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 DOI: 10.1177/019262339402200107

1994 22: 48Toxicol PatholJohn E. Dillberger

Age-Related Pancreatic Islet Changes in Sprague-Dawley Rats  

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TOXICOLOGIC PATHOLOGY 1SSN:O 192-6233 Copyright 0 1994 by the Society of Toxicologic Pathologists

Volume 22, Number 1, 1994 Printcd in U.S.A.

Age-Related Pancreatic Islet Changes in Sprague-Dawley Rats*

JOHN E. DILLBERGER Departinent of Drug Safety, Marion MerreN Dow Inc., P.O. Box 68470,

Indianapolis, Indiana 46268-0470

ABSTRACT To characterize the clinical pathologic and morphologic features of spontaneous age-related changes in

pancreatic islets observed in Crl:CDa(SD)BR rats, I reviewed data from rats used as controls in 10 toxicity studies from 1987 to 1992. Rats were 3.5-26 rno old at necropsy. At necropsy, rats were weighed, and serum, urine, and pancreas samples were collected. Serum was analyzed for glucose, triglyceride, and cholesterol concentrations; urine was analyzed for glucose and ketones; and samples of pancreas were stained with hematoxylin and eosin, Masson's trichrome stain, or for insulin by an immunoperoxidase method with a Masson's trichrome counterstain and examined microscopically. Male rats gained weight more rapidly than females and were visibly obese by 5 mo. Weight gain was accompanied by increased fasting triglyceride and cholesterol concentrations. Triglyceride increased more than cholesterol: from 3.5 to 17 rno ofage, triglyceride concentrations increased 3.4-fold in males and 3.1-fold in females. By 14 mo ofage, rats generally had fasting triglyceride concentrations >200 mg/dl (2.2 PM). Fasting glucose concentrations generally were slightly (<30%) greater in males than females. More males than females had glucose >200 mg/dl (1 1 mM); several males had glucose >300 mg/dl (16.5 mM). Glucosuria was not detected in any rat. Ketonuria was much more common in males than in females, but its incidence did not parallel that of obesity and hypertrigly- ceridemia; instead, ketonuria was most common in young males and decreased with age. Morphologic islet changes were observed in rats as young as 3.5 mo old, and their incidence increased with age. They were much more common in males than in females at all ages. Islet changes began as @-cell hyperplasia causing enlarged islets. With time, fibrosis gradually dissected islets into discreet nests of cells separated by bands of collagenous tissue so that they resembled cirrhotic liver. As the process continued, the ratio of fibrous tissue to islet cells increased until some islets were reduced to scattered @ cells imbedded in a mass of scar tissue. By the time rats were 26 mo old, enlarged islets were rare; instead, remaining islets generally were small. Islet cell neoplasia did not occur in rats 17 mo of age or younger. In 26-mo-old rats, neoplasms were more common in males than in females (incidence of 20% vs lo%, respectively). The pathogenesis of these abnormalities is unclear, but they suggest that male Sprague-Dawley rats fed ad libitum are a less than ideal system in which to conduct long-term toxicity/safety assessment studies or any other research studies that could be influenced by altered metabolism. These results also reveal that male and female Sprague-Dawley rats differ metabolically and that the differences may predispose males to islet cell neoplasms.

Keywords. Fibrosis; hyperplasia; obesity; ketonuria; @ cells; hyperglycemia; hypertriglyceridemia

INTRODUCTION chronic progressive glomerulonephropathy, myo-

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Vol. 22, No. 1, 1994 ISLET CHANGES IN RATS 49

FIG. 1.-Normal islet. H&E. x320.

14, 16). In many strains, islet changes are associated with hyperglycemia, glucosuria, or abnormalities in glucose tolerance in a syndrome that closely resem- bles prediabetic states or diabetes mellitus in people (1 5) . This report describes the clinical pathologic and morphologic features of spontaneous age-relat- ed islet changes in Crl:CD@(SD)BR rats.

METHODS Arzir?ials. Crl:CD@ (SD)BR rats evaluated in this

report were used as controls in 10 toxicity studies (Studies A-J) conducted between 1987 and 1992. They were obtained from Charles River Laborato- ries, Inc. (Portage, MI). Rats were 5-6 wk old when received. Following a 2-wk acclimation period, they were assigned to toxicity studies. During study, rats were identified by ear tags and housed individually in wire-bottomed, stainless-steel cages in light-, hu- midity-, and temperature-controlled rooms. India- napolis municipal water or well water, analyzed pe- riodically for compliance with Environmental Protection Agency potable water standards, and commercial rodent chow (Certified Rodent Chow @

No. 5002; Purina Mills Inc., St. Louis, MO), ana- lyzed by the manufacturer fo,r contaminants rea-

FIG. 2.-Hyperplastic islet. Note increased size, irreg- ular margin, and entrapped exocrine pancreatic cells. H&E. x 320.

sonably expected to be present, were provided ad libiticni. Depending on the nature of the study to which they were assigned, rats were untreated or given distilled water once daily by gavage for the duration of the study. At the end of studies, rats were fasted overnight, weighed, anesthetized by methoxyflurane, euthanized by exsanguination, and necropsied. Studies lasted from 6 wk to 2 years, so rats were 3.5-26 mo old at necropsy.

Rats were cared for and used as recommended in the Guide for the Care arid Use of Laboratory Ani- ritals (3). Study protocols were approved by our In- stitutional Animal Care and Use Committee.

Clinical Pathology. Blood samples were collected from fasted rats at necropsy by cardiocentesis. Ex- cept for rats used in 2-year studies, serum was an- alyzed for glucose, triglyceride, and cholesterol con- centrations using reagents and a Monarch 2000 automated analyzer (Instrumentation Laboratory, Inc., Lexington, MA). Glucose concentration was measured enzymatically using hexokinase and glu- cose-6-phosphate dehydrogenase. Triglyceride con- centration was measured enzymatically using lipase, glycerol kinase, pyruvate kinase, and lactate dehy-

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50 DILLBERGER TOXICOLOGIC PATHOLOGY

TABLE 1.-Mean body weights and glucose, triglyceride, and cholesterol concentrations by age and study.

hlales Females

Age Study

3.5 Mo A

5 M o A

B

C

D

8 Mo D

E

F

G

14 Mo E

F

H

17 Mo E

26 Mo I

J

Mean X

n Mean X

n X

n X

n X

n Mean X

n X

n X

n X

n Mean X

n X

n X

n Mean X

n Mean X

n X

n

u

(I

u

(I

(I

(I

(I

(I

(I

u

U

(I

(I

(I

(I

460 460

34

526 556

52 (15) 513

51 (16) 5 10 50

(18) 528 49

(30) 636 63 1 61

(25) 63 1

64 (30) 620

67 (34) 672

70 (24) 690 719

88 (24) 676

99 (23) 67 1 62

(18) 786 786 103 (13) 709 737 113 (26) 675 137 (22)

(20)

. .

* One rat had glucose = 287 mg/dl; another had glucose = 213 mg/dl. * One rat had glucose = 269 mg/dl; another had glucose = 267 mg/dl. One rat had glucose = 264 mg/dl; another had glucose = 283 mg/dl. One rat had glucose = 240 mg/dl. ' One rat had glucose = 282 mg/dl. 'One rat had glucose = 358 mg/dl; another had glucose = 264 mg/dl. * One rat had glucose = 264 mg/dl; another had glucose = 283 mg/dl.

One rat had triglyceride = 422 mg/dl. ' No rat had triglyceride > 400 mg/dl.

30 1 301 26

(20) 293 330 34

(15) 270 29

(16) 286

26 (17) 292

27 (30) 354 347 36

(25) 345

57 (29) 350

36 (35) 388

52 (18)

434 436

71 (22)

427 61

439 51

(20) 492 492

87

523 512 I08 (14) 532 105 (16)

(24)

(1 1)

. .

J One rat had triglyceride = 561 mg/dl. One rat had triglyceride = 636 mg/dl; another had triglyceride = 435

One rat had glucose = 329 mg/dl. One rat had glucose = 277 mg/dl; another had glucose = 391 mg/dl. One rat had triglyceride = 527 mg/dl. One rat had glucose = 226 mg/dl. One rat had triglyceride = 423 mg/dl.

mg/dl.

* - - not measured.

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Vol. 22, No. 1, 1994 ISLET CHANGES IN RATS 51

TABLE 11.-Incidence of ketonuria.

hlales Females Age

Studv N Affected % N Affected To

3.5 Mo A

5 MO A B C D

8 Mo D E F G

14 Mo E F H

17 Mo E

26 Mo I J

5 5

35 10 10 10 5

36 10 6

10 10 35 10 15 10 6 6

47 25 22

5 5

12 6 1 2 3

16 1 3 6 6 4 2 0 2 0 0

31 19 12

I00 5 0 100 5 0 38 30 3 60 9 2 10 9 0 20 8 0 60 4 1 45 32 0 10 10 0 50 6 0 60 8 0 60 8 0 13 27 3 20 8 1 0 10 1

20 9 1

0 2 0 0 2 0

66 30 6 76 14 3 5 5 16 3

0 0

12 22 0 0

25 0 0 0 0 0

11 13 10 11 0 0

20 21 19

drogenase. Cholesterol was measured enzymatically using cholesterol esterase, cholesterol oxidase, and peroxidase.

Except for rats used in 2-year studies, urine sam- ples were collected by cystocentesis of the urinary bladder at necropsy. Urine samples were analyzed for glucose and ketones using AmesO Reagent Strips (Miles Inc. Diagnostics Division, Elkhart, IN).

Histopathology. Samples of pancreas and other selected tissues were collected at necropsy, fixed in 10% buffered formalin, processed by routine his- tologic methods, imbedded in paraffin, and sec- tioned at 5 pm. Sections were stained with hema- toxylin and eosin and Masson’s trichrome stain. Additional sections were stained for insulin by an immunoperoxidase method and then counter- stained with Masson’s trichrome stain.

RESULTS Body Weight

Rats gained weight rapidly and were visibly obese by 5 mo of age. Rats continued to gain weight well into their second year of age (Table I). Males gained weight more rapidly and were heavier than females at all ages.

Clinical Pathology Weight gain was accompanied by increased fast-

ing triglyceride and cholesterol concentrations (Ta- ble I). Triglyceride concentrations increased more than cholesterol concentrations: from 3.5 to 17 mo

TABLE 111.-Incidence of islet fibrosis.

hlales Females Age

Study N Lesion % N Lesion Yo

3.5 Mo 5 A 5

5 Mo 35 A 10 B 10 C 10 D 5

8 Mo 36 D 10 E 6 F 10 G 10

14 Mo 35 E 10 F 15 H 10

17 Mo 6 E 6

26 Mo 47 I 25 J 22

1 1 9 4 3 2 0

21 7 5 4 5

28 9

13 6 5 5

31 19 12

20 5 0 0 20 5 0 0 23 I 34 0 0 40 10 0 0 30 10 0 0 20 9 0 0 0 5 0 0

61 34 0 0 70 9 0 0 83 6 0 0 40 9 0 0 50 10 0 0 79 35 3 9 90 10 2 20 87 15 1 8 60 10 0 0 83 6 1 17 83 6 1 17 66 30 6 20 76 14 3 21 55 16 3 19

of age, triglyceride concentrations increased 3.4-fold in males and 3.1-fold in females, but cholesterol concentrations increased only 2.0-fold in males and 0.8-fold in females. By 14 mo of age, rats generally had fasting triglyceride concentrations > 200 mudl (2.2 pM).

Fasting glucose concentrations generally were slightly (<30%) greater in males than in females at all ages (Table I). Also, more males than females had glucose concentrations >200 mg/dl (1 1 mM), and several males had glucose concentrations >300 mg/dl (16.5 mM).

Glucosuria was not detected in any rat, even those with blood glucose concentrations >200 mg/dl. Ke- tonuria, however, was detected, and it was much more common in males than in females (Table 11). The incidence of ketonuria did not parallel that of obesity and hypertriglyeridemia; instead, it was highest in young males and decreased with age.

Islet Histopathology Islet changes were observed in rats as young as

3.5 mo old, and their incidence increased with age

TABLE 1V.-Incidence of islet cell neoplasia.

hlales Females N Affected %

Age

26 Mo 99 20 20 99 10 10 I 50 12 24 50 4 8

6 12 J 49 8 16 49

Study N Affected %

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52 DILLBERGER TOXICOLOGIC PATHOLOGY

FIG. 3.-Hyperplastic islet with fibrosis. Note bands of fibrous tissue dissecting islet and entrapped exocrine pan- creatic cells. H&E. ~ 3 2 0 .

(Table 111). Islet changes were much more common in males than in females at all ages; in fact, they were not observed in females younger than 14 mo old.

Islet changes began as @-cell hyperplasia causing enlarged islets (Figs. 1 and 2) with irregular borders. With time, fibrosis began in enlarged islets, gradu- ally dissecting them into discreet nests of islet cells separated by bands of collagenous tissue (Figs. 3- 5). At this stage, islets resembled cirrhotic liver. As the process continued, the ratio of fibrous tissue to islet cells increased until some islets were reduced to scattered small islands of B cells imbedded in a mass of scar tissue (Fig. 6). Accumulations of he- mosiderin-containing macrophages accompanied fi- brosis in some islets (Fig. 5) . Occasionally, islands of exocrine pancreas became entrapped within en- larged and fibrotic islets (Figs. 2 and 3). By the time rats were 26 mo old, enlarged islets were rare; in- stead, remaining islets generally were small.

Islet cell neoplasia did not occur in rats 17 mo of age or younger. In 26-mo-old rats, neoplasms were more common in males than in females (incidence

I

FIG. 4.-Hyperplastic islet with fibrosis. H&E. x 320.

of 20% vs lo%, respectively) (Table IV). One male and 2 females had carcinomas; the rest ofthe tumors were benign. All tumors were composed of insulin- positive cells (Fig. 7).

DISCUSSION Twenty-five years ago, Hajdu and Rona (10) de-

scribed islet changes in male Sprague-Dawley rats that are indistinguishable from those reported here. Coincidentally, the rats they studied came from the same source as those in this report: Charles River Laboratories. However, Hajdu and Rona detected islet changes only in males at least 40 wk (about 9.5 mo) old and did not find them in 20-wk-old rats. They suggested that the pathogenesis of the islet changes begins with an increased need for insulin, which causes compensatory @-cell hyperplasia and islet enlargement (9). With time, islet fibrosis occurs, dissecting islets into multiple discreet nodules of proliferating @ cells, a process they termed nodular regeneration. Indeed, affected islets resemble noth- ing so much as a cirrhotic liver.

Changes similar to those in Sprague-Dawley rats also occur in several other rats strains. Obese Zucker rats develop age-related @-cell hyperplasia, islet en-

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FIG. 5.-Hyperplastic islet with fibrosis. Islet is exten- sively disrupted. Note hernosiderin-containing macro- phages at islet periphery (top of figure). Immunoperoxi- dase stain for insulin with Masson's trichrome counterstain. x 305.

FIG. 6.-Fibrotic islet. Islet consists of discreet nests of /3 cells separated by dense collagenous tissue. Immuno- peroxidase stain for insulin with Masson's trichrome counterstain. x305.

FIG. 7.--Islet cell adenoma. Tumor cells are insulin- positive. Imniunoperoxidase stain for insulin with Mas- son's trichrome counterstain. x 305.

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54 DILLBERGER TOXICOLOGIC PATHOLOGY

largement, and islet fibrosis (12, 16). Islet changes do not develop in lean Zucker rats and can be at least partially reversed by diet restriction that causes weight loss in obese rats. Islet changes begin as hy- pertrophy by 5 wk of age. By 8 wk, @-cell degran- ulation is detectable. Islet fibrosis appears by 24 wk of age and increases with increasing age.

Strain eSS rats spontaneously develop a syndrome similar to non-insulin-dependent diabetes mellitus (NIDDM) as they age (6, 7).'The syndrome occurs more often in males than in females. Abnormalities in glucose tolerance develop as early as 2 mo of age. By 6 mo of age, islets are enlarged and islet structure is disrupted by fibrosis that separates islet cells into discreet nests.

Male LA/N rats (derived from the SHR strain) homozygous for the corpulent gene (cp/cp) develop age-related obesity, hyperinsulinemia, and hyper- lipidemia; normal rats (+/+) or heterozygotes (cp/ +) do not (1). Obese LA/N males develop progres- sive islet hyperplasia that begins as early as 1 mo of age. By 1 year of age, islet structure is disrupted by extensive fibrosis that separates islet cells into nests. Many fibrotic islets have hemosiderin depos- its. Some islets contain exocrine pancreatic tissue. Islets in lean males do not undergo these age-related changes.

Male WBN/Kob rats (derived from the Wistar strain) develop age-related abnormalities in glucose tolerance that progress to hyperglycemia, polyuria, and glucosuria (14). Clinical changes are accom- panied by insulitis with occasional islet hemor- rhages, hemosiderin deposition, and periductular and perivascular edema beginning as young as 3 mo of age (13). With time, islets become disrupted by extensive fibrosis, which also surrounds ducts and blood vessels.

Islet fibrosis without hyperplasia occurs in rats treated with the cytotoxic drug Busulfan and in rats that have part oftheir pancreas removed. Male Wis- tar rats fed Busulfan develop islet hemorrhages that progress to islet fibrosis (1 1). Partial pancreatectomy causes islet degeneration and fibrosis (4). Thirty days after 90% pancreatectomy in male Wistar rats, islets are small, irregularly shaped instead of rounded, and fibrotic (17).

Congenital B-cell hyperplasia with islet enlarge- ment but without fibrosis occurs in spiny mice (Aco- mys) (8). Fed ad libitum, about 50% of these mice become obese, and about a third of these develop diabetes mellitus with hyperglycemia, glucosuria, and ketonuria.

Islet fibrosis is common in people with diabetes mellitus (5 ) . In insulin-dependent diabetes mellitus, it typically occurs in pseudoatrophic islets and rep- resents scarring from insulitis. In NIDDM, it is often

associated with exocrine pancreatic fibrosis, pre- sumably due to vascular sclerosis or pancreatitis. Islet changes similar to those seen in various rat strains have not been described in people with di- abetes.

The results reported here confirm that as male Crl:CD@ (SD)BR rats age, they spontaneously de- velop metabolic abnormalities characterized by obesity, increased triglyceride concentrations, in- creased glucose concentrations, and morphologic changes in pancreatic islets. The pathogenesis of these abnormalities is obscure, but their potential impact on research studies conducted in Sprague-Dawley rats is clear. First, they suggest that male Sprague- Dawley rats fed ad libitlint are a less than ideal sys- tem in which to conduct long-term toxicity/safety assessment studies or any other research studies that could be influenced by altered metabolism. Second, they reveal that male and female Sprague-Dawley rats differ metabolically. Third, the difference in the incidence of islet cell neoplasia between males and females suggests that islet changes which occur in males predispose them to islet cell neoplasms.

Dietary restriction that reduces obesity in male Zucker rats also reduces the incidence of clinical pathologic and islet morphology changes (12), sug- gesting that the same may be true for Sprague-Daw- ley rats. Further studies are required to investigate the relationships among islet changes, serum insulin concentrations, and glucose tolerance in Crl: CD@(SD)BR rats and to evaluate the effect of diet restriction on age-related islet changes.

ACKNOWLEDGMENTS I thank Virginia Stott for her histotechnical ex-

pertise and Dave Loudy for processing and prepar- ing illustrations for this paper.

REFERENCES 1. Ahuja SK, Manickavel V, Amy RM, and Russell JC

(1987). Age-related qualitative and quantitative changes in the endocrine pancreas of the LA/N-cor- pulent rat. Diabetes Res. 6: 137-144.

2. Anver MR and Cohen BJ (1979). Lesions associated with aging. In: The Laboratory Rat, Vol. I, HJ Baker, JR Lindsey, and SH Weisbroth (eds). Academic Press, New York, pp. 377-399.

3. Committee of Care and Use of Laboratory Animals (1985). Guide for the Care and Use of Laboratory Animals. NIH Publication No. 86-23. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Insti- tutes of Health, Public Health Service, U.S. Depart- ment of Health and Human Services.

4. Friedman NB and Marble A (1941). Island hyper- plasia in the partially depancreatized rat. Endocri- nology 29: 577-582.

by guest on July 13, 2011tpx.sagepub.comDownloaded from

Vol. 22, No. 1 , 1994 ISLET CHANGES IN RATS 55

5. Gepts W and LeCompte PM (198 1). The pancreatic islets in diabetes. Am. J. Med. 70: 105-1 15.

6. Gbmez Dumm CLA, Semino MC, and Gagliardino JJ (1989). Quantitative morphological changes in en- docrine pancreas of rats with spontaneous diabetes mellitus. Virchows Arch. B Cell Pathol. 57: 375-381.

7. Gbmez Dumm CLA, Semino MC, and Gagliardino JJ (1990). Sequential morphological changes in pan- creatic islets of spontaneously diabetic rats. Pancreas 5: 533-539.

8. Gonet AE, Stauffacher W, Pictet R, Renold AE, and Mougin J (1965). Obesity and diabetes mellitus with striking congenital hyperplasia of the islets of Lan- gerhans in Spiny mice (Acoinys cahirinzcs). I. Histo- logical findings and preliminary metabolic observa- tions. Diabetologia 1: 162-1 7 1.

9. Hajdu A, Herr F, and Rona G (1968). The functional significance of a spontaneous pancreatic islet change in aged rats. Diabetologia 4: 4447.

10. Hajdu A, and Rona G ( I 967). Morphological obser- vations on spontaneous pancreatic islet changes in rats. Diabetes 16: 108-1 10.

1 1. Kaduk B, Hublein E-M, and Siegfried A (1 987). Mor- phology of the chronic toxicity of Busulfan on the islets of Langerhans in the rat. Hepato-gastroenter- ology 34: 108-1 12.

12. Lanson L-I, Boder GB, and Shaw WN (1977). Changes in the islets of Langerhans’ in the obese Zucker rat. Lab. Inrest. 36: 593-598.

13. Mori Y, Yokoyama M, Nishimura M, Kurala H, Miura J, and Ikeda Y (1990). Diabetic strain (WBN/ Kob) of rat characterized by endocrineexocrine pan- creatic impairment due to dist-inct fibrosis. Pancreas 5: 452459.

14. Nakama K, Shichinohe K, Kobayashi K, Naito K, Uchida 0, Yasuhara K, and Tobe M (1985). Spon- taneous diabetes-like syndrome in WBN/KOB rats. Acta Diabetol. Lat. 22: 335-342.

15. Robbins SL, Cotran RS, and Kumar V (1 984). Patho- logic Basis of Disease, 3rd ed. W. B. Saunders Com- pany, Philadelphia, PA, pp. 972-986.

16. Shino A, Matsuo T, Iwatsuka H, and Suzuoki Z (1 973). Structural changes of pancreatic islets in genetically obese rats. Diabetologia 9: 4 1 3 4 2 1.

17. Yonemura Y, Takashima T, Matsuda Y, Miwa K, Sugiyama K, Miyazaki I, Yamamoto H, and Oka- moto H (1988). Induction of islet B-cell regeneration in partially pancreatectomized rats by poly(ADP-ri- bose) synthetase inhibitors. Int. J. Pancreatol. 3: 73- 82.

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