KOMPARATIVNO-EVOLUCIJSKI PRISTUP FIZIOLOGIJI

Domagoj Đikić Zavod za animalnu fiziologiju, Prirodoslovnomatematički fakultet

ABSTRACT:The differences between comparative physiology and evolutionary physiology are explained. The article refers to Darwin’s opinion on animal experimentation in physiology. Comparative and evolutionary approach to physiological problem solving is illustrated through presentation of two separate but related studies. Development of mice model in hypometabolism is the first presented study and is discussed here in light of other related study of comparative phylogenetic pattern of ganglioside distribution in brain of vertebrates. The potential of mice as a model animal for torpor induction and role of gangliosides in hypometabolic states from the viewpoint of thermoregulation in different wild species are discussed. Two animal models are compared. Both studies in which author participated for years present two different approach in comparative physiology.

Introduction: Physiology is the biological discipline that explores how organisms function. Initially, it was focused mainly on human beings, with the principal ambition to improve biomedical knowledge. Today, animal experimentation is used to gain answers that might help solve medical conditions in which there are no useful in vitro models available [1]. The debates and divided opinions on animal research were present among Darwin contemporaries as they are today. From the correspondence to professor Holmgren of University of Upsala, on april 14, 1881. it is evident that even Charles Darwin, a great advocate of animal rights and human treatment of animals, attributed advances in medical practice to physiological research and states the admiration to anyone that advances the noble science of physiology taking in consideration humanity to animals [2]. To him it was obvious that knowledge of physiological mechanisms allowed deeper insight into possible grounds for evolutionary correlations and that comparative attitude brings new light on biomedical and clinical issues.

INTRODUCTIONComparative approach in physiology starts from the evo–devo assumption of a phylogenetic descending from common ancestors [3]. Identification of comparative similarities among species allowed the possibility that certain animals may be able to serve as model systems for studying basic physiological processes [4]. Based on that assumption physiologist August Krogh noted his acknowledged principle that for any physiological problem there subsist organisms especially well suited for its studies. One of the classical examples of this includes using squid giant axons to understand general principles of nerve transmission [5]. Comparative physiologists utilize a diversity of approaches including molecular, cellular, organ, and organism biochemistry, functional morphology, biomechanics, biophysics, ecology and evolutionary biology to understand physiological processes and traits. Animal (comparative) physiology is an autonomous discipline that allocated with the fields of molecular biology, ecology, zoology integrating the fragmented knowledge of those disciplines into a complete understanding of organism in its environment. Faced with the tremendous diversity of living organisms, both in terms of numbers of species and their behavioural and ecological variation, comparative physiologists have asked what general processes, formed under pressure of natural selection, apply to all or most organisms and how widespread are exceptions to these rules and whether there subsist multiple key to a given adaptive problem.

INTRODUCTIONIn the 1970-ties and 80-ties a new subdiscipline–evolutionary physiology was developed [6]. This novel mechanistic approach focussed on how development of one physiological system under the pressure of natural selection favoured the development of another evolutionary later developed system. That was a guiding principle of evolutionary physiologists who considered a comparative physiological approach to be outdated [7]. However one should be cautious in such scrutiny, in part because many animal groups have been investigated superficially if at all and in part because the incorporation of fundamentally new technologies into the discipline permits us to address previous questions even in intensively studied animal groups. To illustrate the approach of evolutionary physiology the best example are the lung evolution and co-evolution of other systems of the terrestrial animals impelled by fluctuating Phanarezoic oxygen levels. Studies have been supported by an array of experiments [8]. Briefly, Oxygen induces differentiation of alveolar myocytes into alveolar adipocytes [9]. These adipocytes prevent lung injury caused by raised level of oxygen radicals in atmosphere and organisms by production of leptin, which augment pulmonary surfactant activity and alveolar surface area.

INTRODUCTIONDue to higher level of oxidative damage that first terrestrial animals faced, alveolar surfactant and gas exchange co-evolved with organ systems that link lipid metabolism (leptin) and respiration. They simultaneously facilitated the rise of endothermy. Both were accompanied with high-systemic vascular pressure, subsequently compensated by the evolution of the adreno-medullary beta-adrenergic system, again crucial in endothermy. Positive selection for those changes created further selection for the heart, to become more complex, and as such assists in the endothermic metabolism, phylogenetically in tandem with the lung, developmentally, inflicting on the gut mesoderm to induce additional liver development. That evolutionary-developmental interaction was significant since the more efficient liver provided regulated sources of glucose and glycogen to the evolving physiologic system of thermoregulation and increased metabolism. More glucose and oxygen available to all tissues by the help of increased circulation rate, amplified the number of neural cells which led to the evolution of the neocortex. Evolution of neocortical controls furthered integration of physiologic systems [10]..

INTRODUCTIONThe greatest challenge in the postgenomic era is to effectively integrate functionally relevant genomic data in order to derive physiologic first principles, and determine how to use them to decode complex traits. Currently, this problem is being addressed stochastically by analyzing large data sets to identify genesthat are associated with physiologic phenotypes. In lights of what has been said about comparative and evolutionary approach in physiology I will describe studies of two projects I collaborated on. The first one describes the search for the best animal model suited for the postgenomic era in the physiology of hypometabolismbased on evolutionary preserved traits regardless of artificial selection of lab animals. The second study illustrates the comparative interspecies approach of the role of gangliosides in lipid raft stability in the brains of dozen species of animals differently adapted to environmental temperature and evolutionary developed under pressure of natural selection. They are connected with the general question of how the brain regulates its functions through hypometabolicperiods of low body temperature/low metabolism. This has both scientific-fundamental and biomedical significance.

Materials and methods: Detailed experimental conditions and assays used for the two described experiments are explained in the following references [11, 12, 13, 14, 15].Results and Disscussion: Physiological field of manipulating metabolism is important for answering how tissues recuperate after ischemic reperfusion changes, how signal-molecules coordinate metabolic pathways and what physiological changes to expect in induced artificial hypometabolism or suspended animation in biomedicine. In the light of the vast current interest in manipulating metabolism, an obstacle towards advancing the molecular basis of torpor entry, torpor thermoregulation, and arousal, is the limited availability of specific information of the genomic or proteomic background of torpor behaviour in standard torpor-model species (hamsters, deer mice, marmots, squirrels). The genome of the laboratory mouse, on the other hand, has been sequenced [16], and the mouse constitutes the most established mammalian model for comprehensive gene-function analysis. Mice generally do not enter torpor unless forced to do so [17].

Results and Disscussion: In our study we developed a protocol for the induction of torpor in mice. We compared three different strains of laboratory mice (AKR/J, C57BL/6 (B6), SWR/J) to responsiveness of torpor induction. We aimed to further explore the potential of the laboratory mouse as comparative model for molecular pathways in hypometabolism. Detailed protocol is described in Đikić et al., 2008 [11]. Mice were acclimated for ten days at ambient temperature of 30°C, ad libitum, photoperiod set to LD:12:12 (lights off: 6:00 p.m.= 18:00 Central European Time, CET). Mice were transferred into an open respirometric system. During the subsequent 22-24 hours ambient temperature was set to 16°C (±0.5°C) and no food was available to the mouse. O2 consumption and body temperature were recorded every 6 min. Torpor duration was defined as the time spent at a body temperature <30°C. Approximately 6-9 hours after “lights off”, mice shut off their thermogenesis and approached minimal metabolic rates of <25 ml O2*h-1 within the following hour. Arousal frequently occurred within ~2 hours after “lights on” and was characterised by a rapid increase in MR and a return to normothermicbody temperature (>33°C). On average, torpor bouts lasted between 3-7 hours, but in B6 mice individual torpor bouts lasted up to 11 hours (Figure 1). In B6 mice, the occurrence of torpor was most frequent and repeatable (88%), while AKR/J and SWR/J mice had similarly lower torpor frequencies (44% and 50%, respectively). Taken together, the female mouse of the B6 strain is a suitable model for studying the genetic and proteomic changes that tissues undergo when animals enter torpor.

Results and Disscussion: Selective breeding of torpor characterized mice could be employed to identify genetic loci associated with torpor behaviour. Although additional signals and relays may play a role in the seasonal control of spontaneous torpor and hibernation, investigations in inbred mice could lead to novel insights into pathways involved in the active suppression of metabolism. By standardizing this model and with further controlled time shift (darkness and light period signal) in onset of torpor we were able to predictably collect hypothalamic tissues from the brains of group of six or more torpid mice at the same time/conditions and during working hours. We used groups of six torpid and nontorpid mice and analysed the hypothalamic mRNA by Affymetrix chips (B series) for the candidate genes. Out of four candidate genes, Claudine 5 was the most expressed gene in the phase of lowest entry point of the hypometabolism in mice indicating physiological shutting of the BBB in torpid animals (Table 1). The interesting open questions come from hibernating mammals, how neurons in the brain manage normal activity during periods of hypometabolism/low body temperatures. This question is a link to the studies of gangliosides. Hibernating mammals change ration of GD1a and GT1b in favour of GT1b during winter season [18]. Because glycosphingolipids in the brain are present in ten times higher concentrations then in any other extraneural tissue it was assumed that nervous tissues requires some particular functions served by each of known ganglioside structures. It is very likely that the most common complex ganglioside structures serve same functions in rafts of all homothermic mammals [19], because they share same pattern of distribution.

Over the years we conducted a series of interspecies comparative analysis in more than twenty vertebrate species by immunohistochemistry with specific antibodies against major brain gangliosides [12, 13, 14, 15]. The surveys of cross-species differences described a great variation in quantity and pattern of gangliosides in vertebrates, particularly between different species of fish [20, 21, 22]. One current immunohistochemical study showed interspecies difference in expression of GD1a in olfactory bulb and olfactory pathways of two genera of frogs (data in press) which suggested unrelated function of these molecules in a species dependant way [23]. Also, pattern of brain gangliosides is significantly different between warm and cold-blooded animals and can be correlated to the state of thermal adaptation [24]– the lower the environmental or body temperature therefore more polar is the composition of brain gangliosides which corresponded with previous reports from other authors. Future development of experimental tools in further comparative research such as those two presented in the article, might answer some fundamental and practical physiological principles.

-Fiziologija proučava kako funkcioniraju pojedinisustavi u organizmu

-Temeljni fokus fiziologije, u njenim začecima ali idanas, je shvatiti kako zapravo funkcionira ljudskiorganizam

-Kako stečenim spoznajama unaprijediti zdravstveniaspekt biologije čovjeka

-Komparativni pristup u fiziologiji (tj. animalnafiziologija) razvijao se s ciljem boljeg razumijevanja nakoji način pojedini fiziološki sustav funkcionira urazličitih vrsta te može li se pridonijeti rješavanjupojedinog problema u biomedicini

-Područje autonomno ali ima brojne dodirne točke sabitnim područjima u biologiji jer se fiziološki sustaviistražuju na svim razinama organizacije pogotovo upostgenomskoj eri, i koriste znanja molekularnebiologije, ekologije, zoologije i evolucije

-Evolucijska fiziologija-

Novija grana fiziologije (80-tih, 20.st),eksperimentalno pokazuje kako je jedan fiziološkisustav uzrokovao nastanak, razvoj ili unaprjeđivanjedrugog fiziološkog sustava

-U komparativnom pristupu fiziologiji jedna od glavnih stvari je nalaženje prikladnog životinjskog modela i sagledavanje usporedbe kroz evolutivni slijed organizama

-miš, štakor

-ali i ostalih životinjskih vrsta prema tzv.

KROGHOVOM PRINCIPU

August Krogh – Daski fiziolog, dobitnik Nobelove nagrade 1920. za otkriće mehanizma upravljanja cirkulacijom krvi kroz kapilare u koštanom tkivu

-KROGHOV PRINCIP U FIZIOLOGIJI-“ …Za objašnjenje bilo kojeg fiziološkog fenomena postojiživotinjski organizam na kojem je najlakše istraživatipostavljeni problem i čija će građa i ustroj olakšatiobjašnjenje istraživanog fiziološkog fenomena…”

Elektroda za snimanje potencijala

Veliki

akson

Drugii

aksoni

Ganglij

Živci koji sadrže veliki akson

Istraživanje akcijskog potencijala i provođenje živčanog impulsa - Kroghov princip

Traženje prikladnog modela u postgenomskoj eri:

-Obrazloženja pojedinog sustava spuštaju se na subcelularnu razinu interakcije tkiva stanica ili pojedinih molekula i nukleinskih kiselina

Primjerice, neki odgovori u fiziologiji metabolizma itermoregulacije te posebice hipometaboličkih stanja (torpora,hibernacije) razjasnili bi:

1. kako se tkiva nose i rekuperiraju nakon stanja hipoksije uishemično-reperfuzijskim poremečajima pr. moždani ili srčani udar

2. kako se tkiva nose s oksidativnim stresom nastalim nakonreperfuzije

3. koji signali upravljaju metaboličkim putevima uskladištenjaenergije (debljina) ili potroška energije

4. fiziološke prilagodbe ili signali pri induciranj komi ili izazivanjuhipometaboličkih stanja (pr. tijekom operacije) ili transportaranjenika

5. transport u stazi za duga putovanja

KAKO IZABRATI MODEL?

Acomys russatus -Izrael, Egipat, Palestina, Oman, termoneutral 27-33°C

Mesocricetus auratusMarmota marmotaMarmota marmota

Phodopus sungorus

AKR/J 28.6 2.3g n=6BL/6 23.9±1.1g n=8SWR/J 22.2±2.1g n=6

3 soja laboratorijskih miševa, 12-14 tjedana

Implantacija sondi za tjelesnu temperaturuOporavak tri tjednaInduciranje TorporaTb and MR simultano indirektnom kalorimetrijom

Đikić D, Heldmaier G, Meyer CW (2008) Life in the cold 13th ed.

10

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07:12:00 09:36:00 12:00:00 14:24:00 16:48:00 19:12:00 21:36:00 00:00:00 02:24:00 04:48:00 07:12:00

Time

Tb (°

C)

0

20

40

60

80

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120

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Oxy

gen

cons

umpt

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(ml/h

)

Tb

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M4S4B6tsh

lights off

min M (average of minimum metabolic valuses

TIP (Torpor induction point)

End of Entry-Pointfirst point ≥ 20 ml/h;exmpl.=21.3 ml/h

Euthermia (RMR)

ArousalBout plateau (minimal Metabolism)Entry

Euthermia (Night activity h )

EuthermiaTorpor bout

RMR-average of minimal valuse

AKR/J

Long; 3/8 torp (37%) Short; 2/8 torp (25%) Multiple entry; 3/8 torp (37%) No torp 10/18 trials (55%)

B6

Disturbance intorpor; 5/21 torp ( 23%)

Preentry test drop;8/21 torp ( 38 %) No torp; 3/24 trials ( 12 %)Long; all 21 torp (100%)

SWR/J

Short, No B, 9/9 torp (100%) No torp; 9/18 trails (50 %)

Đikić D, Heldmaier G, Meyer CW (2008) Life in the cold 13th ed.

Đikić D, Heldmaier G, Meyer CW (2008) Life in the cold 13th ed. (in press)

91.7% torpid (87.5%)

77.7% torpid (38.8%)

66.7% torpid (38.8%)

#1 #2 #3Torpor Trial

BL/6

SWR/J

AKR/J

tb <25 C or MR <30%RMR tb<30 C or MR<50% RMR tb ≥30 C or MR≥50% RMR

M0S0 13.7. 23.7 27.7M1S1 13.7. 23.7 27.7M2S2 13.7. 23.7 27.7M3S3 13.7. 23.7 27.7M4S4 15.7 23.7 28.7M5S5 15.7M6S6 15.7 24.7M7S7 15.7 24.7 28.7

M1S1 1.9. 10.9.M2S2M3S3M4S4 1.9. 10.9.M5S5 28.8 2.9.M6S6 2.9.

M7S7 28.8 2.9. 11.9.M8S8 28.8. 6.9. 11.9.M9S12

M10S13 29.8M11S0

M12S11

Đikić D, Heldmaier G, Meyer CW (2008) Life in the cold 13th ed.

M3S3

Central European Time

6.00 12.00 18.00 0.00 6.00 12.00M

etab

olic

rate

(ml O

2/h)

0

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Torpor trial #1Torpor trial #2

Tb=26.4°C

M1S1

Central European Time

6.00 12.00 18.00 0.00 6.00 12.00

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te (m

l O2/h

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Torpor trial #1Torpor trial #2

Tb=34.0°C

Tissue sampling

normothermic torpid

Tb: 34-37°CMR: 66-105 ml/h

Tb: 25-29°CMR: 35-15 ml/h

Mozak koji podnosi velike temperaturne razlike mora mijenjati sastav membrane na različitim temperaturama da bi zadržao istu organizaciju membrane...

Kako mozak podnosi velike temperaturne razlike?-TRAŽENJE MODELA U EVOLUCIJSKOM NIZU

Som ima male količine jednostavnih glikosfingolipida u mozgu. GD1a je karakterističan za dijelove mozga za okus, Siluriformes nastali prije 350-400 miliona godina imaju veliki vagalni režanj i veliki facijalni režanj za interpretaciju osjeta koji dolazi putem kemoreceptora smještenih na “brkovima”

U mozgu šarana nalazimo GD1a i GT1b kojih je osobito puno u dijelovima mozga vezanim za interpretaciju okusnih signala te u optičkom tektumu

Cross sections through trout brain (Salmo trutta)

U mozgu pastrve prevladavaju gangliozidi GM1, GD1b i GT1b, a nalaze se u cerebelumu i optičkom režnju

Cross-sections through the brain of green frog (Rana esculenta)

Žaba ima kompleksne gangliozide na regijama mozga za njuh ali i na djelovima iz kojih će se u kasnijih skupina razviti neokorteks

Cross-sections through the brain of long-nosed viper (Vipera amodytes)

Gally Nissl

Sagittal sections through the hen brain

Organizmi sa stalnom tjelesnom temperaturom imaju istu raspodjelu gangliozida...

Organizmi koji žive u velikom temperaturnom rasponu imaju drastično drugačiju raspodjelu gangliozida u mozgu...

DARWINOVO MIŠLJENJE O ŽIVOTINJSKIM MODELIMAU FIZIOLOGIJI.

Pismo profesoru Holmgrenu sa sveučilišta Upsala, koji ga moli da iznese mišljenje o pokusima na životinjama koje se koriste kao usporedni modeli u fiziološkim istraživanjima:

FIZIOLOŠKI POKUSI NA ŽIVOTINJAMA?

"Down, Beckenham, April 14, 1881."Dear Sir,

…….I have all my life been a strong advocate for humanity to animals, and have done what I could in my writings to enforce this duty…….

…….Several years ago, when the agitation against physiologists commenced in England, it was asserted that inhumanity was here practised and useless suffering caused to animals; and I was led to think that it might be advisable to have an Act of Parliament on the subject….

……..I then took an active part in trying to get a Bill passed, such as would have removed all just cause of complaint, and at the same time have left physiologists free to pursue their researches ………….

……..On the other hand, I know that physiology cannot possibly progress except by means of experiments on living animals,

………. he who retards the progress of physiology commits a crime against mankind……..

…………….What improvements in medical practice may be directly attributed to physiological research is a question which can be properly discussed only by those physiologists and medical practitioners …………….. but, as far as I can learn, the benefits are already great.

….Look, for instance, at Pasteur's results in modifying the germs of the most malignant diseases, from which, as it so happens, animals will in the first place receive more relief than man…..

….. As for myself, permit me to assure you that I honour, and shall always honour, every one who advances the noble science of physiology.”

"Dear Sir, yours faithfully,"CHARLES DARWIN."To Professor Holmgren."

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