Official Journal of the Romanian Society of Physiological Sciences

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CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
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IANCĂU MARIAMIHALAŞ GEORGETA
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Fiziologia(80)‑ Physiology 2010supplement
2013.23.4
 Fiziologia- Physiology

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Official Journal of the Romanian Society of Physiological Sciences

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Fiziologia- Physiology 2013.23.4(80)
Fiziologia‑ Physiology 2010supplement

STNETNOC
4................................................................................... recnaC fo skramllaH eht gniylrednU erucsbO revewoH ,romuT fo stnatibahnI – stsalborbiF detaicossA-recnaC :weiveR .1
C ucsetianaP ,V ucsenuaP ,G eisanaT ,M acieD ,F nijoB ,L tanreC
31.................................... sitirhtrA decudnI-tnavujdA fo ledoM taR a ni ecnalaB tnadixoitnA-tnadixO eht no sdicA yttaF detarutasnuyloP 3-agemO dna nituR fo stceﬀE ehT .2
A naşeruM ,D dalV ,R navodloM ,D utsopA ,C uicacoS ,A notraM ,CI şihC ,D uratlaB

CONTENTS

81.................................................................. staR ni stceﬀE evitcetorpoidraC trexE dna aixopyH cirabopyH fo stceﬀE eht esreveR nasotihC dna murabraB muicyL ,nitecreuQ .3
P afloB ,G azroB ,D dalV ,R navodloM ,A notraM ,A naşeruM ,CI şihC ,A icivortimuD
32........................................................................................................................................ yhtapolahpecnE citapeH fo ygolohtapoisyhP eht ni lotisonI-oyM fo ecnatropmI ehT .4
IA uărădăB ,AD noI ,R aecocapaP ,C ienroiC ,M aenuB ,M răzaL ,C uaehcS

CONTENTS

62............................................................................................................................................ ksiR ralucsavoidraC fo noitacﬁitartS eht ni ecnacﬁingiS citsongorP :erusserP esluP .5
S șacnaM ,A rimogarD ,G ucsenoI ,D aţâbiâB
13....................................................................................................................... skramdnaL lacimehcotsihonummI dnA lacigolotsiH - amorbﬁoignA laegnyrahposaN elinevuJ .6
D tahroH ,S aeblutoC ,M atumoD ,MR narihG ,G ucsenavoI ,FD aediG

1. Review: Cancer-Associated Fibroblasts – Inhabitants of Tumor, However Obscure Underlying the Hallmarks of Cancer ...................................................................................4
1.
Review:
Metformin-From
Diabetes
to Cancer
Therapy.C..............................................................................................................................................................4
CernatL, BojinF, Deica M, Tanasie
G, Paunescu
V, Panaitescu
Deica M, Sima R, Cernat L, Bojin F, Tatu C, Tanasie G,Panaitescu C, Paunescu V
53........................................................................................................................................................................................................... sitilucsaV yranomluP eht otni thgisnI weN .7
MI uracojoC ,DC eibarV ,E usuR ,M uracojoC
83............................................................................................................................................................................. ?gnihT laeR a tI sI ,yhtaporueN citpO detaicossA enoradoimA .8
I goloZ ,M naetnuM ,N ucseluciN

SNIRPUC

4................................................................................................................................................. recnac ni uirotinﬁed lor uc elaromut etnenopmoC – elaromutirep eletsalborbiF .1
C ucsetianaP ,V ucsenuaP ,G eisanaT ,M acieD ,F nijoB ,L tanreC

31............................................... tnavujda ed ăsudni ătirtra uc inaloboş al iţnadixoitna-iţnadixo ieţnalab arpusa 3-agemO iţarutasenilop işarg rolizica la iş iulunituR lutcefE .2
A naşeruM ,D dalV ,R navodloM ,D utsopA ,C uicacoS ,A notraM ,CI şihC ,D uratlaB

2. The Eﬀects of Rutin and Omega-3 Polyunsaturated Fatty Acids on the Oxidant-Antioxidant Balance in a Rat Model of Adjuvant-Induced Arthritis ....................................13
2. Adipose-Tissue Derived Stem Cells: State of the Art Tissue Engineering ..................................................................................................................................11
Baltaru D, Chiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
Hurmuz M, Bojin F, Miu C, Ungurean C, Tatu C, Ionac M, Tatu F
81............................ naloboş ed ledom nu-rtnî rotcetorpoidrac snupsăr nu ăticrexe iş erabopih ieixopih eletcefe ăzaeracartnoc lunasotihC iş murabraB muicyL ,lunitecreuQ .3
P afloB ,G azroB ,D dalV ,R navodloM ,A notraM ,A naşeruM ,CI şihC ,A icivortimuD
32............................................................................................................................................................... ecitapeh ieitapolafecne aigolotapoizﬁ nî iululotizoni-oim aţnatropmI .4
IA uărădăB ,AD noI ,R aecocapaP ,C ienroiC ,M aenuB ,M răzaL ,C uaehcS

62................................................................................................................................... ralucsavoidrac iulucsir aeracﬁitarts urtnep acitsongorp aitacﬁinmes :iuluslup aenuiserP .5
S șacnaM ,A rimogarD ,G ucsenoI ,D aţâbiâB

3. Quercetin,
Lycium Barbarum
and ChitosanEffects
ReverseontheActiveEﬀectsBehaviorsof Hypobaric
Hypoxiaand Exert
CardioprotectiveEﬀects in Rats ..................................................................18
3.
Human Recombinant
Inteferon-Alphain theRat Forced
Swimming
Test......................................................................................17
Dumitrovici
A,
Chiş
IC,
MureşanA,
MartonA,
Moldovan
R,
Vlad
D,
Borza
G,
Bolfa
P
Zahiu D, Goganau I, Braga RI, Baca L, Zagrean L
13...................................................................................................................................... ecimihcotsihonumi iş ecigolotsih icitsirecarac – linevuj naignirafosan lumorbﬁoignA .6
D tahroH ,S aeblutoC ,M atumoD ,MR narihG ,G ucsenavoI ,FD aediG
53........................................................................................................................................................................................................................ ăranomlup atilucsav nî iţătilautcA .7
MI uracojoC ,DC eibarV ,E usuR ,M uracojoC

83................................................................................................................................................................ ?etatilaer o etse – anoradoima uc iulutnematart ătaicosa aitaporueN .8
I goloZ ,M naetnuM ,N ucseluciN3ygoloisyhP - aigoloiziF  )77( 1.32.3102

Importance
Myo-Inositol
in thePhysiopathology........................................................................................................................................23
4. TheCortical
ActivityofandHeart Rate
Changes
as MarkersofofHepatic
NoxiousEncephalopathy
Stimulation Response
during Chloral Hydrate Anesthesia....................................................22
PavelCălinM,A,CiorneiRădulescu
Am, RoscaAe,
BragaRi, Zahiu
ScheauB,C,DăneasaLazăr M,A,Bunea
C, Papacocea
R, Ion DA,Bădărău
AI D, Zăgrean L
5.
Arguments
forPrognostic
Using Thrombelastography
in thecation
StudyofofCardiovascularHemostasis Comparedto Other Laboratory Tests..........................................................................26
5. Pulse
Pressure:
Signiﬁcance in the Stratiﬁ
Risk ............................................................................................................................................26
Oniceanu
FM,IonI,
Adumitresi
C,Radulescu
N,
FarcasC,
Stefanescu
AC,
ChiricaR
Bâibâţa D, Ionescu G, Dragomir A, Mancaș S
6. Patient Related Prognostic Factors and Therapeutic Strategy in Larynx Squamous Cell Carcinoma..........................................................................................31
6. Juvenile Nasopharyngeal Angioﬁbroma - Histological And Immunohistochemical Landmarks .......................................................................................................................31
Prodea M, Horhat D,Balica N, Boruga O, Poenaru M
Gidea DF, Iovanescu G, Ghiran RM, Domuta M, Cotulbea S, Horhat D
7. In VitroCharacterisation of Tumor-AssociatedFibroblastsfromDifferent Solid Tumors...............................................................................................................35
7. New L,Insightthe Pulmonary
Vasculitis...........................................................................................................................................................................................................35
CernatBojininto
F, Deica
M, Anastasiu
D, Tanasie
G, Ilina R, Paunescu V, Panaitescu V
Cojocaru M, Rusu E, Vrabie CD, Cojocaru IM
8. Comorbidities and Risk Factors in Heart Failure with Preserved Ejection Fraction....................................................................................................................39
MavreaA, BucurA, Tomescu
8. Amiodarone
AssociatedOpticMNeuropathy, Is It a Real Thing? .............................................................................................................................................................................38
Niculescu N, Muntean M, Zolog I

CUPRINSCUPRINS

1.
Metformin: dela diabet la– terapiaantitumorală........................................................................................................................................................................4
1. Fibroblastele
peritumorale
Componente tumorale cu rol deﬁnitoriu in cancer .................................................................................................................................................4
Deica
SimaF,R,Deica
CernatBojin G,F, Tatu
C, Tanasie
G,Panaitescu
C, Paunescu V
CernatM,
L, Bojin
M, L,Tanasie
Paunescu
V, Panaitescu
C
2.
CeluleleRutinului
stem derivate
din ţesutuladipos:
actualităţiîningineriatisulară.
.................................................................................................................................11
2. Efectulşi al acizilorgraşi polinesaturaţi
Omega-3 asupra balanţei oxidanţi-antioxidanţi
la şobolani cu artrită indusă de adjuvant ...............................................13
Hurmuz M, Bojin F, Miu C, Ungurean C, Tatu C, Ionac M, Tatu F
Baltaru D, Chiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
3. Efecteleinterferonului-alfa recombinant umanasupracomportamentelor active la testulînotuluiforţat, la şobolan................................................................17
3. Quercetinul,
LyciumBarbarumşi Chitosanulefectele hipoxiei hipobare şi exercită un răspuns cardioprotector într-un model de şobolan ............................18
Zahiu
D, Goganau
I, Braga
RI, Baca
L, Zagreancontracarează
L
Dumitrovici A, Chiş IC, Mureşan A, Marton A, Moldovan R, Vlad D, Borza G, Bolfa P
4. Modificărileactivităţii corticale şialeritmuluicardiac ca markeri ai răspunsului la stimulareanociceptivăîntimpulanestezieicucloralhidrat..............................22
Pavel
B, Dăneasa
A, Călin A, Rădulescu
Am, RoscaAe,
Braga hepatice
Ri, Zahiu...............................................................................................................................................................23D, Zăgrean L
4. Importanţa
mio-inozitoluluiîn ﬁziopatologia
encefalopatiei
Scheau C, Lazăr M, Bunea M, Ciornei C, Papacocea R, Ion DA, Bădărău AI
5. Argumentepentruutilizareatrombelastografieiînstudiulhemostazeicomparativ cu alteteste de laborator..............................................................................26
Oniceanu
I, Adumitresi
Farcasstratiﬁ
C, Stefanescu
AC, Chirica
R
5. PresiuneaFM,Ion
pulsului:semniﬁcatiaC,Radulescu
prognosticaN,pentru
carea riscului
cardiovascular...................................................................................................................................26
Bâibâţa D, Ionescu G, Dragomir A, Mancaș S
6. Factori de prognostic legaţi de pacientşistrategiiterapeutice în carcinomullaringian cu celulescuamoase..............................................................................31
Prodea M, Horhat D,Balica N, Boruga O, Poenaru M
6. Angioﬁbromul nasofaringian juvenil – caraceristici histologice şi imunohistochimice ......................................................................................................................................31
Gidea
DF, Iovanescu
Ghiran
RM, Domuta peri-tumorale
M, Cotulbea S, Horhat
7.
Caracterizare
in G,
vitro
a fibroblastelor
izolateDdin diferite tumori solide.............................................................................................................35
Cernat L, Bojin F, Deica M, Anastasiu D, Tanasie G, Ilina R, Paunescu V, Panaitescu V
7. Actualităţi în vasculita pulmonară ........................................................................................................................................................................................................................35
Cojocaru
M, Rusu E, Vrabie CD,IM
8.
Comorbidităţişifactorii
deCojocaruriscîninsuficienţacardiacă
cu fracţie de ejecţieprezervată...............................................................................................................39
Mavrea A, Bucur A, Tomescu M
8. Neuropatia asociată tratamentului cu amiodarona – este o realitate? ................................................................................................................................................................38
Niculescu N, Muntean M, Zolog I
2013.23.4
2013.23.1 (80)(77) 
 Fiziologia
Fiziologia--Physiology
Physiology

33

REVIEWMETFORMIN: FROM DIABETES TO CANCER THERAPYMEDA DEICA, RALUCA SIMA, LAURA CERNAT, FLORINA BOJIN, CARMEN TATU, GABRIELATANASIE, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Metformin was approved for clinical use in treatment of many diseases, including hyperglycemia, polychistic ovary syndrome, andmetabolic syndrome. The most important use of Metformin is as oral antidiabetic in treatment of diabetes mellitus type 2. First evidence
that Metformin could have a role in oncology emerged with the study of possible associations between diabetes and cancer treatment. Several studies showed that diabetic patients have increased mortality due to cancer, comparatively to non-diabetic patients,
while the patients under Metformin treatment have a better prognostic of cancer evolution compared to patients treated with otherantidiabetic drugs. The purpose of this review was to present state-of-the-art regarding multiple roles of Metformin in different typesof cancer, and to suggest the possibility of re-purposing this drug in anti-tumor therapy.
Key words:Metformin, cancer, signaling pathways, anti-tumor therapy

1. Metformin – oral antidiabetic
Metformin history (Figure 1) begins in medieval Europe, when
the botanists noticed that polyuria can be treated using parts ofGalegaofficinalis (French liliac) plant. The active substance was
not known, nor the fact that type 2 diabetes mellitus is responsible
for polyuria. In modern times, the active principle of G. Officinalis– galegine was extracted and new drugs were synthesized – metformin, phenformin, and buformin (biguanides). Clinical studies
demonstrated the efficacy of Metformin in treatment of type 2
diabetes mellitus, and early in the 70’s Metformin was introducedon European market, and in 1995 it was approved for clinical use
in United States (1).Nowadays, Metformin is the most prescribeddrug in treatment of diabetes mellitus type 2, having more than
40 million prescriptions in United States in 2008.
Metformin was approved for clinical use in treatment of hyperglycemia, polycystic ovary syndrome, and metabolic syndrome.
After oral administration, 50-60% is absorbed at intestinal levelin 1-3 hours, and 90% is eliminated at renal level in 12 hours(2). Metformin decreases glucose reabsorption at intestinal level
and hepatic gluconeogenesis, but does not stimulate insulin
secretion. As a result, increases glucose absorption and use bymuscle and adipose tissue. Plasma glucose level decreases only
in diabetic patients, not in non-diabetic patients. Moreover, Metformin increases affinity for insulin of insulin receptors, decreases
hyperinsulinemia, and prevents insulin resistance. After few daysof administration, insulin level decreases with 25-33% in diabetic
and non-diabetic patients. Metformin decreases absorption andoxidation of fatty acids, decreases total cholesterol level, LDL
and triglycerides. So that, Metformin is very well tolerated bypatients and induces a weight loss of approximately 2 kg. Few
side effects may be mentioned: gastro-intestinal, metal taste,decrease of vitamin B12 concentration, and lactic acidosis (3).

Fig. 1. Chemical structure of Metformin (4)2. Antitumoral effects of Metformin
Epidemiological studies
First evidence that Metformin could have a role in oncology
emerged with the study of possible associations between diabetes and cancer treatment. Several studies showed that diabetic
patients have increased mortality due to cancer, comparatively
to non-diabetic patients, while the patients under Metformin treatment have a better prognostic of cancer evolution compared topatients treated with other antidiabetic drugs (5,6).
Evans et al. performed the first epidemiological study, which
demonstrates that Metformin reduces cancer risk in diabetic
patients (7). Bowker et al., in a study performed on 10,309
diabetic patients, compared cancer incidence during 5 year
treatment with insulin, metformin, and sulphonylurea. Theyobserved that patients treated with Metformin presented a lower
mortality rate correlated with cancer, compared with insulin andsulphonylurea-treated patients (8). Curie et al. investigated therisk of solid tumors development in patients with type 2 diabetes
mellitus, correlated with oral antidiabetic therapy, human insulin,

Received 10th of April 2013.Accepted 25th of September 2013. Address for correspondence: MedaDeica, MD, PhD student, Department of Functional Sciences, Physiology, “Victor Babes” University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, Romania, Phone/fax: + 40 256490507;
email: bravameda@yahoo.com

4

Fiziologia - Physiology  2013.23.4 (80)and insulin analogues. Patients under Metformin treatment
presented a lower risk for development of colon and pancreas
cancer, compared to patients under other therapeutic regimens(9). In 2010, a meta-analysis concluded that there is a reductionof 31% in cancer occurrence in patients with Metformin treatment,
and this is a dose-dependent relationship (10).
In 2009, a retrospective study published by Jiralerspong et al.
(11)demonstrated increased efficiency of neoadjuvant chemotherapy in patients with breast cancer undergoing chronic Metformin treatment, both diabetic and non-diabetic. Metformin-treateddiabetic patients presented 3 times increased complete response(24%) compared to other diabetic patients (8%). It is important
to mention that complete response in non-diabetic patients wasof 16%. Although Metformin administration was associated withdecreased risck of breast cancer, survival rate was similar forboth diabetic and non-diabetic patients, regardless of increased
complete response in Metformin-treated group.
It was proven that Metformin use decreases the incidenceof breast cancer. Bayraktar et al. (12) investigated association
between Metformin administration and survival rate in patientswith triple negative breast cancer, characterized by increased
aggressiveness, reduced treatment response, and poor prognostic. Comparing Metformin-treated group with other antidiabetictreated patients group, and non-diabetic patients group, the lasttwo groups presented increased risk for metastasis development.
Study results show that Metformin administration together withchemotherapy do not impact on survival rate in diabetic patientswith triple negative breast cancer.
AMPK/mTOR axis
Beneficial effects of Metformin in diabetic patients are due
to gluconeogenesis regression, which induces a decrease inglucose plasma level. Moreover, it increases insulin sensitivity
and tissue absorption of glucose. The mechanism underlying
these actions is considered to be the inhibition of oxidative
phosphorylation, which induces a misbalance of AMP/ATP, withconsecutive activation of LKB1-AMPK signaling pathway (13).
AMPK is the enzyme regulating metabolic pathways (14), andits activation was correlated with Metformin effect of decreasing
glucose concentration (Figure 3).
In most cases, in tumors with rapid growth, tumor cells should
survive in an environment deprived of nutrients, so that goodfunctioning of AMPK, regulating energetic homeostasis, becomesvital. Activation is triggered when there is a misbalance between
ATP production and consumption, and intracellular AMP/ATPA
ratio increases. As a result of AMPK pathway activation, thecell passes from anabolism to catabolism, decreases ATP consumption and restores energetic homeostasis. Cellular growthand carbohydrates, lipids, and proteins synthesis are inhibited,while fatty acids oxidation and glucose absorption are stimulated.
Thus, tumor cells succeed in survival under metabolic stressconditions (15).
AMPK pathway is under LKB1 control, which is a serine-treonine kinase with tumor suppressor role (16).Mutations of LKB1
were associated with Peutz-Jeghers syndromeand increased
2013.23.4 (80)  Fiziologia - Physiology

risck for tumor development (17). Once activated, LKB1 phosphorylates AMPK. AMPK can also be activated by other kinases
as a response to cellular stress, due to energetic level decrease
and reduced AMP/ATP ratio (14). AMPK activation by LKB1 is
one of possible mechanisms for Metformin action, while othertheories are suggesting that AMPK activation is a side effectof its action on mitochondria. It was demonstrated that Metformin induces moderate and specific inhibition of mitochondrialcomplex I in respiratory chain, while the mechanism underlying
this inhibition is unknown. LKB1 and mitochondrial complex I
are not the only targets of Metformin. Recently, a new regionin ATM gene was discovered (ataxia telangiectasia, mutated),
which controls Metformin response in patients with diabetes
mellitus type 2 (18). ATM phospholylatesLKB1, which activates
AMPK, but can act on AMPK independently of LKB1 (19).ATM
tumor suppressor gene is involved in DNA repair mechanism
and cell cycle control, thus suggesting a possible explanationof Metfromin anti-tumoral effect.
A direct consequence of AMPK activation is inhibition of mTOR
signaling pathway through TCS-2 protein (tuberous sclerosis2) (20). AMPK can act directly, through phosphorylation of aco-signaling molecule binding to mTOR and inhibiting its action(21). mTOR activation occurs frequently in breast cancer andis associated with poor prognostic. Metformin, through AMPK
activation, inhibits mTOR, this being a possible explanation ofanti-tumoraleffect observed in breast cancer patients (22). mTOR
inhibition can be a result of IGF1 inhibition, or insulin receptor,
or AKT (23). Thus, Metformin can inhibit AMPK-dependent
mTOR, through decreased insulin or IGF1 level. mTOR pathway
is involved in regulation of cellular energetic balance, throughmodulation of cellular processes of protein synthesis, or autophagy (18).
Induction of autophagy
Autophagy is a catabolic process in which cell is degrading
its own components with the help of lysosoms. This is a majormechanism of survival, by which cells will relocate the nutrientsfrom less important processes to the essential ones. The mechanism by which AMPK can induce autophagy was not completely
revealed, but there are speculations about Metformin inducing
autophagy by activating AMPK, further activation of p53, whichis involved in cellular metabolism.
Inhibition of endogenous lipogenesis
Another consequence of AMPK activation is inhibition of
lipogenesis in tumor cells, which are relying on increased denovo synthesis of fatty acids. The most aggressive breast cancer phenotypes are endowed with increased lipid metabolism,
involved in cellular proliferation and survival. Moreover, whennormal cells transform into tumor cells, under the action of specific oncogenes, the enzymes required for de novo synthesis offatty acids (acetyl-CoA carboxylase and fatty acid synthase) are
expressed and activated. Starting from this hypothesis, it wasobserved that by inhibiting lipid metabolism in tumor cells, there
is a blockage in expression and activity of certain oncoproteins.
5Metformin effects in normal and tumor cells was characterized byblockage of activation and expression of key enzymes involvedin fatty acids biosynthesis and an increase in expression of
mitochondrial biogenesis regulators (PCG-1α). It is speculated
that these effects have a contribution to the anti-proliferative
effects of Metformin, by inhibiting fatty acids biosynthesis andactivation of catabolic mechanisms (3).
Decrease of estrogen production
AMPK activation is responsible of aromatase inhibition. Metformin inhibits aromatase expression in adipose stromal cells in
vitro, thus inducing local decrease of estrogen production (23).
Thus, Metformin can be investigated as potential drug in breast
cancer prevention, or as adjuvant treatment in cancer types
under hormonal influence.
Blockage of mitotic cell cycle
AMPK is also involved in cellular division. Metformin decreases
expression of some genes involved in mitosis, such as kinesins,
tubulins, histones, polo-like kinases (24). Moreover, by AMPK
activation, p53 phosphorylation is induced, which is requiredfor initiation of AMPK-dependant cell cycle blockage (25). Metformin reduces D1 cyclin level in tumor cells of prostate cancer
in vitroandin vivo and blocks cell cycle in G(0)/G(1) phase (26).
Similar results were obtained in breast cancer cells, in whichcase cell cycle blockage required cyclin D, p27Kip1 or p21Cip(27). Cell cycle blockage was observed in pancreatic cells (28)and triple negative breast cancer cell lines (29). It is important
to mention that these were the only two studies approaching the
apoptotic-inducer effect of Metformin.
Effects on hyperglycemia
Decrease of glucose plasma level can represent an importantanti-tumoral effect. Several studies stressed that type 2 diabetes
mellitus and obesity are associated with increased risk of breast,colon, pancreas and endometrial cancer.
A characteristic of tumor cells is the switch from oxidative
phosphorylation to glycolysis, as main energy source, This
metabolic reprograming is known as Warburg effect, while procuring of energy through glycolysis allows tumor cells to adapt
in hypoxic environment (30).
Tumor cells express insulin and insulin-like growth factor
receptors. Except for metabolic effects, IGF-R stimulates cellular
proliferation and metastasis (31). Tumor cells present increased
glucose absorption, independent of IGF-R activation, due to
Warburg effect. It is considered that IGF-R activation leads to anincreased survival rate and proliferation, independent of glucose
absorption, while hyperglycemia contributes to IGF-R activation and cellular growth, indirectly, due to stimulation of insulin
release. Insulin reduces synthesis of insulin-like growth factor
binding protein (IGFBP)-I, IGFBP-II, and IGFBP-III. IGFBP-I
binds IGF and inhibits its action, so that lack of this protein leads
to increased IGF levels (32).
Hyperinsulinemia can induce an increase of tumor by indirect
mechanisms, such as proliferation of epithelial cells, increased
6synthesis of sexual hormones and IGF, but also inducing disorders in adipokines homeostasis (cytokines involved in cancer
pathology). Moreover, IGF activation promotes angiogenesis,
which can contribute ot tumor growth (32). It was demonstrated
that Metformin inhibits insulin effects on cellular growth by a
AMPK/mTOR-dependent mechanism (33).
Impact on chemotherapeutics effectMetformin increases toxicity of some anti-tumoral drugs, suchas paclitaxel, doxorubicin, and cisplatin. In breast and lung cancer, Metformin acts synergic with paclitaxel and induce blockageof cell cycle, with increased number of cells in G2/M phase and
LKB1-dependent inhibition of tumor cells proliferation. Lliopoulos
et al. demonstrated the existence of a synergic anti-tumoral effectbetween Metformin and cisplatin, doxorubicin, paclitaxel in an
animal experimental model.
Impact on targeted therapy
In vivo studies showed that Metformin inhibits initiation andprogression of tumors in Her-2 transgenic mice (34). In vitro studies showed that Metformin induces a dose-dependent decreaseof Her-2 expression. Thus, low doses block thyrosin-kinase
activity of her2, without affecting its expression, while large
doses can induce inhibition of her-2 expression (35). Suppression of Her-2 expression is performed by inhibition of mTOR,
on an AMPK-partially dependent pathway, by co-incubation withagents blocking oxygen reactive species.
Depending on Her-2 presence or absence on tumor cells
surface, Metformin presents two types of actions (24). In caseof Her-2 negative breast cancer cells, Metformin suppresses
expression of genes involved in mitosis. On the contrary, on
Her-2 positive cancer cells, Metformin induces over-expressionof genes involved in apoptosis.
In vitro studies regarding interactions between Metforminand drugs used in targeted anti-tumoral therapy, demonstrated
that Metformin could be a good adjuvant treatment. A study
performed on Her-2 positive tumor cells showed that Metformin
acts synergic with anti-Her-2 monoclonal antibody Trastuzumab(Herceptin), in order to eliminate tumor stem cells (36). Also,by blocking mTOR action, Metformin prevents occurrence ofTrastuzumab resistance (37). Metformin decreases resistance ofHer-2 positive tumor cells to Her-1/Her-2 tyrosine kinase inhibitor(Lapatinib) (38). By its anti-hyperglycemiant action, Metformin
decreases IGF level, and consequently inhibits activation of
IGF-R, involved in mechanism of resistance to anti-Her-2 therapy(39). We may conclude that Metformin can have a possible
therapeutic role in breast cancer resistant to anti-Her-2 therapy,
due to prevention of IGF-1R activation.
Effects on tumor stem cells
The latest discoveries in the field are supporting the idea thattumors are formed of two subpopulations: tumor cells and tumor
stem cells. The latest subtype is characterized by their ability
to renewal, differentiation into diverse phenotypes, initiation oftumors, and increased resistance to chemotherapy. This is why
Fiziologia - Physiology  2013.23.4 (80)an effective anti-tumor therapy involves tumor destruction andtumor stem cells elimination (40).
In a study performed by Hirsch et al. tumor stem cells sensitivity to Metformin was demonstrated using mouse model withhuman breast cancer xenograft. Interestingly, small doses ofMetformin (0.1 or 0.3 mmol/L), which did not have any effecton tumor cells viability, selectively destroyed tumor stem cells,
characterized by the CD44+/CD24- phenotype. Researchers
demonstrated both in vitro and in vivo that Metformin in combination with doxorubicin can destroy the tumor and prevents cancer
rebound for a longer period of time than any other anti-tumor
agent administered as monotherapy (41).
Considering the Metformin action on tumor stem cells subpopulation, it was supposed that more aggresive cancer types(triple negative and Her2 positive) should present an increased
sensitivity to Metformin due to their similar characteristics tostem cells. Recently, Vasquez et al. demonstrated that tumor
stem cells (CD44+/CD24-) from Her2 positive tumor cell lines
resistant to Trastuzumab have an increased sensitivity to low
doses of Metformin.Trastuzumab and Metformin act synergic
for elimination of tumor stem cell population (42).
Effects on epithelial-mesenchymal transition
Trying to elucidate the mechanism by which Metformin selectively act on tumor stem cells, the same researcher groupobserved that Metformin suppresses the epithelial-mesenchymaltransition (43). During the carcinogenesis process, epithelial cells
lose their epithelial characteristics and acquire mesenchymal cellcharacteristics (positive for vimentin, myosin, invasive motility),
which offers the advantage for survival and invasiveness. Epithelial-mesenchymal transition is a process characterized by loss ofcellular adhesion, E-cadherin expression and increase cellular
motility. Epithelial-mesenchymal transition is often activated during tumor invasion and metastasis, being essential for acquiring
new stem cells and for cellular motility (44). By suppressing
some transcription factors (e.g.: cytokines, TGFbeta), Metformin eliminates CD44+/ CD-(low) cancer cell subpopulations
in case of breast cancer. Metformin exposure impaired loss of
E-cadherin in epithelial cells, induced by TGFbeta activation during epithelial-mesenchymal transition process. Metformin helped
keeping E-cadherin location at the contact zone between cells
and impaired changes associated with their transition towards
mesenchymal cells (size and morphology. A study performed on
Madin-Darby canine renal cells (MDCK) showed that Metformin
reduces TGFbeta-mediated conversion of MDCK cells towards
a spindle-shape aspect. These data suggest that Metformin, byinhibiting TGFbeta, can stop epithelial-mesenchymal transition,
including metastasis (45).
Vitamin B12 deficit
It is known that vitamin B12 deficit is one of the adverse effects of Metformin. In vitro inactivation of B12 vitamin and in vivo
deficiency of this vitamin are associated with increased mortalityof tumor cells and an improved response to chemotherapy. Considering these characteristics, some scientists proposed vitamin2013.23.4 (80)  Fiziologia - Physiology

B12 deficiency as potential anti-tumoral effect of Metfomin (46).
Blockage of HIF-1
AMPK is essential for transcription of hypoxia-inducible factor (HIF-1). HIF-1 represents a key factor in induction of genes
facilitating adjustment and survival under hypoxic conditions(47).mTOR signaling pathway activates HIF-1, so that Metformin can block the signals transmitted by HIF-1 both throughdirect inhibition of mTOR, or indirect activation of AMPK. Thus,
Metformin has the anti-tumoral effects by stopping the mechanism helping the cells to adjust to hypoxia (48).

Fig. 3. Metformin – anti-tumoral effects. Metformin acts on AMPK
pathway and activates autophagy through p53, oxidation of fatty acids in mitochondrial complex I and inhibits mTOR signaling pathway,
thus impairing cellular growth and protein synthesis, blockage of cell
cycle and hypoxia-inducible transcription factor (HIF-1) and inhibition of Her2 expression. Metformin blocks epithelial-mesenchymaltransition. Moreover, Metformin decreases glucose, insulin, and IGF
plasma levels.

3. Antitumoral effect of Metformin in different cancer types
Metformin in breast cancer
Several in vitro and in vivo preclinical trials were performed
related to anti-tumoral action of Metformin in breast cancer(Figure 4). Metformin exhibits its anti-tumoral action in two
ways: direct and indirect. Indirect mechanism consists of activation of hepatic AMPK pathway, inducing a decrease of hepatic
gluconeogenesis and blood insulin level (49). Metformin can
act directly on tumor cells through activation of LKB1/STK11mediated AMPK pathway and concomitant inhibition of mTOR,
thus blocking cellular proliferation and protein synthesis (50).
Other potential anti-tumor mechanisms are: blockage of cell
cycle and colony formation (35), decreased activity of aromatase (applicable in ER positive cancers) (24),and reduced gene
expression of HER2 and HER3 (51). Additionally, Metformindelays the occurrence and development of breast cancer in7

transgenic mice with Her2 positive cancer (34).Different in vitro
studies revealed that Metformin acts synergic with several chemotherapeutical agents. Synergic with monoclonal anti-Her2
antibody (Trastuzumab, HerceptinÔ), Metformin eliminates
stem cell populations of Her2 upregulated gene expression
carcinoma (42). In animal models of mice with breast cancer
xenograft, association with doxorubicin reduces tumor mass
and increases duration of tumor remission (41). In case of cells
resistant to lapatinib (MCF-7/HER2-LapR), Metformin suppresses pro-survival signaling pathways involved in acquiring
auto-resistance (38). A possible involvement in blockage ofepithelial-mesenchymal transition was also noticed. Metformin
suppresses CD44+/CD24low immunophenotype characteristic
to stem cells and decreases expression of factors involvedin epithelial-mesenchymal transition (ZEB1, TWIST1, SNAI2(Slug) and TGFβ) (43). Anti-metastasis action of Metformin
is under investigation. In triple negative cancer cell lines,
Metformin suppresses protein CD24, protein associated withmetastasis (52).

Fig. 4. Metformin – antitumoral effects in breast cancer. Main
action pathway is AMPK, activated by LKB1/STK11. Metformin
has inhibitory action on Her2, epithelial-mesenchymal transition,
cell cycle, aromatase, cell proliferation, protein synthesis andtumor stem cells phenotype. Anti-tumoral effect of some chemotherapeutic drugs (Trastuzumab, Doxorubicin) is augmented by
Metformin

Metformin in colorectal cancer
There are few data regarding Metformin action on colorectal cancer. Epidemiological studies showed a decrease inoccurrence of colorectal cancer in patients with diabetes
mellitus type 2 taking Metformin, compared with patients
not using this treatment (9,53). In vitro, Metformin activates
AMPK pathway and inhibits growth of colon cancer cells(54). Antitumoral effect was also observed in in vivo studies
performed on rodent animal models. Systemic treatment
with Metformin suppresses development of intestinal polyps
in Apc(Min/+) mice with familial adenomatose polyposis (55)and suppresses formation of pathological colorectal cryps
induced by deazoximetan (56). Metformin reduces xenografts
8

development in mice injected with p53-deficient colon cancer
cells (26). Clinical study performed on non-diabetic patientsdemonstrated that Metformin administration in low doses(250mg/day) reduces the number of pathological colorectal
crypts(marker of colorectal cancer) and decreases the proliferative activity of colon epithelium (57). These studies suggest
the need of more detailed investigation of anti-tumoral actionof Metformin in colorectal cancer.
Metformin in prostate cancer
Several epidemiological studies revealed an inverse relationship between diabetes and prostate cancer (58). Thisis related to several factors, such as metabolism alterationinduced by diabetes and anti-tumoralaction of anti-diabetic
drugs. In vitro studies demonstrated that Metformin inhibits
prostate cancer cells growth, without inducing apoptosis andinhibits expression of cyclin D1 and AMPK-dependent pRbphosphorylation. Moreover, Metformin treatment exhibits andanti-neoplasic action in animal models of LNCap prostatecancer cells xenografts (26). Prostate cancer incidence was
44% reduced in a study on Caucasians males taking Metformin (59). In 2010, Patel et al. raised few questions regarding
benefic effects of Metformin in prostate cancer. In a study
performed on 616 patients, Patel demonstrated that diabetes
is associated with cancer rebound after prostatectomy, andMetformin treatment had no beneficial effect in this case (60).
These results discouraged the Metformin use in preclinicaland clinical trials of prostate cancer.
Metformin in ovarian cancer
Recent studies demonstrated that Metformin inhibits proliferation of two cell lines of endometrial cancer, by blocking cell cyclein phase G1. Moreover, Metformin suppresses mRNA expression
for hTERT in the two endometrial cell lines. The effect is mediatedby AMPK activation, with consecutive inhibition of mTOR (61).
Considering that AMPK inhibits mTOR through Akt, AMPK activation by Metformin can be considered an anti-tumoral strategy inovarian cancer, characterized by activation of signaling throughAkt. Increased Akt expression is given by high prevalence of genemutations for PTEN protein (phosphatase and tensin homolog)
in endometrial cancer.
Polycystic ovary syndrome (PCOS) is one of the most common
endocrine disorders, which affect 5-10% of women at fertile age.
Treatment used for this syndrome consists of administration ofdrugs which are increasing insulin sensitivity and mainly, Metformin prescription. This improves reproductive abnormalities of
PCOS and regulates ovulatory processes and menstrual cycle(62). Recently, Metformin was proposed as adjuvant drug intreatment of patients with endometrial cancer (63).
Acknowledgements
This work was supported by the Sectorial Operational Programme for Human Resources Development, financed from theEuropean Social Fund, FSE POSDRU 107/1.5/S/78702 and by
UEFISCDI, PNII-Idei, Project No. 318/2011.
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METFORMIN: DE LA DIABET LA TERAPIA ANTITUMORALAREZUMAT

Metformin a fost aprobat pentru uz clinic in tratamentul a numeroase afectiuni, incluzand hiperglicemia, sindromul ovarelor polichisticesi sindromul metabolic. Cea mai importanta utilizare a Metforminului este ca antidiabetic oral in tratamentul diabetului zaharat tip 2.
Primele dovezi ca Metforminul ar putea avea un rol in oncologie au fost obtinute in studii referitoare la asocierea dintre tratamentul
anti-diabetic si anti-tumoral. Numeroase studii au aratat ca pacientii diabetic prezinta o moratalitate crescuta datorata cancerului,comparative cu pacientii non-diabetici, in timp ce pacientii aflati sub tratament cu Metformin au prezentat un prognostic mai bun incursul evolutiei cancerului, comparativ cu pacientii diabetic aflati sub tratament cu alte medicamente antidiabetice. Scopul acestui
review a fost acela de a prezenta mecanismele si modalitatile de actiune actuale ale Metforminului si de a sugera posibilitatea dere-orientare a acestui medicament pentru terapia anti-tumorala.
Cuvinte cheie: Metformin, cancer, cai de semnalizare, terapie antitumorala.

10

Fiziologia - Physiology  2013.23.4 (80)

ADIPOSE-TISSUE DERIVED STEM CELLS: STATE OF THE
ART TISSUE ENGINEERING
HURMUZ M1, BOJIN F1,2, MIU C1, UNGUREAN C3, TATU C1,2, IONAC M1,2, TATU F1,3
1
“Victor Babes” University of Medicine and Pharmacy Timisoara
2
Emergency Clinical County Hospital Timisoara
3
Emergency Clinical Military Hospital Timisoara

ABSTRACTStem cells represent cells that are capable of differentiating into multiple cell types and to self-renew. Adipose tissue derived stem
cells proved to be a true alternative to bone marrow stem cells, due to its accessibility and abundance. Their capacity to differentiate
into mesodermal, but also ectodermal and endodermal cells, has made them a source for regenerative medicine. The studies inthis field are focusing on trauma, degenerative diseases and even aesthetic surgery. The purpose of this article was to review thelatest published scientific papers regarding the use of ASCs therapies. There are still many problems to be solved, but the increasing
number of studies conducted worldwide will provide many answers in the years to come.
Key words: adipose-derived stem cells, regenerative medicine, differentiation, isolation, tissue engineering

INTRODUCTION
Multipotent mesenchymal stromal cells or mesenchymal
stem cells (MSC) are adult stem cells capable of providing
functional properties through their capacity to differentiate
into multiple cell types (1). The discovery and understanding of the capacities of these cells has led to a new domain:
the regenerative medicine. The MSC have the capability ofmultilineage differentiation, such as adipogenic, osteogenic,chondrogenic, but recently they have been reported to exhibit
immunosuppressive capacities as well (2).
The traditional source of MSC in the adult was the bonemarrow. Although this proved to be a reliable source, thelow number of cells isolated and the high morbidity, led to
the necessity of exploring other sources. These sources
include: muscle (3), adipose tissue (4), connective tissue(5), trabecular bone (6), synovial fluid (7), umbilical cord (8),amniotic fluid and placenta (9, 10).
Although there have been many attempts to generally
accept the specific markers for identifying the MSC, someminimal requirements have been proposed by the International Society for Cryotherapy (11,12):• Adherence to plastic within standard culture conditions;• CD105+; CD73+; CD90+ ;• CD45-; CD34-; CD14-; CD31- ;• HLA-DR- ;• The ability to differentiate into osteoblasts, chondrocytes
and adipocytes.

CHARACTERIZATION OF ADIPOSE TISSUE-DERIVED STEMCELLS (ASCs)
Rodbel, in 1964, was the first scientist to mention the technique of isolating cell populations from adipose blocks harvested
from nude mice. The adipose tissue contains a mixture of adultadipocytes and a stromal vascular fraction (SVF). The SVF contains, among other cell types (circulating blood cells, fibroblasts,smooth muscle cells, pericytes, endothelial cells), the ASCs. The
ASCs represent a small group of these populations (13). The
ASCs, being in fact mesenchymal cells, have the capacity toadhere to plastic and consequently they self-select from the restof the cell populations. Since then, isolating ASCs from adipose
tissue has become a standard protocol.
The adipose tissue proved to be an attractive alternative
source to that of bone marrow stem cells. It has the advantage ofbeing very accessible, minimally invasive, in large quantities andwith a higher proliferation rate (14). At the beginning, the adipose
tissue harvested was manually minced, but currently, due to thedevelopment of technology, the tissue is finely minced directly
during the harvesting procedure (14,15). Usually, the harvestof the adipose tissue samples is performed during abdominal
bariatric surgery, lipoaspiration (15) or more recently during knee
replacement surgery (16,17).
The International Fat Applied Technology Society stated that
“adipose tissue-derived stem cells” (ASCs) should be the term
used in relation to the population of cells obtained from adipose
tissue. This comes after a long debate, since the early discovery

Received 15th of September 2013. Accepted 25th of October 2013. Address for correspondence: Hurmuz Mihai, MD, PhD student, Department of Orthopedics, “Victor
Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania, Phone/fax: + 40 256490507; email: hurmuz_mihai@yahoo.com

2013.23.4 (80)  Fiziologia - Physiology

11

of the cells, regarding the terms used for describing the population: “adipose-derived adult stem (ADAS) cells,” “adipose-derived
adult stromal cells,” “adipose-derived stromal cells (ADSCs),”“adipose stromal cells (ASCs),” “adipose mesenchymal stem cells(AdMSCs),” “preadipocytes,” “processed lipoaspirate (PLA) cells,”
and “adipose-derived stromal/stem cells (ASCs)” (18).

The ASCs differentiated to chondrogenic lineage have a positive cell marker for: CD90, CD73, and CD166 (22).
Adipogenic lineage
The isolation of ASCs from lipoaspirate requires washing with
PBS, collagenase digestion, filtering and then centrifuged. The
differentiation protocol for adipogenic lineage includes: Dexamethasone, isobutyl methylxanthine, indomethacin, insulin andthiazolidinedione (15). Another protocol uses insulin-like growth
factor-1 (IGF-1), growth hormone, glucocorticoids, insulin, fatty
acids and cyclic adenosine monophosphate (21). Yoshimura etal. obtained adipogenic lineage with the help of DMEM, isobutylmethylxanthine, dexamethasone, insulin and indomethacin (20).
The ASCs differentiated to chondrogenic lineage have a positive
cell marker for: CD34 and CD90 (22).
Non-mesenchymal lineage

Fig.1. The Immunophenotype of ASCs

Isolation and differentiation
Mesenchymal lineages
Osteogenic lineage
Under the right conditions, ASCs, like MSCs, can express
genes and proteins according to the osteogenic phenotype. The
protocol used for the ASCs differentiation to osteogenic lineage
includes the isolation protocol (lipoaspirate is washed with PBS,
digested with collagenase, K-NAC medium, then the suspension
is remixed, filtered, plated and incubated) and the differentiation
protocol (ascorbate-2-phosphate, β-glycerolphosphate anddexamethasone) (19). Another protocol used to differentiate
ASCs to ostogenic linieage uses DMEM, FBS, dexamethasone,
ascorbate-2-phosphate and β-glycerophosphate (20). In a study
from Gimble et al. the osteogenic lineage was obtained with
Ascorbic acid, bone morphogenetic protein 2, dexamethasone
and 1α,25-dihydroxyvitamin D3 (15). Locke et al. used dexamethasone, ascorbic acid, β-glycerophosphate and growth factor
bone morphogenetic protein 2 (21).
The osteogenic phenotype has a positive expression for CD106,
CD13, CD49, CD44, CD90, CD105, CD29, and CD166 (22).
Chondrogenic lineage
The isolation of ASCs requires the lipoaspirate to be washedwith PBS, then the cells to be cultured and then to be suspended
in a very low temperature environment. The condrogenic differentiation include: DMEM/F-12, ascorbate-2-phosphate, l-proline,
dexamethasone, sodium pyruvate, ITS, single or combinationsof TGF-β1, TGF-β2 and IGF-I (23). Another protocol for differentiation uses ascorbic acid, bone morphogenetic protein 6,
dexamethasone, insulin, transforming growth factor-β (15). Locke
et al. used for chondregenic differentiation insulin, TGF-β1 andascorbate (21), while Yoshimura et al. used DMEM, FBS, insulin,
TGFβ1and ascorbate-2-phosphate (20).
12For many years, it was thought that ASCs can differentiate
into mesenchymal lineage, but in the last 5 years studies have
proved the ability of ASCs to differentiate into non-mesenchymal
lineage (24). Thus, a number of studies have been published,presenting the capacity of ASCs to differentiate into pancreatic,
neurogenic, cardiac, hepatic, muscular cell types.
Pancreatic lineage
The ASCs have been suspended with Dulbecco’s modified
Eagle’s medium, then collected and washed with Dulbecco’s
modified eagle medium/nutrient mixture. The conditions used toobtain pancreatic lineage were DMEM/F12 medium with glucose,
penicillin/streptomycin, nicotinamide, hepatocyte growth factor,
pentagastrin, B-27activin-A, exendin-4, serum-free supplement
and N-2 Supplement (25).
The pancreatic lineage obtained through differentiation of ACSs
has positive cellular markers for c-kit and stem cell factor (SCF) (22).
Neurogenic lineage
The protocol to differentiate ASCs to neurogenic lineage tested
by Gimble et al. used butylated hydroxyanisole, valproic acid andinsulin (15). An alternative inductive protocol uses serum-free
medium, hEGF, basic FGF, poly-l-lysinated coverslips, BDNF,
FBS and all-trans RA (26).
The neurogenic lineage obtained through differentiation of
ACSs has positive cellular markers for MHC I, CD90, CD73,
CD44, CD29 and CD105 (22).
Cardiac lineage
In order to obtain cardiac lineage from lipoaspirate, it needs tobe washed with PBS, suspended in DMEM and then centrifuged.
The next step is to suspend the pellet in NH4Cl. To induce thedifferentiation to cardiac lineage the protocol requires DMEMLG, insulin, transferrin, sodium selenite, FCS, antibiotics bovine
serum albumin, ascorbate, dexamethasone and linoleic acid (27).
Gimble et al. used transferrin, IL-3, IL-6 and VEGF to induce
differentiation (15).
Fiziologia - Physiology  2013.23.4 (80)The cardiac lineage obtained through differentiation of ACSs
has positive cellular markers for CD105, CD90 and CD73 (22).
Hepatic lineage
Seo et al. used adipose tissue harvested after liposuction,
washed the aspirate with PBS, centrifuged it and then suspended
in medium α-MEM. The differentiation protocol then used
expansion medium without serum, rhOSM dimethyl
sulfoxide and rhHGF (28).
The hepatic lineage obtained through differentiation of ACSs has
positive cellular markers for CD29, CD90, CD105 and CD44 (22).
Muscular lineage
Gimble et al. used to differentiate ASCs to muscular lineage
dexamethasone and horse serum (15), while Locke et al. usedcomplete media, horse serum and glucocorticoid such as hydrocortisone and/or dexamethasone (21).
CLINICAL AND THERAPEUTICAL IMPLICATION OF ASCs
Osteoarticular
The potential of osteogenic lineage of ASCs has been provedby the first studies describing ASCs (2,4,6,7). Thus, many studies
have started analyzing the potential of bone formation in vivo andin vitro in order to obtain bone reconstruction. In the beginning,
ASCs were implanted directly in the bony defect, but recently
scaffolds have been used to assure a support for the ASCs anda proper environment for cell proliferation (29).
Lendeckel et al. reported in 2004 the usage of a scaffold with
ASCs and bone graft in order to obtain the healing of a calvarial
defect in a 7 year old girl. Computer tomography at 3 months
revealed a great reossification of the defect area (30). In 2009,
Mesimäki et al. described a new method to reconstruct a major
maxillary defect, resulted after a hemimaxillectomy, in an adultpatient using autologous human ASCs. Sándor presented a 85%
success in treating 23 patients with craniofacial bony defects using
ASCs and resorbable scaffolds, thus proving the utility of biomaterials and ASCs (31). A more recent report by Pak showed reductionof pain in patients suffering from avascular hip necrosis treatedwith ASCs, percutaneously injected, in association with platelet
rich plasma. At 3 months the patient’s symptoms were improved
and the MRI showed a significant bone filling of the defect (32).
Cartilage
Cartilage defects are becoming a real therapeutic problem,
due to an increase in incidence and also to the difficulties in
obtaining a viable solution. The problem with cartilage is the low
self-renewal capacity. The mesenchymal stem cell’s capacity todifferentiate into chondrogenic lineage has led to many studies
exploring this solution as a therapy (2,4,7,11). In 2009 Zhang etal. used cultured ASCs mixed with calcium alginate to fill a patellofemoral defect created in twenty seven rabbits. The result at 12
weeks revealed full thickness cartilage repair (33). Bahrabi et al.
isolated ASCs from rabbit fat and used it to fill a defect createdin the auricle cartilage. At six months full thickness coverage of2013.23.4 (80)  Fiziologia - Physiology

the defect was observed (34).
Furthermore, a recent study investigated the feasibility oftissue-engineered cartilage in vivo. They concluded that, withthe help of bioreactor culturing of ASCs, it is possible to obtaincartilage engineered in vivo (35).
Cardiac and vascular
Cardiovascular pathologies are very common and they arecharacterized by a high mortality rate. ASCs are capable differentiating into cardiomyocytes in vitro, but in vivo their differentiation
is debatable. Bai et al. concluded that, by injecting ASCs into
infarct areas in mice, the myocardic function would significantly
improve (36). However, Cai et al. observed that intramyocardiallyinjected ACSs differentiated into smooth muscle cells (37).
On the other hand, in a recent study, Wang et al. presented
brown ASCs as new source of cardiomyocytes. By using chitosanhydrogel as a support for ASCs in infracted rat hearts, he wasable to prove an increased angiogenesis and to maintain theheart function (38).
The loss of contractile function of the myocardium, found incongestive heart failure or other cardiomyopathy, could also
benefit from the development of ASCs therapies. When exposed
to ASCs and sildenafil combination therapy, mice sufferingfrom dilated cardiomyopathy revealed a preserved left ventricle
function (39). In a 2014 study on rats suffering from dilated
cardiomyopathy, Liang and Yungfeng managed to prove that by
implanting ASCs to myocardial suffering tissue, the functions of
the heart could improve significantly (40).
Some studies have focused on limb ischemia. So it was determined that ASCs cultured in spheroids improved cell survival,
angiogenic factor secretion, neovascularization and limb survival
in athymic mice (41). In a recent study, Bura et al. enrolled sevenpatients with critical limb ischemia and treated them with intramuscular ASCs. The result was an increase in cutaneous oxygenpressure and an improvement in ulcer wound healing (42).
Neurological and nervous system
The capacity of bone marrow mesenchymal stem cells to
express neuronal markers has been taken into account when
proposing differentiation toward neuronal lineage. Similarly to otherstem cell types, ADSCs have been known to have a differentiation
potential into neuronal and glial cells and to be capable of promoting neuronal healing by secretion of some nerve growth factors.
Following this principles, ACSs have been studied in this manner.
It has been proved that ASCs express neuronal markers SSEA4 and neuronal precursors’ markers nestin and beta-III-tubulin(43). Zhang et al. were among the first to prove the capacity ofASCs to differentiate in vivo and also in vitro intro neuronal-likecells. Yet they were unable to prove a better outcome in spinal
cord injuries (44). On the other hand, Moon et al. in a 2014 study
on rabbits with an induced spinal ischemic/reperfusion episoderevealed that by administrating intrathecal ASCs, the neurologicaloutcome was much improved when compared to the control group(45). Another 2014 study on focal demyelination of rat spinal cordfrom Ghasemi et al. concluded that ASCs present the capacity to13differentiate into oligodendrocyte phenotype cells and improved
remyelination process (46).
Regarding central nervous system injuries, there are studies
investigating the cellular therapeutic potential of ASCs. In a rat
cerebral ischemic-reperfusion injury study, middle cerebral artery
occlusion, ASCs were injected into the cerebral cortex twenty-fourhours after the injury, showed that ASCs promote nerve repair
and inhibit the neuronal cells death (47). Some central nervous
diseases have not been fully understood, so the need to havea decent treatment requires new therapeutically options to be
searched. So is the case of Parkinson’s disease. In a recent studyof a rat model, the administration of ASCs showed a significant
increase in serum TGF-β and MCP-1 levels associated with significant decrease in serum BDNF, brain dopamine, and brain TH
gene expression levels (48). Another neurodegenerative disorder,
Alzheimer, has been studied by Ha et al. regarding the effects ofinjecting intravenously ASCs labelled with multimodal nanoparticle.
The study concluded that nanoparticles could become an important
tool in observing and treating neurodegenerative diseases (49).
Regenerative surgery
The regenerative surgery deals with various types of pathologies, from trauma to congenital and even aesthetic problems.
In regenerative medicine, in order to provide cell infiltration andangiogenesis, it is necessary to combine cell stems, growth factors and even scaffolds. Consequently, the need for cell therapy
was tested by Rigotti et al. who used ASCs in treating severeradiation-induced lesions. He injected ASCs into the injured
areas and the result was neovessel formation and a clinicalimprovement (50). In a study by Yun et al. ASCs subcutaneously
injected in pigs skin scar defects revealed an improved scar, in
relation to its size, colour, texture (51).
An important side-effect of reconstruction operations is the flap
necrosis. Regarding this drawback, Gong et al. tested random
pattern flaps in a rabbit model transplanted with ASCs. The
results were that flaps injected with ASCs presented a longer
survival rate and an increase in the number of capillaries (52).
Another interesting idea tested by Reckhenrich et al., in relation
to wound healing and scaring, was to impregnate bioabsorbablesuture material with ASCs. The results mark a point in the development of surgical suture material (53). Furthermore, in a forty patients
study, Yoshimura et al. performed breast augmentation surgery
using ASCs. The result was an increase in breast circumference
and no major complications, no evidence of fibrosis or adhesions,after twelve months. Another study on eighteen patients sustains
the idea that ASCs are an effective therapy in breast reconstruction
surgery. After 6 months postoperatively the reconstructed breast
had a significantly increased volume and a favourable contour (54).
Respiratory
A new emerging field of research in ASCs cell therapy focuseson the respiratory diseases. In a study conducted on mice sufferingfrom experimentally induced asthma, ASCs were intravenously
injected. Although tracking fluorescent ASCs did not show cell
integration or differentiation into airway cells, the ASCs managed
14

to improve airway hyper-responsiveness and contractile tissue
remodelling (55). However, in a recent study on animals sufferingfrom acute respiratory distress syndrome that received ASCs, thecondition did not improve and the study concluded that, although
not harmful, the cell therapy for this pathology needs further
investigation in order to provide good results (56).
Renal and reproduction
Due to the paracrine capacity of the ASCs and their stimulating
effects on other cells, ASCs have been used in kidney ischemia.
In a murine study, mice suffering from an ischemic-reperfusion
injury, ASCs were injected intra-renal immediate after the injury
and intravenously at six and twenty-four hour after the injury.
The study concluded that ASCs improve the reaction of thesuffering tissues, by diminishing the inflammatory response andthey suppress the oxidative stress (57). In addition, Wang et al.
sustain the idea that ASCs, in the case of ischemic-reperfusion
injury, improve the renal function, by limiting the inflammatory
reactions and the apoptosis (58).
Even the erectile dysfunction can be addressed to ASCs based
therapy. After cavernous nerve crush injury, intracavernous
ASCs injection was performed. The result revieled that ASCs
can stimulate erectile function recovery, presumably throughthe paracrine secretion (59). Hyperlipidemia can lead to erectile
dysfunction causing nerves and endothelium abnormalities. In
a recent study, Huang et al. showed that injecting ASCs into
cavernous corpus of hyperlipidemic rats can improve the erectile
dysfunction (60). Another cause of erectile dysfunction is represented by Peyronie’s disease. It is a connective tissue disordercausing scar tissue formation. In the active phase of the disease
Castiglione et al. injected ASCs directly in the tunica albuginea.
After the ASCs injection, the erectile function has increased andthe fibrous formation has been prevented (61).
FUTURE STUDIES
More than 120 studies are being conducted worldwide regarding ASCs (with) the main poles (areas) being East Asia, Europe
and USA (http://www.clinicaltrials.gov/). These studies are indifferent phases of development and they address all kind ofpathologies.

Fig.2. Worldwide distribution of clinical studies

Fiziologia - Physiology  2013.23.4 (80)CONCLUSION
The cell therapies are probably the most effervescent line ofresearch these days. Potentially giving an answer to chronically,degenerative and even congenital pathologies a great effort
is placed in financing, conducting and publishing the results
regarding ASCs.
There are still many problems to be solved. It is yet to be
determined the ideal place and age to harvest the ASCs. There
are still unclear mechanisms of action, giving room to lots ofspeculations regarding the action of these cell therapies. Still,
the continuous development of the research in this field will
hopefully/probably provide the answers to these questions in
the near future.
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54. Yoshimura K, Asano Y, Aoi N, et al. Progenitor-enriched adipose
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55. Mariñas-Pardo L, Mirones I, Amor-Carro O, et al. Mesenchymal
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56. Zheng G, Huang L, Tong H. Treatment of acute respiratory distresssyndrome with allogeneic adipose-derived mesenchymal stem cells: a
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Eur Urol 2013; 63(3): 551-60.

CELULELE STEM DERIVATE DIN TESUTUL ADIPOS: ACTUALITATIIN INGINERIA TISULARA
RezumatCelulele stem reprezinta celule capabile sa se diferentieze intr-o multitudine de tipuri celulare si care au capacitatea de autoinnoire.
Celulele stem derivate din tesut adipos sunt o adevarata alternativa celulelor stem derivate din maduva osoasa, datorita accesibilitatii
si abundentei. Capacitatea acestora de a se deferentia atat in celule mezodermice, cat si endodermice si ectodermice, le-a transformat
intr-o resursa pentru medicina regenerativa. Studiile in acest domeniu se concentreza pe traumatisme, patologii degenerative si
chiar chirurgia estetica. Scopul acestui articol a fost de a face un rezumat al celor mai noi lucrari stiintifice publicate privind terapiile
celulare pe baza ASCs. Exista inca multe aspecte care trebuie lamurite, dar numarul crescut de studii desfasurate la nivel mondial
vor aduce multe raspunsuri in anii ce vor urma.
Cuvinte cheie: celule stem derivate din tesut adipos (ASC), medicina regenerativa, izolare, diferentiere, inginerii tisulare.

16

Fiziologia - Physiology  2013.23.4 (80)

HUMAN RECOMBINANT INTEFERON-ALPHA EFFECTS ON
ACTIVE BEHAVIORS IN THE RAT FORCED SWIMMING TESTDENISE C.M. ZAHIU*, IOANA GOGANAU, RADU I. BRAGA, LUISA BACA, LEON ZAGREAN
Division of Physiology and Neurosciences, Department of Functional Sciences, “Carol Davila” University of Medicine and Pharmacy,
Bucharest

ABSTRACT

Cytokine-induced depression may be modeled in animals by the administration of interferon-alpha (IFN-α). The behavioral tests
commonly used to assess depression are the forced swimming test (FST) and the tail suspension test.
The aim of the present study was describe the effect of acute and chronic administration of different formulations of recombinant human
IFN-α (Hu-IFN-α) on active behaviors in the FST. Male Wistar rats were randomly allocated into three experimental groups: control,
non-pegylated IFN-α-treated and pegylated IFN-α-treated groups. At the end of the first and the sixth week of treatment, rats were
submitted to a battery of behavioral tests. After one week of treatment, in the FST, both IFN-α formulations decreased significantly
the frequency of swimming counts, but there was no difference in immobility and nor in climbing behavior. No differences have been
noticed between the studied groups regarding the FST behavior at the end of the sixth week of drug administration.
Our study provides data demonstrating that acute administration of both Hu-IFN-α formulations determines changes in active
behaviors during the forced swimming test in rats (swimming). Further studies are needed to proof the implication of serotonergic
neurotransmission impairment in the reduction of swimming.
Keywords: interferon alpha, depression, forced swimming test, active behaviors, cytokines

BACKGROUND
Chronic inflammation is considered today an important factor
in the pathogenesis of depression (25). Increasing amounts ofdata are showing an increase in pro-inflammatory cytokines suchas IL1, IL6 and tumor necrosis factor alpha (TNF-α), in plasma
or in cerebrospinal fluid of patients with major depression, withor without other medical associated comorbidities (1, 18).
Cytokine-induced depression may be modeled by the administration of interferon-alpha (IFN-α), an endogenous cytokine
used in the medical practice for its antiviral and anti-proliferative
actions. The model is based on the observation that a highpercentage of patients receiving IFN-α treatment for chronicviral hepatitis and for malignancies develop depression or otherneuro-psychiatric symptoms (irritability, anxiety, mania, cognitivechanges, loss of appetite, psychosis) (14, 26). These side effects
may appear since the first weeks of treatment and have the highest incidence between the first and the third month (27), making
drug adherence a difficult problem (14). There are two main
IFN-α drug formulations, which have similar antiviral efficacy
and similar neuro-psychiatric adverse events: non-pegylated
IFN-α usually administered 3 times/week and pegylated IFN-α,
once per week (26). In the pegylated formulation a polyethylene
glycol is added to standard IFN-α to delay its clearance and thus
to allow a once-weekly administration (12).
Several mechanisms of IFN-α-induced depression have beensuggested: hyperactivation of hypothalamic-pituitary-adrenal

axis (3), impairments in serotonergic neurotransmission (4,30)
and the enhancement of inflammatory response by stimulating
the synthesis and the release of IL1, IL6 and TNF-α (33).
Due to ethical considerations and the difficult access to humanbrain tissue, animals, especially rodents, have been used to
study the molecular mechanisms of IFN-α-induced behavioral
changes (32). Administration of pro-inflammatory cytokines inanimals induces a behavior pattern called ‘sickness behavior’
which resembles the vegetative symptoms of depression inhumans (10). The classical tests of depression commonly usedin animal models are those based on the learned helplessness
hypothesis, i.e. the forced swimming test (FST) and the tailsuspension test. FST is a behavioral test created by Porsolt in1977 to predict the efficacy of antidepressant treatments (24).
It consisted in measuring the time spent immobile by a rodent
placed in a tank of water for a certain period of time. Since
then, the traditionally FST was improved, scoring not only theimmobility, but measuring also the active behaviors. Different
antidepressant classes selectively modulate the swimming andclimbing active behaviors; selective serotonin reuptake inhibitors
increase selectively swimming score, whereas selective norepinephrine uptake inhibitors increase climbing (8).
The use of a rodent-model for IFN-α-induced depression is stilla subject of debate. Many studies using different IFN-α preparations (human, rat, mouse; pegylated, non-pegylated), doses,
administration routes, durations of therapy, various rodents

Received 22nd of August 2013.Accepted 16th of September 2013. Address for correspondence: Denise CM Zahiu, MD, PhD student, Division of Physiology and Fundamental Neurosciences, “Carol Davila” University of Medicine and Pharmacy, Bd. EroiiSanitari, No. 8, Bucharest, RO-050474; e-mail: denisezahiu@yahoo.com

2013.23.4 (80)  Fiziologia - Physiology

17strains and different assessment tests of depressive behavior
are presenting controversial results (19). Although there is only
a 40 -50% similarity between the human and rodents type I IFN
receptors (15), recombinant human IFN-α (Hu-IFN-α) displays
binding affinity for rat brain tissue membranes (28), peripheral
chronic administration of Hu-IFN-α in rats reduces brain levelsof serotonin (23) and increases IL1 and IL 10 levels (21), intracisternal administration of Hu-IFN-α in mice induces immobility
in the FST (6)AIM
The aim of the present study was to describe the effect of acuteand chronic administration of different Hu-IFN-α formulations ofon active behaviors in the FST.
MATERIALS AND METHODS
All animal procedures were carried out with the approval of
the local ethics committee for animal research ‘‘Carol Davila’’
University of Medicine and Pharmacy (Bucharest, Romania), andin accordance with the European Communities Council Directive
86/609/EEC. Every effort was made to reduce the number ofanimals used in this study.
Animals
Experiments were conducted on 24 male Wistar rats, weighting
150-200g upon arrival, housed in individual cages, at constanttemperature (23°C) and 12-h light-dark cycle, with free accessto food and water. Rats were were randomly allocated into threeexperimental groups: (A, n=8) Hu-IFN-α-2b in aqueous solution(Intron A, Schering Plough, USA, 50000IU/kg for 3 days/ week),
injected intraperitoneally for 6 weeks; (B, n=8) pegylated HuIFN-α-2b (Pegintron, Schering Plough, USA 1.5μg/kg weekly)
administered for six weeks and (C, n=8) control, treated withsaline (1ml/kg, 3 days/ week). The body weight was recorded
once a week in order to adjust the dose of IFN-α.
Behavioral tests
At the end of the first and the sixth week of treatment, 24 hours
after the last drug administration, rats were submitted to a batteryof behavioral tests: open field test (OFT), FST and elevated plus
maze test (EPM), according to Current Protocols in Neuroscience, 2003 (7) recommendations. All tests were performed during
the light phase of the light/dark cycle, between 08:00 and 12:00
a.m., in order to minimize the effect of circadian rhythms.
Experiments for OFT and EPM were recorded and automatically scored using an EthoVision XT Acquisition System (Noldus,
Netherlands). All tracks were manually verified and corrected for
tracking errors. After each behavioral trial, the testing chamber(OFT arena and the plus maze) was cleaned for odor traces
with 70% ethanol.
Rats were tested for locomotor activity (OFT) in an apparatus
consisting in a black plexiglas chamber (40x40x40cm) with anopen top, to permit automated video tracking of the rat. During
the test, spontaneous rat activity was quantified for 10 min bymeasuring the total distance moved in the arena (cm), the velocity(cm/s) and the time (s) spent in a central zone defined virtuallyat 10 cm from the walls.
18

To evaluate the anxiety behavior, rats were put for 5 minutes
in a plus maze (arms 50 cm long and 10 cm wide; two arms enclosed by 40 cm high walls), suspended at 50 cm above the floor.
The total movement distance (cm) and the time (s) spent in the
open arms and in the closed arms were measured automatically.
For the FST, rats were forced to swim for 5 minutes inside a
vertical glass cylinder (height, 45 cm; diameter, 30 cm) filled withwater to a depth of 30 cm and maintained at a temperature of
23-26°C. At this depth of water, the rats were unable to touch thebottom of the cylinder with their hind paws or their tails. During
the trial, behavioral responses were separated into 3 categories: immobility, swimming, climbing (Detke et al. 1995)(8). The
behavioral response was scored at the end of each 5-s period
during the experiment and presented as mean counts (score).
The rat was rated as immobile when it was floating, making only
the necessary movements to keep its head out of water. Climbing
and swimming were defined as escape-oriented behaviors. The
rat was considered to be climbing when it struggled to escapefrom the cylinder by climbing with its forepaws on the glass walls.
When the rat was making active swimming motions, more thanthose movements necessary for floating, it was count as swimming. All of the behavioral scoring for the FST was done by asingle investigator, who was blind to the treatment condition. An
initial 15 minutes pretest session was conducted the day before
drug administration.
Statistical analysis
Each behavioral parameter was analyzed using one-way
ANOVA repeated measures, multiple comparisons. The significance of difference between IFN-treated groups and control
group was determined using Dunnett’s post-hoc test (for thoseanalyses with α=0.05). Descriptive results are presented asmeans ± standard error of the mean (SEM). All statistical tests
and graphs were made in GraphPad Prism 6 (http://www.graphpad.com/scientific-software/prism/).
RESULTS
Acute administration of IFN-α:After one week of treatment, rats receiving IFN-α or saline displayed in the OFT similar behaviors regarding the total distance
moved (F1.7,12.4= 1.61, P>0.05), velocity (F1.9,13.3= 0.50, P>0.05)
or the time spent in the center of the open field arena (F1.3,9.6=
0.47, P>0.05). No differences between groups were recordedin the EPM for total distance moved (F1.23,8.66= 4.45, p>0.05),
time spent in open arms (F1.98,13.87= 1.12, P>0.05), time spent inclosed arms (F1.44,10.07= 0.95, P>0.05) or the arm-entry frequency(F1.10,7.72= 0.60, P>0.05). Detailed descriptive statistics of the OFT
and EPM are shown in Tables I and II.
The effects of acute IFN-α treatment on rat behavior in the
FST are illustrated in Figure 1. Both IFN-α formulations decreased significantly the frequency of swimming counts (F1.53,= 15.48, P<0.01), but there was no difference in immobility
10.72(F1.51, 10.62 = 1.83, P>0.05) and nor in climbing (F1.48, 10.4 = 2.46,
P>0.05) behavior.
Fiziologia - Physiology  2013.23.4 (80)GroupsMean distance
moved (cm)Meanvelocity(cm/s)

Mean timespent in central zone (s)1th week
Control(n=8/group) IFN-α
Peg IFN-α

2381.93±245.01 4.13±0.41 28.22±5.442312.34±256.98 4.03±0.44 43.86±19.68
2843.29±136.90 4.56±0.25 49.26±15.55

6th week
Control(n=8/group) IFN-α
Peg IFN-α

972.68±139.60 1.51±0.21 19.72±5.70
1144.99±84.39 1.89±0.14 22.45±4.68
1431.66±122.53* 2.22±0.21 32.91±5.50*markes significant differences for p<0.05, treated groups compared to control
group *
OFT, open field test; IFN-α, interferon α; PegIFN- α, pegylated interferon α.

at the end of the sixth week of drug administration. Data for all
the behavioral tests after chronic IFN-α treatment is illustrated
in Tables I and II, and Figure 2.
All groups had similar growth curves except for the PegIFNrats that gained significantly less weight compared to control-rats
in the 3rd and 4th week of treatment as shown in Figure 3.
50GroupsMean distance20
10

I m m o b ility

Mean time in

1031±186
1531±137.3

22.77±7.4212.33±5.93

239.3±13.9
265.9±13.49

180
mean
frequency
180
160
11.75±3.24
7.87±1.87
160
8.5±2.58
140

weight increase(%)
weight increase(%)

IFN-α
Peg IFN-α

C lim b in gpresented as mean counts (score) and SEM for immobility, swimmingand climbing. All groups displayed similar behaviors.
Mean time inArm-entry

6 weekControl1095±101
22.11±5.50 257.6±7.45 8.25±1.46th6(n=8/group)
week
Control 1252±175.401095±101 32.44±5.1122.11±5.50
257.6±7.45
IFN-α216.9±9.37* 13.25±0.92*(n=8/group) Peg IFN-α
1252±175.40
216.9±9.37*
IFN-α 1517±150
31.78±5.1232.44±5.11
198.6±8.76* 21.63±1.98*th

S w im m in g

Fig.2. Forced swimming test results after 6 weeks of IFN-α treatment presented as mean cou
Fig.2. Forced swimming test results after 6 weeks of IFN-α treatment
immobility, swimming and climbing. All groups displayed similar behaviors.

moved (cm)arms (s)closed arms (s)1thGroups
week
Control1503±149.6
12.09±3.78open
253.4±13.21
11.75±3.24(n=8/group) IFN-α
1031±186
22.77±7.42 239.3±13.9 7.87±1.87
Peg Control
IFN-α 1531±137.3
12.33±5.9312.09±3.78
265.9±13.49 8.5±2.58
1th week
1503±149.6
253.4±13.21(n=8/group)P e g IF N -0

Mean disMean time Mean timeArm-entry
tance moved
in openin closedmean freTable II. Descriptive statistics for EPM results(cm)
arms (s)arms (s)quencyParameters

IF N -

30

Table II. Descriptive statistics for EPM results
Parameters

C o n tro l

40

sco reTable I. Descriptive data on behavior in OFTParameters

IFN
PegIFN
IFN
Control
PegIFN
Control

8.25±1.46
140
13.25±0.92*
120
Peg IFN-α 1517±150
31.78±5.12
198.6±8.76*
21.63±1.98*
120*markessignificant
differencesfor
p<0.05,treatedgroupscomparedtocontrol
100*markes significant differences for p<0.05, treated groups compared to control group
group
1
2
3
4
5
6
EPM, elevated plus maze; IFN- α, interferon α; PegIFN- α, pegylated interferon α.
100
EPM, elevated plus maze; IFN- α, interferon α; PegIFN- α, pegylated interweeks
1
2
3
4
5
6
feron α.
Fig.3. Rats growth curves during weeks
the IFN-treatment presented as percentage from baseline weight ga
vs. control group).
60
C o n tro l

Fig.3. Rats growth curves during the IFN-treatment presented aspercentage from baseline weight gained each week (* p<0.05, vs.
DISCUSSION
40
group).
P e g IF N -
Data from previous studiescontrol
regardingthe effect of standard IFN-α on behavior ocontroversial results (11; 16; 20). Few studies have tested the pegylated Hu-IF
DISCUSSION
satisfactory
expected results, although clinical studies demonstrate a similar inciden
20
adverseformulations(31).effectDivergent
results might originate
Dataeventsfrom between
previousIFN-α
studiesregarding theof standard
differences.
To our knowledge,
the first to compare**
IFN-α on behaviorof rodents, thishas reportshowniscontroversial
results both IFN-α foactive behavior of rats in the FST, in acute and chronic administration.
0(11; 16; 20). Few studies have tested the pegylated Hu-IFN-α
I m m o b ility
S w im m in g
C lim b in g
In the present study, although acute peripheral Hu-IFN-α administration didn‟t indu
rats, withoutsatisfactory
expected
results,decreased
althoughswimming
clinical behavior. The
ininlocomotor
activity,
both IFN-α
formulations
Fig.1.
scorescorein the inforcedtest after oneof oneIFN-αweekadministration. Data presented as mean counts +Fig.1.Behavioural
Behaviouralthe swimming
forced swimming
testweek
after
studiesdemonstrateasimilarincidenceof
neuropsychiatricimmobility, neither for standard IFN-α, nor for pegylated IFN-α after one week
SEM (* p<0.05, vs. control group).
of IFN-α administration. Data presented as mean counts + SEM (*resultssuggestthatbetween
swimming
behavior
evaluation
more sensitive than imm
adverseeventsIFN-αformulations(31).isDivergent
p<0.05, vs. control group).
assessmentof IFN-α-induced
depression
in rats. differences.
Swimming behavior
is associa
results might
originate due tomethodologicalTo
Chronic administration of IFN-α:
system activity and is enhanced by selective serotonin reuptake inhibitors (8). It
ourknowledge,
thisreport
is
the
first
to
compareboth
IFN-α
After six weeks
of IFN-α administration,
locomotor theactivity
and an (5-HT) uptake, by increasing 5-HT transporter expre
Chronic
administrationof IFN-α: there was an increase in exploratory
upregulates
serotonin
formulations,assessing
behaviorof ratsindolaminein the FST,2,3deoxigenaseinattenuationof anxiety
level forIFN-α treatedthere
rats asshown
by the higher meanvalue5-HTof totaldistancebyactive
regulates
synthesis
activating
the enzyme(IDO
After six weeks
of IFN-αadministration,wasan increase
moved in the OFT for PegIFN-α rats (F1.99, 13.95= 4.22, P<0.05), by thetryptophan,increased
arm-entry
mean to kynurenine, leading to tryptophan depletion (13),
acute
and
chronic
administration.
5-HTprecursor,
inexploratory
locomotor
activity
andanattenuationofanxietyin closed
arms (F1.65,
frequency (F1.36, 9.53= 15.08, P<0.01) and by the reduction of the time spent(30)11.61=
ofneurotoxic
metabolites(22). Hu-IFN-α adInandthesynthesispresent
study,
although
acute peripherallevel
IFN-α treatedas shown
by the higher
mean value
10.48,forP<0.01)
for bothrats
IFN-α
formulations
when compared
to control. However,no significant
Why
in
our
study
Hu-IFN-αdidn‟torincreased
theimmobility
in the FST remains aministration
didn’tinduceanxietychangesin locomotor
activity,differences
have been
noticedbetweenthe PegIFN-α
studied groups
in the open arms of theoftotal distance
moved
in the
OFT forratsregarding(F1.99, 13.95the= time spentfindings
areincontrast
with
two
previous
studies,one
on
Wistarrats and another
EPM and FST behavior at the end of the sixth week of drug administration. Data for all the behavioral
sco reIF N -

both IFN-α formulations decreased swimming behavior. There
4.22,
P<0.05),
byIFN-αthe increasedmeanfrequency
1.36, Figure 2.
tests after
chronic
treatment arm-entry
is illustrated
in Tables
I and II,(Fandwas no increase in immobility, neither for standard IFN-α, nor
15.08,hadP<0.01)by thereductionof thespent in
All=groups
similarand
growthcurvesexcept for
the time
PegIFN-rats
that gained significantly less weight
9.53
for3.pegylated IFN-α after one week of treatment. These resultscompared
to control-rats
the 3rdP<0.01)
and 4th weekof treatment
as shown in Figure
closedarms(F1.65, 11.61=in10.48,
for both
IFN-α formulasuggest that swimming behavior evaluation is more sensitive
tions when compared to control. However, no significant differthan immobility alone in the assessment of IFN-α-induced
ences have been noticed between the studied groups regarding
depression in rats. Swimming behavior is associated with serothe time spent in the open arms of the EPM and FST behavior
2013.23.4 (80)  Fiziologia - Physiology

19tonergic system activity and is enhanced by selective serotonin
reuptake inhibitors (8). It is known that IFN-α upregulates theserotonin (5-HT) uptake, by increasing 5-HT transporter expression (2), and down regulates 5-HT synthesis by activating the
enzyme indolamine 2,3deoxigenase (IDO) that can metabolize
tryptophan, 5-HT precursor, to kynurenine, leading to tryptophan
depletion (13), reduced 5-HT levels (30) and synthesis of neurotoxic metabolites (22).
Why in our study Hu-IFN-α didn’t increased the immobility in
the FST remains an open question. Our findings are in contrast
with two previous studies, one on Wistar rats and another on
mice, that used a similar dose of non-pegylated Hu-IFN-α andreported an increase of the immobility of FST, but after a single
intravenous dose, 15min prior to testing (9; 6). The increasein immobility seemed to be mediated through central µ-opioid
receptors, and not by IFN-α receptors, the effect being reversed
by naloxone injection (6). We must emphasize that in our study
all the behavioral tests were done 24 hours after the last drug
administration and repeated doses.
Another study that developed a murine model of depression
for repeated administration of non-pegylated Hu-IFN-α, and nota single dose, showed that a 400-1600 UI Hu-IFN-α /g/day withdaily administration for 5 days increases immobility in the FST(5). The dose of Hu-IFN-α we used was similar to the doses
administered in patients with chronic hepatitis C (50UI/g/day),and thus, lower than those from Siddegowda et al study (5).
In our study, the FST results of the Hu-IFN-α treated rats
showed that the reduction of swimming counts was associated to an increase in climbing behavior and not to increase
immobility, without reaching a significant level compared tocontrol group, neither for climbing, nor for immobility counts.
The climbing behavior is known to be mediated by enhancementof norepinephrine neurotransmission (8). Acute administrationof IFN-α increases plasma concentration of norepinephrine,
epinephrine and cortisol in humans (17), by causing a marked
activation of hypthalamo-pituitary-aderenal axis (3). In rats, it
was shown that administration of IFN-α for 7 days increases
norepinephrine levels in the cerebral cortex, hypothalamus andmedulla oblongata (17).
Cognitive and behavioral side effects of IFN-α therapy usuallydevelop after a several weeks of treatment (6,29). There arefew animal studies with chronic IFN-α exposure. A study, using
standard IFN-α, observed increased anxiety behavior, without
cognitive impairment, in rats treated for 5 weeks (21). In ourstudy, there were no changes in behavioral parameters after 6
weeks of administration of both IFN-α formulations. As we usedrecombinant human IFN-α, we can’t exclude the formation ofanti-IFN-α antibodies after 4-5 weeks of treatment. Unfortunately,
anti-IFN-α antibodies dosage wasn’t accessible at that moment.
CONCLUSION
Our study provides data demonstrating that acute administration of both Hu-IFN-α formulations, at low doses compared toother studies, determines changes in active behaviors during
the forced swimming test in rats (swimming). Further studies
20

are needed to confirm that the serotonergic system is indeed
involved in the reduction of swimming behavior induced by IFN-α
administration.
Whether the chronic treatment might influence active behaviors in FST is still an open question.
Because histological and immunohistochemical studies suggest a possible neurodegenerative effect of IFN-α, is necessary
the improvement of animal models of IFN-α-induced depression,
to better understand the pathophysiological processes involved
in it and to test possible prophylactic or therapeutic drugs.
DISCLOSURE STATEMENT
This is not an industry supported study. All authors haveindicated no financial conflicts of interest.
Acknowledgement
This work was supported by a CNCSIS grant: BD180/2008.
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EFECTELE INTERFERONULUI-ALFA RECOMBINANT UMAN ASUPRACOMPORTAMENTELOR ACTIVE LA TESTUL INOTULUI FORTAT, LA
SOBOLANREZUMAT

Depresia indusă de cytokine proinflamatorii poate fi reprodusă pe modele animale prin administrarea de interferon alfa (IFN-α).
Cele mai utilizate teste comportamentale pentru evaluarea depresiei pe modele animale sunt testul înotului forţat (FST) şi testul
suspendării de coadă.
Scopul studiului a fost să observe efectele administrării acute şi cornice de interferon-alpha recombinant uman (Hu-IFN-α) asupracomportamentelor active la FST. Şobolani Wistar, masculi, au fost alocaţi aleatoriu în trei grupuri experimentale: control, trataţi cu
IFN-α nepegylat şi trataţi cu IFN-α pegylat. La sfârşitul primei şi a celei de-a şasea săptămâni de tratament, şobolanii au fost evaluaţi
printr-un set de teste comportamentale. La tratamentul acut cu IFN-α, indiferent de tipul de preparat, s-a constat la FST scădereasemnificativă a frecvenţei miscărilor de înot, însă fără diferenţe pentru imobilitate sau încercările de a escalada pereţii cuvei deînot (căţărare; eng. climbing”). La sfârşitul perioadei de şase săptămâni nu au mai fost constatate diferenţe în ceea ce priveşte
comportamentele active la FST.
Studiul nostru a arătat că administrarea acută de IFN-α induce modificări ale comportamentelor active la FST. Studii ulterioare suntnecesare pentru a demonstra implicarea sistemului neurotransmiţător serotoninergic în modificările comportamentale constatate.
Cuvinte cheie: interferon-alfa, depresie, citokine, testul inotului fortat, comportamente active.

2013.23.4 (80)  Fiziologia - Physiology

21

CORTICAL ACTIVITY AND HEART RATE CHANGES AS
MARKERS OF NOXIOUS STIMULATION RESPONSE DURING
CHLORAL HYDRATE ANESTHESIAPAVEL B, DĂNEASA A, CĂLIN A, RĂDULESCU AM , ROSCA AE, BRAGA RI, ZAHIU D,ZĂGREAN L
Division of Physiology and Fundamental Neurosciences, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACTThe aim of our study was to evaluate the importance and applicability of cortical activity and heart rate changes as markers of noxious
stimulation response during chloral hydrate anaesthesia in rats.
In this study we used a group of 11 Wistar rats, which were chronically implanted, with electrodes being placed on the dura materof the right hemisphere, over the olfactory cortex (reference), the frontal and the parietal lobes. A noxious mechanical stimulus wasapplied on the left hindpaw during chloral hydrate anesthesia, at an anesthetic depth of 2-3 Hz, estimated using the median frequency.
We assessed the electrical activity of the cortical areas (evaluated through median frequency, delta power and cortical connectivity
between frontal and parietal cortex) and heart rate response to mechanical noxious stimulation.
After data processing and analysis we observed an increase of median frequency and a decrease of delta power during the application of thenoxious stimulus in both areas (frontal and parietal). No difference between fronto-parietal connectivity during baseline and stimulation tracks
was noted. Tachogram analysis showed a mild increase in heart rate during noxious stimulation, but it did not reach statistic significance.
Monitoring cortical electric activity is a better choice than ECG monitoring for noxious stimulation response assessment during chloral
hydrate anesthesia in rat.
Key words: anesthesia, cortical connectivity, nociception, electrocorticogram

INTRODUCTION
The monitoring of the anesthetic depth has been a primaryconcern for physicians since the dawn of anesthesia. In 1937
anesthetic depth was quantified relying on pupil diameter changes,
respiratory rate, heart rate, and blood pressure (1). In the same
year, the electroencephalogram (EEG) was proposed as a method
for the assessment of anesthetic depth (2). Currently, EEG signal
analysis is used for the monitoring of the hypnotic effect (3); facial electromyogram, analysis of heart rate variability, cutaneous
conductance are among the methods, cited in scientific literature,that are used to measure the reaction to noxious stimuli (4,5,6,7).
Although some data on the subject of EEG changes during noxious
stimulation can be found in scientific literature (8, 9), the main difficulty in using EEG changes as markers of noxious stimulation liesin the occurrence of the phenomenon called paradoxical arousal,
which consists of an increase in anesthesia depth following noxious stimulation, represented by an increase in slow wave activity(10). New data shows that a decrease in cortical connectivity ispresent during anesthesia (11,12). Cortical connectivity decreases
especially between frontal and parietal areas during anesthesia instudies performed in humans and animals. Methods as fMRI andEEG signals correlation algorithms are cited in cortical connectivity
assessment (13,14,15). Recent study investigating simultaneous
cortical connectivity assessment using fMRI and EEG has proveda similar change trend (16).

The main aim of this study is to investigate if changes in thefunctional connectivity between frontal and parietal areas could
be used as marker of a reaction to noxious stimulation. As far
as we know such an inquiry has not been yet attempted. Other
aims of this study are to investigate if the activations of the frontal
and parietal areas differ one from another and to find out what
relationship exists between cortical activation and heart ratechanges during noxious stimulation.
For these issues we used a chronically implanted rat model,
who received a painful stimulus under chloral hydrate anesthesia.
Cortical area (frontal and parietal) activity was measured as medianfrequency (MEF) and delta power variation, during stimulation. For
the evaluation of the functional connectivity, electrocorticogram(ECoG) synchronicity between cortical areas, using correlation
coefficient was performed (17). The electrocardiogram (ECG) wasrecorded and heart rate changes were analyzed.
MATERIALS AND METHODS
Animals
In this study a group (n=11) of Wistar rats was used, with weights
ranging from 250 to 300 grams, which were kept in a room with constant temperature (230 Celsius) and 12-h light-dark cycle (lights on at
07:00 h), with free access to food and water. Rats were chronicallyimplanted, under chloral hydrate (Sigma-Aldrich Gmbh, Munich,Germany) anesthesia (400 mg/kg, injected intraperitoneal), using

Received September 10th 2013. Accepted October 12th 2013. Address for correspondence: Pavel B, MD, Division of Physiology and Fundamental Neurosciences,
University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania, phone: +40213180762; e-mail: pavelbogdan2009@gmail.com

22

Fiziologia - Physiology  2013.23.4 (80)Nickel-Chrome (Ni80Cr20, Ø 0.15 mm, Goodfellow, UK) electrodes
which were placed directly on the dura mater, through burr holes
obtained by trephination, and were finally fixed with cement. Threeelectrodes were implanted, one reference electrode was placed over
the olfactory cortex (7 mm anterior to bregma and 1 mm lateral to
the frontal suture) and two active electrodes were placed over theright hemisphere, in the frontal lobe (3 mm anterior to bregma and 3
mm lateral to the frontal suture) corresponding to the motor cortex,
and in the parietal lobe (3 mm posterior to bregma and 3 mm lateral
to the sagital suture), corresponding to the somatosensory areas ofthe left hindpaw (18). The rats underwent experimentation after aminimum period of 7 days after the intervention. This study has been
approved by the Ethics Committee of the University of Medicine andPharmacy Carol Davila Bucharest.
Anesthesia and stimulation procedure
Anesthesia was induced and maintained with chloral hydrate(300 mg/kg administrated intraperitoneally). After a steady state of
20 minutes from the induction, we started recording, but only whenthe median frequency of the ECoG, recorded in the frontal lobe,
reached 2 – 3 Hz, indicating an appropriate anesthetic depth. The
stimulus consisted in placing the skin from the posterior left hindpaw
between the arms of a Pean type forceps, which was then closed,
locking only the first ratchet. It has already proven in human andanimal that peripheral noxious stimulation induced predominantly
right hemisphere activation, phenomena called lateralization (19,20). So, we chose the left hindpaw to be stimulated and right hemisphere for ECoG recordings. The mechanical noxious stimulus wasapplied for 10 seconds. During the experiment rats temperature wasmaintained at 370-380 Celsius using a heating pad.
Data acquisition and signal processing
The electrocortigram (ECoG) signal acquisition was madeusing BIOPAC MP-150 system (Biopac Systems, CA, USA) at asample rate of 1000 Hz, a gain of 1000, with filters set at 0.5 Hz(high pass) and 30 Hz (low pass). Median frequency and delta
power of the spectrum were computed for each channel separately, using AcqKnowledge 4.2 software. Epoch length was set
at 10 seconds. Functional connectivity between the ECoG signals
from the two areas was computed using correlation coefficient
function provided by the AcqKnowledge 4.2. The analyzed dataconsisted of 1 minute baseline signal and 10 seconds of signal
corresponding to the noxious stimulation. Because the acquisition frequency was set at 1000 Hz, we obtained 60.000 pointsof data for one baseline track of 1 minute, summing 660.000
points of data for 11 rats. This large amount of data was notsupported by our statistics program, so we decided to computea correlation coefficient for each of the 11 tracks and calculate
the mean. We compared the mean values of the correlation
coefficients during baseline track versus stimulation track (17).
ECG signal acquisition was also made using BIOPAC MP-150
system at the same sample rate and using the same filters as
for the ECoG, but with a gain of 500.
Statistical analysis
Data were presented as mean value ± standard deviation(SD). Statistical analysis was performed with the Wilcoxon rank
2013.23.4 (80)  Fiziologia - Physiology

sum test, using SPSS (version 11.0, SPSS Inc., Chicago, IL). P
values ˂ 0.05 were considered statistically significant.
RESULTSAn increase of the median frequency in the frontal area during
noxious stimulation was observed, from a value of 2.66 ± 1.06
Hz to 5.37 ± 1.7 Hz (p<0.05)(Figure 1). Parietal somatosensory
area activation follows a similar trend during noxious stimulation,
median frequency rose from 2.92 ± 1.01 Hz during baseline to
5.11 ± 1.78 Hz (p <0.05) (Figure 2).

Fig. 1. Median frequency (Hz)variation during mechanicalnoxious stimulation comparedwith baseline, in the frontal
lobe (p<0.05)

Fig. 2. Median frequency (Hz)variation during mechanicalnoxious stimulation comparedwith baseline, in the parietal
lobe (p<0.05)

Delta power (μV2/Hz) followed an inverse trend, resulting ina decrease in delta power during noxious stimulation in bothareas, as a consequence of the increase in cortical activity.
So, we got a decrease of delta power from 8.65 ± 6.77 duringbaseline to 4.36 ± 1.90 (p<0.05) during mechanical clamp, infrontal area (Figure 3). In parietal area delta power decrease
from a baseline of 17.84 ± 14.27 to 8.42 ± 5.56 during noxious
stimulation (Figure 4).

Fig. 3. Delta power (μV2/Hz)
variation during mechanicalnoxious stimulation comparedwith baseline, in the frontal
lobe (p<0.05)

Fig. 4. Delta power (μV2/Hz)
variation during mechanicalnoxious stimulation comparedwith baseline, in the parietal
lobe (p<0.05)

Analysis of the functional connectivity between frontal and parietal lobe did not yield statistically significant results, correlation
coefficient was 0.63 ± 0.17 during baseline recording and 0.61
± 0.16 during noxious stimulation with a p = 0.7. Heart rate
maintained a steady value of 394.8 ± 44.15 beats per minute(bpm) during baseline recording and a value of 399 ± 46.18 bpm
during stimulation with a p = 0.4.
DISCUSSION
Cortical activation and connectivity
Change of cortical electric activity during noxious stimulation23

are reported in humans, as well as in animals (8, 21, 22).
The novelty of our study is represented by the analysis of corticalconnectivity, using ECoG signals, during noxious stimulation underchloral hydrate anesthesia, which has not been reported until now.
Following data processing and analysis, an increase in corticalelectric activity in parietal and frontal lobe is noticed. Similarly, medianfrequency increases in the parietal and the frontal lobe comparedwith baseline. Increase in median frequency following mechanicalnoxious stimulation is not a constant finding during anesthesia. Twoother studies where mechanical stimulation was realized by clamping, under halothane anesthesia, revealed two different responses(23, 24). In one study MEF increased and in the other one it did not.
The explanation for these contradicting results could be found bylooking at the types of stimulation used, because MEF increases
during oscillating clamp application compared with stationary clamp,
which does not produce MEF changes. In our study we obtained
an increase of MEF during stationary clamping. The factors in ourstudy that could account for this result are a longer stimulation time,
namely 10 seconds versus the 5 seconds used in the previous study,
and the different anesthetic that was used.
Delta power analysis showed a similarly decrease during
noxious stimulation compared with baseline in the parietal lobe
and in the frontal lobe. This data is not concordant with previous findings, in which a greater decrease in delta power wasobserved in the frontal lobe(8), in a study performed in humans
under isofluran anesthesia. We interpret these different results
as being specific to chloral hydrate anesthesia.
Some studies maintained the idea that MEF increase reflects
the noxious character of the stimulus, in horse under halothane
anesthesia (25) and total power spectrum decreases following
noxious stimulation as a consequence of anesthetic depth
inadequacy (24, 26). We observed an increase in MEF anda decrease in delta power during noxious stimulation in both
cortical areas, therefore, following the same line of reasoning as
above, we could conclude that the observed cortical activation isdue to a combination of supramaximal noxious stimulation andinadequate anesthetic depth.
Recent studies have proven that anesthetics reduce cortical and cortical-subcortical connectivity during anesthesia(11, 12), including fronto-parietal connectivity (27). In order toevaluate the connectivity between the two areas we measured
the functional cortical connectivity, analyzing the synchronicity
between the two cortical electrical signals (ECoG) using correlation coefficient (17). We did not obtain a statistically significant
difference between correlation coefficient during stimulationcompared with baseline. We considered that noxious stimulation
was supramaximal and as such it reduced anesthetic depth, as
was indicated by the MEF and delta Power changes, therefore
a higher than expected connectivity between the parietal andfrontal lobe can be explained. The reason for which we obtained
a correlation coefficient during baseline that was very similar tothe one during stimulation, in spite of a significant change incortical electrical activity (assessed by MEF and delta power)
is an open question for future studies. A low specificity of thecorrelation coefficient in the functional connectivity assessment
24

could partially explain this finding.
ECG changes
It is known that noxious stimulation leads to change in heart rate,change which is present in the awake as well as in the anesthetizedpatient; nevertheless a tight correlation between the change in heart
rate and noxious stimulation during anesthesia could not be established. A higher sensitivity parameter derived from ECG analysis, isthe heart rate variability (HRV), which is indicative of the sympathetic– parasympathetic balance. In human studies, a good correlation
between noxious stimulation during anesthesia and the sympathetic– parasympathetic balance has been established. Despite of thisgood correlation between anesthesia and ECG changes in humans,
ECG changes during anesthesia are poorly understood in rats (28).
We have chosen for heart rate analysis instead of HRV analysis
because the rats in our study where breathing spontaneously andtherefore the effects of sinus arrhythmia could not be excluded. It is
worth mentioning that chloral hydrate, compared to other anesthetics(ketamine, pentobarbital), has no known pharmacodynamic effect on
the myocardium (29), so it seems to be the ideal anesthetic for heart
rate variation assessment during noxious stimulation. Tachogram
analysis showed that heart frequency mildly varies during noxious
stimulation, without reaching statistical significance. How noxious
stimuli can activate the cerebral cortex but not the sympathetic
nervous system, knowing there is a strong correlation between
frontal cortex activity and sympathetic activity in human (30) is stilla matter of debate.
CONCLUSIONS
Cortical activity increases in both areas during noxious stimulation, most likely due to a combination of supramaximal noxious
stimulation and inadequate anesthetic depth. Our hypothesis
regarding the change of cortical functional connectivity during
noxious stimulation, by measuring correlation coefficient, is notconfirmed by our results. Heart rate does not change during
stimulation, so we conclude that monitoring cortical electric activity is a better choice than ECG monitoring for noxious stimulation
response assessment during chloral hydrate anesthesia in rat.
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MODIFICARILE ACTIVITATII CORTICALE SI ALE RITMULUI CARDIAC CA
MARKERI AI RASPUNSULUI LA STIMULAREA NOCICEPTIVA IN TIMPUL
ANESTEZIEI CU CLORALHIDRATREZUMAT

Scopul studiului nostru a fost sa evaluam importanta si aplicabilitatea utilizarii modificarilor activitatii corticale si ale ritmului cardiac,ca markeri de raspuns la stimularea nociceptiva in timpul anesteziei cu cloralhidrat la sobolani.
In acest studiu am folosit 11 sobolani Wistar, care au fost implantati cronic, cu electrozi plasati pe duramater la nivelul emisferului
drept, deasupra cortexului olfactiv (referinta), a lobului frontal si a lobului parietal. In timpul anesteziei cu cloralhidrat, la o profunzimede 2-3 Hz, estimata prin frecventa mediana, a fost aplicat un stimul nociceptiv mecanic pe laba posterioara stanga a sobolanului.
Noi am evaluat activitatea electrica a ariilor corticale (evaluata prin frecventa mediana, puterea delta si conectivitatea corticala intre
cortexul frontal si parietal) si raspunsul frecventei cardiace la stimularea nociceptiva.
Dupa procesarea si analiza datelor am constatat o crestere a frecventei mediane si o scadere a puterii delta in timpul aplicarii stimulului nociceptiv in ambele arii (frontal si parietal). Nu s-a observat nici o modificare a conectivitatii fronto-parietale in timpul stimularii
fata de traseul martor. Analiza tahogramei a evidentiat o usoara crestere a frecventei cardiace in timpul stimularii nociceptive dar
fara semnificatie statistica.
Monitorizarea activitatii electrice corticale este o optiune mai buna decat monitorizarea EKG pentru evaluarea raspusului la stimularea
nociceptiva in timpul anesteziei cu cloralhidrat la sobolan.
Cuvinte cheie: anestezie, conectivitate corticala, nociceptie, electrocorticograma.
2013.23.4 (80)  Fiziologia - Physiology

25

ARGUMENTS FOR USING THROMBELASTOGRAPHY
IN THE STUDY OF HEMOSTASIS COMPARED TO OTHERLABORATORY TESTSFLORINA-MADALINA ONICEANU, ILEANA ION, CECILIA ADUMITRESI, NINELA RADULESCU,CRISTINA FARCAS, ANABELLA-CRISTIANA STEFANESCU, CHIRICA R
Faculty of Medicine, “Ovidius” University Constanta

ABSTRACTThe authors showcase clinical manifestations and lab work in the evolution of haemostatic pathology in children. Classic haemostasis
tests were used, along with more specific and sensitive ones, like determination of some coagulation factors and thrombelastography.
The authors aimed to identify the mechanisms involved in this pathology: immunological mechanisms, deficiency of production,
thrombocytopenia produced by medication, endothelial lesions due to capillary fragility, but some mechanisms remained unrevealed.
Also, the authors compared basic coagulation test with thrombelastography to emphasize the utility and rapidity of this last test toevaluate the hypo or hypercoagulable state in children. For all the patients, there were determined complete blood count, prothrombin
time (PT), activated partial thromboplastin time (APTT), International Normalised Ratio (INR), total calcium level, ionized calcium,
fibrinogen. The objective was to evaluate the risk for thrombotic or bleeding manifestations using thrombelastography. Classicalmethods in the study of haemostasis are not able to highlight clot formation or the changes produced after thrombus formation andalso they cannot suggest predisposition for thrombosis the way thrombelastography does.
Key words: children, thrombelastography, haemostasis

INTRODUCTION
Bleeding syndromes in children represent an extensive pathology in medical practice. Lab work in these cases represents,
along with precise clinical examination, the key in ascertainmentof the proper diagnosis. Based on standard coagulation tests,
lab work includes also more specific and sensitive analysis, likethrombelastography, a method which is not used in Romania,
but applied in international clinics and research fields, a methodthat enhances the diagnosis in bleeding, but also thrombotic
pathology (1).
Using thrombelastography for these patients, the authors wantto highlight the utility and the necessity of this method in clinic toestablish a tendency towards hypo or hypercoagulation in several
bleeding or thrombotic syndromes in children.
MATERIALS AND METHOD
The present study was performed from January 2010 toJanuary 2013 on a group of 36 patients admitted in the OncoHemathology Department, Paediatric Clinic, Constanta County
Hospital. From this group of patients, 12 were admitted forthrombotic events and 24 of them had hemorrhagic manifestations. This last subgroup present characteristic clinical manifestations for thrombocytopenia, like petechiaes, bruising, epistaxis,
gingival bleeding, metrorrhagia,hematuria. The authors tried to

identify the mechanisms involved in this pathology: immunological mechanisms, deficiency of production, thrombocytopenia produced by medication, endothelial lesions with capillary fragility,
but some mechanisms remained unrevealed (2).
Also, the authors compared basic coagulation test with thrombelastography to emphasize the utility and rapidity of this last
test to evaluate the hypo or hypercoagulable state in children.
For all the patients, we determined complete blood count,prothrombin time (PT), activated partial thromboplastin time
(APTT), International Normalised Ratio (INR), serum calcium
level and ionic part, fibrinogen. We tried to evaluate the risk for
thrombotic or bleeding manifestations using thrombelastography.
The results were shown in tables and graphics.
RESULTS
Thrombotic manifestations were represented by stroke, with anincidence of 16.6%, deep venous thrombosis (DVT) representing
50%, and the rest of 33.3% are patients with high risk for thrombotic events. Hemorrhagic manifestations were represented byclinical characteristic manifestations for thrombocytopenia, like
petechiaes, bruising, epistaxis, gingival bleeding, metrorrhagia,
hematuria. Among this patients, 7 had immunological thrombocytopenic purpura (PTI), representing 29% of this subgroup; themechanism that produces thrombocytopenia is immunological,
with shortage of the platelets life span.

Received 15th of September 2013.Accepted 25th of October 2013. Address for correspondence: Adumitresi Cecilia, MD, PhD, Department of Physiology, Faculty of
Medicine, “Ovidius” University Constanta,Universităţii Street, Campus 2 Constanţa, Romania, phone / fax: 0241- 605000; email:cadumitresi@yahoo.com

26

Fiziologia - Physiology  2013.23.4 (80)Another mechanism of bleeding production is represented
by deficiency of production and was identified on 5 patients,
representing 21% from the subgroup. Two of these patients werediagnosed with neoplastic disorders. Thrombocytopenia secondary to medication was registered in 2 patients, representing 8%of the bleedings; these patients had received anti inflammatory
or antiepileptic treatment. Hemorrhagic manifestations regarding ear-nose-throat areas due to endothelial injury with capillaryfragility were recorded in 5 patients, representing 21% from thesubgroup. Another 21 % couldn’t be classified based on the
production mechanism.
We evaluated primary haemostasis for the patients withhemorrhagic manifestation by the number of platelets. Patientsdiagnosed with PTI have low level of platelets, which induce theseverity of the hemorrhagic manifestations: a level of platelets
under 30.000/mm3 represents a risk of spontaneous bleeding;over this limit, the haemorrhage is secondary to small mechanicaltraumas. Myeloproliferative disorders are often accompanied bya reduction in platelet reactivity, along with bone marrow inhibition
and inefficient thrombopoiesis.

Fig.3. Graphic representation for MPV (fL) and PDW (%) values forthe patients with bleeding manifestations
Table I.Mean values for MPV (fL) and PDW (%) for the patients withbleeding manifestations

MPV (fl)PDW%

min max
7
13
8
19

mean
11.12513SD
1.54
2.686183p0.00011424

For evaluation of secondary haemostasis in patients withhemorrhagic manifestation, we performed QT, APTT and INR
tests. We determined the plasma concentration of several coagulation factors, like total plasmatic calcium, ionized calcium
and fibrinogen.
For all ages, the values obtained for QT are in normal ranges,
same as the values for INR. The values obtained for APTT forchildren of 13-16 years have increased value of approximately
20% compared to normal values. QT and APTT numbers havestatistic value, with p-0.0132962.

Fig.1.Evaluation of primary haemostasis in patients with hemorrhagic
manifestation by determination of platelets count/mm3

Fig.4.Graphic representation for evaluation of secondary haemostasis in patients with haemorrhagic manifestation using QT (sec), APTT(sec) and INR
Table II.Mean values for QT and APTT for the patients with bleeding
manifestations

Fig.2. The number of platelets/mm3 correlated with the severity of
bleeding manifestations

Mean platelet volume (MPV) and platelet distribution width(PDW) are simple platelet indices, which are known to increaseduring platelet activation (3). Data, expressed as mean values,
show no modification compared to the normal range, with
p<0.05.
2013.23.4 (80)  Fiziologia - Physiology

Age

Mean values for
QT(sec)Mean values for
APTT (sec)

0-4 years

1432

5-8 years1337

9-12 years

12.4

36

13-16 years

13.3

55

SD

0.6652067

10.230673

p0.0132962
27

Trombocytopeniaandhypocalcemia enhance coagulation disorders, characterised by cutaneous and mucosal manifestations,also cerebral involvement which can be fatal for the patients’ life.

Fig.5.Graphic representation of mean values for fibrinogen (mg/dl), total
calcium (mg/dl) and ionized calcium (mg/dl) on age groups

High numbers for mean values of fibrinogen along with increasing age can be observed. This can be explained maybe due tothe increased incidence of infectious inflammatory diseases inage group 9-12 years and 13-16 years.
Total and ionized calcium levels are in normal range for allgroup age, suggesting that calcium as coagulation factor doesn’t
influence bleeding disorders for the patients in the study group.
The numbers have statistical significance.
Table III.Mean values of total and ionized calcium on age groups forthe patients with haemorrhage

Age group

Total Calcium Ionized Calcium(mg/dl)(mg/dl)

0-4 y

9.3

3.9

5-8 y

9.2

3.6

9-12 y7.8

3.8

13-16 y

9.1

4.3

SD

0.7047458

0.294392p0.0001995

Fig.7.Graphic representation for mean MPV and PDW values for thepatients with thrombotic events

For MPV, we calculated a minimum of 8.6 fL, a maximum of
13.4 fL and mean value 10.558 fL. For PDW, the minimum valueis 9.2%, maximum is 13.4%, with mean value 11.141%.
Table IV. Mean values for MPV and PDW in patients with thrombotic
manifestations

min

max

mean

SD

p

MPV (fL)

8.6

13.4

10.5581.34

0.32607

PDW
(%)

9.2

13.4

11.141

1.49

For the patients with thrombotic events, we evaluated secondary haemostasis by testing QT, APTT, INR and also several
coagulation factors: calcium and fibrinogen.

For the patients diagnosed with thrombotic manifestation,
we evaluated primary haemostasis using platelet count, MPV
and PDW.

Fig.8. Graphic representation for mean vales of QT (sec), APTT (sec)and INR for the patients with thrombotic manifestations

Fig.6. Graphic representation for platelet count (/mm3) in patientswith thrombotic manifestations

28

For age group 0-4 years, QT values are found in normal range,same as in INR, but with a slight increase compared to the meanvalues for the other age groups.
APTT numbers for all age groups are in normal range. The
values have no statistic significance.
Fiziologia - Physiology  2013.23.4 (80)Table V.Mean values for QT and AP in patients with thrombotic
manifestations

Age group

QT (sec)

APTT (sec)0-4years

14.1

33.9

5-8 years

12.6

33.5

9-12 years

12.6

33.9

13-16 years

13.3

34.4

SD

0.7141428

0.3685557

p

2.081

Evaluation of secondary haemostasis using coagulation
factors calcium and fibrinogen point out the same phenomena
recorded in patient with bleeding disorders: the rising of meanvalue in fibrinogen as the children grow old.

Fig.9.Graphic representation for mean values of fibrinogen, ionized
calcium, total calcium in patients with thrombotic events

Calcium values, both ionized and total are in normal ranges,
suggesting that this coagulation factor doesn’t influence thehaemostatic equilibrium for the patients in the study group.
Table VI. Mean values for total and ionized calcium in patients withthrombotic events

Age group

Ionized Calcium(mg/dl)

Total
Calcium(mg/dl)

0-4 years

4.4

10.3

5-8 years

4.5

10.2

9-12 years

4.4

9.2

13-16 years

4.8

9.8

SD

0.1892969

0.499166

p

4.932

To evaluate the risk for thrombotic events or bleeding, we performed thrombelastography for all patients in the two subgroups.
2013.23.4 (80)  Fiziologia - Physiology

Fig.10. Graphic representation comparing mean values for thethrombelastographic parameters R, K, MA and α-angle in the two
subgroups

Mean value for parameter R in patients with bleedings is 36.5
mm. This number is 2.6 times bigger compared to the normal
values. This can be a sign that haemorrhage condition is about
to happen and soon it will be clinically distinguished.
The same increase is recorded for the mean value for K
parameter, approximately three times the normal range; thissuggests a prolonged time for clot formation in patients withhaemorrhagic manifestations.
For the subgroup with thrombotic tendencies, we can observe
prolonged reaction time and prolonged clot formation time, but
not with the same amplitude as in the first subgroup.
The value for maximum amplitude for the patients with hemorrhagic manifestations is 46.5 and suggests lack of elasticity forthe forming clot.
The same friable clot is formed in the patients with thrombotic
manifestations, with high risk of an acute event. The accidental
thrombosis can have the origin at the level of endothelial lesions
which can possibly trigger coagulation cascade in this case, lesions produced by pre-existing hypercoagulable state.
CONCLUSION
Haemostasis testing using classical methods doesn’t enhancechanges after thrombotic events. Thrombelastography evaluatesvery accurately tendencies towards hypo or hypercoagulable
state.
Classical methods in the study of haemostasis are not able
to highlight clot formation or the changes produced after thrombus formation and also they cannot suggest predisposition for
thrombosis.
Primary hypercoagulable state remains an extremely sensitivelab diagnosis, referring to the high specialization for the personnel
working in the lab, but also regarding the costs.
In such pathology, we should always consider the factorsthat could trigger thrombotic events, such as: severe varices,
oncologic and hematologic pathology which provides hyperviscosity, sevre infections, nephrotic syndrome, congestive heart
failure, severe haemorrhage, dehydration. Thromboprophylaxis
must be initiated knowing all these aspects, including the risk
for adverse reactions.
29

REFERENCES
1. Serban M, Schramm W. Hemostazeologieclinica, Ed. Brumar,
Timisoara, 2001
2. Montgomery RR, Scott JP. Hemoragic and thrombotic disease. In

Nelson Textbook of Pediatrics, ed. 17, 2004
3. Vagdatli E, Gounari E, Lazaridou E, et al. Platelet distribution
width: a simple, practical and specific marker of activation of coagulation. Hippokratia. 2010; 14(1): 28-32.

ARGUMENTE PENTRU UTILIZAREA TROMBELASTOGRAFIEI IN STUDIULHEMOSTAZEI COMPARATIV CU ALTE TESTE DE LABORATORREZUMAT

Autorii prezinta manifestarile clinice si testele de laborator in evolutia sindroamelor hemoragipare la copii. Au fost utilizate testeclasice de hemostaza, impreuna cu alte teste specifice, precum determinarea unor factori ai coagularii si trombelastografia. S-a
incercat identificarea mecanismelor implicate in aceasta patologie: mecanisme imunologice, deficite de productie, trombocitopenia
indusa medicamentos, leziuni endoteliale secundare fragilitatii capilare, dar unele mecanisme au ramas neidentificate. De asemenea, autorii au comparat teste clasice de hemostaza cu trombelastografia pentru a evidential utilitatea si rapiditatea acestui ultim
test pentru evaluarea statusului hipo sau hipercoagulabil la copii. Pentru toti acesti pacienti, am determinat hemograma, timpul deprotrombina, timpul de tromboplastina partial activata, INR, calciu total si ionic, fibrinogenul. Am incercat sa evaluam riscul pentru
evenimente hemoragice sau trombotice folosind trombelastografia. In concluzie, metodeleclasice de studiu ale hemostazei nu pot
evidential formarea cheagului si modificarile produse dupa formarea acestuia; de asemenea, ele nu pot sugera predispozitia pentru
tromboza asa cum o face trombelastografia.
Cuvinte cheie: copii, trombelastografie, hemostaza.

30

Fiziologia - Physiology  2013.23.4 (80)

PATIENT RELATED PROGNOSTIC FACTORS AND THERAPEUTIC
STRATEGY IN LARYNX SQUAMOUS CELL CARCINOMAMIHAELA PRODEA1,2,3, DELIA HORHAT3,4, NICOLAE BALICA1,3, OVIDIU BORUGA3,5, MARIOARAPOENARU1,3
1
MD PhD, E.N.T. Department, Municipal Emergency Hospital Timişoara
2
MD PhD Student UMF Victor Babes Timişoara
3
UMF Victor BabeşTimişoara
4
MD PhD, E.N.T. Department, Emergency Hospital Timişoara County
5
MD PhD, Ophtalmology Department, Municipal Emergency Hospital Timişoara

ABSTRACT

Aim: This report is trying to achieve a detailed analyze about oncological and functional results of total and reconstructive surgery oflarynx cancer. Identifying certitude and probable prognostic factors (clinic, pathologic and biologic) in head and neck squamous cell
carcinoma (HNSCC) is a big step in the patient evaluation. A modern oncologic vision provides a more sophisticated and advanced
diagnosis evaluation which is correlated with the therapy indications often integrated and multimodal. The initiative to find certitude
prognostic markers which do not need advanced molecular biology techniques is useful in making a more accurate “classificationof those factors”. This will give the opportunity of a multidisciplinary approach, with a tight collaboration between otolaryngologist,
pathologist, radiotherapist and oncologist, to improve local control, survival rate and quality of life in HNSCC patients (1,2). These
factors may be characterized as follows: prognostic factors related to patient (age, gender, associated pathology etc.); prognostic
factors related to the primary tumor (staging, cervical metastatic lymph nodes, capsular integrity, grading, molecular biology etc.);
prognostic factors related to therapy.
Methods: In our retrospective study on 332 patients treated in the ENT Clinic Timişoara, from 2008-2010, we took into consideration
patient related factors such as age, tobacco intake, alcohol intake and localization of the tumor (supraglottic, glottic, and transglottic), histopathological differentiation of tumor, T stage, subglottic extension, anterior commissure invasion, vocal cord mobility, thesurgical margins, the type of surgery and the post-operative radiotherapy (pRT) applied. Local control and 3-year survival rates were
estimated. The local control was calculated in the same way as the survival using the life table method. In order to measure thestatistical comparison between local recurrence and the data of the 213 patients with primary surgery and we used a Chi-square test.
All the analyses were made with MedCalc for windows. Statistical significance was set at the P < 0.05 level. Results: In this period we
performed as a first choice treatment modality: 73 total laryngectomies, 5 horizontal supraglotticlaryngectomies with epiglottectomy, 9vertical hemi-laryngectomies, 6 supracricoid partial laryngectomies 25 classic cordectomies, 62 endoscopic laser CO2 microsurgicalinterventions, 5 suspended microlaringoscopies with excision, in 28 patients we performed tracheostomy followed by radiotherapy
or salvation surgery and RT. In 44 patients we performed tracheostomy without other treatment in our unit and the patientswere lostfor follow up, in 62 cases we performed microlaringoscopy with biopsy of the tumor, the patients being lost for follow the treatment
options, 13 patients of these refused any treatment modality, they were also excluded from the study. After primary surgery weperformed 20 re-interventions, due to local and lymph node recurrences, 11 total laryngectomies, 6 tracheostomy, 2 fronto-laterallaryngectomies, 1 radical neck dissection and in one case a phonator prostheses was inserted. In our study we had one case withpulmonary metastases which followed radio-chemotherapy. Conclusions: In our series, of the 213 patients managed with surgical treatment, 20 developed local recurrence (9.38%). The average time for the local recurrence was 10.5 months (1–21 months).
Glottic cancer is the most common form of larynx cancer consists of 60% of cases, followed by supraglottic cancer (40%). In ourseries, we found 137 (64%) cases with glottis localization, 60 (28%) cases of supraglottic cancer and 16 (8%) of subglottic cancer.
We used Kaplan-Mayer method to calculate free of disease survival in our series for three years period and we had in the first 12
months 96.24% survival, in 24 months 92.44% and in 36 months 90.14% free of disease survival.
Key words: larynx cancer, prognostic factors, partial and reconstructive larynx surgery, radiotherapy.

INTRODUCTION
Despite of the surgical techniques improvement, complementary therapies and modern treatments, the Head and Neck
Squamous Cell Carcinoma (HNSCC) survival has been almost

the same in the last 25 years. It is difficult to explain why similar
tumors for stage and site have different clinical outcome. In fact,
nowadays there are not certain predictive factors that indicate
the prognosis for one type of cancer stage (1).
In the light of these considerations, studies of molecular biology

Received 15th of September 2013.Accepted 25th of October 2013. Address for correspondence: MihaelaProdea, MD, PhD Student, ENT Department, “Victor Babes”
University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, Romania; phone: +40723036805; e-mail: mihaelaprodea@umft.ro2013.23.4 (80)  Fiziologia - Physiology

31

have been developed new markers useful in addition to the traditional
ones to indicate the prognosis, to orientate the therapeutic strategy
and improve the outcome. These new markers should be able toidentify cancer subgroups with particular evolution and provide major
information on the cancerous cell development in order to upgrade
the therapeutic strategies for preventive and specific treatment basedon an accurate biological characteristic definition (2).
Identifying certitude and probable prognostic factors (clinic,
pathologic and biologic) in larynx cancer is a big step in patient
evaluation. A modern oncologic vision provides a more sophisticated and advanced diagnosis evaluation which is correlatedwith the therapy indications often integrated and multimodal.
The initiative to find certitude prognostic markers which do notneed advanced molecular biology techniques is useful in making
a more accurate “classification of those factors”. This will givethe opportunity of a multidisciplinary approach, with a tight collaboration between otolaryngologist, pathologist, radiotherapist
and oncologist, to improve local control, survival rate and qualityof life in larynx cancer patients (1,2).
These factors may be characterized as follows: prognostic factors related to patient (age, gender, associated pathology etc.);
prognostic factors related to the primary tumor (staging, cervicalmetastatic lymph nodes, capsular integrity, grading, molecularbiology etc.); prognostic factors related to therapy.
In our retrospective study on 332 patients treated in the ENT
Clinic Timişoara, from 2008-2010, we took into consideration
patient related factors such as age, tobacco intake, alcohol
intake and localization of the tumor (supraglottic, glottic, andtransglottic), histopathological differentiation of tumor, T stage,
subglottic extension, anterior commissure invasion, vocal cord
mobility, the surgical margins, the type of surgery and the postoperative radiotherapy (pRT) applied. Local control and 3-year
survival rates were estimated. The local control was calculated in
the same way as the survival using the life table method. In order
to measure the statistical comparison between local recurrence
and the data of the 213 patients with primary surgery and weused a Chi-square test. All the analyses were made by MedCalc
for windows. Statistical significance was set at the P < 0.05 level.

Alcohol and tobacco consumption: 88.26% (188) of our patients wereusing alcohol and tobacco, which has been recognized
as important risk factors for the development of HNSCC. Exposure
to these two carcinogens produces specific molecular insults that
promote neoplasia. Cigarette smoking has been associated withover expression of the proto-oncogene bcl-2, a protein that inhibit
apoptosis (2) and the association of cigarette smoking and alcohol
consumption has been associated with higher rate of nonspecificmutations in the tumor suppressor gene p53 (3).
Comorbidities: The patients with HNSCC have often otherdiseases, some of them correlated to the use of alcohol andtobacco. Rogers et al used the Adult Comorbidity Evaluation(ACE-27) and found that 53% of 157 patients with HNSCC had
at least one comorbidity (4). Piccirillo et al studied the comorbidityof 7131 HNSCC patients from the Surveillance Epidemiology and
End Results (SEER) database in the U.S.A. and found prevalence rates of comorbidity to be 34.6%, 21.2%, 12.7%, 9.5%,
and 22.1%, respectively for CCI (Charlson comorbidity index)
scores of 0, 1, 2, 3 and 4, respectively (5). Cardiovascular andpulmonary diseases are the most common comorbid conditions
in data on Caucasians, other frequent comorbid condition wasliver disease, followed by gastrointestinal disease (6). Most
frequent in our patients were cardiovascular diseases, liverdisease and pulmonary affections.
Socio-economic status: Almost 44% (146 patients) of ourpatients were from rural area, which could explain the fact that
their presentation to the doctor and actual diagnosis was delayedcompared to patients with medium and high level of education.
Nutrition status: Our patients who developed squamous cell
carcinoma of the larynx were often malnourished because of poor
dietary habits (72%), excessive alcohol consumption (92.77%),
local tumor effects, tumor-induced cachexia (4%). Logistic regression analysis identified a weight loss of more than 10% to
be the most prominent predictive parameter for the occurrenceof major postoperative complications (7).

MATERIALS AND METHODSIn this period we performed as a first choice treatment modality: 73 total laryngectomies, 5 horizontal supraglotticlaryngectomies with epiglottectomy, 5 vertical hemilaryngectomies, 6
supracricoid partial laryngectomies 25 classic cordectomies, 62
endoscopic laser CO2 microsurgical interventions, 5 suspended
microlaryngoscopies with excision, in 57 patients we performed
tracheostomy but the patients didn’t followed further treatment
in our unit, in 62 cases we performed microlaringoscopy withbiopsy of the tumor, the patients being lost for follow the treatment options, and 13 patients of these refused any treatment
modality, they were also excluded from the study.
The overall objective of all surgical oncology procedures is
to excise all tumor extensions with sufficient surgical margins.
In our study we had positive margins only in two cases, both
treated with postoperative radiotherapy. Oral cavity and salivary
glands tumors, which are accessible and relatively non-sensitive

Prognostic factors related to patient
Demographic parameters
Age: the average age in these types of tumors is 63 years old,
it is known to influence the outcome in certain types of cancers
and therefore to be considered a negative prognostic factor innumerous studies (1,2). Patient age and associated diseases
may influence the systemic immune response and patient ability
to tolerate a maximal therapy. In our patients these tumors weremore frequent in the interval 55-64 years old and the averageage is 59.8 years old.
Gender: In our study of patients with primary surgery, gender
was taken into consideration, and we had 206 (97%) males and7 (3%) females treated in Timişoara ENT Clinic.
32

Prognostic factors related to the therapy: Surgery,radioteraphy(RT) and chemotherapy (CT)

Fiziologia - Physiology  2013.23.4 (80)

to radiation, are managed surgically. Patients with HNC have a
4% risk per year of developing a second primary tumor evenafter complete tumor ablation. Prevention (smoking and alcohol
cessation) and early detection should be a principal objective of
postoperative patient care (8). Computed tomography, MRI andPET imaging can be used for early detection of recurrence in thispopulation (9). To follow-up our patients we performed routine
head and neck examination and yearly chest X-ray imaging andabdominal ultrasonography after surgery.
After primary surgery we performed 20 re-interventions, due
to local and lymph node recurrences, 11 total laryngectomies,
6 tracheostomy, 2 fronto-lateral laryngectomies, 1 radical neckdissection and in one case a phonator prostheses was inserted.
In our study we had one case with pulmonary metastases which
followed radio-chemotherapy.
Surgery after (chemo)radiation (RCTX/RTX) therapy is felt
to be plagued with a high incidence of wound complications
as the consequence of radiation induced wound bed changes(10). Salvage laryngectomy was more frequently associated withpostoperative complications after chemoradiotherapy comparedwith primary total laringectomy. Problems related to local woundhealing, especially the development of pharyngocutaneous fistula, constituted the most common postoperative complicationin these patients. Multivariate analysis showed that primarychemoradiotherapy was an independent predictor of local woundcomplications and pharyngocutaneous fistula (11).
Major peri- and postoperative complications upon surgery after
RCTX or RTX are reported to be up to 73% for i.e. salvage
laryngectomies (12).
Prognostic factors related to Radiotherapy and Chemotherapy
Studies in the last 25 years tried to define precise indications
for Radiotherapy (RT) and Radio-chemotherapy. This technology
advanced, radiant substances doses were adjusted and newpharmaceuticals were developed. It is obvious that RT and CT
with improper indication or inadequate doses will not ensure thebest local control and becomes a negative prognostic factor.
In our series we had 20 cases of postoperative radiotherapy and2 cases of radio-chemotherapy.
RESULTS AND DISCUSSIONSIn our series, of the 213 patients managed with surgical treatment, 20 developed local recurrence (9.38%). The average time
for the local recurrence was 10.5 months (1–21 months).
Glottic cancer is the most common form of larynx cancer
consists of 60% of cases, followed by supraglottic cancer (40%).
In our series, we found 137 (64%) cases with glottis localization,
60 (28%) cases of supraglottic cancer and 16 (8%) of subglottic
cancer. Dufour (13) and Gallo (14) reported that localization oftumor does not correlate with local recurrence and our results
also support their theory.
We used Kaplan-Mayer method to calculate free of disease2013.23.4 (80)  Fiziologia - Physiology

in our series for three years period and we had in the first 12
months 96.24% survival, in 24 months 92.44% and in 36 months
90.14% free of disease survival.
CONCLUSIONS
The increasing knowledge about clinical oncology and biologyof head and neck carcinomas, together with the improvementof statistical methods for studying the prognostic role of clinicaland biological factors, have outlined several prognostic factors,
based on scientific data. These factors can have a routine clinicalapplication in head and neck oncology.
Moreover clinical and histopathological factors (i.e. age, stage,concomitant diseases, data obtained from pTNM as pN+, capsular breakings, intra-vascular emboli, etc) have been more clearly
defined and reinforced. As a result it has been accepted that the
prognostic factors listed above might have a practical application in the case history of these patients. Studies demonstratethat different prognostic factors may influence the choice of thetherapeutic treatment.
Owing to the well documented negative effects of tobacco andalcohol use, in addition to data suggesting that abstinence fromthese substances may improve outcomes. Substance abusecounseling and cessation programs should be integrated into
the care of patients with larynx cancer.
Lavaf A et al. reported in their large analysis of adjuvant RT
in patients with locally advanced HNSCC, that adjuvant RT
resulted in an approximately 10% absolute increase in 5-year
cancer-specific survival and overall survival for patients withpositive lymph nodes HNSCC compared with surgery alone.
Despite combined surgery and adjuvant RT, outcomes in thishigh-risk population remain suboptimal, emphasizing the need forcontinued investigation of innovative treatment approaches (15).
Besides, new research on biological factors may acknowledge
on the tumor sensitivity to radiotherapy and radiochemotherapy.
Treatment of head and neck cancer requires accurate risk
stratification in order to determine its type and expected clinicaloutcome. Physical examination, diagnostic imaging studies,and pathologic review enable the clinician to determinate theprimary tumor size and extent, lymph nodes status and distantmetastases (2).
Further studies of these prognostic factors are required forprevention, early detection and to provide a complex treatment
recommendation for patients with head and neck squamous cell
cancer to improve local control and patient life quality.
REFERENCES
1. Cortesina G, et al. Oncogenes and carcinogenesis of head andneck carcinomas, Ed. 2002. ISBN 88-7711-424-X
2. Gallo O, Bianchi S, Porfirio B. Bcl-2 overexpression and smoking history in head and neck cancer [letter]. J Natl Cancer Inst 1995; 87: 1024-5.
3. Brennan JA, Boyle JO, Koch WM, Goodman SN, Hruban RH,
Eby YJ, Couch MJ, Forastiere AA, Sidransky D. Association between
cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck. N Engl J Med. 1995; 332(11): 712-717.

33

4. Rogers SN, Aziz A, Lowe D, Husband DJ. Feasibility study of theretrospective use of the Adult Comorbidity Evaluation index (ACE-27)
in patients with cancer of the head and neck who had radiotherapy. Br
J Oral MaxillofacSurg 2006; 44: 283-8.
5. Piccirillo JF, Spitznagel EL, Vermani N, Costas I, Schnitzler M.
Comparison of comorbidity indices for patients with head and neckcancer. Med Care 2004; 42: 482-6.
6. Liu CT, Chiu TJ, Huang TL, Chien CY, Fang FM. Impact of comorbidity on survival for locally advanced head and neck cancer patientstreated by radiotherapy or radiotherapy plus chemotherapy. Chang
Gung Med J. 2010; 33(3): 283-91.
7. vanBokhorst-de van der Schueren MA1, van Leeuwen PA, Sauerwein HP, Kuik DJ, Snow GB, Quak JJ. Assessment of malnutrition
parameters in head and neck cancer and their relation to postoperativecomplications. Head Neck 1997;19(5):419-25.
8. Gil Z, Fliss DM. Contemporary management of head and neckcancers. Isr Med Assoc J. 2009; 11(5): 296-300.
9. Gil Z, Even-Sapir E, Margalit N, Fliss DM. Integrated PET/CT
system for staging and surveillance of skull base tumors. Head Neck

2007; 29(6): 537-45.
10. Schultze-Mosgau S, Grabenbauer GG, Radespiel-Troger M, et al.
Vascularization in the transition area between free grafted soft tissues
and pre-irradiated graft bed tissues following preoperative radiotherapy
in the head and neck region. Head Neck 2002, 24: 42-51.
11. Ganly I, Patel S, Matsuo J, et al. Postoperative complications ofsalvage total laryngectomy. Cancer 2008; 103:2073-2081.
12. Relic A, Scheich M, Stapf J, et al. Salvage surgery after induction
chemotherapy with paclitaxel/cisplatin and primary radiotherapy foradvanced laryngeal and hypopharyngeal carcinomas. Eur Arch Otorhinolaryngol 2009, 266: 1799-1805.
13. Dufour X, Hans S, De Mones E, et al. Local control after supracricoid
partial laryngectomy for ‘advanced’ endolaryngeal SCC classiWed as
T3. Arch Otolaryngol Head Neck Surg 2004; 130(9): 1092-99.
14. Gallo A, Manciocco V, Tropiano ML, et al. Prognostic value of resection
margins in supracricoidlaryngectomy. Laryngoscope 2004; 114: 616-621.
15. Lavaf A, Genden EM, Cesaretti JA, Packer S, Kao J. Adjuvant radiotherapy improves overall survival for patients with lymph node-positive
head and neck squamous cell carcinoma. Cancer 2008; 112(3): 535.

FACTORI DE PROGNOSTIC LEGAŢI DE PACIENT ŞI STRATEGII TERAPEUTICEÎN CARCINOMUL LARINGIAN CU CELULE SCUAMOASEREZUMAT

Scop: În acest studio ne propunem să analizăm rezultatele oncologice şi funcţionale în cazul intervenţiilor chirurgicale totale şi
reconstructive, ale laringelui. Identificarea factorilor prognostici probabili şi de certitudine (clinici, biologici şi morfopatologici)
în cancerele capului şi gâtului reprezintă un pas important în evaluarea pacienţilor. O viziune modern prevede o evaluare diagnostic
mai avansată, corelată cu indicaţiile de terapie multimodală. Iniţiativa de a găsi markeri de prognostic de certitudine care nu necesită
tehnici avansate de biologie molecular este utilă în a face o mai corectă clasificare a acestor factori. Acest lucru oferă posibilitatea
de abordare multidisciplinară, cu o colaborare strânsă între otorinolaringolog, patolog, radioterapeut, şi oncolog, pentru a îmbunătăţi
controlul local, rata de supravieţuire şi calitatea vieţii la aceşti pacienţi. Aceşti factori pot fi caracterizaţi după cum urmează: factori deprognostic legaţi de pacient (vârsta, sex, patologie asociată etc); factori de prognostic legaţi de tumora primară (stadiu, ganglion limfatici
metastatici, integritatea capsulară, clasificare, biologie molecular etc); factori de prognostic legaţi de tratament. Material şi metodă:Am realizat un studio retrospective pe 332 de pacienţi trataţi în Clinica ORL Timişoara, din perioada 2008-2010, am luat în considerare factorii prognostici legaţi de pacient, cum ar fi vârsta, consumul de tutun, consumul de alcool şi localizarea tumorii (supraglotică,glotică, şi subglotică) , diferenţierea histopatologică a tumorii, stadiul T, prelungirea subglotică, invazia comisurii anterioare, mobilitatea
corzilor vocale, marginile chirurgicale, tipul de chirurgie şi radioterapia postoperatorie (pRT) aplicată. Au fost evaluate controlul local şi
supravieţuirea la 12, 24 si 36 de luni. Controlul local a fost calculate în acelaşi mod ca supravieţuirea folosind metoda Kaplan-Mayer.
Pentru a efectua comparaţia statistic între recidiva locală şi datele obţinute de la cei 213 de pacienţi cu intervenţii chirurgicale primare,am folosit un test Chi-pătrat. Toate analizele au fost efectuate cu programul Med Calc pentru Windows. Semnificaţie statistică a foststabilită P< 0,05. Rezultate: În această perioadă s-au realizat ca tratament de primă alegere: 73 laringectomii totale, 5 laringectomii
orizontale supraglotice cu epiglotectomie, 9 hemilaringectomii verticale, 6 laringectomii parţiale supracricoidiene, 25 cordectomii clasice,
62 de intervenţii microchirurgicale endoscopice cu laser CO2, 5 microlaringoscopii suspendate cu excizia tumorii, la 28 de pacienţi s-apracticat mai întâi traheostomie de necessitate şi ulterior intervenţia chirurgicală curativă. Au fost excluşi din studiu 44 de pacienţi
la care s-a practicat traheostomie fără ca aceştia să mai urmeze tratamentul în unitatea noastră şi au fost pierduţi din evidenţă, în 62
de cazuri s-au efectuat microlaringoscopii cu biopsie a tumorii, fără ca aceştia să mai urmeze alt tratament, dintre aceştia, 13 pacienţi
au refuzat orice modalitate de tratament, acestea au fost, de asemenea, excluşi din studiu. După intervenţia chirurgicală primară s-au
efectuat 20 de reintervenţii, datorită recidivelor locale şi metastazelor limfonodale: 11 laringectomii totale, 6 traheostomii de necesitate,
2 laringectomii fronto-laterale, 1 evidare ganglionară radical şi într-un caz inserţia de proteză fonatorie. În studiul efectuat, metastazele
pulmonare au fost evidenţiate doar într-un caz, care a urmat ulterior tratament radio-chimioterapic. Concluzii: Din cei 213 de pacienţi
cu tratament chirurgical, 20 au dezvoltat recurenţe (9,38%). Intervalul de apariţie a recurenţelor locale a fost de 10,5 luni (1-21 luni).
Cancerul cu localizare glotică este forma cea mai des întâlnită, 60% din cazuri, urmat de tumori cu localizare supraglotică (40%). În
seria noastră, am avut 137 (64%) cazuri cu localizare la nivelul glotei, 60 (28%) de cazuri de cancer supraglotic şi 16 (8%) de cancer
subglotic. Am folosit metoda Kaplan-Mayer pentru a calcula supravietuirea fara recidiva pe perioada de trei ani şi am avut în primele
12 luni 96,24% supravieţuire, la 24 de luni 92,44% şi 90,14% la 36 luni.
Cuvinte cheie: cancer de laringe, factori de prognostic, laringectomie parţială şi reconstructivă, radioterapie.

34

Fiziologia - Physiology  2013.23.4 (80)IN VITRO CHARACTERISATION OF TUMOR-ASSOCIATED
FIBROBLASTS FROM DIFFERENT SOLID TUMORSLAURA CERNAT1, FLORINA BOJIN1, MEDA DEICA1, DIANA ANASTASIU1, GABRIELA TANASIE1,
RAZVAN ILINA2, VIRGIL PAUNESCU1, CARMEN PANAITESCU1
1
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara
3
Oncologic Surgery Department, Municipal Emergency Hospital Timisoara

ABSTRACT

Despite the uncertain origin, tumor-associated fibroblasts (TAFs) are crucial stromal cellular components of solid tumors. Their rolein tumor development, progression and metastasis is well known. Establishment of molecular phenotype of TAFs and some specificmarkers and also the examination of behavior of TAFs derived from different solid tumors could provide priceless information for
considering these cells as serious targets for novel therapeutic approaches. In our experiment we attended characterization by flow
cytometry of TAFs derived from 2 types of solid tumor (uterus and breast) and make a comparison regarding markers expressed.
Both types of TAFs expressed CD90, CD73, CD44, CD29 and CD26 (in small extent). Results of this study revealed some similarities between the two types of isolated TAFs, suggesting that it is possible to materialize a possible common phenotype of TAFs andmake them really efficient targets for therapy.
Key words: TAFs, microenvironment, flow cytometry, markers, FAP

INTRODUCTION
In the complex research of cancer disease, last decades arecharacterised by studies of elaborated molecular mechanisms
which underlie the cancer pathogenesis. Heterogeneous andintricated biological networks are involved in neoplasm initiation, progression, invasion and metastasis. Among these, thecomplicated tumor-stroma interactions are primarily, because weknow now that the involvement of microenvironmental elements
are crucial for disease. There are multiple environmental stimuli
which produce effects in cells and are capable to convert cells
fate by phenotypic changes. During tumorigenesis, stromal
cellular components are active players and deeply implicated incancer evolution. Researchers put questions about the capabilityof tumoral stromal cells of being potential therapeutical target
and markers of prognosis. Accordingly, they focused on thistumor compartment, especially on tumor-associated fibroblasts(TAFs), cells which appear to be an active and powerful anchor
for tumoral cells.
Tumor stroma contains many cellular types (fibroblasts, immune cells, inflammatory cells, smooth muscle cells). Multiple interactions between them results in changes in intercellular media(ECM, extracellular matrix), creating a permissive environment
for tumoral develop and progression, so-called „stromogenesis”(1). Multiple alterations in this stroma are giving the state ofphenotipically activated stroma(2,3) which directly contributes tocancer stages of progression. It seems that,althoughfibroblasts
are the preponderant population in tumor stroma, they are foundin various proportions in different types of carcinomas. The origin
of TAFs is still uncertain, many studies are assigning them todifferent sort of cells: resident activated fibroblasts, MSC-derived
TAFs, trans-differentiating epithelial/endothelial cells throughepigenetic transitions (4,5,6).
This is an important reason of difficulties in TAFs phenotypicalcharacterization, especially when considering the tissue fromwhich the tumor originates and also the histological type of tumor.
The marked heterogeneity of TAFs phenotype arise serious
questions and issues for considering these cells as potential
therapeutic targets. Molecular markers of activated state ofstromal fibroblasts are required in order to identify these cells
and to find targeted therapies. Within different tumors there areseveral subpopulations of TAFs. The expression of activation
markers of these subpopulations is partial (4). Some studies
identified fibroblast-specific protein (FSP), PDGF receptor β andfibroblast activation protein (FAP) as overexpressed markers in
TAFs from some solid tumors (7,8), along with expression of
α-SMA expression as myofibroblast marker.
FAP is a dipeptidyl peptidase of the serine protease family
which possess a restrictive motif of protein expression and activity (in stromal fibroblasts and tisular fibroblasts during granulation
process, but not in fibroblasts from normal stroma) (9,10,11) It
seems that FAP is involved in tumorigenesis in animal modeland the treatment with anti-FAP directed antibodies diminished
FAP-dependent tumor growth(10).
In our study, we intended to isolate and phenotypical characterise TAFs from solid tumors derived from breast and uterus
and to investigate whether TAFs are expressing FAP and othermarkers and if there is a pattern regarding the origin tissue oftumor or an interindividual variation of marker expression.

Received 21st September 2013.Accepted 15th October 2013.Address for correspondence: Laura Cernat, MD, PhD student, Department of Functional Sciences, “Victor
Babes” University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256220479; e-mail: mlaura_73@yahoo.com

2013.23.4 (80)  Fiziologia - Physiology

35

MATERIAL AND METHODS
1. Isolation and culture of TAFs
TAFs were isolated from 10 breast cancer and 5 uterus cancer
surgical pieces using collagenase type IV-S from Clostridium
histolyticum(Sigma, St. Louis, MO, USA) method. Cells isolated
from tissues were washed several times with phosphate bufferedsaline (PBS, Sigma) solution and passed through 0.70/0.40 μm
strainer filters and then plated as suspension in adherent plastic
culture plates using Dulbecco’s modified Eagle Medium (DMEM;
Sigma), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 2% Penicillin/Streptomycin
mixture (Pen/Strep, 10,000 IU/ml; PromoCell), and incubatedincubation at 37oC in 5% CO2 atmosphere. Every 3 days, medium
was replaced with freshly prepared medium.
2. Immunophenotyping
At different passages, TAFs were trypsinized (Trypsin-EDTA,
Sigma), centrifuged 7 min at 1500 rpm, and washed twice with
PBS. After resuspension inPBS, 100 μl of cellular suspension
was mixed with 4 μl of each antibody (mouse anti-human fluorochrom conjugated). Cells were vortexed and put in the darkfor 30 minutes at the room temperature. After incubation, cells
were washed with 2 ml Cell-Wash Solution (BD Biosciences),
centrifuged 1 min at 1500 rpm.Supernatant was removed andcells were re-suspended in 500 μl Cell-Wash. Flow cytometricanalysis was performed using FACSCalibur (Becton-Dickinson).
The antibodies used for flowcytometric assay were conjugatedwith fluorochroms as follows:- PE - CD31, CD73, CD49d, CD109, CD117, Bcrp1 ABCG2(BD Pharmingen™), CD29 (R&D Systems)- FITC - CD34, CD44, CD90, CD106, HLA-DR, CD26, CD95,
HLA-A2 (BD Pharmingen™), Cytokeratin (R&D Systems)- APC - CD45 (BD Pharmingen™)

uterus			breast		

3. Statistical analysis
Was performed using Excel Microsoft Office 2003 (Microsoft
Corporation) software.
RESULTS
Phenotypical examination of TAFs
The flow cytometric analysis revealed a high variability
between samples regarding the percentage of positive markers
expressing cells for both types of neoplasms examined. However,
we could observe a similar pattern of expression between the 2types of TAFs. The markers and average percentages of markers
expressed are illustrated in Table I and Figure 1.
All samples were negative for CD34, CD45, CD106 and CD117.
We also didn’t detect expression of HLA-DR, Bcrp1 ABCG2. The
presence of HLA-A2 noticed in both types of TAFs but in higher
degree in samples derived from uterus cancer. CD26 (FAP) was
expressed on very low level in both types of specimens. This may
be important as it is known that the level expression of markers
decreases as number of passages of cells increases.
36

uterus			breast

Fig. 1. Markers expression by TAFs from uterus and breast cancer
specimens

Fiziologia - Physiology  2013.23.4 (80)Table I. Average expression of TAF markers

CD90
CD73
CD44
CD29
CD26
HLA-A2TAFs derived fromTAFs derived fromuterus cancer samples breast cancer samples(%) (n=5)(%) (n=10)
37.24
25.03
47.27
38.21
21.5
12.8
43.45
38.38
2.51
2.33
27.08
5.22

DISCUSSION. CONCLUSIONSTAFs affect in an important degree cancer development andprogression. As key players in this complex process, TAFs areneeded to be fully understand and characterised. This meansthat TAFs are an attractive therapeutic target and identificationof specific markers of TAFs and signaling pathways involved intheir activation and function within the tumor has to be an esential
prerogative. Ideally, it would be valuable to find some universal„badges” of TAFs, which could be reachable in any tumor, regardless of histologic type. In our work we tried to characterise,by flow cytometry, TAFs derived from 2 types of solid tumor
and make a comparison. We found some similarities regarding
the type of markers expressed, even there were quantitative
variations between samples.
1.
CD90 (Thy-1) is suggested as biomarker for several
types f cancer and cancer stem cells. It is expressed ont only
by TAFs, but also by bone-marrow derived stem cells, T-cells,
endothelial cells (12,13). CD90 seems to be an interesting factor for distinguishing TAFs from benign stromal cells in prostatecancer (14).
1.
CD73 (5’-nucleotidase) is a potent immunosuppressive molecule and is overexpressed in different types of cancer(leukemia, melanoma, breast, colon) (15,16). CD73 positive
cells are capable to evade anti-tumor immune mechanisms. The
prognostic value of this marker is still controversial, especiallyfor breast cancer.
2.
CD44 is a glycoprotein involved in cell proliferation,
migration, angiogenesis, cell survival, tumor metastasis – essential aspects in cancer progression. CD44 expression by
TAFs may be engaged in sustenance of cancer stem population
inside the tumor (17).
3.
CD29 (Integrin beta-1) is involved in cell adhesion,
immune response and metastasis.
4.
HLA-A2 is one of the most frequent HLA class I allospecificities and is considered a potential prognostic factor insome malignancies. The presence of HLA-A2 antigen is crucial
for tumor recognition by T-cells; down-regulation of this antigen
expression delineates one of tumor strategies of escape fromhost immune defense.
5.
CD26 (FAP) seems to be restricted as expression to
TAFs, granulation tissue of healing wounds, soft tissues sarco2013.23.4 (80)  Fiziologia - Physiology

mas, although the biological role in tumor microenvironment is notwell known (it is thought to be involved in EMT during embryogenesis and carcinogenesis). Some authors revealed that silencingof FAP induced the inhibition of TAFs growth and decreased thetumor growth in ovarian cancer xenograft models (18).
6.
Although the markers mentioned above are highly
seductive candidates for therapeutically targeting in cancer, mostof them are not specific for TAFs; many types of cells (normal
cells from different origins, tumoral cells, immune cells etc.) are
expressing these markers. CD26 could be a serious nominee
as a specific TAF marker. However, our experiment revealed aweak expression of this marker by TAFs derived from both uterus
cancer and breast cancer samples. Further studies are required
for establish the conditions in which it could be determined if
this marker is constantly expressed by TAFs in solid tumors.
Furthermore, targeting TAFs with active functional CTLs against
CD26 protein could be an interesting approach.
Acknowledgements
This work was supported by the Sectorial Operational Programme for Human Resources Development, financed from theEuropean Social Fund, FSE POSDRU 107/1.5/S/78702 and by
UEFISCDI, PNII-Idei, Project No. 318/2011.
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1. Tuxhorn JA, Ayala GE, Rowley DR. Reactive stroma in prostatecancer progression. J Urol 2001;166(6): 2472-83.
2. De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion.
J Pathol 2003;200: 429-47.
3. Weaver VM, Gilbert P. Watch thy neighbor: cancer is a communal
affair. J Cell Sci 2004;117:1287-90.
4. Cirri P, Chiarugi P. Cancer associated fibroblasts: the dark side of
the coin. Am J Cancer Res 2011; 1(4): 482-497.
5. Anderberg C, Pietras K. On the origin of cancer-associated fibroblasts.
Cell Cycle 2009; 8(10):1461-2.
6. Paunescu V, Bojin FM, Tatu CA, Gavriliuc OI, Rosca A, Gruia AT,
Tanasie G, Bunu C, Crisnic D, Gherghiceanu M, Tatu FR, Tatu CS, Vermesan S. Tumour-associated fibroblasts and mesenchymal stem cells:
more similarities than differences. J Cell Mol Med 2011; 15(3):635-646.
7. Kalluri R, Zeisberg M Review. Fibroblasts in cancer. Nat Rev Cancer
2006; 6(5):392-401.
8. Micke P, Ostman A. Review: Tumour-stroma interaction: cancerassociated fibroblasts as novel targets in anti-cancer therapy? Lung
Cancer 2004; 45(2):S163-75.
9. Kunz-Schughart LA, Knuechel R. Tumor-associated fibroblasts (partI): active stromal participants in tumor development and progression.
Histol Histopathol 2002;17: 599-621.
10. Cheng JD, Dunbrack Jr RL, Valianou M, Rogatko A, Alpaugh RK,
Weiner LM. Promotion of tumor growth by murine fibroblast activation
protein, a serine protease, in an animal model. Cancer Res 2002;62:4767-72.
11. Huber MA, Kraut N, Park JE, Schubert RD, Rettig WJ, Peter RU,
et al. Fibroblast activation protein: differential expression and serine
protease activity in reactive stromal fibroblasts of melanocytic skintumors. J Invest Dermatol 2003;120: 182-8.
12. Dennis JE, Esterly K, Awadallah A, Parrish CR, Poynter GM et al.
Clinical-scale expansion of a mixed population of bone-marrow-derived
stem and progenitor cells for potential use in bone-tissue regeneration.

37

Stem Cells 2007; 25: 2575-82.
13. Herrera MB, Bruno S, Buttiglieri S, Tetta C, Gatti S et al. Isolationand characterization of a stem cell population from adult human liver.
Stem Cells 2006; 24: 2840-50.
14. True LD, Zhang H, Ye M, Huang CY, Nelson PS, von Haller
PD, Tjoelker LW, Kim JS, Qian WJ, Smith RD, Ellis WJ, Liebeskind
ES, Liu AY. CD90/THY1 is overexpressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker. Mod
Pathol2010;23(10):1346-56.
15. Serra S, Horenstein AL, Vaisitti T, et al. CD73-generated extracellularadenosine in chronic lymphocytic leukemia creates local conditions

counteracting drug-induced cell death. Blood 2011; 118: 6141-52.
16. Spychala J, Lazarowski E, Ostapkowicz A, Ayscue LH, Jin A, Mitchell
BS. Role of estrogen receptor in the regulation of ecto-5′-nucleotidase andadenosine in breast cancer.Clinical Cancer Research 2004; 10: 708-717.
17. Kinugasa Y, Matsui T, Takakura N. CD44 expressed on cancer-associated fibroblasts is a functional molecule supporting the stemness anddrug resistance of malignant cancer cells in the tumor microenvironment.
Stem Cells 2014;32(1):145-56.
18. Lai Ma, Fangyuan Wang. Fibroblast activation protein regulates
tumor-associated fibroblasts and epithelial ovarian cancer cells.Int J
Oncol 2012; 41: 541-550.

CARACTERIZARE IN VITRO A FIBROBLASTELOR PERI-TUMORALE IZOLATEDIN DIFERITE TUMORI SOLIDEREZUMAT

Cu toate ca originea acestora nu este deplin lamurita, fibroblastele peritumorale sunt componentele celulare esentiale ale stromei
peri-tumorale. Este cunoscut deja rolul acestora in initierea, dezvoltarea si metastazarea tumorala. Stabilirea unui profil molecularal fenotipului si a unor markeri specifici ai fibroblastelorperitumorale izolate din tumori solide cu origini diferite ar aduce informatii
esentiale privind considerarea acestor celule ca tinte terapeutice de succes. In experimentul nostru am tentat caracterizarea prin flow
citometrie a fibroblastelor peritumorale izolate din piese rezecate chirurgical de cancer de uter si de san, pentru a depista eventuale
similitudini privind markerii exprimati. Ambele tipuri de fibroblaste peritumorale au exprimat CD90, CD73, CD44, CD29 si CD26
(FAP)(acesta din urma in grad foarte redus). Rezultatele au relevat similaritati in ceea ce priveste tipurile de markeri exprimati, ceea
ce ar putea sugera posibilitatea stabilirii unui fenotip general al fibroblastelor peritumorale aplicabil indiferent de originea acestora.
Cuvinte cheie: fibroblaste peritumorale, microclimat tumoral, markeri de expresie, FAP

38

Fiziologia - Physiology  2013.23.4 (80)

COMORBIDITIES AND RISK FACTORS IN HEART FAILURE
WITH PRESERVED EJECTION FRACTIONADELINA MAVREA, ADINA BUCUR, MIRELA TOMESCU
University of Medicine and Pharmacy “Victor Babes“ Timisoara

ABSTRACT

Almost half of heart failure patients have heart failure with preserved ejection fraction (HFPEF). Patients are elderly with multiple
comorbidities.
We aimed to evaluate the incidence of comorbidities and their influence on risk factors and clinical status, in patients with heart failure
with preserved ejection fraction (HFPEF).
Material and methods:We included 78 patients who were diagnosed for the first time with HFPEF. We evaluated the incidence ofcomorbidities and their connection with prognostic markers (represented by pro-BNP), with inflammatory status (represented by
TNF - tumor necrosis factor) and with clinical status (represented by quality of life and exercise capacity) at baseline and after 1 year.
Results:There were statistically significant differences between the amount of pro-BNP in patients with chronic kidney disease bothat baseline and after 1 year in patients, in patients with coronary heart disease and metabolic syndrome after 1 year of follow up,
and for diabetes patients between pro-BNP values at baseline. In any category of patients were not clinically significant differencesin relation to clinical status.
Conclusion: Comorbidities in HFPEF can influence patient evolution through influence on prognostic factors.
Key words: comorbidities, HFPEF, prognostic markers

INTRODUCTION
During the last 15 years it has increasingly recognized thatmany patients with heart failure (HF) have normal left ventricularejection fraction. Current studies have reported a prevalence ofheart failure with preserved ejection fraction (HFPEF) ranging
from 30% to 70% among patients with heart failure. Actually, theprevalence of HFPEF relative to heart failure with reduce ejection fraction (HFREF) is rising at a rate of approximately 1% peryear (1).The investigators concluded that distinct risk profiles for
HFPEF and HFREF exist, which would support differential pathophysiological mechanism for both HF subtypes. High prevalenceof comorbidities in patient with heart failure suggested common
risk factors or a causal relation. Compared with HFREF patients,
those with HFPEF were older, and had a higher prevalence of hypertension, diabetes, chronic renal disease, ischemic coronaries
disease (2).HFPEF is a syndrome disease with multiple etiologies
and phenotypic expressions. Left ventricular diastolic dysfunction
is a key pathophysyological element and relates to increased
myocytes stiffness and extracellular matrix changes. Systolicabnormalities, left atrial dysfunction, chronotropic incompetence,
vascular stiffening, pulmonary hypertension, contribute to HFPEF(3).Exercise incompetence of affected patientscan be related to
striking hemodynamic abnormalities in HFPEF at rest, and evenmore pronounced, during exercise. Common comorbidities suchas diabetes, hypertension, renal dysfunction, may aggravate theclinical picture and play a role in precipitating the disease (4).

We aimed to compare the presence of comorbidities withclinical status, with prognosis marker and with clinical evolution
for patients diagnosed with HFPEF.
MATERIAL AND METHODS
Seventy-eight patients admitted to the Ascar Cardiology Clinic
in 2011/2012 years, whohad clinical signs of heart failureandprimary diagnosis of heart failure with preserved ejection fraction,
were included in the present study.
Inclusion criteria:
̶ Sinus rhythm̶ Clinical signs and symptoms of heart failure, according to
Framingham criteria
̶ FE ≥ 45 %
̶ E/E´ ≥ 15
̶ Pro-BNP >150pg/ml
Exclusion criteria:
̶ Atrialfibrillation
̶ Acute coronary syndrome within the last 30 days
̶ Cardiac stimulator implant̶ Severe valvular heart diseases
̶ Severe respiratory dysfunction
̶ Chronic kidney disease ≥4 KDOQI̶ Mental disorders
̶ Cancer

Received 21st September 2013.Accepted 15th October 2013. Address for correspondence: Adina Bucur, MD, PhD, Department of Functional Sciences, “Victor Babes”
University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256490507; e-mail: adina.bucur@gmail.com

2013.23.4 (80)  Fiziologia - Physiology

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Presence of sinus rhythm was considered as mandatory criterionin order to eliminate the effect of atrial fibrillation on cardiac
function (the diastolic one, especially), clinical symptomatology
andheart failure evolution.All the patients have signed Informed
Consent forms.Laboratory constants were determined at patients’ admission to clinic.BNP was determined by MEIA Abbott
method. Ultrasound evaluation has been performed usinga Vivid
S5 cardiac ultrasound system, as follows:assessment of LVTD,
LVTS and LV wall thickness in M-Mode; measurement of ejection
fraction by Simpson method; assessment of transmitral diastolic
flow (E and A waves, e/a ratio, EWDT- E wave deceleration time,
IVRT - isovolumetric relaxation time), assessment of pulmonary
venous flow; tissue Doppler with recording of early and latediastolic mitral annulus velocities (E´ - maximum early diastolicvelocity and A´ - maximum late diastolic velocity); assessment ofleft atrium sizes – determination of LA area in apical 4-chamber
One year after patients’ inclusion, inflammatorymarkers, along
withproBNPand clinical status components were re-assessed.
Minnesota quality of life questionnaire was filled in by patientsthe day before discharge. Twelve months after patients’ inclusion, inflammatory markers, along with proBNP, NT-proBNP andclinical status components were re-assessed.

Table I.Features of patient groups according to NYHA class

Grup I
N=31

Grup IIN=47p

65 ±9.0

66± 9.2

0.63

Male sex

8 (26%)

15 (32%)0.75

CI

17 (55%)33(70%)

0.26

HTA

29 (94%)42 (89%)0.72

DZ

9 (29%)

16 (34%)

0.82

Metabolic syndrome

23 (74%)20 (42.5%)

0.0123Age

BCR
10 (32%) 19 (40%)
0.0066Regarding the patients diagnosed with diabetes and those
who are not diabetic, has revealed a statistically significant difference in BNP at patients inclusion in the study (p=0.046, 95%
confidence interval of the difference). We are not able to certify
the same about proBNP value after 1 year (pro-BNP1), because
there are not statistically significant differences between patientswith diabetes and those without diabetes(p =0.09)(Figure 2).

RESULTSOf all patients, 50 (64.10%) patients were diagnosed with ischemic heart disease (IC), 29 (37.17%) patients with chronic kidneydisease (KCD) and 25(32%) with diabetes(D). From coronary
patients, 14 patients (28%) are diagnosed with chronic kidneydisease, and 12 patients (24%) are diabetic. Only 5 patients(6.4%) are diagnosed with all three comorbidities (Figure 1).
Fig. 2. Pro-BNP evolution for diabetic patients

In patients diagnosed with ischemic heart disease were statistically significant differences for proBNP after 1 year of follow
up (p=0.03). Moreover, it can be observed that in patients withischemic heart disease, the pro-BNP is growing after 1 year
follow-up compared with patients who were not diagnosed withthe disease (Figure 3).

Fig.1.Prevalence of comorbidities

Two groups of patients were formed, according to NYHA class:
NYHA III (group1) and NYHA II(group2). We must mention that no
NYHA I or NYHA IV patients were included in the studied group.
Table I shows the features of the two patient groups.As one
can see, there are no statistically significant differences between
the two groups, except the presence of metabolic syndrome(p=0.0123) and chronic kidney disease (p=0.0066).
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Fig. 3. Pro-BNP evolution for patient diagnosed with ischemic
cardiopathy

Fiziologia - Physiology  2013.23.4 (80)

Statistically significant differences are noted in pro-BNP value
both initially (p=0,028) and after 1 year of follow-up (p=0,048)
in patients with chronic kidney disease (GFR<60ml/kgc/min)(Fig.4).
In case of metabolic syndrome were not significant differencesof biomarkers and clinical status in patients included in the study.

Fig. 4. Pro-BNP evolution for patient diagnosed with renal chronic
diseaseAfter one year of monitoring, there were significant differences
for proBNP value (p <0.0001) and TNF value (p = 0.004) (Figure 5).

DISCUSSION
It is expected that the incidence and prevalence of heart
failure, as well as of comorbidities, will increase exponentially inthe following decades due to an aging population. An increased
incidence ofcomorbiditiesresulted in an increasein the number
ofhospitalizationsdue to non-cardiovascular andnon-HF
hospitalization (5).
We can see that there are non-cardiovascular comorbidities(diabetes, metabolic syndrome), which have significant
differences in prognostic markers after one year of follow up,
which may negatively influence the evolution of these patients.
Although a short period of monitoring, these comorbidities did
not influence the clinical status of patients; patients with morecomorbidityare more likely to have clinical signs of congestion.
Systemically raised venous pressure with ancillary neurohormonal
responses may negatively impact non-cardiac tissues andorgans, thus facilitating the occurrence of so called comorbidities.
Therefore, comorbidities and heart failure are conditions that can
influence each other in terms of their evolution.
Over the past decade, myocardial structure, cardiomyocyte
function, and intramyocardial signaling were shown to be
specifically altered in heart failure with preserved ejection fraction(HFPEF). A new paradigm for HFPEF development is therefore
proposed, which identifies a systemic pro-inflammatory state
induced by comorbidities as the cause of myocardial structuraland functional alterations. (6)CONCLUSION
The new HFPEF paradigm proposes comorbidities, plasma
markers of inflammation, or vascular hyperemic responses tobe included in diagnostic algorithms .

Fig.5. Pro-BNP and TNF value for patients diagnosed with metabolic
syndrome, after 1 year.
2013.23.4 (80)  Fiziologia - Physiology

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1. Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL, Redfield
MM. Trends in prevalence and outcome of heart failure with preserved
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COMORBIDITĂŢI ŞI FACTORII DE RISC ÎN INSUFICIENŢA CARDIACĂ, CUFRACŢIE DE EJECŢIE PREZERVATĂREZUMAT

Aproape jumătate din pacienţii cu insuficienţă cardiacă au insuficienţă cardiacă cu fracţie de ejecţie prezervată (ICFEP).Pacientii
sunt în vârstă, cu mai multe comorbidităţi.
Ne-am propus pentru a evalua incidenţa comorbidităţilor şi influenţa lor asupra factorilor de risc şi asupra stării clinice la pacienţii cu
insuficienţă cardiacă cu fractie de ejectieprezervată (ICFEP).
Material şimetode: Am inclus 78 de pacienti, care au fost diagnosticaţi pentru prima dată cu ICPEF. Am evaluat incidenţa comorbidităţilor
şi legătura lor cu markeri de prognostic (pro - BNP), cu statusul inflamator (TNF-factor de necroză tumorală) şi cu starea clinică,reprezentată de calitatea vieţii şi capacitatea de efort, la includere şi după 1 an de urmărire.
Rezultate: Au fost diferenţe semnificative statistic între valoarea pro-BNP la pacienţii cu boală cronică de rinichi, atât la momentul iniţial
şi după 1 an, la pacientii cu boala coronariana si sindrom metabolic după 1 an de urmărire, precum şi pentru pacienţii cu diabet între
valoarea pro-BNP la momentul iniţial. În orice categorie de pacienţi nu au fost diferenţe semnificative în ceea ce priveşte statusul clinic.
Concluzie: Comorbidităţile în ICFEP pot influenţa evoluţia pacienţilor prin influenţa asupra factorilor de prognostic.
Cuvinte cheie: comorbidităţi, ICFEP, markeri de prognostic.

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