Comparative Biochemistry and Physiology Part A 134(2003) 21–26

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Liver and kidney structure and iron content in romanian brownbears(Ursus arctos) before and after hibernation

Carol-Constantin Prunescu *, Nicolae Serban-Parau , Jeremy H. Brock , Diane M. Vaughan ,a, a b b

Paula Prunescua

Institute of Biology, Center-Animal Experimental Morphology, P.O. Box 56-53, Bucharest, Romaniaa

Department of Immunology, Western Infirmary, Glasgow G11 6NT, UKb

Received 6 December 2001; accepted 14 May 2002

Abstract

The annual cycle of the brown bear(Ursus arctos) in the Carpathian Mountains(Romania) consists of an activeperiod from April to November, and an inactive period(hibernation) of approximately 4–5 months between Novemberand March. During hibernation, the brown bears sleep continually and do not feed or drink water. Analyses of liver andkidney of male brown bears showed that liver iron content was 3 times higher in bears at the end of hibernation than atthe end of the active period. A possible trend towards a decrease in iron content was noted for the kidney. The presenceof iron in the liver was confirmed by the presence of the Perls-positive granules in the cytoplasm of Kupffer cells, inother non-parenchymal cells and also in some hepatocytes. The hepatic veins of the bear liver samples obtained in earlyspring showed narrower lumens with pleated walls, compared to the normal outline of the hepatic veins in the liverfrom the bears sampled during autumn. Also in the early spring bears, the renal glomeruli were partially fibrosed. Renalglomerular fibrosis was sometimes observed in samples from the prehibernation period. The tissue iron values from thelivers and kidneys of brown bears in early spring or autumn might provide useful data on iron metabolism underconditions of hibernation and accompanying starvation.� 2002 Elsevier Science Inc. All rights reserved.

Keywords: Brown bear; Carpathian mountains; Fibrosis; Hibernation; Histology; Kidney; Liver; Tissue iron;Ursus arctos

1. Introduction

The biology of the brown bear(Ursus arctos)of the Carpathian Mountains has been little stud-ied. The Carpathian population of this species ischaracterized by a 4–5 months of winter sleep inthe period November–March(Micu, 1998).According to Hissa(1997), Hissa et al.(1998a,b)which referring to the brown bears of Finland, theanimals do not eat, drink water, defecate or urinateduring the 6–7 months of long winter sleep

*Corresponding author. Tel.:q40-1-223-9072; fax:q40-1-221-9071.

E-mail address: prunescu@biochim.ro(C.-C. Prunescu).

A previous study of the biology and physiologyof a different species of bears from North America(Folk et al., 1976), also noted that during hiber-nation the bears sleep continually and do not eator drink water. Similar observations have beenmade with black bears(Ursus americanus) inVirginia—North Carolina region of southern USA(Hellgren et al., 1989).

A previous unpublished histological study onbrown bear visceral organs, carried out in theInstitute of Biology from Bucharest(Romania),noted that stainable ferric iron(Perls-positive) wasmore frequently observed in the liver of the bearsin the spring, compared with the bears in autumn.Comparison of the tissue iron values from the

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Table 1List of the bears.

Nr.of bear Sex Date of shooting District of HistoRomania

2585 M 05.04.1998 Fagaras–Brasov L., K.2586 M 09.04.1998 Vladeni–Brasov L. -2587 M 16.04.1998 Dosca–Brasov L. -2588 M 19.04.1998 Fagaras–Brasov L., K.2593 M 19.04.1998 Fagaras–Brasov L. -2594 M 24.04.1998 Fagaras–Brasov L. -2629 F 10.10.1998 Bistrita–Nasaud. L., K.2630 F 10.10.1998 Bistrita–Nasaud L., K.2631 M 10.10.1998 Bistrita–Nasaud. L., K.2633 F 11.10.1998 Bistrita–Nasaud. L., K2634 M 11.10.1998 Bistrita–Nasaud. L., K.2635 M 11.10.1998 Bistrita–Nasaud. L., K.2671 M 19.04.1999 Bistrita–Nasaud L., K2672 M 10.04.1999 Bistrita–Nasaud L., K.2673 M 09.04.1999 Bistrita–Nasaud L, K.2675 M. 27.03.1999 Fagaras–Brasov L., K.2676 M. 04.04.1999 Fagaras–Brasov L., K.2698 M. 02.10.1999 Domnesti–Arges L., K.2699 M. 02.10.1999 Domnesti–Arges L., K.2700 M 03.10.1999 Domnesti–Arges L., K.

Abbreviations: Msmale; Fs female; Lsliver; Kskidney.Fig. 1. Central vein lumen of the hepatic lobule is unfibrosedand loaded with blood cells.(Autumn). PSR–H, 560=.

livers and kidneys of brown bears in the earlyspring or in the autumn might provide useful dataon the effects of hibernation and the absoluteabsence of nutrition, on iron metabolism. Thebiological and medical significance of the hiber-nation phenomenon on normal metabolism is notwell understood and led us to carry out thisresearch. We here report that levels of tissue ironin kidney and liver differ between bears pre- orpost-hibernation, and present some observations onthe structural features of the liver and kidney inpre- and post-hibernation bears.

2. Materials and methods

Liver and kidney samples were collected frombrown bears shot by hunters in the autumn orspring, at various localities in the CarpathianMountains of Romania(Table 1). The bears whenshot weighed between 120 and 240 kg, accordingto estimates provided by the hunters.

Liver samples were obtained from 6 male and3 female brown bears in autumn and from 11bears, all male, in early spring. Kidney sampleswere obtained from all 6 male brown bears inautumn and from 7 in spring. The time betweenthe death of the bears and collection and fixationof the liver and kidney histological samples was

of 2–12 h. Liver and kidney samples were fixedin 8% formaldehyde in saline. Histological sec-tions, 5 mm thick, were stained with hemalum–eosin (H–E) for anatomic microarchitecture andwith picro-sirius red-Hemalum(PSR–H) to dem-onstrate collagen in the fibrous structures. Bothtechniques were combined with staining by Perlsmethod to show the presence of ferric iron.

Tissue iron analyses were performed by thestandard ferrozine method following acid digestionof the tissues(Bothwell and Torrance, 1980).

Statistical analyses were performed using Stu-dent’s t-test.

3. Results

3.1. Liver

Regardless of when the bears were sampled, thehistological structure of the liver was characterizedby the existence of hepatic lobules lacking inter-lobular fibrous septae. The capsule of Glisson wasrelatively thin. The main portal tracts were highlyfibrosed.

The liver structure in the brown bears in autumnshowed the central veins of the lobules to be

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Fig. 2. The central vein wall is collapsed and pleated; the con-tent of the vessel is homogenous with few blood cells; peri-venular fibrosis is moderate.(Early spring). PSR–H, 560=.Insert.: Perls-positive material in the Kupffer cells.(Earlyspring). Perls method, 560=.

Fig. 3. Renal cortical zone. Note the normal structure of theMalpighian corpuscle.(Autumn). PSR–H, 224=.

unfibrosed and normally loaded with erythrocytes(Fig. 1).

In the livers of brown bears in early spring thecentral veins, in particular the sublobar veins werepartially collapsed and pleated. The veins of thehepatic venous system showed a decreased diam-eter of the lumen. A few red cells embedded in ahomogenous material were present in the lumen(Fig. 2). The partial restriction of the lumen ofthese vessels created an impression of superfibros-is. In the lining of these vessels, endothelial cellswere not always evident.

In the livers of brown bears in early spring,evidence of a moderate amount of Perls-positivematerial was noted(Fig. 2, insert). Fine Perls-positive granules were present especially in thecytoplasm of Kupffer cells. These granules werealso observed in endothelial cells of hepatic sinu-soids but seldom in the perisinusoidal stellate cells.Also, siderosomes in the hepatocytes wereobserved.

The great portal tracts also presented morpho-logical modifications due to the long period ofinactive liver function. The portal veins werefibrosed and lacked blood cells.

The tissue iron values in the brown bears livershowed considerable variation. Using only thesamples from male bears, the mean concentrationof iron in spring bears(64.6"34.7 mg Feygtissue), ns11, range: 11.4–113.2) was neverthe-less approximately 3 times higher than in theautumn livers(21.8"6.6 mg Feyg tissue,ns6,range: 16.0–30.8). This difference was significant(P-0.01). The mean concentration in the liversof the 3 females was 34.3"23.2 mg Feyg tissue,range: 15.0–60.0, suggesting no obvious differ-ence from the male, although the number of femalesamples was too small for meaningful comparison.There was no correlation between the iron-deter-mined biochemically and the ferric iron seen byPerls method on histological sections.

3.2. Kidney

Kidney of the brown bear consisted of manylobes. Each lobe, pyramidal in shape, was formedby a cortex region, surrounded at its base andsides by a medullary region(Anderson et al.,1988).

Histological sections through the kidney fromthe brown bears in early autumn revealed a normalstructure of the renal corpuscle. There was rela-tively unimportant picro-sirius red positive mate-

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Fig. 4. Renal cortical zone. Note the large spaces between therenal corpuscle and Bowman’s capsule and important amountof picro-sirius red positive material around the podocytes andmesangial cells.(Early spring). PSR–H, 224=.

rial among the podocytes and the mesangial cells.(Fig. 3).

The kidney from the brown bears in spring, likeof some bears shot during the autumn, followingpicro-sirius red staining, showed considerableamounts of collagen in the Malpighian corpuscle.The collagen, not yet organized into fibres, wassituated among the podocytes and mesangial cells(Fig. 4). Between the renal corpuscle and Bow-man’s capsule a large space was noted(Fig. 4).

Staining did not reveal the presence of Perl’s-positive ferric iron in kidney histological sectionsin either autumn or spring brown bears.

The results of the tissue iron analyses againshowed considerable variation. The mean concen-tration of iron in autumn male bears was47.3"34.3 mg Feyg tissue(ns6, range: 10.2–105.4), and in spring male bears was 23.7"10.5mg Feyg tissue(ns7, range: 9.8–34.3). Althoughthe figures suggest that there might be more ironin the autumn bear kidneys, this difference did notachieve significance(Ps0.08). The iron contentof the kidneys of the three female autumn bearswas 36.7"14.9 mg Feyg tissue (range: 19.4–47.5).

4. Discussion

Earlier reviews on liver microanatomy in mam-mals reported the structure of the hepatic lobulesin the family of Ursidae(Dorst, 1973), or in polarbear(Gabe, 1973) as being similar to that of thepig. The hepatic lobules in the pig are isolatedfrom each other by perilobular connective septa(Elias et al., 1954; Wunsche, 1985). The American¨black bear was also included in the list of mam-mals with hepatic lobulation similar to the pigliver (Ekataksin and Wake, 1991).

As we report here, we could not confirm theexistence of perilobular connective septa in brownbear liver. In the histological sections, the brownbear hepatic lobule appears similar to the hepaticlobular structure of the majority of mammals(human, rodents etc.), but not to the structure ofthe hepatic lobule of the pig.

During hibernation sleep, the bear’s normaltemperature is reduced by a few degrees(Hissa etal., 1998a,b). This small decrease of body temper-ature led Hock(1960) to consider the winter sleepof the black American bear ascarnivorean lethargyrather than true hibernation. Folk et al. (1976) inan eight years study, concluded that North Amer-ican bear species experience mammalianhibernation.

Regardless of whether it should be consideredhibernation or lethargy, the brown bears sleepcontinually for 4–5 months, corresponding to theduration of winter in the Carpathian Mountains(Micu, 1998).

The histological structure of the livers of thebrown bears in the autumn, i.e. in a period ofmetabolic activity, was considered as representingnormal histological structure. Comparison of theliver microanatomy from the bears in autumn andspring showed that during winter the hepatic veinsshowed a tendency towards restriction of the lumen(Fig. 2). In this period, the bears stop drinkingwater and urinating, hence necessarily the bloodvolume must decrease due to loss of water vaporduring breathing. The amount of blood filling thelumen of hepatic veins was greater in autumn thanin spring. The histological structure of the wall ofthe medium sized andyor the terminal hepaticveins did not permit the preservation of the ‘nor-mal’ shape, if the blood content of these vesselswas reduced. Thus in spring, the veins wallsappeared pleated.

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Lack of feeding leads to the disappearance ofnutrients in the intestine and portal blood. Mayer(1960) demonstrated that during the hibernationcycle of the arctic ground squirrel, intestinal epi-thelial cells are embedded in a protective layer ofmucus. At the same time production and releaseof digestive enzymes stopped. A reduction inportal flow can be deduced from the restriction ofthe hepatic veins lumen. Physiological data indi-cate that during the winter sleep of grizzly bearsthe heart rate decrease(Folk et al., 1976).

The decrease in blood circulation through thekidney of the brown bear before and during hiber-nation seems to be severe. Anderson et al.(1988)described morphological structures that served tolimit blood circulation through the kidney duringhibernation. These consisted of vessel sphincterssituated in the muscular layer of the afferentarterioles.

Our research demonstrated the existence of apericapillary fibrosis in the renal corpuscle, in thespring bears. This was also sometimes observed inautumn bears, suggesting preparation of the kidneyfor the hibernation period. The pericapillary fibro-sis would lead to a drastic reduction of the absorb-tive capacity of the glomerular podocytes.

The livers of the brown bears in early springcontained significantly more iron than those ofbears in the autumn. The idea that exogenous ironmight contribute to this can be excluded becausethe complete cessation of feeding during the hiber-nation period. In Svalbard reindeer, seasonal ironloading of the liver was considered to be a con-sequence of the decreases in body weight duringwinter (Borch-Iohnsen and Nilssen, 1987).

During the winter sleep of the small hibernatinganimals, the body weight decreases by approxi-mately 30%, due to the minimal food intake(Rathsand Kultzer, 1976). In hibernating bears the lossof body mass, mainly due to consumption of fatreserves, varies from 250 to 500 g per day, depend-ing on the size of the animal and ambient temper-ature(Hissa et al., 1998a,b).

The considerable variation in the liver iron couldreflect the different regional origins, age, unknownlife-history, etc. of the animals studied.

The main forms of intracellular iron storage areferritin and haemosiderin, with the distribution ofthe latter in normal human subjects being restrictedto reticuloendothelial cells(Crichton, 1991). Liveriron may be acquired from old red cells phagocy-tosed by Kupffer cells and from other physiologi-

cal sources. It seems that under the conditions ofhibernation, the iron was not recycled from theliver to other organs. This also could account forthe higher iron levels in the livers of the springbears.

The iron estimated chemically did not correlatewith the presence of Perls positive iron, whichrepresents only a part of the liver iron.

The tendency towards lower iron levels in thekidneys of the spring brown bears comparativelywith the liver iron levels requires further samplesto determine whether a statistically significantdifference can be detected.

Acknowledgments

We thank Dr Mark Wareing(University ofManchester) for helpful discussion.

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