous
studies have fo*used on
a**omplishing this same out*ome% mainly through dire*t ele*tri*al stimulation. A study by Dr.
Stephen Smith ,as one of the first to investigate the effe*ts of using an implantable ele*trode in
frogs to stimulate regeneration. He used adult Rana pipiens frogs that had entered into the post=
metaphori* period of gro,th. He (ept these frogs in individual plasti* bo2es housed in a 9I.
in*ubator ,ith a t,elve hour light *y*le. In addition% he fed the animals three times a ,ee( on
meal,orm larvae. Frogs ,ere divided into 9 separated treatment groups of 5 frogs ea*h%
anesthetiGed% and then amputated ,ith s*issors at the mid=forearm level. &he ".75 ? mer*ury *ell
,as pla*ed into an in*ision in the dorsal midline of the frog. 1e2t they fed a &eflon=insulated
&he results of SmithJs e2periment indi*ated a large amount of regeneration *aused by the
implantation of this ele*trode. In all 9 treatment groups the vast ma0ority of frogs demonstrated
at least a grade regeneration. In addition% treatment groups II 4 I? demonstrated several grade
9 regenerates. In fa*t% all " surviving frogs of group I? produ*ed some regenerative response.
He reports that one of his grade 9 regenerates produ*ed a *ompletely organiGed regenerate that
,as Eabsolutely indistinguishable from a normal one.F Ho,ever% this individual frog represented
an e2treme *ase and all other grade=9 regenerates ,ere not nearly to the same degree.
Interestingly% both of the posta2ial treatment groups produ*ed a higher proportion of regenerates%
espe*ially grade 7 and 9 regenerates than the prea2ial groups. &hese results *an be seen in &able
".
Smith su**essfully demonstrated that a very small ele*tri*al *urrent is *apable of having
a profound effe*t on the stimulation of regeneration% but spe*ifi*ally in a non=regenerating
spe*ies. In addition% the position of the implant played a *riti*al role in the development of the
regenerate. Smith *on*luded that stimulation of the posta2ial stump must mimi* the embryoni*
state more a**urately than prea2ial stimulation. &hese results provide a great deal of information
about the possibility to indu*e stump regeneration% ho,ever there is a large amount of
information missing. For instan*e% Smith introdu*ed no *ontrol samples to demonstrate that the
regenerative effe*ts ,ere not due to an additional fa*tor. Spe*ifi*ally% he needed t,o non=gro,th
*ontrols# 8ne group that *ontained no ele*trode and ,as left untreated% as ,ell as an additional
group that *ontained a sham ele*trode. .ontrols li(e these are imperative to provide a means to
*ompare the results. In addition% it is possible that the addition of the antiba*terial and antifungal
po,der *ould have some measured effe*t. Although they used only healthy and vigorous frogs%
pla*ing the frogs in individual *ontainers in an in*ubator is an interesting approa*h to frog *are%
as frogs in high stress or un*omfortable situations may be less li(ely to fo*us all energy on limb
regeneration.
In "<::% three years after
SmithJs ,or(% a resear*h team led
by /. )orgens sought to follo,=
up on Dr. Smiths initial
observations. &hey used a nearly
identi*al e2perimental setup% as
sho,n in figure 6. &herefore they
,ere able to improve upon several
aspe*ts that greatly improved the
quality of their e2perimental
pro*edure. 8ne of the most important adaptations that )orgens used ,as the introdu*tion of a
,i*( stimulator that is able to dra, *urrent through the stump ,ithout introdu*ing signifi*ant
ele*trode produ*ts into the tissue. &his ,as made possible by using Ag=Ag.- ele*trodes
*onne*ted via a ,i*( bridge% instead of an ele*trode made using an unrea*tive metal su*h as
platinum% as used by Smith. &he only to2i* produ*t of this system is the AgA ions that are
released from the interfa*e bet,een the silver ele*trodes and the Ag.l aqueous environment
prote*ting it. )orgens maintained large adult Rana pipiens in individual plasti* bo2es ,ith
moistened to,eling at the bottom. &he frogs ,ere fed three times a ,ee( on live meal,orm
larvae% and ,ere inspe*ted for ,ellbeing before surgi*al pro*edures. )orgens used MS % as
did Smith% to anesthetiGe the frogs and amputated at the right forearm. &,enty frogs ,ere
implanted ,ith these ,i*( stimulators that ,ere *apable of delivering !.":=!. KA of negative
*urrent% and ten ,ere implanted ,ith anodal stimulators Bpositive *urrentC. In addition% )orgens
in*luded ten more frogs that ,ere implanted ,ith a sham stimulator to a*t as a *ontrol group. All
units ,ere removed after three to four ,ee(s% but frogs ,ere allo,ed to *ontinue gro,th for up
to one year.
&he results from )orgenJs e2periment sho,ed mar(ed and *onsistent differen*es bet,een
the three e2perimental groupsD *athode stimulate% anode stimulated% and sham stimulated.
Although there ,ere a fe, atypi*al responses% *athode stimulated animals sho,ed an organiGed
e2tension of the radio=ulna. In addition there ,as some gro,th of nerve and mus*le tissue. Sham
stimulated animals sho,ed formation of disorganiGed *alluses% ,ith little to no nerve or mus*le
gro,th. In *omparison% anode stimulated frogs sho,ed e2tensive degeneration% a *onfirmation of
their earlier ,or( ,hi*h suggested *urrent dire*ted into the stump *urrent *ould de*rease or
inhibit stump gro,th. In addition% both sham and anodally stimulated frogs demonstrated no
*hange in e2ternal appearan*e after 5=; ,ee(s post amputation. .athodally stimulated frogs
sho,ed grossly s,ollen and red ,ound sites at 9=6 ,ee(s post amputation% and formed a
blastema. &hese bulbous masses *ontinued to gro,th and *hange form for up to "" months. &he
.athode treated frogs ,ere the only ones to e2hibit gro,th of nerve trun(s% regenerated mus*le
and organiGed bone e2tension. Ho,ever% there ,as one atypi*al anode stimulated frog that
developed some nerve and mus*le regeneration. A s*hemati* diagram of the differen*e bet,een
ne,t s(in batteries and implanted frog batteries is sho,n in figure :.
Although )orgenJs results *learly delineated a differen*e bet,een out flu2 and influ2 of
*urrent% the frogs in his study did not e2hibit nearly as high a degree of regeneration as did
SmithJs study. Although these differen*es bet,een the t,o studies *ould be due to a variety of
fa*tors% it raises some questions about the regeneration of Rana, parti*ularly due to the presen*e
of t,o atypi*al regenerates% the fully regenerated spe*imen des*ribed by Smith% and )orgenJs
anodally treated frog that sho,ed minor regeneration. Although both ,ere e2treme *ases ,ithin
their treatment groups% it is interesting to note that this atypi*al event may be a *ommon
o**urren*e. Despite these differen*es% both studies demonstrated that relatively small *urrents
*an evo(e limb regeneration in a typi*ally non=regenerative spe*ies. &hese studies demonstrated
that dire*t ele*tri*al stimulation is *apable of *ausing this regenerative responseD ho,ever the
implantation of su*h a devi*e requires t,o pro*edures and is very invasive. &herefore% a
pharma*ologi*al means of indu*ing a regeneration response ,ould represent an interesting
approa*h to regeneration
d. .urrent Studies
/e*ent ,or( by Mi*hael -evinJs Developmental )iology lab BA. &seng !"!C has
indi*ated that vertebrate regeneration *an be indu*ed by transient sodium *urrents into spe*imens
that gro, a nonpermissive ,ound epithelium% as most non=regenerating spe*ies do. &his is
possible through dire*t modulation of the voltage=gates sodium *hannels to in*rease the sodium
transport and thus the stump *urrent. &he resear*h team used the larval stage spe*imens of the
aquati* frog spe*ies Xenopus laevis% ,hi*h has ,idely been impli*ated as a po,erful study
organism that uses several regeneration path,ays *onserved by mammals B)e*( !!<C. In the
larval stages it is *apable of *omplete repair of all appendages upon in0ury% and even sho,s
regenerative ability in adult stages.
&he resear*h team first investigated the importan*e of sodium flu2 in regeneration by
blo*(ing the voltage=gated sodium *hannels during larval development. In addition% through the
use of /1A interferen*es of the Xenopus 1a
?
". gene% they demonstrated that these sodium
*hannels are a requirement for the establishment and outgro,th phases of regeneration in
Xenopus. &his partially e2plains ,hy in*reasing sodium *on*entration of frog baths *aused a
great in*rease in the stump *urrents of the frogs B)orgens "<::C. &hese *hannels are *riti*ally
a*tive during the initial stages of the regenerative response% *ausing a large influ2 of sodium
thusly *reating a strong *urrent leaving the stump site. &hey ne2t investigated the effe*ts of the
sodium ionophore monensin to dire*tly modulate sodium transport ,ithout geneti* manipulation.
&o a**omplish this tas(% they amputated larval tails that had entered into the refra*tory
period. During this stage the Xenopus tadpoles have lost their regenerative potential and ,ill no
longer be able to regenerate a tail. At a similar time=point to the native 1a
v
". e2pression% the
tadpoles ,ere treated ,ith monensin in a medium *ontaining elevated levels of sodium BSodium
>lu*onateC. 'sing a .oro1a
>reen indi*ator dye to visualiGe sodium *ontent% they *onfirmed that the monensin treatment
in*reased intra*ellular sodium *ontent Bfigure ;C. In addition% this ,as *apable of returning the
tadpole to a regenerative state in ,hi*h full regeneration is *apable. &heir results indi*ated that it
,as the effe*t of the indu*ed sodium influ2% rather than monensin or high e2tra*ellular sodium
treatment alone.
&hese results represent a novel role for the ?oltage=gated sodium *hannels in the
mediation of sodium transport. &hey ,ere able to demonstrate that 1a
v
a*tivity ,as required
during the initiating stages of limb regeneration. In addition% monensin has the *apability of
indu*ing a large sodium influ2 that *an effe*tively mimi* the a*tion of these *hannels ,hen they
are either blo*(ed or have lost fun*tion and guide regenerative outgro,th through *ell
proliferation and gene e2pression. It has long been thought that on*e *ells rea*h the refra*tive
period they lose all ability to regenerate. Ho,ever% these results indi*ate that the *ells maintain
their intrinsi* ability and represent an e2*iting pharma*ologi*al approa*h to restore regeneration.
e. Monensin
Monensin is an ionophore antibioti* that is *apable of *ollapsing 1a
A
and H
A
gradients.
Monensin is an open *hain mole*ule BFig. <C% that is *apable of sele*tively binding sodium at an
affinity ten times greater than its nearest biologi*al *ompetitor $
A
BMollenhauer "<<!C. It is
*omposed of non=polar hydro*arbons ,hi*h allo, it the *omple2 to be freely soluble in the lipid
membranes BMollenhauer B"<<!CC. &hrough this binding% monensin is *apable of transversing the
membrane in this monensin=ion *omple2. 8n*e it rea*hes the interior membrane% it releases the
ion and binds to a proton. It then returns through the membrane and e2*hanges the proton to the
e2ternal medium as sho,n in Figure < BMollenhauer "<<!C. 8n a *ellular level monensin has
f. 8ur Approa*h
&he ,or( by the -evin >roup demonstrated an intriguing approa*h to indu*e stump
*urrents during non=regenerative gro,th periods. Although they used the aquati* Xenopus laevis
model ,hi*h e2hibits regenerative *apabilities throughout its life *y*le% the previous ,or( by
Smith and )orgens B&able Chas suggested that even non=regenerative spe*ies su*h as Rana
pipiens are *apable of a regeneration response. Xenopus laevis are a very effe*tive study
organism that requires fairly minimal Bless involvedC *are% ,e ,anted to study the effe*ts of
Monensin on the Rana pipiens frog due to its non=regenerative abilities. &his ,ould allo, for
greater a**ura*y ,hen determining the degree of regenerative gro,th of the frogs% be*ause
endogenous regeneration ,ill not be *onfused ,ith indu*ed regeneration. In addition% previous
studies that have used Rana as a study organism have ta(en minimal effort into their *are and
,ellbeing. &herefore% it ,as of paramount importan*e to our study that ,e provided the most
*omfortable and natural habitat for our study organisms. Poor *onditions ,ill indu*e a stress
response in the frogs *ausing lots of e2*retion and s(in shedding. 'nder these *onditions% the
frogs ,ill be*ome un*omfortable and less li(ely to feed properly% de*reasing their health and
re*overy abilities. &able belo, outlines the results and methods of several previous studies.
Studies &ethod Time 'oint Species (utcome
)orgens B"<::C Implantable )attery Post=amputation Adult Rana Slight /egeneration
.e*il B"<;6C ?itamin A treatment Post=amputation Juvenile Rana +nhan*ed regeneration
Smith B"<:9C Implantable battery Post=amputation Adult Rana Some full regeneration L
&seng B!"!C Monensin Post=refra*tory Xenopus tadpoles full regeneration
&herefore this presents a situation in ,hi*h the regenerative response of Rana *an be
determined in response to pharma*ologi*al treatment instead of dire*t ele*tri*al stimulation
requiring implantation and removal operation. In addition% be*ause the monensin treatment is
applied after a normal ,ound healing response% it represents a ne, method of regeneration
indu*tion that is free of the pre*on*eived regeneration timeframes. Although the dire*t
me*hanisms Bdo,nstream pro*essesC through ,hi*h sodium *urrent *ontributes to the
regenerative pro*ess is the sub0e*t of many *urrent studies% it has *learly been impli*ated as
having an important role in the indu*tion of regeneration B-evin !"!C. In addition% be*ause the
degree of differen*e bet,een the regeneration events e2perien*ed by the )orgenJs study and Dr.
SmithJs study% additional studies should investigate this regeneration in order to *ompare the
results.
)$ *ypothesis""""""""""""""""""""""""""""""""""""""""""""
In this study ,e e2amine the ability of treatment ,ith monensin and sodium to the
amputated forelimbs of non=regenerating post=metamorphi* Rana pipiens. Previous studies have
indi*ated that regeneration *an be indu*ed in Rana frogs as demonstrated in &able . Ho,ever%
,e present a novel timeframe in ,hi*h regeneration *an o**ur after the normal ,ound healing
response. @e hypothesiGe that treatment ,ith Monensin A sodium ,ill result in a higher degree
of regeneration than sham treated and Monensin only treated individualsD ho,ever% full
regeneration of a fun*tional limb ,ill not o**ur. /egeneration events in the e2perimentally
treated individuals ,ill indi*ate that a regeneration event *an be indu*ed outside of the refra*tory
period.
+$ Specific
Aims"""""""""""""""""""""""""""""""""""""""""""
&he spe*ifi* aim of this study ,as to determine the response of Rana pipiens to the sodium
ionphore monensin. Previous studies by &seng et al demonstrated that monensin ,as *apable of
indu*ing regeneration of a full tail after the refra*tory period of tadpole tail gro,th. Previously%
regeneration ,as assumed impossible after the refra*tory period had passed. &herefore% by
e2amining the ability of monensin and sodium treatment to indu*e regeneration in a typi*ally
non=regenerative spe*ies ,e see( to further develop an understanding of the role of monensin in
regeneration. &his study see(s to evaluate the regenerative response of these treated spe*imens
in *omparison to *ontrol treated spe*imens. &hese *ontrols should e2hibit no outgro,th and
*onfirm that Rana pipiens is a non=regenerative spe*ies in the adult form. In addition this study
see(s to provide the best *are *onditions for the study organisms. @e believe that by providing
the most natural habitat and providing full *are for the frogs that the most natural response to the
treatment ,ill be a*hieved. )e*ause the frogs ,ill be *omfortable in their environment and ,ell=
fed% they ,ill be *apable of fo*using all energy on regeneration instead of to,ards a stress
response and hypera*tivity.
,$ &ethods""""""""""""""""""""""""""""""""""""""""""""""
a. +2perimental Pro*ess
b. Animal .are
All spe*imens ,ere *ared for in a**ordan*e to submitted proto*ols for IA.'. an D-AM.
I. Study 8rganism M Rana pipiens
Rana pipiens is a fairly large spe*ies of frog that rea*hes a length of about 9.5 in*hes BFig.
""C. &hey are a member of the /anidae family of frogs. &his family is referred to as E&rue
FrogsF be*ause they have smooth moist s(in% ,ebbed feet and po,erful hind legs BHalliday
"<;6C. In addition their s(in is *apable of *hanging hue to improve its *amouflage depending
on its environmental surroundings. &heir natural habitat is lo*ated around permanent sour*es
of ,ater ,ith aquati* vegetation that is abundant. Most of their time ,ill be spend in marshy
or grassy land% but they ,ill return to the ,ater for breeding and hibernation. Although they
are opportunisti* feeders% they are very ,ell adapted to hunting due to their great 0umping
ability as ,ell as their sti*(y tongue ,hi*h allo,s them to grab and hold onto prey su*h as
*ri*(ets BHofri*hter B!!!CC. .5F frogs ,ere a*quired from 1AS.8. &he frogs ,ere rush
shipped to our lab and ,ere immediately unpa*(ed and *he*(ed for si*(ness or any
noti*eable *hange in appearan*e. &hey ,ere then pla*ed into tan(s at a ma2imum of ": per
tan(% or else over*ro,ding *an be*ome an issue.
II. .age Setup
&opFin glass aquariums ,ere used to house all of the frogs. ?arying aquarium siGe ,as used
depending on the amount of frogs *ontained ,ithin ea*h tan(. )e*ause the frogsJ ideal
habitat is aquati* based but ,ith lots of surrounding dry terrain% I attempted to *reate a
terrarium setup that in*luded both parameters BFig "C. Smooth aquarium ro*(s ,ere first
added to the empty tan( to provide a base for the substrate. /o*(s ,ere pla*ed about an in*h
thi*( for the first half of the tan(. &he substrate used ,as Noo=MedJs +*o +arth *o*onut fiber
substrate. &his substrate has the ability to absorb and brea(do,n ,aste produ*ts in these
amphibian habitats% therefore it (eeps the tan(s from smelling li(e frog urine. In addition%
this substrate does not allo, for ba*terial or fungal gro,th% ma(ing it an ideal solution. &he
substrate *omes in *ompressed bri*(s that are soa(ed ,ith ,ater to allo, them to separate.
III. Feeding
All frogs are fed live *ri*(ets three times a ,ee( ad libitum. .ri*(ets are removed from the
*age and pla*ed into a *ri*(et sha(er. Here they ,ere *oated ,ith *al*ium po,der to
supplement the frogJs diet. In addition to providing *al*ium supplementation% the *al*ium
po,der made the normally bro,nish *ri*(ets a pure ,hite *olor. &his eliminated their natural
*amouflage in the substrate and made them easier to see and therefore *at*h by the frogs.
After the *ri*(ets ,ere ,ell *oated in *al*ium% they ,ere dropped into the frog tan(s.
&ypi*ally ea*h frog ,ould eat 7=5 *ri*(ets per feeding time% but in some of the larger tan(s it
is nearly impossible to assure a *ompletely balan*ed distribution. .ri*(ets ,ere *ontinually
added until frogs did not sho, any more interest in eating. After the frogs appeared to slo,
do,n their eating% any *ri*(ets that remained in the tan(s ,ere hand fed to individual frogs
using tongs. Although most frogs ,ere initially afraid of the tongs% after *ontinued use they
be*ame a**ustomed to it and did not 0ump a,ay. Frogs ,ere never for*e fed.
I?. Frog &an( .are
After ea*h feeding% the ,ater in the frog tan(s ,as *hanged and the tan( ,as gently *leaned.
'sing a siphon atta*hed to the sin( in our room% the ma0ority of the ,ater in the tan( ,as
removed. In addition be*ause the siphon ,as atta*hed to the sin(% ,e ,ere able to *reate very
po,erful su*tion that ,as useful to remove ,aste% fe*es and s(in% from the tan(. 8n*e the
ma0ority of the ,ater had been removed% the tan(s ,ere allo,ed to sit as the frogs finished
the remaining *ri*(ets. During this time% ,ater and ,aste that had been absorbed by the
*o*onut substrate ,as allo,ed to filter out into the little ,ater than remained in the tan(. &his
remaining ,ater ,as s*ooped out via a small *apsule so that there ,as essentially no ,ater
remaining. &ongs ,ere used to remove remaining s(in and ,aste that ,as either on the dry
terrain portion or ,as stu*( toOin the ro*(s. After the tan(s had been *leaned% Poland Spring
,ater ,as added ba*( into the tan( to its original ,ater level BFig. "7C
8ften times the a*tivity of the frogs *auses the ro*( slope to degrade and e2poses the leading
edge of the *o*onut substrate to the ,ater. &he tidal a*tion *aused by frogs moving in and out
of the ,ater slo,ly erodes a,ay the substrate% de*reasing the amount of dry land spa*e.
@hen these situations o**ur% the eroded *o*onut is removed from the bottom of the tan( and
the slope is rebuilt. Additional substrate is added to return the land terrain to its original form.
After a month of use% tan(s ,ere *ompletely *leaned by removing all substrate and the ro*(s
and s*rubbing the tan(. &his ensures that the substrate does not be*ome oversaturated ,ith
,aste produ*ts and (eeps the frog habitats relatively fresh.
In the event that a frog sho,s sign of infe*tion or si*(ness% it is immediately separated from
the other frogs and pla*ed into a solitary *age *ontaining a lo, level of ro*(s and substrate
but *overed *ompletely in ,ater. Antibioti*s ,ere administered to the ,ater to improve the
infe*tion. Si*( frogs ,ere isolated for up to a ,ee(% at ,hi*h time their appearan*e typi*ally
returned to normal. 8n*e they returned to normal appearan*e% they ,ere returned to their
original tan(.
?. .ri*(et .are
.ri*(ets ,ere ordered through and delivered by >hannJs .ri*(et Farm B,,,.ghann.*omC% on
a ,ee(ly basis. Appro2imately "%5!! *ri*(ets in the siGe ,ere ordered. &he *ri*(ets ,ere
equally distributed into *ri*(et *ages BFig. "7C to prevent over*ro,ding. &he *ri*(et *ages
*ontained t,o feeding troughs filled ,ith >hannJs *ri*(et *ho,. In addition% *ri*(ets require
,ater% ho,ever they *an easily dro,n in large bodies of it% so *ri*(et pillo,s ,ere used.
8n*e added to ,ater% the pillo,s rapidly absorb moisture and balloon up. &,o to three of
these pillo,s ,ere pla*ed in ea*h *age. Finally% *ri*(ets require a dar( habitat in ,hi*h to
hide% or else they ,ill stress out and die. +gg *rates are *ommonly used% but for the siGe of
our *ages they ,ere rather in*onvenient. &herefore ,e used long bla*( tubes ,ith a *overed
outside end that had been gauged on the interior to allo, *ri*(ets to *limb into them. 1ot
only did these tubes provide an adequate pla*e to hide% but they also made the feeding
pro*ess very easy. If egg=*rates ,ere used% it ,as very diffi*ult to effe*tively dump them into
the *ri*(et sha(er. &hese tubes allo,ed for easy removal from the *ri*(et *ages and then
subsequent dumping into the *ri*(et sha(er. In addition% this prevented me from having to
individually pi*( up ea*h *ri*(et ,ith tongs% as ,as ne*essary ,hen *leaning the *ages.
?I. Post=operative *are
Follo,ing surgi*al pro*edure% as ,ill be dis*ussed ne2t% frogs ,ere maintained in individual
,eigh boats ,ith a small amount of ,ater. &he amputated limb ,as slightly elevated out of
the ,ater so that it *ould su**essfully *lot. Frogs ,ere periodi*ally sprayed ,ith ,ater to
ensure that they ,ould not dry out. @hen the frogs are anesthetiGed% they are e2tremely limp%
therefore any movement of the frog must be made *arefully or else the ,ound site *ould be
reopened. Frogs ,ere allo,ed to re*over in their individual areas until the bleeding had
stopped and they ,ere *apable of supporting themselves. 8n*e they ,ere *apable of free
movement% they ,ere returned to a re*overy tan( ,ith the other frogs that ,ere also
re*overing.
*. +2perimental Setup
I. .ontrol and +2perimental >roups
All frogs under,ent the same e2a*t surgi*al and *are pro*edure. About 9; days after the date
of amputation the frogs ,ere bro(en into different treatment groups. &he +2perimental group
of frogs ,as treated in a sodium and monensin bath. Several *ontrol groups ,ere used to not
only to ensure that the frogs did not possesses natural regenerative *apabilities% but also to
ensure that if regeneration o**urred in the e2perimental treatment groups% that it ,as due to
monensin and sodium. In addition% it provides a point of *omparison for the e2perimental
treatments% and ensures that monensin itself is not *hemi*ally responsible for the
regeneration indu*tion% but rather stump *urrent *reated through the a*tion of the sodium
ions. &hese treatment groups are displayed in &able 7.
Treatment -umber .roup
Monensin A Sodium A Poland Spring 5 +2perimental
Monensin A Poland Spring 5 .ontrol
Poland Spring 5 .ontrol
II. Surgery
For ea*h surgery day% a ne, tan( is prepared to house the post=amputation frogs. About "5
frogs that ,ere in good health and had been a**limated to the lab environment for at least
,ee(s ,ere amputated on ea*h surgery day. Frogs ,ere not fed ,ithin 9hrs of the surgi*al
pro*edure. Frogs are removed from their original housing and in0e*ted ,ith =9m-
Bdepending on siGeC of tri*aine mesylate BMS=C the same anestheti* used by both Smith
and )orgens. Frogs ,ere allo,ed to go *ompletely under the affe*ts of anesthesia before an
operation ,as to o**ur. &he frogs ,ere pla*ed on their stoma*h in a spra,led out position
and observed for any sort of movement. If the frogs *ontinued to t,it*h and sho, slight leg
movements% then an additional in0e*tion of &ri*aine ,as used. 8n*e frogs ,ere *ompletely
anesthetiGed% typi*ally after a P hour% their right forearm ,as amputated ,ith a single *lean
*ut through tissue and bone ,ith a 1o. "" surgi*al grade s*alpel BFig. "5C. Although some
spe*imens e2hibited e2tensive bleeding% most bled relatively minor amounts. After
amputation the frogs ,ere pla*ed into their individual ,eigh boats as outlined by the post=
operative *are pro*edure
III. &reatment )aths
All treatments ,ere applied 9; days post amputation% after the frogs have endured a normal
,ound healing event% and the amputated site is largely *overed by non=permissive ,ound
epithelium. &he e2perimental group of frogs is introdu*ed to the monensin treatment. Small
*ontainers% *apable of *omfortably holding =7 frogs are filled ,ith about an in*h of solution%
or enough to *over the ,ound area. &he solution *ontains about !! KM Monensin and ";!
mM 1a> 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
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%
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
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 adult Rana 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
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.
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 monensinJs 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
B-evin !!<C% ,here a variety of physiologi*al fa*tors may be a*ting to signal the *ells to
dedifferentiate. Additionaly% the role of ioni* *urrents has been demonstrated to play a *riti*al
role in the limb development pro*ess of mammalian vertebrates. A !!" study by AltiGer et al.%
as part of )orgens group% demonstrated the importan*e of these *urrents in limb development of
*hi*( and mouse embryos. 'sing a vibrating probe they monitored the e2terior portion of the
embryo for ioni* *urrents. &hey dis*overed that the emergan*e of the murine limb bud ,as
asso*iated ,ith a steady out,ardly dire*ted ioni* *urrent. In addition% in,ardly dire*ted flan(
*urrents ,ere observed at flan( regions of the embryo indi*ating the *ompletion of a d* *ir*uit.
&he resear*hers ,ere able to indu*e developmental defe*ts in these embryos by applying an
in,ardly dire*ted *urrent to the sites of limb development. &hese results of this resear*h team
indi*ated that ioni* *urrents are *ru*ial to the development of a normal limb. If monensin is
*apable of indu*ing regeneration at a timepoint after the normal regeneration response is
*onsidered a*hievable% then it may be a*ting to *reate *onditions that are very similar to the
developmental stages. Although humans are in*apable of large s*ale regeneration% the
developmental pro*esses used are similar through most animals. Monensin may be *apable of
indu*ing the mammalian *ells into a developmental=li(e state of regeneration.
As previously dis*ussed% proper frog *are ,as *onsidered to be a *ru*ial *omponent of
this study. @hen frogs initially arrived they ,ere very startled by any sort of human intera*tion
and ,ould rapidly 0ump around their habitat attempting to es*ape. After a ,ee( or t,o of
a**limitiGation in the lab environment their fear subsided and they ,ere be*ame more
*omfortable ,ith human intera*tion. In addition% they be*ame a**ustomed to the ,ee(ly feeding
*y*le and their a*tivity and a,areness ,ere mar(edly in*reased shortly before feeding time.
During this period leading up to feeding% as ,ell as during and immediately after% the frogs
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( $o0i* in the Spring of !"!% and it ,asnJt until the late fall of
that same year that our first frog spe*imens arrived. It has been a very long and time *onsuming
pro*ess from starting off ,ith a fe, Xenopus laevis frogs to transitioning to in*lude over !!
Rana pipiens. Ho,ever% the *ontinued effort has led to a fantasti* learning e2perien*e%
spe*ifi*ally for e2perimentation using live animals% as ,ell as a great opportunity to learn from
1i( and Punita. Although my ,or( on this pro0e*t is shortly *oming to a *lose% there remains a
great amount of ,or( and resear*h that ,ill be *ontinue to be *ondu*ted on this on=going study.
2$ onclusion""""""""""""""""""""""""""""""""""""""""""""
"
a. &his Pro0e*t
1o definitive *on*lusions *an be dra,n at this time point be*ause it is still at a very early
stage in this study. Although some of the initial results indi*ate that Monensin and sodium are
indu*ing a Eregeneration=li(eF event in the amputated frogs% a greater degree of reprodu*ibility is
required. As has been demonstrated by previous studies% these frogs have demonstrated a ,ide
range of regeneration responses for similar to identi*al treatments. If Monensin and sodium
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!!<C.
@ith the promise demonstrated by the initial results% a strong *ontinued effort on this
investigation ,ill hopefully indi*ate the ability of monensin to a*t as a regeneration indu*er.
.urrent ,or( is fo*used on providing information regarding the effe*t of environmental and
*hemi*al fa*tors on the frogJs regeneration. It is e2pe*ted that these treatments ,ill not
demonstrate regeneration and thus ,ill further impli*ated the role of sodium and monensin.
b. Potential of /egeneration
'sing regeneration as a means of therapeuti* treatment still remains a distant possibility.
/esear*hers are still ,or(ing to analyGe all the fa*tors that enable many organisms to
demonstrate the ability to regenerate lost limbs and organs. 'ntil all these fa*tors *an be
determined and understood% use on a human model is not possible% although that remains the
ultimate goal. Mu*h of these resear*hers ,or( is spent determining *onne*tions and similarities
bet,een pro*esses in regenerating spe*ies and in humans. A study by A. AltiGer et al. indi*ated
that the lo*ation of flan( *urrents in the larvae of *hi*(s and mouse are indi*ative of the site of
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 of Xenopus frogsD 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 dayD 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 pipiens are not available
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
vitro studies% 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 promisingD 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
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 that Rana pipiens are 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.
SmithJs 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
through the use of ion spe*ifi* vital dyes. &hese dyes allo, for the in vivo monitoring 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.
"!. 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<"=9!".
AltiGer A% Ste,art S% Albertson )% 4 )orgens / B!!C. S(in flaps inhibit both the *urrent of
in0ury at the amputation surfa*e and regeneration of that limb in ne,ts. Journal of +2perimental
Noology <7# 96:=9::.
)e*( .% )elmonte J% 4 .hristen ). B!!<C. )eyond early development# Xenopus as an emerging
model for the study of regenerative me*hanisms. Developmental Dynami*s 7;# "6="9;.
)ely A% 4 1yberg $. B!!<C. +volution of animal regeneration# re=emergen*e of a field. &rends
in +*ology and +volution. Arti*le in Press.
)orgens /% ?anable J% 4 Jaffe -. B"<::C. )ioele*tri*ity and /egeneration# I. Initiation of Frog
-imb /egenertion by Minute .urrents. Journal of e2perimental Goology !!# 9!7=9"6.
)orgens /% ?anable J% 4 Jaffe -. B"<::C. )ioele*tri*ity and /egeneration# -arge *urrents leave
the stumps of regenerating ne,t limbs. Developmental )iology :9."!# 95;=957.
)orgens /% ?anable J% 4 Jaffe -. B"<:<C. /ole of subdermal *urrent shunts in the failure of frogs
to regenerate. Journal of e2perimental Goology !<# 9<=56.
)orgens /% ?anable J% 4 Jaffe -. B"<:<C. Small artifi*ial *urrents enhan*e Xenopus limb
regeneration. Journal of e2perimental Goology !:# ":=6.
)orgens /% /ouleau M% 4 De-anney - B"<;7C. A steady efflu2 of ioni* *urrent predi*ts hind
limb development in the A2olotol. Journal of +2perimental Noology ;#9<"=5!7.
)orgens /% M*>innis M% ?anable J% 4 Miles + B"<;9C. Stump *urrents in regenerating
salamanders and ne,ts. Journal of +2perimental Noology 7"# 9<=56.
)ro*(es J. B"<<:C. Amphibian limb regeneration# rebuilding a *omple2 stru*ture. S*ien*e :6#
;"=;:.
)ro*(es J. 4 $umar A. B!!5C. Appendage regeneration in adult vertebrates and impli*ations
for regenerative medi*ine. S*ien*e 7"!# "<"<="<7.
.allaerts P. 4 De -oof A. B"<<7C. Developmental effe*ts of monensin on Drosophilia
Melanogaster. /ou2Js Ar*hives of Developmental )iology. !7#;7=<".
.arlson ). B!!:C. Prin*iples of /egenerative )iology. 1e, Ror(# +lsevier In*.
Duffield &% /abiee A% 4 Jean I B!!;C. A Meta=Analysis of the Impa*t of monensin in la*tating
dairy *attle# Part . Produ*tion effe*ts. Journal of Dairy S*ien*e. <".9#"79:="76!.
>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"<;6C. &he +n*y*lopedia of /eptiles and Amphibians. 1e, Ror(% 1.R.#
Fa*ts on File. Print.
Hofri*hte / B!!!C. Amphibians# the @orld of Frogs% &oads% Salamanders and 1e,ts. )uffalo%
1.R.# Firefly. Print.
Illing,orth .. B"<:9C. &rapper fingers and amputated finger tips in *hildren. Journal of Pediatri*
Surgery <# ;57=;5;
Jen(ins -% Duersto*( )% 4 )orgens / B"<<6C. /edu*tion of the *urrent of in0ury leaving the
amputation inhibits limb regeneration in the red spotted ne,t. Developmental )iology ":;# 5"=
6
$rag M et al. B!!<C. .ells (eep a memory of their tissue origin during a2olotl limb
regeneration. 1ature 6!# 6!=6:.
-i*htshtein D% Dunlop $% $aba*( H% 4 )lume A B"<:<C. Me*hanism of monensin=indu*ed
hyperpolariGation of neuroblastoma=glioma hybrid 1>"!;="5. Pro*. 1atl. A*ad. S*i :6.6# 5;!=
5;9
-evin M. B!!:C. -arge=s*ale biophysi*s# ion flo,s and regeneration. &rends in .ell )iology
B":C 6# 6"=:!.
-evin M. B!!<C. +ditorial# /egeneration# /e*ent advan*es% ma0or puGGles% and biomedi*al
opportunities. Seminars in .ell 4 Developmental )iology !# 5"5=5"6.
M*>innis M 4 ?anable J B"<;6C. @ound epithelium resistan*e *ontrols stump *urrents.
Developmental )iology ""5# ":9=";7.
Mollenhauer H% Morre D% 4 /o,e - B"<<!C. Alteration of intra*ellular traffi* by monensinD
me*hanism% spe*ifi*ity and relationship to to2i*ity. )io*hemi*a et )iophysi*a A*ta. "!7"# 5=
96.
8viedo 1 4 )eane @ B!!<C. /egeneration# &he 8rigin of *an*er or a possible *ureS Seminars
in .ell 4 Developmental )iology !# 55:=569.
8G(u*ur 1% +pperlein HH% Fun( / B!"!C. Ion imaging during a2olotl tail regeneration in vivo.
Developmental dynami*s 7<# !9;=!5:.
/ose S.M B"<99C. Methods of initiating limb regeneration in adult anura. J. +2p. Nool.% <5#"9<=
":!.
S*hotte + 4 @ilber J B"<5;C. +ffe*ts of adrenal transplants upon forelimb regeneration in normal
and in hypophyse*tomiGed adult frogs. J. +mbryol. +2p. Morphol.% 6# 9:=6".
Smith S.B"<:9C. +ffe*ts of ele*trode pla*ement on stimulation of adult frog limb regeneration.
Annals of 1e, Ror( A*ademy of S*ien*es. 7)% 5!!=5!:
Song )% Nhao M% Forrester J% 4 M*.aig . B!!C. +le*tri*al *ues regulate the orientation and
frequen*y of *ell division and the rate of ,ound healing in vivo. P1AS vol <<."# "75::="75;.
Sto*um D. B!!6C. /egenerative )iology and Medi*ine. 1e, Ror(# +lsevier In*.
&ana(a +. B!!7C. /egeneration# If they *an do it% ,hy *anJt ,e. .ell ""7# 55<=56.
&seng AS% )eane @% -emire J% Masi A% 4 -evin M B!"!C. Indu*tion of ?ertebrate /egeneration
by a &ransient sodium *urrent. &he 0ournal of 1euros*ien*e 7!B7<C# "7"<="7!!.
Ra(ushi0i 1% Ro(oyama H% 4 &amura $. B!!<C. /epatterning in amphibian limb regeneration# A
model for study of geneti* and epigeneti* *ontrol of organ regeneration. Seminars in .ell 4
Developmental )iology !# 565=5:9.
Nhang R 4 -evin M B!!<C. Parti*le tra*(ing model of ele*trophoreti* morphogen movement
reveals sto*hasti* dynamis of embryoni* gradient. Developmental Dynami*s 7;# "<7="<75.